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Testing for Phytochemical Synergism: Arthropod
Community Responses to Induced Plant Volatile
Blends Across Crops
Joseph Braasch &Gina M. Wimp &Ian Kaplan
Received: 16 May 2012 /Revised: 12 September 2012 /Accepted: 17 September 2012 /Published online: 23 October 2012
#Springer Science+Business Media New York 2012
Abstract Using herbivore-induced plant volatiles (HIPVs)
to attract specific natural enemies in the field has proven
challenging, partly because of a poor understanding of: (i)
which compound(s) to manipulate to attract specific taxa,
and (ii) the ecological conditions over which HIPVs are
effective. To address these issues, we quantified the re-
sponse of a complex arthropod community to three common
HIPVs (methyl salicylate, cis-3-hexen-1-ol, and phenylethyl
alcohol) as individual compounds and equal part blends in
corn and soybean fields. Of 119 arthropod taxa surveyed,
we found significant responses by four species in corn fields
(2 parasitoids, 1 herbivore, and 1 detritivore) and 16 in
soybean fields (8 parasitoids, 3 predators, 4 herbivores,
and 1 detritivore), with both attractive and repellent effects
of the HIPVs observed. For example, tachinid flies were
highly attracted to cis-3-hexen-1-ol (ca. 3-fold increase), but
repelled by methyl salicylate (ca. 60 % decrease).
Surprisingly, we found very few cases in which HIPVs acted
synergistically; only two arthropod groups (ichneumonid
wasps and phorid flies) were more attracted by a blend of
the HIPVs than by the individual compounds composing the
blend. Crop type, however, had a strong impact on the
strength of arthropod responses to HIPVs. A few arthropod
species were broadly affected across both crops (i.e., the
herbivore Halticus bractatus was repelled by most of our
treatments, regardless of crop background), but overall more
arthropod groups responded to HIPVs released in soybean
fields compared with corn. This was true despite the fact that
taxa responding to HIPVs were present and abundant in
both systems, suggesting that crop-based outcomes were
likely driven by the plant matrix rather than mere differences
in taxonomic composition of the arthropod community in
corn vs. soybean fields. As a whole, these results suggest
that: (i) repellent effects of HIPVs on natural enemies of
herbivorous insects can be observed as frequently as attrac-
tive effects; (ii) odor blends may be no more effective than
single-compound lures for some taxa; and (iii) crop back-
ground alters the magnitude of attraction to HIPVs, depend-
ing on the species being targeted.
Keywords HIPV .Herbivore induced plant volatile .Methyl
salicylate .Natural enemies
Introduction
When fed upon by herbivorous arthropods, plants undergo
physiological changes that result in the activation of bio-
chemical pathways associated with antiherbivore defense
(Levin, 1976; Karban and Baldwin, 1997), including the
emission of volatile compounds (Vet and Dicke, 1992).
These herbivore-induced plant volatiles (HIPVs) then are
utilized by predaceous and parasitic arthropods to locate
herbivore-infested plants (Turlings et al., 1990;Thaler,
1999; Kessler and Baldwin, 2001). Because these com-
pounds attract carnivores, HIPVs have long been viewed
as an ideal tool for modifying the behavior and colonization
time of beneficial arthropods in agricultural fields (Turlings
and Ton, 2006; Khan et al., 2008; Kaplan, 2012). Efforts are
now underway to genetically modify crops that release more
attractive odor blends (Kappers et al., 2005; Degenhardt et
Electronic supplementary material The online version of this article
(doi:10.1007/s10886-012-0202-y) contains supplementary material,
which is available to authorized users.
J. Braasch (*):I. Kaplan
Department of Entomology, Purdue University,
901 West State Street,
West Lafayette, IN 47907, USA
e-mail: jbraasch@purdue.edu
G. M. Wimp
Department of Biology, Georgetown University,
Washington, DC 20057, USA
J Chem Ecol (2012) 38:1264–1275
DOI 10.1007/s10886-012-0202-y
al., 2009), but most current studies manipulate volatile com-
position in the field with synthetically produced compounds
emitted from controlled-release dispensers. To date, synthet-
ic HIPVs have been deployed in a range of agricultural
systems including, but not limited to, hops (James, 2003a),
grapes (James and Price, 2004), cranberries (Rodriguez-
Saona et al., 2011), soybean (Zhu and Park, 2005;
Mallinger et al., 2011), turnip (Orre et al., 2010), apples
(Jones et al., 2010), strawberries (Lee, 2010), and broccoli
(Simpson et al., 2011).
Initial studies that measured the effects of HIPV deploy-
ment on natural enemy recruitment focused on commonly
emitted volatiles (Flint et al., 1979; James, 2003a), and were
further propelled by early field experiments showing broad
scale attraction to a few notable compounds such as methyl
salicylate (hereafter, MeSA), indole, cis-3-hexen-1-ol, cis-3-
hexenyl acetate, and geraniol (James, 2005). In these experi-
ments, each volatile was considered individually to evaluate
which exerts the strongest ‘pull’on predators and parasi-
toids (James, 2003a,b;Yuetal.,2008; Simpson et al.,
2011). Notably, these single-compound manipulations dra-
matically differ from insect-damaged plants, which almost
always release a complex and highly specific odor blend
consisting of multiple HIPVs. This has led to speculation
that blends of plant volatile compounds consisting of two or
more chemicals may be preferable to the standard single-
compound lures that dominate the empirical literature on
this topic (Szendrei and Rodriguez-Saona, 2010; Kaplan,
2012). In their meta-analysis of arthropod responses to
volatiles, Szendrei and Rodriguez-Saona (2010) found that
individual volatiles were attractive overall, but increased
blend complexity corresponded with stronger attraction.
For example, Jones et al., (2010) increased the attractiveness
of iridodial through the addition of MeSA, doubling the
number of lacewings captured. Tóth et al. (2009) obtained
similar results for lacewings by adding MeSA to a blend of
phenylacetaldehyde and acetic acid. In both cases, MeSA
acted synergistically since MeSA addition increased trap
catch but was itself unattractive.
A second factor expected to mediate arthropod attraction
to HIPVs in agricultural landscapes is the crop matrix in
which lures are embedded. In cotton fields, for example,
Flint et al. (1979) found that initial attraction of chrysopids
to synthetic caryophyllene waned as the plants grew and
produced the compound themselves, thus reducing the abil-
ity of insects to differentiate lure from crop. Similarly,
attraction of the wasp, Closterocerus ruforum,to(E)-β-
farnesene depended on background pine volatiles, with no
attraction occurring when the volatile was presented alone
(Beyaert et al., 2010). Recent work also suggests that syn-
thetic HIPVs signal to neighboring plants (Rodriguez-Saona
et al., 2011; von Merey et al., 2011), and thus crop background
does not simply mask the lure; surrounding plants facilitate
attraction. This interplay between crop volatiles and lure com-
position may also explain why synthetic HIPVs are attractive
in certain studies but not others, despite working with the
same species complex (see ‘context-dependent responses’—
Kaplan, 2012). Because the vast majority of field studies test
responses to volatiles in only one crop, however, the contri-
bution of the plant matrix cannot be directly evaluated.
To better understand predatory and parasitic natural ene-
my responses to plant volatiles in the field, we manipulated
HIPV diversity in a factorial design experiment across two
crop systems (corn and soybean). Specifically, we: (1) com-
pared arthropod attraction to three HIPVs; (2) tested for
response additivity when HIPVs are sequentially added to
a complex odor blend from individual compounds; and (3)
assessed the role of crop background in mediating the
strength of attraction to lures.
Methods
Volatiles and Release Devices
We tested three HIPVs: (i) MeSA, (ii) phenylethyl alcohol
(syn. 2-phenylethanol), and (iii) cis-3-hexen-1-ol. These
compounds are emitted by corn and soybean plants, and
thus are ecologically relevant compounds shared by our
two crop systems (Buttery and Ling, 1984; Takabayashi et
al., 1995; Turlings et al., 1998; Zhu et al., 1999; Damiani et
al., 2000; Engelberth et al., 2004; van den Boom et al.,
2004; Zhu and Park, 2005; Rostas and Eggert, 2008). For
example, MeSA is a key volatile released from soybean
upon feeding by soybean aphid, Aphis glycines, which sub-
sequently attracts predaceous lady beetles (Zhu and Park,
2005). Similarly, corn plants rapidly emit cis-3-hexen-1-ol
when leaves are chewed by beet armyworm larvae
(Engelberth et al., 2004). In addition to being common to
corn and soybean, there is ample evidence linking these
three chemicals with physiological and behavioral responses
in several important natural enemy groups (see below).
Methyl Salicylate and phenylethyl alcohol are shikimic
acid pathway derived aromatic organic compounds (Mann,
1987; Croteau and Karp, 1991), and are common compo-
nents of leaf and/or floral odors (Honda et al., 1998). Methyl
salicylate emission is inducible by sap-feeding (e.g., aphids,
mites) and chewing (e.g., caterpillars, beetles) herbivores in
a wide diversity of plants ranging from grasses to trees
(Bolter et al., 1997;Scutareanuetal.,1997,2003;
Agrawal et al., 2002; van den Boom et al., 2004; Lou et
al., 2006; Zhu and Park, 2005; Kigathi et al., 2009).
Phenylethyl alcohol also is a component of insect-
damaged leaf odors in a diversity of plants including mul-
berry (Tanaka et al., 2009), potato (Weissbecker et al.,
2000), and pear (Scutareanu et al., 2003). Notably, both
J Chem Ecol (2012) 38:1264–1275 1265
volatiles constitute the active ingredients for commercially
marketed carnivore attractants (MeSA: Predalure, AgBio Inc.,
Westminster, CO, USA; phenylethyl alcohol: Benallure,
MSRTS Technologies, Ames, IA, USA). A recent meta-
analysis documented the broad-scale attractive properties of
MeSA in the field to numerous entomophagous arthropods
(Rodriguez-Saona et al., 2011). Phenylethyl alcohol also is
known to elicit behavioral and electrophysiological responses
by several key predator groups such as lacewings, coccinell-
ids, and syrphids (Zhu et al., 1999; Zhu and Park, 2005). The
third compound, cis-3-hexen-1-ol, is a green leaf volatile
known to attract braconids, syrphids, Anagrus daanei
(Mymaridae), Stethorus punctum picipes (Coccinellidae),
Orius tristicolor (Anthocoridae), Macrocentrus linearis
(Braconidae), and Anaphes iole (Mymaridae) (James, 2005;
Williams et al., 2008;Yuetal.,2008). Since cis-3-hexen-1-ol
is rapidly released after chewing damage to the leaves of both
crops, we anticipated that it would act synergistically with
MeSA, which is emitted later in the induction process
(Engelberth et al., 2004).
Release devices were constructed from capped, UV-
resistant polyethylene vials (1 ml) (Wheaton Science
Products, Millville, NJ, USA), containing 1 ml of neat
compound(s) per vial (Sigma-Aldrich, St. Louis, MO,
USA). To measure release rates of volatiles, we
deployed single compounds and all blends (described
below) in a soybean field from July 2nd until August
1st. Release devices were returned to the lab and
weighed weekly to estimate mass loss per unit time.
Field Experiment
Arthropod responses to MeSA, phenylethyl alcohol, and cis-
3-hexen-1-ol were tested in corn and soybean fields during
the 2010 growing season. Treatments consisted of a blank
control (i.e., a polyethylene vial with no HIPV) and each of
the three HIPVs in all possible one, two, or three equal part
blends (e.g., 0.5 ml MeSA and 0.5 ml cis-3-hexen-1-ol), for
8 total treatments.
The experiment was performed at the Meigs-Purdue
Agricultural Center in Tippecanoe County, Lafayette,
IN, USA. Six fields were selected, 3 in corn and 3 in
soybean, with each field <3 km apart and possessing at
least one edge bordering a wooded area to serve as a
source for overwintering parasitic and predatory arthro-
pods. All fields received similar herbicide treatments,
and no post-planting insecticides were applied. Corn
fields were planted during the 3rd and 4th weeks of
April, while soybean fields were planted in the 2nd and
4th weeks of June.
In each field, release devices were placed along two
transects; one 5 m into the field along the woodlot border,
the second 50 m into the field, parallel to the first. Each
transect consisted of 8 trapping locations and is thus con-
sidered one complete experimental replicate of the above-
described HIPV manipulations. Volatile treatments were
randomly assigned along transects, separated by at least
5 m. Release devices were attached to the top of a 1 m
bamboo stake along with a 3×5 in. yellow sticky card
to capture arthropods. Sticky cards were collected and
replaced once a week for the duration of the experi-
ment; June 9th through September 7th in corn, June
25th through September 10th in soybean. All arthro-
pods, excluding some selected taxa (e.g., Nematoceran
dipterans), were counted from each card and identified
at least to family or “morphospecies”based on morpho-
logical differences between individuals, an appropriate
analogue for species when gathering community data
(Oliver and Beattie, 1996).
When more than one treatment was missing from a
site as the result of destructive anthropogenic (e.g.,
tractors) or environmental factors (e.g., wind/rain), all
treatments for that site and week were excluded from
the analysis. If only one treatment was absent, however,
data for the missing card were estimated as the means
from all other treatments for that site on that date. This
is considered to be a conservative method for retaining
observations, as it decreases the variability that might be
explained by any one treatment. Data were summed
across weeks for each arthropod group and divided by
the total number of dates for which cards were collected
to obtain the average catch per sticky card per week,
the dependent variable used in our analyses.
Arthropod abundance data were square root trans-
formed to meet parametric assumptions, and analyzed
using a customized general linear model (GLM) (SAS,
Version 9.2). Each of the three HIPVs was used as a
predictor variable, and we tested for the main effect of
each compound, along with statistical interactions be-
tween compounds. Non-significant interactions were se-
quentially removed from our model starting with the
highest order 3-way interaction (i.e., MeSA × phenylethyl
alcohol × cis-3-hexen-1-ol), followed by 2-way interac-
tions. Field site and transect were included as random
effect variables. We analyzed corn and soybean arthropods
separately due to differences in collection dates. When a
significant HIPV effect was detected, the model was re-run
for that taxon as a univariate ANOVA with a post-hoc
Tukey’s HSD test, using HIPV blend as a single treatment
factor to identify pairwise differences.
Effects that were significant in the initial GLM were
subsequently used to evaluate additive vs. non-additive
responses. The mean abundance value of each odor blend
for each arthropod was calculated with 95 % confidence
intervals and compared with the average value for the
individual components of the blend, which represents the
1266 J Chem Ecol (2012) 38:1264–1275
expected additive result of blending HIPVs. For example,
to test the additivity of wasp attraction to our 3-part
blend, the average response to the three individual
HIPV treatments was estimated as the expected additive
model. The observed response to the 3-part blend was
considered to be significantly higher, and thus synergis-
tic, if the 95 % confidence interval did not bracket this
expected additive mean (see Fig. 4b).
In addition to impacts on natural enemy abundance,
differences in volatile compounds may also alter the
species composition of natural enemies. In particular,
we examined the effects of volatile compounds and crop
type (corn or soybean) on the composition of
Hymenopteran parasitoids using NMDS (Non-Metric
Multidimensional Scaling), which is a robust ordination
technique for community data (Minchin, 1987). We used
the average abundance of each taxon across all time
periods for our analysis, so time was not included as
a factor. By averaging across time periods, we were
able to examine patterns of species assortment among
our treatments that were robust to seasonal changes in
species abundance through time. We used the ordination
program DECODA (Database for Ecological Community
Data, Minchin, 2001) to create a dissimilarity matrix
among volatile treatments and crop types using the
Bray-Curtis dissimilarity coefficient (Faith et al., 1987),
andthentestedfordifferencesincommunitycomposi-
tion among treatments and crop types using ANOSIM
(Analysis of Similarity), which uses 1,000 random reas-
signments of species to groups and determines if the
generated dissimilarity matrix is significantly different
than chance (Warwick et al., 1990)
Results
Release Rates
Of the three volatiles, MeSA released at the highest rate,
37.4 mg/week, with cis-3-hexen-1-ol and phenylethyl alco-
hol releasing slower, 2.2 and 0.3 mg/week, respectively. All
release rates were relatively constant for the duration of the
experiment, allowing us to compare across mixtures con-
taining different volumes (Supplementary Fig. 1).
Field Summary
Over the sampling period, we collected in total 271,335
arthropods that were identified to 119 different taxonomic
groups, 113 of which were collected in both corn and
soybean fields (Tables S1, herbivores and S2, natural ene-
mies). Fifty-nine of these groups were Hymenoptera, with
46 classified as microhymenopteran morphospecies
(Table S2). Rare taxa (i.e., those with <50 total individuals
in corn and soybean fields combined) were not statistically
analyzed in the GLM due to low sample size. In total, 63
groups passed this threshold and thus were used in the
analysis.
In corn fields, four arthropod groups responded to HIPVs
(Table 1); two carnivorous taxa (Ichneumonidae and
Sarcophagidae), one herbivore species (Halticus bractatus;
Hemiptera: Miridae), and one detritivorous species
(Phoridae; represented by Gymnophora luteiventris,
Megaselia sp., and Metopina sp.). Sarcophagids (Fig. 1b)
and H. bractatus (Fig. 1d) were generally repelled by
HIPVs. The blend of MeSA and cis-3-hexen-1-ol repelled
sarcophagids, while all treatments except MeSA + phenyl-
ethyl alcohol repelled H. bractatus. Methyl salicylate was
broadly attractive to both ichneumonids (Fig. 1a) and phor-
ids (Fig. 1c) (i.e., compare abundance in the four ‘M’-
marked bars vs. the four ‘M’-free bars).
In soybean fields, sixteen arthropod groups responded to
HIPVs (Table 2)—11 carnivorous taxa (Ichneumonidae,
Mordellidae, Tachinidae, Thripidae, Orius insidiosus, cera-
phronid morphospecies A, encyrtid morphospecies A, and
four unidentified Hymenopteran morphospecies), four her-
bivorous taxa (Cynipidae, Derbidae, Thripidae, and the
mirid H. bractatus), and detritivorous phorid flies.
Ceraphronid A, cynipids, derbids, herbivorous thrips,
encyrtid A, ichneumonids, tachinids, mordellids, phorids,
and O. insidiosus generally were attracted to at least some
of the HIPV treatments, while predatory thrips, all
Hymenopteran morphospecies, and H. bractatus were re-
pelled by one or more of the volatiles. Methyl salicylate
Table 1 Significance (P-values)
of arthropod taxa showing at-
traction to methyl salicylate
(MS), cis-3-hexen-1-ol (HEX),
phenylethyl alcohol (PA), and all
possible equal part blends in
corn fields. Bold values indicate
significant effects (p<0.05)
a
Significant site effect
Taxa MS HEX PA MS*HEX MS*PA PA*HEX MS*PA*HEX
Halticus bractatus
a
0.081 0.033 0.793 0.649 0.408 0.341 0.018
Ichneumonidae <0.001 0.760 0.167 n.s. n.s. n.s. n.s.
Phoridae
a
<0.001 0.396 0.970 n.s. n.s. n.s. n.s.
Sarcophagidae
a
0.074 0.377 0.948 0.321 0.599 0.046 n.s.
Predator total 0.146 0.797 0.665 n.s. n.s. n.s. n.s.
Parasitoid total 0.568 0.227 0.278 n.s. n.s. n.s. n.s.
Natural enemies 0.353 0.287 0.341 n.s. n.s. n.s. n.s.
J Chem Ecol (2012) 38:1264–1275 1267
was broadly attractive to encyrtid A (Fig. 2b) and phorids
(Fig. 2j), but repelled tachinid flies (Fig. 2i), Hymenoptera
C (figure not shown, MeSA absent00.116± 0.028, MeSA
present00.043± 0.022), Hymenoptera AC (Fig. 2d), and
predatory thrips (Fig. 2h). Phenylethyl alcohol was attrac-
tive to derbids (Fig. 3b) and mordellids as a single-
compound when compared in post hoc analysis (control 0
0.113±0.075, phenylethyl alcohol00.339±0.124), but
unattractive to herbivorous thrips as part of a blend
(Fig. 3c). Cis-3-hexen-1-ol was only attractive to tachinids
(Fig. 2i), an effect that was revealed in post hoc analysis
and not the original model. Attraction to the blend of all
three volatiles was found for ceraphronid A (Fig. 2a) and
ichneumonids (Fig. 2c) in the original model, while post
hoc analysis showed attraction of O. insidiosus (Fig. 2g)to
the blend of cis-3-hexen-1-ol and phenylethyl alcohol,
Ichneumonidae (no./sample)
0.0
0.2
0.4
0.6
HP HPMHMMPMHP
b
a
ab
ab
ab
ab
ab
ab
A
Sarcophagidae (no. /sample)
0.0
1.0
2.0
3.0
MHP MPMH HP MHP
a
a
b
ab
ab
ab ab
ab
B
Phoridae (no./sample)
0.0
2.0
4.0
6.0
8.0
MHP MPMH MHP
c
a
ab
ab
a
bc
c
bc
HP
C
H. bractatus (no./sample)
0
4
8
12
MHP MPMH HP MHP
a
b
bb
b
b
ab
b
D
Fig. 1 Response of arthropod
taxa (mean ± SE) to HIPVs in
corn fields, including a
ichneumonid wasps, b
sarcophagid flies, cphorid flies,
and dthe mirid, Halticus
bractatus. Bars are shaded
according to the number of
HIPVs in the blend, with no
HIPVs (control) in white and a
blend of three volatiles shaded
darkest. M 0methyl salicylate,
H0cis-3-hexen-1-ol, P 0phe-
nylethyl alcohol. Letters above
means represent significant dif-
ferences between treatments
determined by univariate
ANOVA
Table 2 Significance (P-values) of arthropod taxa showing attraction to methyl salicylate (MS), cis-3-hexen-1-ol (HEX), phenylethyl alcohol (PA),
and all possible equal part blends in soybean fields. Bold values indicate significant effects (P<0.05)
Taxa MS HEX PA MS*HEX MS*PA PA*HEX MS*PA*HEX
Ceraphronidae –A
a
0.760 0.351 0.940 0.732 0.607 0.936 0.016
Cynipidae
a
0.016 0.024 0.033 n.s. n.s. 0.020 n.s.
Derbidae
a
0.307 0.556 0.047 n.s. n.s. n.s. n.s.
Encyrtidae A 0.004 0.614 0.80667 n.s. n.s. n.s. n.s.
Halticus bractatus
a
0.064 0.546 0.215 n.s. 0.027 n.s. n.s.
Hymenoptera C
a
0.027 0.831 0.084 n.s. n.s. n.s. n.s.
Hymenoptera AC
a
0.004 0.212 0.334 0.258 0.408 0.580 0.006
Hymenoptera AF 0.619 0.492 0.876 0.646 0.285 0.751 0.029
Hymenoptera AQ 0.883 0.391 0.880 0.021 n.s. n.s. n.s.
Ichneumonidae 0.732 0.927 0.144 0.711 0.509 0.584 0.040
Mordellidae
a
0.987 0.096 0.488 0.592 0.128 0.229 0.021
Orius insidiosus
a
0.996 0.649 0.599 0.012 n.s. n.s. n.s.
Phoridae
a
<0.001 0.729 0.140 n.s. n.s. n.s. n.s.
Tachinidae
a
0.039 0.041 0.688 n.s. n.s. n.s. n.s.
Thysanoptera, herbivorous
a
0.868 0.211 0.047 n.s. n.s. n.s. n.s.
Thysanoptera, predatory
a
0.012 0.477 0.244 n.s. n.s. n.s. n.s.
Predator Total 0.239 0.530 0.722 n.s. n.s. n.s. n.s.
Parasitoid Total 0.260 0.647 0.903 n.s. n.s. n.s. n.s.
Natural enemies 0.303 0.558 0.943 n.s. n.s. n.s. n.s.
a
Significant site effect
1268 J Chem Ecol (2012) 38:1264–1275
cynipids to the blend of MeSA and phenylethyl alcohol
(Fig. 3a), and a repellent effect of all two part blends and
MeSA alone on H. bractatus (Fig. 3d).
None of the treatments affected the pooled response of all
predators, parasitoids, or both (predators + parasitoids 0nat-
ural enemies of herbivores) in either crop system (Tables 1and
2). Of the 33 total significant effects detected, 19 were main
effects of a single volatile, whereas 14 were interactive effects
involving two or more compounds. Moreover, MeSA was the
most influential of the HIPVs, being involved in 23 of the 33
significant effects, followed by cis-3-hexen-1-ol (16 effects)
and phenylethyl alcohol (14 effects).
Synergism and Antagonism of Volatile Blends
Only six arthropod taxa demonstrated non-additive responses
to volatile blends, with three groups showing synergism
(Fig. 4a, b, f) and three showing antagonism (Fig. 4c, d,e).
Ceraphronid A (no./sample)
0.0
1.0
2.0
3.0
MHP MPMH HP MHP
b
b
ab ab
aaa
a
Encyrtid A (no./sample)
0.0
1.0
2.0
3.0
MHP MPMH HP MHP
a
a
a
a
a
a
a
a
Ichneumonidae (no./sample)
0.0
0.1
0.2
0.3
0.4
MHP MPMH HP MHP
b
a
a
a
ab ab
ab
ab
Hymenoptera AC (no./sample)
0.0
1.0
2.0
3.0
MHP MPMH HP MHP
a
bb
ab ab
ab
ab ab
Hymenoptera AF (no./sample)
0.0
0.2
0.4
MHP MPMH HP MHP
b
a
ab
ab ab
ab
ab
ab
Hymenoptera AQ (no./sample)
0.0
0.2
0.4
0.6
MHP MPMH HP MHP
b
aa
a
ab
ab
ab
ab
O. insidiosus (no./sample)
0.0
2.0
4.0
MH P MPMH HP MHP
b
a
ab
ab
ab
ab
ab
ab
Predatory Thrips (no./sample)
0.0
0.2
0.4
0.6
MHP MPMH HP MHP
a
b
b
ab
ab
ab ab
ab
Tachinidae (no./sample)
0.0
1.0
2.0
MHP MPMH HP MHP
a
c
a
b
a
aa
a
Phoridae (no./sample)
0
5
10
15
MHP MPMH HP MHP
bc
a
abc
c
ab
abc
aa
A
CD
EF
G
J
I
H
B
Fig. 2 Response of predatory
and parasitic arthropods to
HIPVs in soybean fields,
including aceraphronid A, b
encyrtid A, cichneumonid
wasps, dHymenoptera AC, e
Hymenoptera AF, f
Hymenoptera AQ, gOrius
insidiosus,hthrips, itachinid
flies, and jphorid flies. Bars are
shaded according to the number
of HIPVs in the blend, with no
HIPVs (control) in white and a
blend of three volatiles shaded
darkest. M 0methyl salicylate,
H0cis-3-hexen-1-ol, P 0phe-
nylethyl alcohol. Letters above
means represent significant dif-
ferences between treatments
determined by univariate
ANOVA
J Chem Ecol (2012) 38:1264–1275 1269
Surprisingly, ichneumonid wasps in corn fields displayed two
of the three synergistic responses, whereas mordellids in soy-
bean fields displayed two of the three antagonistic responses.
Only one of the six cases (Fig. 4b), involved an emergent
outcome of the three-part blend; the remainder are two-part
HIPV interactions.
Effects of Crop System
In total, three taxa responded to HIPV deployment in both
corn and soybean systems; phorid flies, ichneumonid wasps,
and H. bractatus. Phorids were consistently attracted to
MeSA in both crops (Figs. 1c and 2j), while H. bractatus
Cynipidae (no./sample)
0.0
1.0
2.0
MHP MPMH HP MHP
b
ab ab ab
aa
aa
Derbidae (no./sample)
0
2
4
MHP MPMH HP MHP
b
a
a
ab
ab ab
ab ab
Herbivorous Thrips (no./sample)
0
40
80
120
160
M H MH HP MHPMPP
a
b
ab
ab
ab
ab
ab
ab
H. bractatus (no./sample)
1
2
3
4
MHP MPMH HP MHP
a
ab
ab
ab
bbb
b
A
DC
B
Fig. 3 Response of
herbivorous arthropods to
HIPVs in soybean fields,
including aCynipidae, b
Derbidae, cThysanoptera, and
dHalticus bractatus. Bars are
shaded according to the number
of HIPVs in the blend, with no
HIPVs (control) in white and a
blend of three volatiles shaded
darkest. M 0methyl salicylate,
H0cis-3-hexen-1-ol, P 0phe-
nylethyl alcohol. Letters above
means represent significant dif-
ferences between treatments
determined by univariate
ANOVA
CMHMH
Ichneumonidae (sqrt no./sample)
0.6
0.7
0.8
0.9
1.0
CMHPMHP
Ichneumonidae (sqrt no./sample)
0.6
0.8
1.0
CMPMP
Hymenoptera AC (sqrt no./sample)
0.8
1.2
1.6
CMPMP
Mordellidae (sqrt no./sample)
0.6
0.7
0.8
0.9
CHPHP
Mordellidae (sqrt no./sample)
0.6
0.8
1.0
CMPMP
Phoridae (sqrt no./sample)
2
3
4
A
CD
EF
B
Fig. 4 Arthropod taxa that
showed a non-additive (syner-
gistic or antagonistic) response
to the deployment of volatile
blends; aIchneumonidae to
methyl salicylate and cis-3-
hexen-1-ol in corn, bIchneu-
monidae to methyl salicylate,
cis-3-hexen-1-ol, and phenyl-
ethyl alcohol in corn, cHyme-
noptera AC to methyl salicylate
and phenylethyl alcohol in soy-
bean, dmordellids to methyl
salicylate and phenylethyl alco-
hol in soybean, emordellids to
cis-3-hexen-1-ol and phenyl-
ethyl alcohol in soybean, and f
phorids to methyl salicylate and
phenylethyl alcohol in soybean.
The dashed reference line
denotes the expected response
to the blend, calculated as the
average of the blend compo-
nents when tested individually.
Error bars indicate 95 % con-
fidence intervals. C 0HIPV-
free control, M 0methyl salic-
ylate, H 0cis-3-hexen-1-ol, P 0
phenylethyl alcohol
1270 J Chem Ecol (2012) 38:1264–1275
was consistently repelled by all HIPVs and HIPV blends across
the two systems (Figs. 1d and 3d). In contrast, while ichneu-
monid wasps were attracted to the three-part odor blend in corn
and soybean fields (Figs. 1b and 2c), they also demonstrated
system-specific responses; namely, strong attraction to MeSA
in corn (main effect of MeSA, P<0.001) but no corresponding
response in soybean (main effect of MeSA, P00.732).
We also found significant differences in the composition of
Hymenopteran parasitoids present in corn and soybean fields
(ANOSIM R00.723, P<0.001; Fig. 5). While the composi-
tion of parasitoid communities was different between the two
crop types, we did not find any significant differences in the
response of these parasitoids to the different volatile treat-
ments (ANOSIM R00.0725, P00.999; Fig. 5).
Discussion
Previous studies have identified the need for more field-
based and mechanistic approaches to understand natural
enemy responses to plant volatiles (Hunter, 2002). Our work
builds on recent experiments by: (i) evaluating responses at
the community level, including non-target effects; (ii) teas-
ing apart differences between single-compounds vs. odor
blends; and (iii) quantifying the importance of the plant
matrix in mediating attraction to HIPVs.
Community-Scale Patterns
Although most published HIPV field studies quantify the
response of more than one arthropod group, the majority of
‘community’experiments only report the outcome for 10 to
20 taxa. To our knowledge, the 119 groups identified here is
the most extensive community tested to date. Of the 63
arthropods statistically analyzed, 10 taxa (15.9 % of the
community) responded to at least one of the HIPV treat-
ments, with MeSA eliciting the greatest number of
responses, which may have resulted from its substantially
greater release rate. None of the treatments, however, in-
duced responses when individuals were pooled into broader
functional groups, i.e., natural enemies of herbivorous
insects (parasitoids, predators). Thus, our results stand in
contrast with other studies that have found broadly attractive
community-level effects of HIPV deployment, particularly
those performed with MeSA (James, 2005; Rodriguez-
Saona et al., 2011) including in soybean fields (Mallinger
et al., 2011). This was unexpected due to similarities in
trapping techniques (i.e., yellow sticky cards) and release
rates of the focal compounds. Some of these discrepancies
likely are driven by differences in taxonomic composition
across study systems and sites. For example, two of the
dominant predators in the above mentioned studies that
displayed notably strong attractive responses to MeSA were
hoverflies (Syrphidae) and lacewings (Chrysopidae), two
groups that were rare in our samples.
Our findings further deviate from other field experiments
in that certain natural enemies of herbivores were repelled
by volatiles (studies typically report either attraction or no
response), an effect most apparent in two microhymenop-
teran morphospecies (Figs. 2e, f). Virtually all field HIPV
manipulations treat parasitic wasps as a single unit or over-
look all but one or two select species (but see James and
NMDS Axis 2
NMDS Axis 1
Corn Control
Corn MeSa
Corn Cis-3
Corn PhAl
Corn Mixture
Soybean Control
Soybean MeSa
Soybean Cis-3
Soybean PhAl
Soybean Mixture
Fig. 5 Composition of the
Hymenopteran parasitoid
communities found in corn and
soybean fields across five
different volatile compound
treatments. The composition of
the hymenopteran communities
differed significantly between
crop types, but not among
volatile treatments.
Compositional dissimilarity
was based on 54 parasitoid
morphospecies. Shown is the
two-dimensional representation
of the parasitoid community
found on 60 different crop/vol-
atile treatments. Solid shapes
represent samples from corn,
while open shapes represent
soybean samples. Volatile treat-
ment abbreviations: MeSa 0
methyl salicylate; Cis-3 0cis-3-
hexen-1-ol; PhAl 0phenylethyl
alcohol; Mixture 0mixture of
all three volatiles
J Chem Ecol (2012) 38:1264–1275 1271
Grasswitz, 2005). By examining this highly diverse group at
a finer taxonomic scale, species- or family-specific
responses can be teased apart. Simpson et al. (2011) ana-
lyzed responses of microhymenopteran families in vine-
yards and, and as with our study, observed repellent effects
at finer taxonomic levels, which would have gone over-
looked had hymenopterans been analyzed as a group. A
recent meta-analysis of predator and parasitoid responses
to MeSA found that attraction of parasitic wasps was less
pronounced than attraction of predators, suggesting that
parasitoids may be less amenable to HIPV manipulations
than predaceous arthropods (Rodriguez-Saona et al., 2011).
Laboratory experiments also show that inclusion of MeSA
to an otherwise attractive HIPV blend can weaken responses
by parasitic wasps (Snoeren et al., 2010; Pierre et al., 2011).
We strongly emphasize, however, that making broad gener-
alizations about specioise taxa like Hymenopterans is bound
to fail. Although several of the morphospecies noted above
were repelled, two of the most distinct attractive responses
in the entire community were that of ichneumonid and
encyrtid wasps to MeSA (Figs. 1a,2b, respectively).
Similar to predators and parasitoids, non-target herbi-
vores also displayed a combination of attractant and repel-
lent responses to our treatments. Gall wasps (Cynipidae;
Fig. 3a) and planthoppers (Derbidae; Fig. 3b) were attracted
to several treatments in soybean; however, neither group is a
known pest of this system, and it is likely that both are
instead exploiting resources provided by weedy plants with-
in or adjacent to the fields. Two polyphagous herbivores and
potential consumers of crop plants, thrips and the mirid H.
bractatus, were repelled by volatiles, an encouraging out-
come, given that HIPVs are hypothesized to be attractive to
some herbivores because of increased plant apparency
(Dickens, 2000; Halitschke et al., 2008; Dicke, 2009). The
response by H.bractatus was particularly impressive be-
cause of its magnitude and the fact that it occurred in
response to all treatments in both crops (Figs. 1d,3d).
One of the strongest and most consistent responses across
the community came from a detritivore (phorid flies) rather
than an herbivore or carnivore; phorids were highly attracted
to MeSA in corn and soybean fields. Although much of their
general ecology and foraging behavior are unknown, adults
are sugar feeders (Chen et al., 2005), and members of the
genus Megaselia have been observed pollinating inflores-
cences of Artolochia littoralis in Florida (Hall and Brown,
1993). Thus, we speculate that responses of phorids to
MeSA are a consequence of sugar-limited adult flies search-
ing for a nectar meal, as MeSA is also a common component
of floral blends in addition to acting as an HIPV (Honda et
al., 1998). Given the paucity of flowering plants in and
around field crops at our study site, this response is likely
either innate, or adults are moving long distances between
crop and non-crop habitats. Furthermore, this outcome
reinforces the danger of viewing HIPVs such as MeSA
purely as odors released from damaged plants. That being
said, unintentional attraction of phorid flies to agricultural
fields will almost certainly have little to no detrimental
impact on pest outbreaks and/or crop production.
Blend Composition and Complexity
Overall, we found neither strongly positive nor negative
repercussions to deploying HIPVs as blends, since both
synergism and antagonism were rare and had no effect on
the group of predators and parasitoids as a whole. Two
arthropod groups were overrepresented among the few
non-additive responses; four of the six cases involved ich-
neumonid wasps and tumbling flower beetles (Mordellidae).
We initially hypothesized that blends combining cis-3-
hexen-1-ol, a cue indicative of recent feeding damage, with
MeSA would be highly attractive, as this could correspond
to natural cues indicating large or persistent herbivore pop-
ulations. This hypothesis was only partially supported for
ichneumonids. Nevertheless, it might be instructive for fu-
ture studies to consider how major classes of volatiles (e.g.,
green leaf volatiles, terpenoids, phenylpropanoids) may
combine to generate synergistic outcomes, as opposed to
mixtures containing compounds all derived from the same
biosynthetic pathway.
Why odor blends were not particularly attractive is some-
what perplexing. A meta-analysis by Szendrei and
Rodriguez-Saona (2010) found that arthropod attraction in-
creased in proportion to the number of compounds emitted,
an effect they attribute to an increased likelihood of deploy-
ing a single, highly attractive compound as complexity
increases (i.e., the ‘sampling effect’). Our results do not fit
this model though, and are perhaps better explained by
recent work that suggests that arthropods perceive odors as
a whole rather than a mixture of individual compounds (van
Wijk et al., 2011). When this is the case, any odor that
moves the environment away from the “ideal”scent should
decrease attraction. If the addition of a second or third
volatile in a blend acts in this way, a likely scenario when
blends are not tailored to the arthropod or system, repellent
effects would be expected. This concept is supported by
Webster et al. (2010) who recently found strong attraction
of the black bean aphid, Aphis fabae, to a fifteen-part
mixture of volatiles constituting the scent of the aphid’s
host-plant. Yet, aphids were repelled by each of the fifteen
volatiles when presented individually. In this case, there was
no single compound accounting for aphid attraction; only
the complete odor blend elicited a positive response. It also
is important to point out that our experiment was substitu-
tive in nature (total volatile volume was held constant),
while most experiments testing the effects of blends tend
to be additive (total volume increases with addition of
1272 J Chem Ecol (2012) 38:1264–1275
volatiles), which could lead to decreased attraction relative
to other studies. Another possibility is that a blend of three
volatiles is not sufficiently complex to improve attraction,
and a greater subset of the full complement of compounds is
necessary to generate this effect.
System Specificity
Of the 119 arthropod groups surveyed, 113 were found in
both systems, allowing us to directly compare responses
across crops due to taxonomic similarity of the two com-
munities. Compositional shifts between corn and soybean
were clearly apparent in the Hymenoptera (Fig. 5), however,
illustrating that the relative dominance of those species was
quite different in each crop, and thus the two communities
are not identical in a functional sense.
There was a clear difference in the number of arthropod
taxa affected by HIPVs in the two crops with far more
groups responding in soybean (16) than corn (4). If arthro-
pods were simply more abundant in soybean, this might
explain the difference; however, the abundance of natural
enemies responding to HIPVs in soybean was comparable to
that of corn (Table S2). Thus, it seems more likely that
ecological differences inherent to the two crops, rather than
possible external factors, were ultimately responsible for
variation in attraction. One possible exception to this was
the greater availability of inflorescences by weedy plants
(especially Solidago sp.) in and around soybean fields. With
greater sugar availability, parasitoids and predatory arthro-
pods are expected to live longer (Hagley and Barber, 1992),
produce more eggs (Heimpel et al., 1997), and once satiated,
spend more time in search of oviposition sites (Desouhant et
al., 2005). In the laboratory, this correlates with increased
attraction to host associated cues (Wäckers, 1994), includ-
ing those released by the host plant (Takasu and Lewis,
1993). It is also possible that arthropods were in search of
floral nectar, since both MeSA and phenylethyl alcohol are
commonly found in floral odor blends (Honda et al., 1998).
Last, it should be noted that soybeans reduce production of
herbivore-induced plant volatiles once the reproductive stage
is reached (Rostas and Eggert, 2008). Deployed volatiles then
might be more apparent to foraging arthropods in this system
and thus act as a more attractive (or repellent) cue.
Summary and Implications
Our results lead us to two separate conclusions pertaining to
the deployment of synthetic HIPVs for the purpose of con-
servation biological control. First, our data suggest that
attraction of predatory and parasitic arthropods to synthetic
volatiles is potentially dependent upon factors specific to the
crop system (context-dependency), to the local arthropod
community, and to the specific field studied, which makes
the implementation of HIPVs across systems challenging.
Second, odor blends were no more effective than single
compounds for attracting beneficial arthropods. Third, de-
spite the small spatial scale over which our experiment was
conducted, we observed large variation between individual
fields (i.e., highly significant site effects were prevalent
across taxa), possibly the result of heterogeneity in alterna-
tive, non-crop resources. Therefore, synthetic HIPV deploy-
ment might be more effective when combined with other
forms of conservation biocontrol, especially those that ma-
nipulate the surrounding landscape to maintain populations
of beneficial insects (Landis et al., 2000; Khan et al., 2008).
Future work would benefit from identifying the environ-
mental factors that shape the response of parasitic and
predatory arthropods to plant volatiles to more consistently
and reliably achieve attraction of these taxa in the field.
Acknowledgments We thank Brian Brown (Natural History Museum
of Los Angeles County) for his help with phorid identifications, Purdue
University, and USDA (NIFA, Grant No. 2011-67013-30126) for funding
this work. Thanks also to Gina Angelella, Carmen Blubaugh, Matthew
Ginzel, Ulianova Vidal-Gómez, Jessica Kelly, Christian Krupke, Cesar
Rodriguez-Saona, and two anonymous reviewers for help and insight in
designing the project and writing the manuscript.
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