Specificity in Induced Plant Responses Shapes Patterns of Herbivore Occurrence on Solanum
dulcamara
Author(s): Danush V. Viswanathan, Anita J. T. Narwani, Jennifer S. Thaler
Source: Ecology, Vol. 86, No. 4 (Apr., 2005), pp. 886-896
Published by: Ecological Society of America
Stable URL: http://www.jstor.org/stable/3450842
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Ecology, 86(4), 2005, pp. 886-896
? 2005 by the Ecological Society of America
SPECIFICITY IN INDUCED PLANT RESPONSES SHAPES PATTERNS OF
HERBIVORE OCCURRENCE ON SOLANUM DULCAMARA
DANUSH V. VISWANATHAN,' ANITA J. T. NARWANI, AND JENNIFER S. THALER2
Departmentof Botany, Universityof Toronto,25 WillcocksStreet, TorontoM5S 3B2, Canada
Abstract. Interspecific competition between phytophagous insects can occur when plant
responses induced by an early-season herbivore alter host quality for later colonizers. Recent
evidence for specificity in the elicitation of induced plant responses by different attackers
suggests that dynamics of host use in the field may be more complex than previously
anticipated, because host suitability for colonizing herbivores may depend on which herbivore species has initially damaged a plant. In each of two years, we manipulated the first
herbivore to attack Solanum dulcamara plants in an experimental population using several
different arthropod species and subsequently monitored colonization by natural herbivores
over the course of the growing season. We additionally performed weekly herbivore counts
in wild S. dulcamara populations following natural variation in herbivore arrival. Plantmediated interactions occurred primarily between two leaf-feeding beetles, Psylliodes affinis
and Plagiometriona clavata. In both manipulative and observational experiments, P. clavata
oviposition was reduced on plants initially damaged by P. affinis (or a third leaf-feeding
beetle, Lema trilinea) relative to plants that were initially undamaged. Lowered P. clavata
occurrence continued through subsequent life-history stages, resulting in decreased emergence of second-generation P. clavata adults on these plants. The occurrence of P. affinis
was also lowered on plants damaged by conspecifics in both manipulative and observational
experiments. Resistance against P. affinis also followed applications of jasmonic acid, an
elicitor of plant defensive responses. Conversely, early-season damage by P. clavata did
not influence plant quality for either later conspecifics or P. affinis. Initial herbivory by
the spittlebug Aphrophora saratogensis or generalist taildropper slugs (Prophysaon sp.)
likewise had no influence on P. clavata and P. affinis colonization, whereas L. trilinea
damage did not affect later arriving P. affinis. Hence, only a subset of early-season damagers
influenced herbivore occurrence on S. dulcamara. Preference tests examining P. affinis
feeding and P. clavata oviposition confirmed that specificity in elicitation of induced plant
responses produced the divergent herbivore occurrence patterns observed in the field. Overall, the existence of plant-mediated competitive asymmetry between herbivore species on
S. dulcamara highlights the dynamic nature of plant resistance and its potential role in
organizing herbivore communities.
Key words: bittersweetnightshade;competition;herbivory;induced responses;phytophagous
insect communities;Plagiometrionaclavata;plant-mediatedinteractions;Psylliodes affinis;Solanum
dulcamara;specificity.
INTRODUCTION
Increasingly widespread documentation of induced
plant responses to herbivory has fostered interest in the
possibility of plant-mediated interactions between different herbivores (Faeth 1986, Denno et al. 1995). Such
indirect effects may occur when physical or chemical
plant responses induced by a given herbivore affect
resource quality for subsequent foragers (Karban and
Baldwin 1997). Indeed, a review by Denno et al. (1995)
found that induced responses were involved in over
half of the published studies in which competition occurred between mandibulate herbivores. More recent
received13 February
2004;revised15 June2004;
Manuscript
Editor:K. C. Larson.
accepted23 August2004. Corresponding
I E-mail:danush@
botany.utoronto.ca
2 Present address:
Departmentof Entomology, Cornell
University, 3128 Comstock Hall, Ithaca, New York 14853
USA.
work has begun to extend these results to other feeding
guilds (Denno et al. 2000), and further suggests that
induced responses may play a part in structuring herbivore communities (Tscharntke 1999, Gonzalez-Megias and Gomez 2003, Van Zandt and Agrawal 2004).
Plant-mediated interactions between herbivore species are therefore emerging as an important mechanism
of competition in many systems. To date, however,
most experimental field studies have examined only
whether a single early-season inducing agent can influence later foragers (e.g., Haukioja and Niemela
1979, Faeth 1986, Hanhimaki 1989, Karban 1993, Thaler et al. 2001, Wise and Weinberg 2002). This approach mimics natural conditions in systems where a
particular species is the exclusive or predominant earlyseason damager. In many systems, however, several
different herbivore taxa feed abundantly on a common
host plant species during its early growth phase. The
886
SPECIFICITYOF PLANT RESPONSES
April2005
dynamics of host use over a growing season may be
substantially more complex than previously expected
if the host plant exhibits specificity in induced responses to different early-season attackers.
Specificity in plant response has two components:
effect and elicitation. Specificity of effect occurs when
herbivore species are differently affected by a given
induced response (Stout et al. 1998, Agrawal 2000; see
also Hartley and Lawton 1987). Experiments assessing
colonization of treated and untreated plants by natural
assemblages of herbivores following early-season induction frequently find some degree of specificity of
effect (e.g., Hunter 1987, Inbar et al. 1998, Agrawal
1999, Agrawal and Sherriffs 2001, Thaler et al. 2001,
Riihimaki et al. 2003). Conversely, specificity of elicitation occurs when herbivore species differentially induce phenotypic responses in a particular host plant
(Stout et al. 1994, 1998, Agrawal 2000, Traw and Dawson 2002). In the field, specificity of elicitation is evident when a given attacker varies in abundance across
plants initially damaged by different herbivore species.
Using lab preference tests, Hartley and Lawton (1987)
revealed specificity of elicitation in field-collected
birch leaves damaged by different herbivores. Additionally, Inbar et al. (1998) showed that different chemical elicitors of induced plant responses varied in their
ability to influence the colonization of tomato plants
by specific herbivores. Otherwise, however, field colonization of plants treated with different damage types
has been explicitly compared only between plants with
herbivore vs. mechanical damage (e.g., Agrawal 1999,
Agrawal and Sherriffs 2001; but see Van Zandt and
Agrawal 2004).
In this paper, we present two years of field data from
manipulative experiments examining whether the identity of the initial attacker on Solanum dulcamara plants
can influence patterns of colonization by subsequent
foragers. Regular counts of herbivore abundance on
plants over the growing season enabled us to assess the
duration and strength of specific, plant-mediated interactions between different herbivores. We also used
early- through late-season damage estimates to distinguish between the direct effects of initial damage per
se on later colonization and the indirect effects of early
damage type mediated through differences in subsequent levels of herbivory. We additionally performed
weekly herbivore counts in wild S. dulcamara populations following natural variation in herbivore arrival
to determine whether similar patterns occur in natural
conditions. Finally, we conducted preference tests using a subset of treatments from manipulative experiments to expressly investigate specificity in the elicitation of induced plant responses as the mechanism
underlying results from the field.
METHODS
Study system
Solanum dulcamara L. (bittersweet nightshade) is a
perennial vine in the Solanaceae that has been shown
887
?
PLATE. 1.
'4
Plagiometriona clavata (tortoise beetle) adult
feeding on Solanumdulcamara(bittersweetnightshade)leaf.
Photo credit:D. Viswanathan.
to induce pathogenesis-related proteins, peroxidases,
polyphenol oxidases, and cell necrosis in response to
herbivory by gall mites (Bronner et al. 199 la, b). These
responses reduce intraspecific gall mite damage to the
plant (Westphal et al. 1991), exert negative interspecific
effects on rust mite fecundity (Westphal et al. 1991),
and positive interspecific effects on spider mite fecundity (Westphal et al. 1992). Additionally, herbivory by
Lema trilinea (three-lined potato beetle) induces
changes in polyphenol oxidase and proteinase inhibitor
activity, which can influence the growth rate of L. trilinea larvae (Viswanathan and Thaler 2004).
Natural populations of S. dulcamara occur at the
Koffler Scientific Reserve at Jokers Hill (hereafter Jokers Hill), King City, Ontario, Canada, where all field
experiments were conducted. Several herbivore species
feed on S. dulcamara at Jokers Hill, five of which were
examined in this study: two univoltine native specialists on solanaceous plants, Lema trilinea White and
Plagiometriona clavata Fab. (tortoise beetle; see Plate
1); an introduced univoltine solanaceous specialist,
Psylliodes affinis Paykull (European potato flea beetle);
Aphrophora saratogensis Fitch (Saratoga spittlebug),
a native that feeds on understory herbs as a nymph in
the spring and later specializes on pine trees as an adult
froghopper; and native generalist taildropper slugs
(Prophysaon sp.).
Manipulative experiments
Manipulative experiments using transplants of S.
dulcamara were conducted in June-August of both
2001 and 2002 (n = 400 and 500, respectively). Seeds
had been collected previously from wild S. dulcamara
populations at Jokers Hill, and seedlings were grown
in ProMixBX soil (Premier Horticulture, Riviere-duLoup, Qu6bec, Canada) contained in 131-mL pots under a 16:8 L:D greenhouse cycle for three weeks prior
to transplantation. Both manipulative experiments were
conducted in the semi-shaded understory of a young
forest where S. dulcamara occurs naturally, with transplants spaced -30 cm apart in a square grid.
Following transplantation, each plant was protected
from ambient herbivores with a spun polyester sleeve
888
DANUSH V. VISWANATHANET AL.
(Rockingham Opportunities Corporation, Reidsville,
North Carolina, USA) and was allowed to acclimate to
field conditions for a week. In 2001, eight different
treatments were then randomly imposed, with 50 replicates of each treatment. Five treatments consisted of
inoculations with either adult L. trilinea, P. clavata, P.
affinis, Prophysaon sp., or A. saratogenis nymphs. The
remaining three treatments included a I-mL spray of
a 0.5 mmol jasmonic acid (an elicitor of plant defensive
responses) solubilized in 1%acetone solution (see Thaler et al. [1996] for dosage), and two controls (plants
undamaged and sprayed with a control solution of water and 1% acetone, or plants mechanically damaged
with a hole punch to produce leaf loss without specific
induction cues). In 2002, the experiment was repeated
at an adjacent site without the A. saratogensis and mechanical damage treatments, resulting in -92 replicates
for each of the remaining six treatments.
Over a one-week initial damaging phase, the amount
of tissue consumed was equalized at 15-20% across
leaf-feeding herbivore treatments using herbivore additions and removals. ANOVA results compared damage measurements using an acetate grid on a subset of
20 plants from each treatment (for 2001, F495 = 0.71,
P = 0.56; for 2002,
F376
= 0.08,
P = 0.97).
Equal-
ization of tissue removal was used to prevent dosedependent differences between treatments in induced
responses. On average, 2-4 herbivores per plant were
required to produce this level of herbivory. For the A.
saratogensis treatment, four individuals were placed
on each plant, which was sufficient to cause wilting in
10% of replicates. After one week, -15% leaf tissue
was removed per plant in the mechanical damage treatment, and the jasmonic acid treatment was applied.
After the treatments had been imposed, all sleeves
and treatment herbivores were removed. Colonization
by naturally occurring herbivores was then assessed
through regular counts of the abundance and identity
of all aboveground herbivores on each plant. Because
herbivore colonization may be influenced by plant size,
plant height was recorded at each sampling interval in
both years, along with the number of leaves in 2002.
In both years, visual estimates of total percentage damage were also recorded, because colonizing herbivores
may be sensitive to differences between treatments in
levels of herbivory following initial damage. In 2001,
plants were sampled weekly throughout June, and subsequently every second week until mid-August, for a
total of seven sampling dates. In 2002, plants were
sampled every 10 days, with three sampling dates in
each of June, July, and August.
Observational study
In May-August of 2002, observational studies were
conducted at Jokers Hill in two populations of S. dulcamara. Site 1 consisted of 200 second-year seedlings,
transplanted into an open forest understory in the previous year. Site 2 consisted of a large natural S. dul-
Ecology,Vol. 86, No. 4
camara population occurring in a pine stand, where
100 ramets were randomly selected for use in the study.
Ramets occurred in clumps spaced 0.5-3 m apart, with
clumps varying in size from 10 to 50 ramets. Only one
ramet was selected per clump to reduce the probability
of sampling within a genet. Selected ramets varied in
size from 20 to 100 cm, measured from their point of
connection with other ramets, and were sampled over
this entire length.
At the end of May, when S. dulcamara herbivores
were first appearing, the species that had first attacked
each seedling or ramet was assessed; S. dulcamara herbivores generally have a characteristic pattern of damage that can be used to identify early-season attackers
when levels of damage are relatively low. A number
of plants were still undamaged at this point. Plant
height, number of leaves, and visual estimates of percentage tissue removed were also recorded on the initial
sampling date. At weekly intervals for the next two
months, these three variables, as well as herbivore
abundance and identity, were measured on each plant.
Preference tests
In 2003, P. affinis feeding and P. clavata oviposition
preference tests were conducted using potted plants,
because these two herbivores discriminated between
treatments in field experiments (see Results). For these
tests, plants were grown in the greenhouse for three
weeks as previously described, after which experimental treatments were imposed. A no-choice feeding assay
was used to examine the absolute preference of P. affinis for different induced plant phenotypes. For this
assay, four treatments were randomly assigned across
44 plants: control, or herbivory by adult L. trilinea, P.
clavata, or P. affinis. Each plant was covered with a
spun polyester sleeve, and the amount of tissue consumed across herbivore treatments was equalized at
15-20% using herbivore additions and removals, as per
the manipulative experiments. Following a one-week
damaging phase, all herbivores and sleeves were removed. After a subsequent week of growth, a sleeve
was used to enclose the bottom (damaged) portion of
each plant, while a second larger sleeve was used to
contain the entire plant. Single P. affinis adults were
placed in each large sleeve, and the leaf area consumed
was recorded after two days, along with available leaf
area and total plant height. Because assay herbivores
did not have access to damaged portions of the plant,
we interpret differences in feeding between treatments
to indicate specificity in the elicitation of induced plant
responses.
A choice assay was used to examine the oviposition
preference of P. clavata females for different induced
phenotypes relative to uninduced plants. A choice assay
was used instead of a no-choice assay because P. clavata females seem to oviposit on low-quality plants
when higher quality resources are not available (D. V.
Viswanathan and J. S. Thaler, unpublished data). Two
April2005
889
SPECIFICITYOF PLANT RESPONSES
trials of the P. clavata oviposition choice assay were
conducted, using 82 and 64 plants, respectively. For
each trial, half of the plants were left as undamaged
controls. Every control plant was paired with another
plant assigned to one of three treatments: adult L. trilinea, P. clavata, or P. affinis herbivory. All four treatments were imposed as previously described. After a
week of growth following the initial damage phase, the
bottom (damaged) portion of each plant was covered
with a sleeve. Each pair of damaged-control plants was
then enclosed together in a larger sleeve. A single gravid P. clavata female was placed in every large sleeve,
and the differences between the two plants in eggs laid,
available leaf area, and total plant height were calculated after four days. Each damaged-control pair
served as a replicate, with 41 and 32 in each trial,
respectively. As with the P. affinis feeding test, bioassay P. clavata did not have access to damaged portions of the plant. Hence, we likewise interpret differences in relative oviposition across the different treatment-control combinations to indicate differences in
plant quality induced by initial herbivory.
Data and statistical analyses
Across all field experiments, L. trilinea, P. clavata,
P. affinis, and several slug species colonized the plants.
However, only P. clavata and P. affinis occurred at high
enough frequencies to perform statistical analyses. For
P. clavata, overwintering adults oviposit on S. dulcamara leaves over 3-4 weeks in early summer, with
the entire juvenile stage subsequently being completed
on leaf surfaces. Hence, we were able to record all life
stages for this species: adults, eggs, larvae, pupae, and
exuviae (the presence of a pupal molt can be used to
infer successful eclosion; D. V. Viswanathan, personal
observation). P. affinis larvae feed on roots, so only
the occurrence of adults could be scored for this species.
Herbivore occurrence data were not normally distributed; there were many zero and one values. Dichotomous, binomially distributed data are analyzed using
logistic regression, which can be generalized for polychotomous dependent variables with a multinomial distribution (Trexler and Travis 1993). Hence, generalized
estimating equations were used to perform repeatedmeasures poisson regressions on herbivore counts
(GEE-PROC GENMOD in SAS System Version 8;
Allison 1999). Poisson regression calculates likelihoodratio chi-square statistics, with degrees of freedom equal
to one minus the number of levels of each factor. It also
produces an odds ratio for each level of every factor
adjusted for other terms in the model, indicating the odds
(or likelihood) that the response will occur. The odds
ratio for each level is calculated relative to a reference
level, with a value of one indicating an equal likelihood
of the event taking place. Values above or below one
indicate greater or lesser odds, respectively, when compared to the reference level.
In the current study, repeated-measures Poisson regressions were used to compare the relative odds of
herbivore occurrence across treatments over several
weeks. If there was a significant week by treatment
interaction in the repeated-measures analysis, individual weeks were also examined separately. When the
effect across individual weeks was similar, only the
repeated-measures analysis were reported. In the observational study, treatments were classified by which
herbivore species had first damaged a plant, with undamaged plants as "controls." At each observational
site, the number of plants damaged by different herbivore species, or initially undamaged, was similar.
Plant height and leaf number were used as covariates,
because these variables may additionally influence herbivore foraging choice. In the observational study, the
amount of damage imposed by initial damagers was
used as a covariate for similar reasons. Covariates that
were not significant were removed from the analysis.
Initial damage type may directly influence later herbivore occurrence, or may act indirectly through differences in subsequent levels of herbivory. Hence, we
also used damage estimates from each previous sampling interval as a covariate to distinguish between
direct and indirect effects of initial damage type. Poisson regressions were performed with and without the
damage estimate covariate. If inclusion of damage levels from the previous week eliminated a significant
main effect of treatment, we conclude that effects on
herbivore occurrence resulted from differences between treatments in levels of damage following initial
herbivory, and not directly from lasting effects of initial
damage type per se (cf. Van Zandt and Agrawal 2004).
For the P. affinis no-choice preference test, leaf area
consumed was analyzed using a one-factor ANOVA,
with damage type as the main effect and available leaf
area and total plant height as covariates. For the P.
clavata choice preference test, the difference in number
of eggs laid between damaged and control plants was
analyzed using a two-factor ANOVA, with damage
type and trial as the main effects and differences in
both available leaf area and total plant height as covariates. Significant ANOVA results were further examined using Tukey post hoc comparisons.
RESULTS
Manipulative experiments
For both 2001 and 2002, the repeated-measures analysis revealed a significant week by treatment interaction on the odds of Psylliodes affinis occurrence (Table
1). For the 2001 data, examination of individual sampling dates revealed that initial treatment had a significant effect only in the second week after initial damage (X2= 47.7, df = 7, P < 0.001). Similarly, treatment
in 2002 had a significant effect only one week following initial damage (X2 = 68.7, df = 5, P < 0.001). The
effect of each specific treatment was consistent across
890
DANUSH V. VISWANATHANET AL.
TABLE 1. Results from repeated-measuresPoisson regres-
Ecology, Vol. 86, No. 4
stages. There were strong effects of initial treatment
sions examiningthe occurrenceof Psylliodesaffinisadults
P. clavata oviposition in 2002 (Table 1; egg number
on
and Plagiometrionaclavata eggs on Solanumdulcamara
was not recorded in 2001). Both Lema trilinea and P.
plants following differentearly-seasondamagetypes.
affinis damage decreased the odds of P. clavata oviP
df
Effect
X2
position by -50% relative to undamaged controls, with
no influence of Prophysaon or mechanical damage
adults
a) Psylliodes affinis
(Fig. lb; see the Appendix for raw means). AdditionManipulativeexperiment2001
7
0.28
8.6
Treatment
ally, the occurrence of P. clavata larvae was signifi5
<0.001
88.7
Week
cantly affected by initial treatment in both years (for
35 <0.001
75.8
Treatmentx Week
2001, x2 = 16.1, df = 7, P = 0.02; for 2002, x2 =
1
<0.001
12.4
Percentagedamage
28.45, df = 5, P < 0.001). As with egg numbers, odds
Manipulativeexperiment2002
of occurrence for P. clavata larvae were -50% lower
5
0.004
17.3
Treatment
<0.001
6
159.9
Week
on L. trilinea and P. affinis damaged plants relative to
74
30 <0.001
Treatmentx Week
undamaged controls (Fig. 1).
1 <0.001
Plant height
28.3
We further examined whether decreased larval oc1
0.027
4.92
Leaf number
was a consequence of lower relative ovipocurrence
Observationalsite 2
L.
trilinea and P. affinis damaged plants rather
on
sition
2
<0.001
Initial damager
23.8
7
4
0.78
Week
than lasting effects of initial treatment. Counts of lar14
0.12
20.2
Initial damagerx Week
vae in the week when they were maximally abundant
b) Plagiometrionaclavata eggs
in 2002 were analyzed using Poisson regression, with
Manipulativeexperiment2002
egg number from the last week of oviposition as an
5
0.004
17.4
Treatment
additional covariate. The 2-3 weeks between final ovi2
<0.001
64.8
Week
10
0.59
8.9
Treatmentx Week
position date and maximal larval abundance is long
1 <0.001
14.9
Percentagedamage
enough for all P. clavata eggs to have hatched, yet too
Observationalsite 1
soon for pupation to have occurred. Egg number sig3
0.034
8.7
Initial damager
nificantly influenced larval occurrence, with initial
1 <0.001
11
Week
treatment remaining marginally significant (Table 2).
3
1.1
0.78
Initial damagerx Week
1
In 2002, similar effects of treatment on the occurrence
0.02
5.4
Percentagedamage
of P. clavata pupae (X2 = 30.2, df = 5, P < 0.001)
Observationalsite 2
2 <0.001
14.4
Initial damager
and exuviae (X2 = 32.6, df = 5, P < 0.001) were
5
<0.001
69.1
Week
entirely driven by lower occurrences in previous life
8
0.013
19.4
Initial damagerx Week
stages (Table 2). Hence, adult P. clavata oviposition
1
0.006
7.5
Plant height
determined occurrence in all other life stagpreference
Notes: In manipulativeexperiments,total percentagedamexuviae were too few to perform Poisson
and
es.
Pupae
age in the week previousto each samplingintervaland plant
in
2001.
as
week
were
used
in
current
and
leaf
number
the
regressions
height
covariates. Percentageof leaf area consumedon each plant
Observational study
by initial herbivoreswas used as an additionalcovariatein
the observationalstudy.Covariatesnot significantat an alpha
At
site
1, damage levels on the initial sampling date
value of 0.05 were removed from the analysis and are not
0% to 15%, with P. affinis and Prophysaon
from
reported.
ranged
sp. as the main initial damagers (plants damaged by
both species are not included in this study). Both of
years: P. affinis damage and jasmonic acid decreased these herbivores were subsequently found at extremely
the odds of P. affinis occurrence relative to undamaged low frequencies, while later arriving P. clavata were
controls by >50% and 75%, respectively, whereas Pla- more abundant. Initial damage type significantly afgiometriona clavata damage increased the odds of P. fected the number of P. clavata eggs (Table 1), with
affinis occurrence by >50% relative to controls, al- the odds of oviposition being 47% lower on plants
though this comparison was not statistically significant damaged by P. affinis relative to plants that were iniin 2001 (Fig. 1; see the Appendix for raw means). tially undamaged. However, no such influence of ProConversely, Aphrophora saratogensis, Prophysaon sp., physaon damage was apparent (Fig. 2a; see the Apor mechanical damage did not influence P. affinis oc- pendix for raw means). Significant overall effects of
currence (Fig. 1). Effects of initial treatment were not early-season damager on the occurrence of larvae (X2
= 3, P = 0.02; see Fig. 3), pupae (X2 =
significant in subsequent weeks, despite the continued = 9.57, df
16.2, df = 3, P = 0.001), and exuviae (X2 = 17.1, df
presence of P. affinis adults throughout each season.
A similar consistency was apparent across years for = 3, P < 0.001) were due to the previous distribution
P. clavata. Although adult occurrence was not influ- of eggs (Table 2). Adult P. clavata occurred at too low
a frequency to allow Poisson regression.
enced by initial treatment (for 2001, x2 = 12.1, df =7,
At site 2, damage levels on the initial sampling date
P = 0.10; for 2002, X2 = 2.7, df = 5, P = 0.75), Poisson
regressions revealed significant effects on other life also ranged from 0% to 15%, with P. clavata and P.
SPECIFICITYOF PLANT RESPONSES
April 2005
891
2.5
a) 2001
O Plagiometriona clavata larvae
A Psylliodesaffinis
n
Z.u
1.5
0
1.0
0
()
0.5
*
*A
0
C
U
Mechanical Aphrophora Psylliodes PlagiometrionaPryphasaon
affinis
clavata
sp.
damage
saratogensis
Lema
trilinea
Jasmonic
acid
()
0
D
.0
2.5
1)
03
-0
0
* Plagiometriona
clavata eggs
clavata larvae
? Plaaiometriona
A
Psylliodesaffinis
b) 2002
2.0-
1.5
................
1.0-
.... ..... . ........
....
.............
0.5
*9
*A
A
u
Psylliodes PlagionmetionaPryphasaon
clavata
affinis
sp.
Lema
trilinea
Jasmonic
acid
FIG. 1. Results from repeated-measures Poisson regression showing relative odds of herbivore occurrence on Solanum
dulcamara plants following several different early-season manipulations in 2001 and 2002, calculated relative to undamaged
controls (dotted line). Psylliodes affinis data are from week 2 only. Symbols and bars show the mean + 1 SE for each treatment.
Values above and below 1 indicate greater and lesser odds, respectively, of herbivore occurrence when compared to the
control. An asterisk indicates a significant difference at an alpha value of 0.05. Note that odds of occurrence for Plagiometriona
clavata larvae in panel (b) result from a similar distribution of P. clavata eggs, with continuing effects on P. clavata pupation
and eclosion (see Results). For treatment details, see Methods: Manipulative experiments.
affinis as the main initial damagers. Subsequently, these
same two species were the main colonizers. Initial damage type significantly affected colonization by P. affinis
(Table 1), with 60% lower odds of occurrence on plants
receiving conspecific herbivory relative to plants that
were initially undamaged (Fig. 2b; see the Appendix
for raw means). There was no such effect of P. clavata
herbivory (Fig. 2b). P. clavata oviposition was also
significantly influenced by initial damage type (Table
1), with 27% lower odds of oviposition on P. affinis
damaged plants relative to controls. However, there was
no influence of P. clavata herbivory on oviposition by
conspecifics (Fig. 2b). Similarly, significant effects of
initial damage type on larvae (X2 = 15.5, df = 2, P <
0.001; see Fig. 2b), pupae (X2 = 9.01, df = 2, P =
0.011), and exuviae (X2 = 15.4, df = 2, P < 0.001)
were again due to the previous distribution of eggs
(Table 2). The occurrence of P. clavata adults was not
affected by differences in early-season attacker (X2 =
1.7, df = 2, P = 0.43).
Direct vs. indirect effects
For the results just reported, significant effects of
initial damage type on herbivore occurrence were never
eliminated when damage estimates from the previous
week were included in Poisson regressions. Nevertheless, percentage damage was a significant covariate in
one-third of the analyses, whereas plant height and
number of leaves only occasionally influenced foraging
herbivores (Tables 1 and 2). Percentage damage by
initial herbivores never significantly affected later herbivore occurrence in the observational study (Tables 1
892
TABLE2.
DANUSH V. VISWANATHANET AL.
Ecology,Vol. 86, No. 4
Results from Poisson regressionsexaminingthe ber of
eggs between damaged and control plants (F265
occurrenceof Plagiometrionaclavata larvae, pupae, and
=
P = 0.016), with P. clavata females strongly
4.44,
exuviae on Solanumdulcamaraplants following different
early-seasondamagetypes.
disfavouring plants damaged by P. affinis (Fig. 3b). No
effect of trial was apparent (F, < 0.001, P = 0.99),
Effect
df
P
X2
nor was there a trial x treatment interaction (F2,65 =
Larvae
0.52, P = 0.598).
a)
Manipulativeexperiment2002
Treatment
11
5
0.052
DISCUSSION
183.9
1
<0.001
Egg number
5.7
1
0.017
Percentagedamage
Experimental demonstrations of induced plant reObservationalsite 1
sponses influencing herbivore success are one of the
Initial damager
3.7
3
0.3
main reasons for renewed interest in competition be126.1
1
<0.001
Egg number
tween insects (Denno et al. 1995). A number of field
Observationalsite 2
studies examining plant-mediated competition have, in
Initial damager
2
1.9
0.39
shown that early-season herbivory can decrease
number
fact,
97.1
1
<0.001
Egg
the
size of subsequent colonizing species,
population
b) Pupae
including phytophagous mites (English-Loeb et al.
Manipulativeexperiment2002
Treatment
9.1
5
0.11
1993), microlepidoptera (Karban 1993), and sap-feedLarvalnumber
414.5
1
<0.001
ing planthoppers (Denno et al. 2000). In this study, we
Observationalsite 1
similarly show strong influences of early-season inInitial damager
3.7
3
0.29
duction
on occurrence across herbivore generations. In
Larval number
21
1
<0.001
both experimental and natural patterns indiaddition,
Observationalsite 2
cate that such lasting effects of initial herbivory may
Initial damager
1.9
2
0.39
Larvalnumber
97.1
1
<0.001
depend on the order of herbivore arrival when plants
exhibit specificity in the elicitation of induced plant
Exuviae
c)
responses.
Manipulativeexperiment2002
Treatment
8.9
5
0.11
Across both manipulative and observational exper479.2
1
<0.001
Pupal number
iments, heterospecific herbivory by P. affinis consisObservationalsite 1
tently decreased P. clavata oviposition when compared
Initial damager
5
3
0.17
to plants that initially received no damage. Decreased
46.5
1
<0.001
Pupal number
6.8
1
0.009
Percentagedamage
egg number resulted in decreased occurrence of larvae
and pupae, with subsequently fewer second-generation
Observationalsite 2
Initial damager
4.5
2
0.1
adults emerging on P. affinis damaged plants. The same
44.2
1
<0.001
Pupal number
effect was observed following L. trilinea damage in
Notes: Analyses were performedon data from the week the manipulative experiments, whereas conspecific herwhen each life stage was maximallyabundant,with numbers
never influenced P. clavata occurrence. Interfrom the final week of the previouslife stage as a covariate. bivory
In manipulativeexperiments,total percentagedamagein the estingly, adult P. clavata occurrence was never affected
week previousto the samplingintervaland plant height and by the identity of early-season attackers, suggesting
leaf numberin the currentweek were also used as covariates. that adults
forage independently of initial damage type,
Percentageof leaf area consumed on each plant by initial but nevertheless assess host
plant quality when making
herbivoreswas used as an additionalcovariatein the observational study. Covariatesnot significantat an alpha value oviposition decisions.
of 0.05 were removedfrom the analysisand are not reported.
Early-season damage by conspecifics consistently
decreased P. affinis occurrence relative to plants that
initially received no herbivory. However, heterospeand 2), whereas in the manipulative experiments, her- cific damage did not have a similar effect. In the mabivore occurrence was never different between me- nipulative experiments, only P. affinis herbivory had
chanically damaged plants and undamaged controls negative effects on conspecifics 1-2 weeks after initial
(Fig. 1).
damage (with weak evidence for a short-term positive
effect of P. clavata herbivory on P. affinis occurrence).
Preference tests
At site 2 of the observational study, negative effects
In the no-choice assay, P. affinis feeding rate was of conspecific damage on P. affinis lasted the entire
season (with neither short- nor long-term effects of P.
significantly affected by initial damage type (F339 =
4.64, P = 0.007), with 47% less tissue consumed on clavata herbivory). Because the length of the sampling
plants damaged by conspecifics relative to controls interval exceeded the recorded generation time for this
(Fig. 3a). A similar reduction did not occur following
species by several weeks (Wheeler and Hoebeke 1983),
P. clavata or L. trilinea herbivory (Fig. 3a). In the P. P. affinis adults at the end of the season most likely
clavata oviposition choice assay, there was a significant included progeny of early-season colonizers. Hence,
effect of initial damage type on the difference in num- effects of initial P. affinis herbivory on conspecifics
SPECIFICITYOF PLANT RESPONSES
April 2005
893
I
1.0
a) Site 1
0.8
--
0.6
0
c
o
0
0
0.4
cavata
Plagiometriona
a)
0?
0.2
Plagiometrionaclavata
clavata larvae
larvae
Plagiometriona
(a)
I
Psylliodes affinis
(D
0
a)
o
>
Prophysaon sp.
1.6
1.4
b) Site 2
._
) 1.2
U) 1.0
.. .... .... ...............
...........................
. .............................
v
0
0.8
0.6
0.4
~* Ay/2\
0.2
Psylliodes affinis
* Plagiometrionaclavata eggs
0 Plagiometrionaclavata larvae
A
Psylliodes affinis
Plagiometrionaclavata
FIG. 2. Results from repeated-measures Poisson regression showing relative odds of herbivore occurrence on Solanum
dulcamara plants following natural early-season damage by different herbivore species at site 1 and site 2, calculated relative
to plantsthat initially received no herbivory(dotted line). Symbols and bars show the mean + 1 SE for each damagetype.
Valuesabove and below 1 indicategreaterand lesser odds, respectively,of herbivoreoccurrencewhen comparedto initially
undamagedplants. An asteriskindicates a significantdifferenceat an alpha value of 0.05. Note that odds of occurrencefor
P. clavata larvae result from a similar distributionof P. clavata eggs, with continuingeffects on P. clavata pupationand
eclosion (see Results).For treatmentdetails, see Methods:Observationalstudy.
may have persisted across generations in the observational study.
Several lines of evidence together suggest that specificity in the elicitation of induced plant responses
caused the divergent herbivore occurrence patterns observed in the field. Most importantly, findings from the
preference assays match those from field experiments.
P. affinis had lower feeding rates on undamaged tissue
from conspecifically, but not heterospecifically, damaged plants when compared to controls. P. clavata females also showed a relative oviposition bias against
undamaged tissue from P. affinis damaged plants, although the expected bias against L. trilinea damaged
plants was not found. Secondly, herbivore occurrence
never differed between mechanically damaged plants
and undamaged controls, indicating that removal of
tissue in the absence of herbivore-specific induction
cues did not influence plant quality for subsequent foragers. Finally, treatment with jasmonic acid consistently lowered the occurrence of P. affinis, showing
that induction in the absence of any external cues left
by herbivores is sufficient to cause changes in the suitability of S. dulcamara.
The pattern of induced resistance in this study may
have resulted from specific changes in the quality of
plant tissues (Stout et al. 1994, 1998). In addition, specific induction of volatile compounds may have influenced either host use by herbivores (e.g., Pallini et al.
1997) or the attraction of predators and parasitoids
(e.g., De Moraes et al. 1998, Meiners et al. 2000).
Preliminary analysis of tissue collected from the manipulative experiments following initial damage has
shown that P. affinis did, in fact, induce qualitatively
different chemical responses in S. dulcamara leaves
than did P. clavata (D. V. Viswanathan, O. A. Lifchits,
and J. S. Thaler, unpublished data). The responses induced by P. clavata seemed simply not to have influenced later colonizers. Although it is possible that herbivory by subsequent attackers further altered the
strength or quality of initial plant responses, the sim-
DANUSH V. VISWANATHANET AL.
894
Ecology, Vol. 86, No. 4
50
cm
E
E
4a)
1
0a) 300)
a) 25
0c
0)
-J
ab
* *
35-
b
2015
10
Control
Psylliodes Plagiometriona
clavata
affinis
Lema
trilinea
t0
)
C
0)
4
2
E
I
o
CO-2
a)
c -4
C
0) -6
0) -8
]Q
FIG. 3.
Psylliodes
affinis
Plagiometriona
clavata
Lema
trilinea
(a) Amount of Solanum dulcamara leaf area consumed by a single Psylliodes affinis adult over two days of feeding
on systemic tissue producedfollowing one of four initial treatmentsin a no-choice feeding experiment.(b) Differencein the
numberof eggs laid by single gravidPlagiometrionaclavata females over four days between pairs of damagedand control
Solanumdulcamaraplants.Damagewas imposedusing one of threedifferentherbivorespecies, andovipositionwas restricted
to tissue producedafter the damagingphase. In both panels (a) and (b), boxes and bars show the mean + 1 SE for each
treatment.Treatmentsthat do not share a common letter above boxes are significantlydifferentat an alpha value of 0.05,
calculatedwith Tukey'spost hoc comparisonsfollowing ANOVA. For treatmentdetails, see Methods:Preferencetests.
ilarity of results between preference tests and field experiments suggests that responses to initial damagers
were, by themselves, sufficient to determine host plant
quality.
Specificity in induced responses produced lasting
differences in herbivore occurrence in the field, but
whether this stemmed from equivalently long-term alterations in plant phenotype was not always clear. Despite an early-season difference between treatments in
oviposition quality for P. clavata, subsequent effects
of initial damage type on the relative survival of larvae
and pupae were not apparent. Although these results
might suggest a mid-season diminution in the strength
of induced responses or lowered sensitivity of later life
stages, several unmeasured life-history characteristics
could have in fact been influenced by damage type,
including time to pupation, pupal mass, or emergence.
Conversely, results for P. affinis occurrence were more
conclusive, although slightly inconsistent: definite season-long effects in the observational study contrasted
with solely short-term effects in both manipulative experiments. This difference may have been caused by
transplantation shock preventing the induction of
strong responses with lasting effects in the manipulative experiments. Additionally, relatively close spacing
of individuals in manipulative experiments may have
promoted competition as plants grew, which can attenuate the expression of plant responses (Cipollini and
Bergelson 2001).
Because induced plant responses are a product of the
interaction between plant and herbivore, it is not clear
from the current data whether the observed pattern of
specificity is adaptive for either S. dulcamara or its
attackers. Alternatively, it may be solely a functional
April2005
SPECIFICITYOF PLANT RESPONSES
consequence of different herbivore feeding styles or
salivary elicitors. Whether or not there is a clear adaptive explanation, however, plant-mediated interactions
on S. dulcamara took place largely between its most
abundant colonizers, P. affinis and P. clavata. These
effects were unidirectional: P. affinis feeding had negative effects on both P. clavata and conspecifics,
whereas P. clavata damage did not influence either
itself or P. affinis. This result is not unusual; interactions between insects are often asymmetric, particularly
among mandibulate herbivores (Denno et al. 1995).
However, asymmetric competition between herbivores
caused by specificity in the elicitation of induced plant
responses is a novel possibility in the study of plantinsect interactions. Similar specificity in prey responses
to multiple predator species has recently received much
experimental attention from behavioral and evolutionary ecologists (Sih et al. 1998, Relyea 2003), following
earlier theoretical work showing that trade-offs among
predator-specific defenses in prey can promote the coexistence of different predator species (Matsuda et al.
1993) and overall community complexity (Matsuda et
al. 1994). Hence, our results (and similar findings by
Van Zandt and Agrawal 2004) suggest that comparable
attention to specificity in induced plant responses may
provide related insights into the dynamics of herbivorous insect communities.
In sum, there was a strong similarity of results across
experiments in this study. Manipulative experiments
indicated that initial damage type could affect herbivore occurrence in the field, and observational studies
verified that herbivore distribution in natural populations of S. dulcamara is indeed influenced by the order
of arrival of herbivore species. Subsequent preference
tests showed that specificity in induced plant responses
underlay these patterns. Overall, we therefore found
evidence for specificity in the elicitation of induced
plant responses that was consistent across time and
space, with lasting effects on herbivores.
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APPENDIX
A table showing raw means for (A) Psylliodes affinis adult and (B) Plagiomatriona clavata egg abundance on Solanum
dulcamara plants following different early-season damage types is available in ESA's Electronic Data Archive: Ecological
Archives E086-047-A 1.