Oecologia (2005)
DOI 10.1007/s00442-005-0008-5
P L AN T A N IM A L I NT E R AC TI O NS
Kathleen L. Prudic Æ Jeffrey C. Oliver Æ M. Deane Bowers
Soil nutrient effects on oviposition preference, larval performance,
and chemical defense of a specialist insect herbivore
Received: 1 September 2004 / Accepted: 11 January 2005
Springer-Verlag 2005
Abstract This study examined the effects of increased
leaf nitrogen in natural host-plants (Plantago spp.) on
female oviposition preference, larval performance, and
larval chemical defense of the butterfly Junonia coenia.
Increased availability of soil nutrients caused the hostplant’s foliar nitrogen to increase and its chemical defense to decrease. Larval performance did not correlate
with increases in foliar nitrogen. Larval growth rate and
survival were equivalent across host-plant treatments.
However, larvae raised on fertilized host-plants showed
concomitant decreases in chemical defense as compared
to larvae reared on unfertilized host-plants. Since most
butterfly larvae cannot move long distances during their
first few instars and are forced to feed upon the plant on
which they hatched, J. coenia larval chemical defense is
determined, in large part, by female oviposition choice.
Female butterflies preferred host-plants with high
nitrogen over host-plants with low nitrogen; however,
this preference was also mediated by plant chemical
defense. Female butterflies preferred more chemically
defended host-plants when foliar nitrogen was equivalent between host-plants. J. coenia larvae experience
intense predation in the field, especially when larvae are
not chemically well defended. Any qualitative or quantitative variation in plant allelochemical defense has
fitness consequences on these larvae. Thus, these results
indicate that females may be making sub-optimal
oviposition decisions under a nutrient-enriched regime,
when predators are present. Given the recent increase in
Communicated by Oswald Schmitz
K. L. Prudic (&) Æ J. C. Oliver Æ M. D. Bowers
Ecology and Evolutionary Biology, University of Colorado,
334 UCB, Boulder, CO 80309, USA
E-mail: [email protected]
Present address: K. L. Prudic
Ecology and Evolutionary Biology and the Bio5 Institute,
University of Arizona, PO Box 210088, Tucson, AZ 85721, USA
Present address: J. C. Oliver
Interdisciplinary Program in Insect Science, University of Arizona,
PO Box 210036, Tucson, AZ 85721, USA
fertilizer application and nitrogen deposition on the
terrestrial landscape, these interactions between female
preference, larval performance, and larval chemical defense may result in long-term changes in population
dynamics and persistence of specialist insects.
Keywords Nutrient enrichment Æ Lepidoptera Æ
Herbivore performance Æ Female preference Æ
Host-plant quality
Introduction
Recent anthropogenic nutrient availability changes in
terrestrial systems have had a significant effect on ecosystem dynamics (Jefferies and Maron 1997; Whittaker
2001; Matson et al. 2002; Vitousek et al. 2002). Nutrient
enrichment from agricultural and atmospheric sources
has the potential to alter plant–insect interactions via
changes in plant growth and defense (Whittaker 2001;
Coviella et al. 2002; He et al. 2002; Richardson et al.
2002). Soil nutrient augmentation has been shown to
change plant community structure (Pauli et al. 2002;
Richardson et al. 2002), as well as the corresponding
plant–herbivore dynamics (Kinney et al. 1997; Kerslake
et al. 1998; Whittaker 2001; Richardson et al. 2002),
although the direction of these changes can be systemspecific. While changes in the plant allelochemistry due
to nutrient enrichment have been demonstrated (e.g.
Oyeyele and Zalucki 1990; Fajer et al. 1992; Hugentobler and Renwick 1995; Bezemer et al. 2000; Coviella
et al. 2002), few studies have investigated the effect of
nutrient enrichment on specialist insects’ chemical defenses (Rank et al. 1998).
Host-plant nitrogen content is viewed as the ultimate
limiting nutrient for most chewing insects (Mattson
1980). Augmenting soil nutrients often increases plant
nitrogen concentration and reduces production of some
allelochemicals, resulting in higher growth and consumption rates in generalist phytophagous insects (e.g.
Schafellner et al. 1996; Lindroth and Kinney 1998).
Because host-plant nitrogen limits phytophagous insect
growth and development, specialist phytophagous insects should also demonstrate increased larval performance on high nutrient plants. However, these
presumed benefits may be offset by other changes in
plant chemistry which may affect specialist phytophagous insects differently from generalist phytophagous
insects. Specialist phytophagous insects commonly use
plant allelochemicals for host-plant location and hostplant identification, and these allelochemicals may
influence female oviposition choices (Chew 1979; Feeny
et al. 1983; Honda 1986; Pereyra and Bowers 1988).
Some specialist phytophagous insect species also
sequester plant allelochemicals for protection against
their own predators (Bowers 1990). Lower levels of plant
chemical defense often correlate with lower levels of
insect chemical defense (e.g. Pasteels et al. 1988; Malcolm 1995) with potential consequences on insect palatability (Brower et al. 1970; Camara 1997b). As a
result, variation in plant allelochemicals has potential
fitness consequences on specialist insects that rely on
these compounds for host-plant location, oviposition
cues, or chemical defense.
The present study examines the effects of soil
nutrient enrichment on the interactions between a hostplant and a specialist insect. We used the buckeye
butterfly, Junonia coenia Hübner (Nymphalidae) and
two principal host-plants, narrow-leafed plantain,
Plantago lanceolata L., and common plantain, Plantago
major L. (Plantaginaceae) to evaluate how soil nutrient
changes affect plant chemistry, larval performance,
larval defensive chemistry, and female oviposition
preference. Previous experiments demonstrated that
one of the major groups of secondary metabolites in P.
lanceolata, iridoid glycosides, decreases with an increase in soil nutrients (Fajer et al. 1992; Jarzomski
et al. 2000). Also, J. coenia larvae reared on P.
lanceolata, which has high levels of iridoid glycosides,
are rejected more often by an invertebrate predator
than larvae reared on P. major, which has low levels of
iridoid glycosides (Theodoratus and Bowers 1999).
High larval predation in the field (90%) and local
adaptation to plant allelochemicals by J. coenia demonstrate the importance of larval chemical defense for
larval survival (Camara 1997b, c)
Most butterfly larvae cannot move long distances
during their first few instars and feed upon the plant on
which they hatched (Mayhew 1997); therefore, J. coenia
larval chemical defense will be determined, at least in
part, by female oviposition choices. In J. coenia butterflies, host-plant iridoid glycoside concentration influences female oviposition preference (Pereyra and Bowers
1988; Klockars et al. 1993). Females prefer to lay eggs
on substrates containing relatively high levels of the
iridoid glycoside catalpol than on those containing relatively low, or no catalpol (Pereyra and Bowers 1988).
We designed a set of experiments to answer three
questions about the potential effects of increased soil
nutrient availability on the dynamics of plant chemistry,
larval performance, larval chemical defense, and female
oviposition choice in a specialist phytophagous insect:
(1) How does soil nutrient enrichment affect plant performance and chemistry? (2) How do host-plant chemical changes resulting from soil nutrient enrichment
influence larval performance and defensive chemistry?
and (3) How do host-plant chemistry changes in response to soil nutrient enrichment influence female oviposition choice?
To address questions 1 and 2, we performed a
greenhouse experiment using J. coenia and P. lanceolata.
Since both insect and plant growth are generally nutrient
limited, we expected P. lanceolata and J. coenia to grow
and develop better under increased soil nutrient regimes.
Also since iridoid glycosides are carbon based allelochemicals, we predicted plant defensive chemistry would
decrease with increasing soil nutrient availability (e.g.
Fajer et al. 1992; Jarzomski et al. 2000). Since J. coenia
larvae sequester iridoid glycosides from their larval hostplants, we predicted the larval defensive chemistry
would also decrease with increasing soil nutrient
enrichment.
To address question 3, we evaluated the relative
roles of both host-plant nutrient availability and hostplant defensive chemistry for female oviposition preference. To investigate host-plant nutrient availability,
we compared J. coenia oviposition behavior on fertilized P. lanceolata (high nitrogen, low defensive chemistry) to unfertilized P. lanceolata (low nitrogen, high
defensive chemistry) in the greenhouse. To investigate
the role of host-plant chemical defense on female
oviposition choice, we compared female oviposition
preference between unfertilized P. lanceolata (high
defensive chemistry) to unfertilized P. major (low
defensive chemistry) in the greenhouse. If predation is a
strong selective agent against larval survival, then we
expected females to preferentially oviposit on more
chemically defended host-plants (P. lanceolata, unfertilized > P. lanceolata, fertilized = P. major, unfertilized). If nitrogen is a limiting resource for larval
development, we expected females to preferentially
oviposit on higher nutrient host-plants regardless of
host-plant defensive chemistry (P. lanceolata, fertilized > P. lanceolata, unfertilized = P. major, unfertilized). By combining all experimental results, we were
able to evaluate the relative importance of host-plant
nitrogen uptake and chemical defense on female
oviposition choice, larval performance, and larval
chemical defense in a specialist phytophagous insect.
Methods
Study system
Plantago lanceolata and P. major are cosmopolitan,
herbaceous weeds, which are annual or facultative
perennials (Cavers et al. 1980; Kuiper and Bos 1992).
They were introduced in North America approximately
200 years ago, thriving in disturbed habitats such as
gardens and agricultural areas (Thomas et al. 1987).
Both species contain iridoid glycosides, a group of cyclopentanoid monoterpene-derived compounds found in
about 50 plant families (El-Naggar and Beal 1980; Boros
and Stermitz 1990). P. lanceolata primarily contains two
iridoid glycosides, aucubin and catalpol (Duff et al.
1965; Bowers and Stamp 1992, 1993); although trace
amounts of other iridoid glycosides (which are not
sequestered by J. coenia larvae) have been identified
(Willinger and Dobler 2001; Taskova et al. 2002).
Aucubin is the biosynthetic precursor to catalpol
(Damtoft et al. 1983), and it is less toxic to herbivores
than catalpol (Bowers 1991). P. major contains aucubin
but not catalpol, and has lower amounts of total iridoid
glycosides than P. lanceolata (Massa and M.D. Bowers,
unpublished data; this study).
The insect herbivore used in our experiments, J.
coenia Hübner (Lepidoptera: Nymphalidae), consumes
only plants containing iridoid glycosides (Bowers 1984),
and P. lanceolata and P. major are commonly used hostplants (Shapiro 1974; Scott 1986). Iridoid glycosides
serve as larval feeding and female oviposition stimulants
for J. coenia (Bowers 1984; Pereyra and Bowers 1988,
respectively). J. coenia larvae sequester iridoid glycosides
from their host-plants (Bowers and Collinge 1992), and
these compounds deter predation by invertebrate (Dyer
and Bowers 1996) and vertebrate (Bowers and Farley
1990) predators.
Plant treatments
Our nutrient enrichment regime was based on three
factors. First, since both the butterfly and the host-plant
are commonly found in agriculture and other nutrient
enriched areas, our fertilization treatments should fall
within range applied by farmers. In Colorado, the
average farmer applies 140 kg/ha/year with a range of 0–
340 kg/ha/year (USDA 2002); we applied the equivalent
of 89 kg/ha/year. We also wanted our nutrient treatments to be ecologically relevant. Based on previous
nutrient experiments, this regime produced P. lanceolata
foliar nitrogen levels within the documented range of
forbs under natural conditions (Joern and Behmer
1998). Finally, our treatment had to be potent enough to
result in significant changes in both plant foliar nitrogen
and iridoid glycosides.
Plantago lanceolata and P. major seeds were collected
from two sites on the University of Colorado, Boulder
campus. Three-week-old (first true leaves) plants were
transplanted into 4-l pots containing Fafard Nursery
mix#2 (Amherst, MA, USA). Half of the P. lanceolata
seedlings were transplanted into normal potting soil, and
the other half were transplanted into normal potting soil
with 300 mg of Osmocote 14N:14P:14K fertilizer:
150 mg mixed in the soil and 150 mg applied to the soil
surface to more closely simulate incidental fertilization
caused by agricultural runoff. All P. major seedlings
were transplanted into normal potting soil without
additional fertilizer. Plants were grown in the greenhouse at an average daytime temperature of 24C with
60% RH and 10light:14dark. Plants were watered every
third day.
Herbivory treatments
Three-week-old (first true leaves) P. lanceolata plants
were transplanted into 4-l pots with Fafard Nursery
mix#2 (Amherst, MA, USA) with the fertilization
treatments described in ‘Plant treatments’. Plants
were then randomly assigned to one of the four
treatments:
1. Unfertilized, no herbivory: each plant (n=10) was
watered to saturation every third day. Neither fertilizer enrichment nor herbivory occurred in this plant
treatment.
2. Fertilized, no herbivory: each plant (n=10) was watered to saturation every third day. With regular
watering, fertilizer nutrients were released at a constant rate.
3. Unfertilized, herbivory: each plant (n=25) was watered to saturation every third day. A single second
instar J. coenia caterpillar was placed on an 8-weekold plant.
4. Fertilized, herbivory: each plant (n=25) was watered
to saturation every third day. Fertilization regime
was the same as described for the ‘‘Fertilized, no
herbivory’’ treatment (no. 2 above). A single second
instar J. coenia caterpillar was placed on an 8-weekold plant.
Throughout the experiment, the plants were kept in
a greenhouse randomly arranged on a single 2·4 m
raised bench. The average daytime temperature was
24C with 60% RH and 10light:14dark. The experiments began at week 8 (average leaf height of 20 cm)
and lasted until the caterpillars molted into the fifth
instar (n=70). After the larvae reached fifth instar, the
plant above ground biomass was harvested and prepared for chemical analyses.
Plant performance and chemical analyses
At the end of the experiments, biomass and iridoid
glycosides were measured in plants from all experiments
(n=123). Plants were oven dried at 50C, and total
above ground dry biomass was measured as a plant
performance proxy. Dried plants were weighed, ground
with a Wiley Mill (#40 mesh), and stored in a glass jar at
40C until further analysis. Iridoid glycosides were
extracted from each sample and analyzed by gas chromatography (Gardner and Stermitz 1988; Bowers 1991).
Total foliar nitrogen for a subset of P. major and P.
lanceolata (n=68) was quantified with a copper sulfate
catalyst procedure using a Lachat colorimetric autoanalyzer (Bowman et al. 1993).
Insect performance and iridoid glycoside sequestration
Survival, larval mass gain, rate of larval mass gain,
larval final mass and number of days to the fifth instar
were measured for all larvae (n=50) from the herbivory
experiment. A second instar caterpillar, taken from a
laboratory colony raised on P. lanceolata, was weighed
and then placed on a single plant described previously in
the plant herbivory treatment. All plants were covered
with netting secured at the bottom with a large twist tie.
Every second day, each larva was removed from the
plant, weighed and returned to the plant. Larvae were
collected after their fifth instar molt because their
chemical defense steadily decreases from this ontological
point through pupation (M.D. Bowers, unpublished
data). Because of this limitation, the larvae were not
allowed to pupate, and we could not use the measure of
relative growth rate used in previous Lepidoptera performance experiments (Wiklund et al. 1991; Fischer and
Fiedler 2000).
To quantify larval iridoid glycosides, newly molted,
fifth instar caterpillars were collected and starved for
12 h to ensure empty guts. Aucubin and catalpol were
measured using methods similar to those used for plants,
except that the caterpillars were lyophilized instead of
oven dried (Gardner and Stermitz 1988; Bowers and
Collinge 1992). To determine whether caterpillar iridoid
glycoside content depended on host-plant content, we
tested for a correlation between host-plant and caterpillar iridoid glycosides.
Female oviposition preference
Females were simultaneously offered two 10-week-old
plants, one each from two of the following treatments:
unfertilized P. lanceolata (high iridoid glycoside content,
n=19), fertilized P. lanceolata (low iridoid glycoside
content, n=20), and unfertilized P. major (low iridoid
glycoside content, n=21). There were three experiments
in all: fertilized P. lanceolata versus unfertilized P.
lanceolata (n=9); fertilized P. lanceolata versus unfertilized P. major (n=11); and unfertilized P. lanceolata
versus unfertilized P. major (n=10).
Female and male J. coenia were taken from a laboratory colony, separated by sex, and placed into cylindrical net bags (40 cm H·30 cm D). The butterflies were
provided with honey water as a food source, but no
suitable oviposition substrate. After 48 h of isolation
from the opposite sex, males and females were placed
together in net bags, in a 2 male:1female ratio (three or
six individuals total). Following 72 h of potential mating
time, females were removed from the net bag and used in
the oviposition experiment. For each choice test, individual plants from two of the three plant treatments
were selected randomly and each plant was used only
once. Plants were placed 20 cm apart in a clear plastic
container (80 cm W·35 cm D·35 cm H) in the greenhouse. A honey water source was placed in the middle of
the container, equidistant from both plants. The container was covered with fine mesh and secured with
twist-ties. A single female butterfly was then placed upon
the honey water source in the container, and allowed to
oviposit for 48 h. The container was checked after 24 h
to refill the food supply and to water the plants if necessary. After 48 h of oviposition, the plants were removed from the container and the eggs were counted as
individual leaves were removed from the plants. These
leaves were then prepared for subsequent chemical
analysis.
Statistical analyses
The percentage based data (aucubin, catalpol and total
iridoid glycoside concentrations) were arcsine transformed to normalize the data prior to analysis. In order
to assess the effect of plant treatment on oviposition,
the proportion of eggs laid on each plant was transformed, and these transformed data were then used to
calculate a difference between eggs laid on each treatment:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
r1 þ ð3=8Þ
r2 þ ð3=8Þ
Difference in eggs ¼
n þ ð3=4Þ
n þ ð3=4Þ
where r1 and r2 are the number of eggs laid on each of
the treatments, respectively, and n is the total number of
eggs laid (Judd and McClelland 1989). t-tests were performed to determine if females preferred one plant
treatment over another. The effects of fertilization and
herbivory on plant performance and plant chemical
defense were analyzed using a two-way ANOVA. The
effects of soil fertilizer on plant nitrogen concentration,
insect performance, and insect defensive chemistry were
analyzed using one-way ANOVAs. Larval survival was
compared using a chi-square test. Relationships between
plant defensive chemistry and insect defensive chemistry
(percent total iridoid glycosides, percent aucubin, and
percent catalpol) were tested for using regression analyses. All statistical analyses were conducted with SAS
version 8.1 (SAS 2000).
Results
How does soil nutrient enrichment influence plant
performance and chemistry?
Soil fertilization significantly increased the foliar nitrogen concentration (measured as percent dry mass) in P.
lanceolata (Table 1; Fig. 1), as well as plant above
ground biomass (Table 1). However, from the larval
performance experiments, herbivory also significantly
Percent dry mass
3.5
Catalpol
2.5
2.0
Nitrogen
1.5
1.0
0.5
P. lanceolata
Unfertilized
Fertilized
P. major
Unfertilized
Plant species and treatment
Fig. 1 The effect of soil fertilization on chemical defenses (iridoid
glycosides aucubin and catalpol) and foliar nitrogen of Plantago
lanceolata and P. major measured in percent dry mass. Mean ± SE
presented
iridoid glycosides
6.0
How do host-plant chemistry changes influence larval
performance and defensive chemistry?
There was no significant effect of fertilization on any
measure of larval performance. Neither total larval mass
gain, rate of larval mass gain, larval final mass, nor days
to fifth instar (n=40) was affected by fertilization
treatment (Table 1). Larval survival (n=50) did not
differ between fertilized and unfertilized plants (fertilized=84%, unfertilized=76%, v2 =0.503, P=0.4783).
Iridoid glycoside concentrations were significantly
lower in larvae reared on fertilized P. lanceolata than
those reared on unfertilized P. lanceolata (Table 1;
Aucubin
Iridoid glycosides
3.0
P. lanceolata
Larval percent
affected foliar dry mass (Table 1). On average, foliar dry
mass in the herbivory treatment was 1.3 g greater than
foliar dry mass of the plants in the non-herbivory
treatment. The interaction between herbivory and fertilization was not significant (Table 1).
For P. lanceolata, total iridoid glycoside concentration was negatively affected by fertilization (Table 1).
On average, fertilized plants contained about half the
total iridoid glycosides of unfertilized plants (Fig. 1).
This effect was attributable to differences in aucubin, not
catalpol (Table 1). Aucubin concentrations (percent dry
mass) of non-fertilized plants were, on average, 1.7 times
higher than fertilized plants (Fig. 1). P. major contained
no catalpol, but did have significantly less aucubin than
plants in either of the P. lanceolata treatments (Fig. 1;
ANOVA n=123, F1,121=35.52, P<0.0001). There was
no significant difference in plant foliar nitrogen content
between unfertilized P. major and unfertilized P.
lanceolata (Fig. 1; ANOVA n=68, F1,65=0.02,
P=0.8865).
5.0
Aucubin
4.0
Catalpol
3.0
2.0
1.0
Fertilized
Unfertilized
Host-plant treatment
Fig. 2 The effect of host-plant soil fertilization on buckeye
(Junonia coenia) larvae chemical defenses (iridoid glycosides
aucubin and catalpol) measured in percent dry mass. Mean ± SE
presented
Table 1 Summary of statistical tests of fertilization effects on plant foliar chemistry and subsequent larval performance and chemical
defense profiles
Source
Trait
Soil fertilization
Plantago lanceolata traits
Foliar nitrogen
Foliar dry mass
Total foliar iridoid glycosides
Foliar aucubin
Foliar catalpol
Junonia coenia traits
Larval mass gain
Rate of larval mass gain
Larval final mass
Days to fifth instar
Total larval iridoid glycosides
Larval aucubin
Larval catalpol
Plantago lanceolata traits
Foliar dry mass
Total foliar iridoid glycosides
Foliar aucubin
Foliar catalpol
Larval herbivory
n
df
F
P
Direction (mean)
40
110
110
110
110
1,
1,
1,
1,
1,
38
108
108
108
108
117.95
11.63
16.74
15.71
1.35
<0.0001
0.0009
<0.0001
0.0001
0.2470
F(2.76%) > U(1.00%)
F(13.7 g) > U(8.1 g)
F(1.30%) < U(2.90%)
F(1.13%) < U(2.63%)
F(0.17%) = U(0.27%)
40
40
40
40
40
40
40
1,
1,
1,
1,
1,
1,
1,
38
38
38
38
38
38
38
0.18
0.21
0.16
2.81
6.54
7.64
1.11
0.6710
0.6500
0.6910
0.1030
0.0155
0.0094
0.2989
F(0.281 g) = U(0.266 g)
F(0.018/day) = U(0.019/day)
F(0.282 g) = U(.268 g)
F(15.1 days) = U(13.5 days)
F(0.74%) < U(4.78%)
F(0.24%) < U(4.08%)
F(0.50%) = U(0.70%)
70
70
70
70
1,
1,
1,
1,
66
66
66
66
5.06
1.12
1.20
0.28
0.0287
0.2335
0.2360
0.7838
H(6.1 g) > N(4.8 g)
H(2.1%) = N(1.9%)
H(4.08%) = N(4.49%)
H(0.28%) = N(0.25%)
All are ANOVA results with significant effects labeled in bold. Direction of effects and mean of treatment shown F fertilized Plantago
lanceolata, U unfertilized P. lanceolata, H P. lanceolata with herbivory, N P. lanceolata without herbivory
Table 2 Correlation results between host-plant defensive chemistry and larval defensive chemistry
Number of correlations
df
F
P
R2
Total foliar iridoid glycosides versus total larval iridoid glycosides
Foliar aucubin versus larval aucubin
Foliar catalpol versus larval catalpol
40
40
40
1, 38
1, 38
1, 38
26.03
23.40
25.13
0.001
0.001
0.001
0.441
0.414
0.432
Proportion of
total eggs laid
Correlation
1.0
0.8
0.6
0.4
0.2
A
P. lanceolata P. major
Unfertilized Unfertilized
B
P. lanceolata P. major
Fertilized
Unfertilized
C
P. lanceolata P. lanceolata
Unfertilized
Fertilized
Host-plant and treatment
Fig. 3 The effect of host-plant species and soil fertilization on female buckeye (J. coenia) oviposition preference. Original data was arcsin
transformed for statistical analyses; proportion data is shown for illustrative purposes only. Mean ± SE presented
Fig. 2). On average, the larvae raised on unfertilized
plants contained 4.1 times more total dry mass iridoid
glycosides as compared to larvae raised on fertilized
plants (Fig. 2). This effect is attributable to differences in
aucubin, not catalpol (Table 1). Larvae reared on
unfertilized plants, on average, sequestered 3.9 times
more aucubin than larvae reared on fertilized plants
(Fig. 2).
Host-plant iridoid glycoside concentration showed
significant positive correlation with insect iridoid
glycoside concentration (Table 2). This was the case
for aucubin, catalpol and total iridoid glycosides
(Table 2).
How do host-plant chemistry changes influence female
oviposition choice?
These results are presented as a series of three pair-wise
experiments. In all three tests, females exhibited a clear
preference for plants in one treatment over those in
another plant treatment (Fig. 3). In the unfertilized P.
lanceolata versus unfertilized P. major trials, females
preferred unfertilized P. lanceolata (t test, n=10,
t1,8=30.25, P=0.0004), and only rarely laid eggs on
unfertilized P. major. In the fertilized P. lanceolata versus unfertilized P. major trials, females preferred fertilized P. lanceolata (t test, n=11, t1,9=89.11, P<0.0001),
almost to the exclusion of unfertilized P. major. Finally,
in the fertilized P. lanceolata versus unfertilized
P. lanceolata, females again preferred to lay eggs on
fertilized P. lanceolata, although the effect was not as
strong as in the previous two experiments (t test, n=9,
t1,7=5.62, P=0.0452). Female oviposition preference
was not predicted by foliar biomass (ANOVA n=31,
F1,29=0.02, P=0.8820).
Discussion
How does soil nutrient enrichment influence plant
performance and chemistry?
Soil nutrient enrichment increased P. lanceolata above
ground biomass and plant foliar nitrogen as predicted.
Previous experiments demonstrated an increase in
nitrogen concentration with fertilization in P. lanceolata
(Jarzomski et al. 2000; Tosserams et al. 2001). Fertilized
P. lanceolata also had lower total iridoid glycosides
concentrations and lower aucubin concentrations than
unfertilized plants, in agreement with previous observations (Fajer et al. 1992; Jarzomski et al. 2000).
One unanticipated result was that plant biomass response to herbivory was dramatic: herbivory by J. coenia caterpillars increased plant biomass by over 25%,
indicating significant overcompensation. Although in
some experiments, compensation has been observed in
P. lanceolata in response to herbivory (e.g., Stamp and
Bowers 1996; Jarzomski et al. 2000), there was no evidence of compensation in others (e.g. Bowers and Stamp
1993; Darrow and Bowers 1999).
How do host-plant chemistry changes influence larval
performance and defensive chemistry?
Host-plant nitrogen content is viewed as the ultimate
limiting nutrient for chewing insects (Mattson 1980), but
in this study, additional fertilization did not increase
J. coenia larval performance (Table 1). In herbivorous
insects, higher foliar nitrogen levels generally increase
larval developmental and growth rates (e.g. Slansky and
Feeny 1977; Tabashnik 1982; Ravenscroft 1994);
although, there is accumulating evidence for negative
correlations between insect performance and host-plant
nitrogen concentrations (Schroeder 1986; Joern and
Behmer 1998; Fischer and Fiedler 2000). Within a certain range, lepidopteran herbivores have been observed
to compensate for reduced host-plant nitrogen content
by increasing their food consumption or by concentrating their feeding on the most nitrogen rich plant
parts (e.g. Slanksy and Feeny 1977; Mattson 1980;
Ravenscroft 1994; Hättenschwiler and Schafellner 1999).
Compensatory feeding in J. coenia has been observed in
response to CO2 enrichment, which decreases nitrogen
concentration in foliage (Fajer 1989).
Our result may also be explained by reduced larval
feeding rates in the fertilized treatment due to decreased
iridoid glycosides, which are phagostimulants for
J. coenia caterpillars (Bowers 1984; Adler et al. 1995;
Camara 1997a). At high levels of plant iridoid glycoside
concentration (ca. 10%), there is evidence of a small
larval physiological cost of iridoid glycoside sequestration (Camara 1997a); although, the high plant iridoid
glycoside levels in this study were much lower (ca. 3%)
than those reported in the Camara study. Finally, foliar
nitrogen concentrations in this study are relatively low
compared with other P. lanceolata studies, which used
different soil enrichment regimes (Jarzomski et al. 2000;
Tosserams et al. 2001). Our nitrogen levels may have
been too low to significantly affect larval performance.
Following the pattern of the plant iridoid glycosides,
J. coenia showed a decrease in total iridoid glycoside and
aucubin concentrations with increased host-plant fertilization (Fig. 2). Larvae raised on fertilized P. lanceolata,
on average, sequestered 4.1 times less total iridoid glycosides than larvae reared on unfertilized P. lanceolata.
Therefore, as predicted, soil nutrient enrichment decreases both plant and larval defensive chemistry.
Overall, there was a significant positive correlation
between plant and larval iridoid glycosides (Table 2).
Thus, plant iridoid glycoside content is an important
predictor of caterpillar iridoids and the degree to which
they may be protected from predators. Although such
comparisons have been made between caterpillars fed on
different host-plant species or on artificial diets with
different concentrations of iridoid glycosides (Dyer and
Bowers 1996; Camara 1997a, c), correlations between
individual plants and the caterpillars feeding on them
have not been previously made.
How do host-plant chemistry changes influence female
oviposition choice?
Two clear patterns emerged from the oviposition results:
female J. coenia preferred to oviposit on Plantago
lanceolata over P. major, regardless of fertilization
treatment, and females oviposited more on fertilized P.
lanceolata versus unfertilized P. lanceolata (Fig. 3). The
former result is likely attributable to qualitative differences between the two Plantago species: P. lanceolata
contains both aucubin and catalpol, while P. major
contains only aucubin. Also, the two plant species differ
substantially in total iridoid glycoside concentrations
(Duff et al. 1965; Massa and Bowers, unpublished data;
this study). Since P. major lacks catalpol, a known oviposition stimulant for J. coenia (Pereyra and Bowers
1988), females may have perceived P. lanceolata as the
only suitable host-plant when offered a choice between
P. lanceolata and P. major.
The second result, female oviposition preference for
fertilized over unfertilized P. lanceolata, suggests that
female oviposition behavior is affected by host-plant
traits other than iridoid glycoside concentration. In
other butterfly species also, female oviposition preferences are not solely determined by host-plant defensive
chemistry (Zalucki et al. 1990; Camara 1997c), and plant
nutritive quality may also influence female oviposition
decisions (Honda 1995). The two P. lanceolata treatments did not differ in their catalpol concentrations
(Fig. 1); therefore, females may have perceived both
treatments’ iridoid glycoside profiles as chemically
equivalent, at least for oviposition purposes. If so, females are able to discern between low- and high-nutrient
plants when an oviposition stimulant (catalpol) does not
vary. Since females seldom oviposited on P. major, a low
level aucubin species, their preference for low aucubin
host-plants is probably not attributable to the co-variation between nitrogen and iridoid glycosides in
P. lanceolata. Irrespective of the precise mechanism,
females laid more eggs on high-nutrient, low iridoid
glycoside plants than on low-nutrient, high iridoid
glycoside plants.
Given these results, the obvious question is, ‘‘Are
females making good oviposition choices?’’. Phytophagous insects should preferentially oviposit on host-plants
which promote offspring survival (Courtney and Kibota
1990; Mayhew 1997). Our results showed that J. coenia
females preferred to oviposit on fertilized (high foliar
nitrogen) plants over unfertilized (low foliar nitrogen)
ones. However, there was no corresponding increase in
larval performance on the fertilized, high foliar nitrogen
P. lanceolata. Those larvae which fed on fertilized P.
lanceolata also had significantly lower iridoid glycoside
content. Yet, total iridoid glycoside concentration
determines predator avoidance or acceptance (Dyer and
Bowers 1996; Camara 1997b; Theodoratus and Bowers
1999). Females appear to be exhibiting suboptimal oviposition decisions because these decisions influence larval chemical defense, larval chemical defense deters
predation, and larval predation pressure is high.
This discrepancy between preference and performance could be explained by insufficient time to adapt,
undetected pre- and post-pupation benefits, or reduced
larval feeding time. J. coenia has probably had enough
time to adapt to Plantago species, because it has adapted
locally to other non-native host-plant species (Shapiro
1978; Singer et al. 1993; Radtkey and Singer 1995;
Camara 1997c). Differences in larval chemical defense
between individuals reared on fertilized, low iridoid
glycoside plants and unfertilized, high iridoid glycoside
plants were significant, suggesting that there would also
be differences in pre-pupation performance. These prepupation differences would likely be highly significant to
compensate for the predation cost of low larval chemical
defenses (Dyer and Bowers 1996; Camara 1997b). Because iridoid glycosides in J. coenia larvae decline during
the fifth instar, we could not quantify both post-pupation benefits and chemical defense. These post-pupation
benefits would likewise need to be extraordinary to
counter the survival cost of low chemical defense (Dyer
and Bowers 1996; Camara 1997b, c). If high-nutrient
plants afforded J. coenia more time to hide from natural
enemies (via increased feeding efficiency), then this escape from predators may offset any decrease in chemical
defense (Bernays 1997). Unfortunately, we did not
measure larval feeding time.
Highlights and implications
Rising levels of available nutrients have altered the
globalnutrient cycle substantially, with consequential
changes in terrestrial and aquatic systems (Aber et al.
2003; Fenn et al. 2003; Vitousek et al. 1997). Herbivorous insects and their predators are also affected by increased nutrient availability. Our results showed
unexpected effects of increased fertilization on the relationships between a specialist insect, J. coenia, and its
larval host-plant, Plantago lanceolata. Female J. coenia
preferred to oviposit on host-plants with higher foliar
nitrogen and lower chemical defense while the larvae
consuming these plants did not demonstrate higher
performance but did have lower chemical defense levels.
Given the high larval predation rates and the role
defensive chemicals play in reducing this predation (e.g.
Dyer and Bowers 1996; Camara 1997b), females appear
to be making sub-optimal decisions in an increased
nitrogen regime. Even though our foliar nitrogen levels
where within the documented range of forbs under
natural conditions (Joern and Behmer 1998), this
experiment demonstrates how increased soil nutrients in
a controlled environment affect plant performance,
plant chemistry, female oviposition preference, larval
performance, and larval defensive chemistry. Future
efforts should focus on how nutrient enrichment affects
plant and specialist insect dynamics in a natural setting.
Acknowledgements We would like to thank W. Bowman for foliar
nitrogen analyses, and J. Ramp for greenhouse assistance. K.
Barton, E. Bernays, W. Bowman, J. Bronstein, G. Davidowitz, Y.
Linhart, K. Mooney, J. Ness, D. Papaj and M. Singer provided
helpful manuscript comments. Our handling editor, O. Schmitz,
and two anonymous reviewers also greatly improved this manuscript. This research was supported by an NSF graduate research
fellowship to KLP, an Ecology and Evolutionary Biology graduate
student research award to KLP, and a grant from the University of
Colorado Graduate School to JCO. These experiments, to the best
of our knowledge, fully comply with the current laws of the United
States and the state of Colorado.
References
Aber JD, Goodlae CL, Ollinger SV, Smith M-L, Mahill AH,
Martin ME, Hallet RA, Stoddard JL (2003) Is nitrogen deposition altering the nitrogen status of northeastern forest? Bioscience 53:375–389
Adler LS, Schmitt J, Bowers MD (1995) Genetic variation in
defensive chemistry in Plantago lanceolata (Plantaginaceae) and
its effect on the specialist herbivore Junonia coenia (Nymphalidae). Oecologia 101:75–85
Bernays EA (1997) Feeding by lepidopteran larvae is dangerous.
Ecol Entomol 22:121–123
Bezemer TM, Jones TH, Newington JE (2000) Effects of carbon
dioxide and nitrogen fertilization on phenolic content in Poa
annua L. Biochem Syst Ecol 28:839–846
Boros CA, Stermitz FR (1990) Iridoids. An updated review, part I.
J Nat Prod 53:1055–1147
Bowers MD (1984) Iridoid glycosides and hostplant specificity in
larvae of the buckeye butterfly, Junonia coenia (Nymphalidae).
J Chem Ecol 10:1567–1577
Bowers MD (1990) Recycling plant natural products for insect
defense. In: Evans DL, Schmidt JO (eds) Insect defenses:
adaptive mechanisms and strategies of prey and predators.
State University of New York Press, Albany, pp 352–386
Bowers MD (1991) Iridoid glycosides. In: Rosenthal G, Berenbaum M (eds) Herbivores: their interaction with secondary
plant metabolites, vol 1, 2nd edn. Academic, Orlando, pp 297–
325
Bowers MD, Collinge SK (1992) Fate of iridoid glycosides in different life stages of the buckeye, Junonia coenia (Lepidoptera:
Nymphalidae). J Chem Ecol 18:817–831
Bowers MD, Farley SD (1990) The behavior of gray jays (Perisoreus canadensis) toward palatable and unpalatable Lepidoptera.
Anim Behav 39:699–705
Bowers MD, Stamp NE (1992) Chemical variation within and
between individuals of Plantago lanceolata (Plantaginaceae).
J Chem Ecol 18:985–995
Bowers MD, Stamp NE (1993) Effects of plant age, genotype, and
herbivory on Plantago performance and chemistry. Ecology
74:1778–1791
Bowman WD, Theodose TA, Schardt JC, Conant RT (1993)
Constraints of nutrient availability on primary production in
two alpine tundra communities. Ecology 74:2085–2097
Brower LP, Pough FH, Meck HR (1970) Theoretical investigations
of automimicry. I. Single trial learning. Proc Natl Acad Sci
USA 66:1059–1066
Camara MD (1997a) Physiological mechanisms underlying the
costs of chemical defense in Junonia coenia Hübner (Nymphalidae): a gravimetric and quantitative genetic analysis. Evol
Ecol 11:451–469
Camara MD (1997b) Predator responses to sequestered plant
toxins in buckeye caterpillars: are tritrophic interactions locally
variable? J Chem Ecol 23:2093–2106
Camara MD (1997c) A recent host range expansion in Junonia
coenia Hübner (Nymphalidae): oviposition preference, survival,
growth, and chemical defense. Evolution 51:873–884
Cavers PB, Bassett IJ, Crompton CW (1980) The biology of
Canadian weeds. 47. Plantago lanceolata L. Can J Plant Sci
60:1269–1282
Chew FS (1979) Community ecology and Pieris-crucifer coevolution. NY Entomol Soc 87:128–134
Courtney SP, Kibota TT (1990) Mother doesn’t know best:
Selection of hosts by ovipositing insects. In: Bernays EA (ed)
Insect–plant interactions, vol II. CRC, Boca Raton, pp 161–
187
Coviella CE, Stipanovic RD, Trumble JT (2002) Plant allocation to
defensive compounds: interactions between elevated CO2 and
nitrogen in transgenic cotton plants. J Exp Bot 53:323–331
Damtoft S, Jensen SR, Nielsen BJ (1983) The biosynthesis of
iridoid glycosides from 8-epi-deoxyloganic acid. Biochem Syst
Ecol 25:1–11
Darrow K, Bowers MD (1999) Effects of herbivore damage and
nutrient level on induction of iridoid glycosides in Plantago
lanceolata. J Chem Ecol 25:1427–1440
Duff RB, Bacon JSD, Mundie CM, Farmer VC, Russell JD, Forrester AR (1965) Catalpol and methyl catalpol: naturally
occurring glycosides in Plantago and Buddleia species. Biochem
J 96:1–5
Dyer LA, Bowers MD (1996) The importance of sequestered iridoid glycosides as a defense against an ant predator. J Chem Ecol
22:1572–1539
El-Naggar LJ, Beal JL (1980) Iridoids: a review. J Nat Prod
43:649–707
Fajer ED (1989) The effects of enriched CO2 on plant-insect herbivore interactions: growth responses of larvae of a specialist
butterfly, Junonia coenia (Lepidoptera: Nymphalidae). Oecologia 81:514–520
Fajer ED, Bowers MD, Bazzaz FA (1992) The effect of nutrients
and enriched CO2 environments on production of carbon-based
allelochemicals in Plantago: a test of carbon/nutrient balance
hypothesis. Am Nat 140:707–723
Feeny P, Rosenberry L, Carter M (1983) Chemical aspects of
oviposition behavior in butterflies. In Ahmad S (ed) Herbivorous insects: host-seeking behavior and mechanisms. Academic,
New York
Fenn ME, Baron JS, Allen EB, Rueth HM, Nydick KR, Geiser L,
Bowman WD, Sickman JO, Meixner T, Johnson DW, Neitlich
P (2003) Ecological effects of nitrogen deposition in the western
United States. Biosciences 53:404–420
Fischer K, Fiedler K (2000) Response of the copper butterfly Lycaena tityrus to increased leaf nitrogen in natural food plants:
evidence against the nitrogen limitation hypothesis. Oecologia
124:235–241
Gardner DR, Stermitz FR (1988) Hostplant utilization and iridoid
glycoside sequestration by Euphydryas anicia individuals and
populations. J Chem Ecol 14:2147–2168
Hättenschwiler S, Schafellner C (1999) Opposing effects of elevated
CO2 and N deposition on Lymantria monacha larvae feeding on
spruce trees. Oecologia 118:210–217
He JS, Bazzaz FA, Schmid B (2002) Interactive effects of diversity,
nutrients and elevated CO2 on experimental plant communities.
Oikos 97:337–348
Honda K (1986) Flavanone glycosides as oviposition stimulants in
a papilionid butterfly, Papilio protenor. J Chem Ecol 12:1999–
2010
Honda K (1995) Chemical basis of differential oviposition by lepidoperous insects. Arch Insect Biochem Physiol 30:1–23
Hugentobler U, Renwick JAA (1995) Effects of plant nutrition on
the balance of insect relevant cardenolides and glucosinolates in
Erysimum cheiranthoides. Oecologia 102:95–101
Jarzomski CM, Stamp NE, Bowers MD (2000) Effects of plant
phenology, nutrients and herbivory on growth and defensive
chemistry of plantain, Plantago lanceolata. Oikos 88:371–379
Jefferies RL, Maron JL (1997) The embarrassment of riches:
atmospheric deposition of nitrogen and community and ecosystem processes. TREE 12:74–78
Joern A, Behmer ST (1998) Impact of diet quality on demographic
attributes in adult grasshoppers and nitrogen limitation
hypothesis. Ecol Entomol 23:174–184
Judd CM, McClelland GH (1989) Data analysis: a model comparison approach. Harcourt, Brace, Jvanovich Publishers, San
Diego
Kerslake JE, Woodin SJ, Hartley SE (1998) Effects of carbon
dioxide and nitrogen enrichment on a plant–insect interaction:
the quality of Calluna vulgaris as a host for Operophtrea brumata. New Phytol 140:43–53
Kinney KK, Lindroth RL, Jung SM, Nordheim EV (1997) Effects
of CO2 and NO2 availability on deciduous trees: phytochemistry and insect performance. Ecology 78:215–230
Klockars GK, Bowers MD, Cooney B (1993) Leaf variation in
iridoid glycoside content of Plantago lanceolata (Plantaginaeae)
and oviposition of the buckeye, Junonia coenia (Nymphalidae).
Chemoecology 4:72–78
Kuiper PJC, Bos M (1992) Plantago: a multidisciplinary study.
Springer, Berlin Heidelberg New York
Lindroth RL, Kinney KK (1998) Consequences of enriched
atmospheric CO2 and defoliation for foliar chemistry and gypsy
moth performance. J Chem Ecol 24:1677–1695
Malcolm SB (1995) Milkweeds, monarch butterflies and the ecological significance of cardenolides. Chemoecology 5(6):101–117
Matson P, Lohse KA, Hall SJ (2002) The globalization of nitrogen
deposition: consequences for terrestrial ecosystems. Ambio
31:113–119
Mattson WJ Jr (1980) Herbivory in relation to plant nitrogen
content. Annu Rev Ecol Syst 11:119–161
Mayhew PJ (1997) Adaptive patterns of host-plant selection by
phytophagous insects. Oikos 79:417–428
Oyeyele SO, Zalucki MP (1990) Cardiac-glycosides and oviposition
by Danaus plexippus on Asclepias fruticosa in southeast
Queensland (Australia), with notes on the effect of plant
nitrogen content. Ecol Entomol 15:177–185
Pasteels JM, Rowell-Rahier M, Raupp MJ (1988) Plant derived
defense in chrysomelid beetles. In: Barbosa R, Leourneau D
(eds) Novel aspects of insect-plant relationships. Wiley, New
York, pp 235–272
Pauli D, Peintinger M, Schmid B (2002) Nutrient enrichment in
calcareous fens: effects on plant species and community structure. Basic Appl Ecol 3:255–266
Pereyra PC, Bowers MD (1988) Iridoid glycosides as oviposition
stimulants for the buckeye butterfly, Junonia coenia (Nymphalidae). J Chem Ecol 14:917–928
Radtkey RR, Singer MC (1995) Repeated reversals of host-preference evolution in a specialist insect herbivore. Evolution
49:351–359
Rank NE, Kopf A, Julkunen-Tiitto R, Tahvanainen J (1998) Host
preference and larval performance of the salicylate-using beetle
Phratora vitellinae. Ecology 79:618–631
Ravenscroft NOM (1994) The ecology of the chequered skipper
butterfly Carterocephalus palaemon in Scotland. 2. Foodplant
quality and population range. J Appl Ecol 31:623–630
Richardson SJ, Press MC, Parsons AN, Hartley SE (2002) How do
nutrients and warming impact on plant communities and their
insect herbivores? A 9-year study from a sub-arctic heath.
J Ecol 90:544–556
SAS Institute (2000) SAS/STAT user’s guide, release 8.0. SAS
Institute, Cary
Schafellner C, Berger R, Mattanovich J, Führer E (1996) Variations in spruce needle chemistry and implications for the little
spruce sawfly, Pristiphora abietina. In: Mattson WJ, Niemelä P,
Rousi M (eds) Dynamics of forest herbivory: quest for pattern
and principle. US Forest Service General Technical Report Nc183, St. Paul, pp 248–256
Schroeder LA (1986) Protein limitation of a tree feeding lepidopteran. Entomol Exp Appl 41:115–120
Scott JA (1986) The butterflies of North America: a natural history
and field guide. Stanford University Press, Stanford
Shapiro AM (1974) Butterflies of the Suisun Marsh, California.
J Res Lepid 13:191–206
Shapiro AM (1978) A new weedy host for the buckeye, Precis
coenia (Nymphalidae). J Lepid Soc 32:224
Singer MC, Thomas CD, Parmesan C (1993) Rapid human-induced evolution of insect–host associations. Nature 366:681–
683
Slanksy F Jr, Feeny P (1977) Stabilization of the rate of nitrogen
accumulation by larvae of the cabbage butterfly on wild and
cultivated food plants. Ecol Monogr 47:209–228
Stamp NE, Bowers MD (1996) Consequences for plantain chemistry and growth when herbivores are attacked by predators.
Ecology 72:535–549
Tabashnik BE (1982) Responses of pest and non-pest Colias butterfly larvae to intraspecific variation in leaf nitrogen and water
content. Oecologia 55:389–394
Taskova R, Evstatieva L, Handjieva N, Popov S (2002) Iridoid
patterns of genus Plantago L. and their systematic significance.
Z Naturforsch 57c:42–50
Theodoratus DH, Bowers MD (1999) Effects of sequestered iridoid
glycosides on prey choice of the prairie wolf spider, Lycosa
carolinensis. J Chem Ecol 25:283–295
Thomas CD, Ng D, Singer MC, Mallet JLB, Parmesan C, Billington HL (1987) Incorporation of a European weed into the
diet of a North American herbivore. Evolution 41:892–901
Tosserams M, Smet J, Magendans E, Rozema J (2001) Nutrient
availability influences UV-B sensitivity of Plantago lanceolata.
Plant Ecol 154:159–168
United States Department of Agriculture (2002) Agriculture
chemical usage: 2001 field crop summary. National Agriculture
Statistics Service, Agriculture Statistics Board
Vitousek PM, Aber JD, Howarth RW, Likens GE, Matson PA,
Schindler DW, Schlesinger WH, Tilman DG (1997) Human
alteration of the global nitrogen cycle: sources and consequences. Ecol Appl 7:737–750
Vitousek PM, Hattenschwiler S, Olander L, Allison S (2002)
Nitrogen and nature. Ambio 31(2):97–101
Whittaker JB (2001) Insects and plants in a changing atmosphere.
J Ecol 89:507–518
Wiklund C, Nylin S, Forsberg J (1991) Sex-related variation in
growth rate as a result of selection for large size and protandry
in a bivoltine butterfly, Pieris napi. Oikos 60:241–250
Willinger G, Dobler S (2001) Selective sequestration of iridoid
glycosides from their host-plants in Longitarsus flea beetles.
Biochem Syst Ecol 29:335–346
Zalucki MP, Brower LP, Malcolm SB (1990) Oviposition by
Danaus plexippus in relation to cardenolide content of three
Asclepias species in the southeastern USA. Ecol Entamol
15:231–240.