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Interactions between Spinacia oleracea and Bradysia impatiensA role for phytoecdysteroids.

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204
Schmelz et al.
Archives of Insect Biochemistry and Physiology 51:204–221 (2002)
Interactions Between Spinacia oleracea and Bradysia
impatiens: A Role for Phytoecdysteroids
Eric A. Schmelz,1,3* Robert J. Grebenok,2 Thomas E. Ohnmeiss,2 and William S. Bowers1
Plant produced insect molting hormones, termed phytoecdysteroids (PEs), are thought to function as plant defenses against
insects by acting as either feeding deterrents or through developmental disruption. In spinach (Spinacia oleracea), 20hydroxyecdysone (20E) concentrations in the roots rapidly increase following root damage, root herbivory, or methyl jasmonate
(MJ) applications. In this inducible system, we investigated the plant defense hypothesis by examining interactions of roots,
20E concentrations, and larvae of the dark-winged fungus gnat (Bradysia impatiens). Root herbivory by B. impatiens larvae
resulted in a 4.0- to 6.6-fold increase in root 20E concentrations. In paired-choice tests, increases in dietary 20E stimulated
B. impatiens feeding deterrency. B. impatiens larvae preferred control diets, low in 20E, to those constructed from induced
roots and those amended with 20E (25 to 50 mg/g wet mass). When confined to 20E-treated diets, concentrations as low as
5 mg/g (wet mass) resulted in significantly reduced B. impatiens survivorship compared to controls. The induction of root 20E
levels with MJ resulted in a 2.1-fold increase in 20E levels and a 50% reduction in B. impatiens larval establishment. In a
paired-choice arena, untreated control roots were damaged significantly more by B. impatiens larvae than MJ-induced roots
that contained 3-fold greater 20E levels. Based on dietary preference tests, the 20E concentrations present in the MJ-induced
roots (28 mg/g wet mass) were sufficient to explain this reduction in herbivory. Interactions between spinach roots and B.
impatiens larvae demonstrate that PEs can act as inducible defenses and provide protection against insect herbivory. Arch.
Insect Biochem. Physiol. 51:204–221, 2002. Published 2002 Wiley-Liss, Inc.†
KEYWORDS: Bradysia impatiens; Spinacia oleracea; 20-hydroxyecdysone; phytoecdysteroid; defense
INTRODUCTION
Complexities in host plant selection by phytophagous insects have plagued investigations of
phytoecdysteroids (PEs) as potential mediators of
plant-insect interactions. On the one hand, insects
that include PE-containing plants in their host
range have evolved efficient detoxification mechanisms (Feyereisen et al., 1976; Zhang and Kubo,
1993) and are thus unaffected by dietary molting
hormones (Carlisle and Ellis, 1968; Blackford et
al., 1996). On the other hand, insects with narrow
host ranges that do not normally feed on PE-containing plants often exhibit elevated mortality
when fed dietary PEs (Kubo and Klocke, 1983;
Blackford and Dinan, 1997). When PEs are exogenously applied to otherwise suitable host plants,
non-adapted insects often display reduced feeding
1
Department of Entomology, University of Arizona, Tucson
2
Department of Biology, Canisius College, Buffalo, New York
3
Center of Medical, Agricultural, and Veterinary Entomology, USDA, Agricultural Research Service, Gainesville, Florida
The use of trade, firm, or corporation names in this publication (or page) is for the information and convenience of the reader. Such use does not constitute an
official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of
others that may be suitable.
*Correspondence to: E.A. Schmelz, Center of Medical, Agricultural, and Veterinary Entomology, USDA, Agricultural Research Service, 1600/1700 Southwest 23rd
Drive, Gainesville, FL 32608. E-mail: eschmelz@gainesville.usda.ufl.edu
Received 6 May 2002; Accepted 13 August 2002
of America.of Insect Biochemistry and Physiology
Published 2002 Wiley-Liss, Inc. †This article is a US Government work and, as such, is in the public domain in the United StatesArchives
DOI: 10.1002/arch.10062
Published online in Wiley InterScience (www.interscience.wiley.com)
Induced Phytoecdysteroids and Plant Defense
and developmental abnormalities resulting in death
(Blackford and Dinan, 1997). However, endogenous
PEs in intact plants have not been clearly demonstrated to cause feeding deterrence or host plant rejection by non-adapted insects. Insects may reject
plants for a variety of reasons, including the lack of
required phagostimulants (Hsiao and Fraenkel,
1968; Bowers, 1984), improper nutritional balance
(Champaign and Bernays, 1991), and the presence
of other toxic or deterrent secondary metabolites
(Kubo et al., 1984). For these reasons, the demonstration of PE-mediated plant-insect interactions in
natural systems has proven elusive.
The sensitivity of Diptera to applications of
molting hormones was recognized long before
ecdysteroids were isolated and structurally identified (Horn, 1989). In fact, a sclerotization bioassay using mature larvae of the blowfly Calliphora
erythrocephala was instrumental in the first isolation of ecdysteroids (Butenandt and Karlson,
1954). Shortly after the discovery of ecdysteroids
in plants, Ohtaki et al. (1967) utilized a flesh fly
(Sarcophaga peregrina) puparium formation bioassay to screen relative activities of seven common PEs. Robbins et al. (1968) considered the
potential negative effect of PEs and synthetic analogs on insect development by incorporating them
into insect diets. PEs clearly inhibited ovarian development and yolk deposition in female houseflies (Musca domestica). Singh and Russell (1980)
demonstrated that 20-hydroxyecdysone (20E) at
100 ppM caused up to 84% mortality in M.
domestica fed on semi-synthetic diets. Given the
historical significance of Diptera in ecdysteroid
research and the interest in PEs as putative plant
defenses, it is curious that interactions of PEs with
phytophagous Diptera have not been more closely
examined.
The larvae of the dark-winged fungus gnat
(Bradysia impatiens Johannsen) feed primarily on
fungi; however, when this food source is exhausted
they will actively feed on plant roots and stems
(Kennedy, 1974; Springer, 1995). B. impatiens larvae attack a diverse array of greenhouse and crop
plants, including citrus, geranium, lupine, clover,
corn, and tobacco (Harris et al., 1996). Plants,
December 2002
205
however, are not defenseless against insect herbivory. Following damage, either chemical (Pearce
et al., 1991) or electrical (Wildon et al., 1992)
wound signals travel systemically to surrounding
tissues and modulate gene expression, often resulting in induced increases in chemical defenses. Most
wound signals either stimulate or interact with the
production of jasmonic acid (JA) and the subsequent activation of wound responsive genes (Creelman and Mullet, 1997). McConn et al. (1997)
demonstrated the significance of JA signaling in
the defense of Arabidopsis against B. impatiens attack. Mutant plants unable to synthesize linolenic
acid (a JA precursor), could not produce JA, failed
to produce marker wound gene transcripts, and suffered significantly higher mortality than plants with
intact signaling pathways (McConn et al., 1997).
However, the actual mechanism of the JA-mediated
root-defense response in Arabidopsis is currently
unknown. This is not surprising, as only a few insect-induced responses have been described in
roots (Birch et al., 1990; Mayer et al., 1995; Zangerl
and Rutledge, 1996) compared to the vast literature on induced defenses in foliage (Karban and
Baldwin, 1997).
Spinach (Spinacia oleracea L.) is known to synthesize and accumulate PEs, predominantly 20hydroxyecdysone (20E), in the apical leaves and
stems (Grebenok and Adler, 1991). We recently described the rapid induction of root 20E levels following wounding and applications of JA analogs
(Schmelz et al., 1998). In soil-grown plants, herbivory by the root feeding weevil larvae (Otiorhynchus sulcatus) also stimulates increased root 20E
levels, a response driven by increased root 20E biosynthesis (Schmelz et al., 1999). A role for endogenous JA signals was implied as salicylic acid
analogs, potent inhibitors of the JA biosynthetic
enzyme allene oxide synthase (Pan et al., 1998),
inhibit the wound-induced production of 20E
(Schmelz et al., 1998).
In an experimental system using B. impatiens larvae and spinach plants, we investigate the interactions of induced accumulations of PEs with below
ground herbivores. Specifically, we examine spinach roots, 20E concentrations, and B. impatiens lar-
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Schmelz et al.
vae to address the following questions: (1) Does
root damage by larval feeding stimulate the induction of root 20E accumulation? (2) Can induced
20E concentrations influence larval preference in
artificial diets? (3) Does dietary 20E affect larval
development and survivorship? (4) Do elevated
root 20E levels affect the establishment of B. impatiens? and (5) Are plants with induced root 20E
levels better protected against larval attack than previously unchallenged control plants?
MATERIALS AND METHODS
Insects
Adult fungus gnats were isolated from infested
growth chambers (Feldman Laboratory, The University of Arizona) and identified as Bradysia impatiens
Johannsen using morphological and developmental characters (Wilkinson and Daugherty, 1970;
Steffan, 1981). The method for rearing Sciaridae developed by Gillespie (1986) was used for B. impatiens in our studies. At a constant 23°C, individual
colonies were propagated every 18 days by transferring approximately 50 newly emerged males and
females to new diet. Newly emerged adults were
transferred with a camel hair brush after being
chilled for 5 min in a 5°C environmental chamber.
The B. impatiens were returned to 23°C immediately
after the manipulations were completed. On culture diets, peak emergence of adults from the diet
occurred 18 days after the initial inoculation and
egg laying. This was intermediate between the 19
days and 15–16 days previously reported for the species (Wilkinson and Daugherty, 1970; Kennedy,
1974). The developmental time was often delayed
when reared in potting soil with plants, likely due
to a reduced food source. Larval head capsule measurements for the four instars were in accordance
of those reported by Kennedy (1974). Forceps were
used to transfer live larvae in experiments. The larval cuticle would normally adhere to the smooth
outside edge of the forceps, allowing transfers to be
made without the danger of the larvae actually being pinched. All larvae that died within the first 24
h were excluded from analyses.
Plant Growth and PE Analysis
Spinach (S. oleracea L. var. Avon) seeds from
W. Atlee Burpee Co. (Warminster, PA) were germinated in vermiculite for 10 days, and transferred
to individual 1-L clear plastic containers (Reynolds
Co.) containing approximately 100 ml of a 1:1:1
peat moss:vermiculite:Shultz Seed Starter Mix
(Schultz Company, St. Louis, MO) mixture. The soil
mixture was first passed through a no. 6 standard
sieve (3,360 mm mesh) to remove large debris.
Spinach grows poorly in acidic soils and prefers a
pH range between 6.5 and 8.0 (Rubatzky and
Yamaguchi, 1997); thus, the initial pH of the soil
was adjusted from 5.5 to 7.0. Immediately following the transfer, all plants received a 1-L nutrient
equivalent of the hydroponic solution (see Schmelz
et al., 1998). All plant containers had hundreds of
perforations on the undersides that enabled bottom
watering through capillary action. Other nutrient
additions are detailed within the experimental designs. The growth of hydroponic plants and 20E
analysis of plant tissues via C18 reverse-phase
HPLC followed exactly from Schmelz et al. (1999).
Induction of Root 20E Levels by Larval Attack
Experiment 1. In spinach, root 20E concentrations
are induced following mechanical damage and insect herbivory (Schmelz et al., 1998, 1999). In
three separate trials, the effect of B. impatiens herbivory on 20E induction was examined 21 days after adult female oviposition and subsequent larval
development. Initially, each plant was surrounded
by mesh enclosures, enabling selective infestation.
All adult B. impatiens deceased within 4 days, at
which point all mesh enclosures were removed.
In the colony, males typically surround newly
emerged females, which emerge one day earlier
(Harris et al., 1996), and thus are rapidly mated.
Female B. impatiens were only utilized if their abdomens were clearly swollen indicating that oviposition had not yet taken place. Oviposition by
females and larval herbivory on plant roots were
occasionally observed during these experiments. In
trial one, twenty soil-grown plants (40 days postArchives of Insect Biochemistry and Physiology
Induced Phytoecdysteroids and Plant Defense
germination) were randomly placed into four
groups of five. At this time, each plant received an
additional 1-L nutrient equivalent of the standard
hydroponic solution (see Schmelz et al., 1998).
Four days later, 0, 2, 4, or 8 gravid females were
caged on selected plants. The number of larvae and
pupae in the soil were not quantified upon harvesting roots for 20E analysis. In trial two, eight
plants were separated into two groups of four
plants consisting of uninfested controls and a
heavy infestation using 24 gravid B. impatiens females per plant. These plants were 44 days old at
the start of the experiment and no additional nutrients added. Upon harvesting the plants, soil from
infested plants contained from 20 to 200 final instar larvae depending on the replicate. In trial three,
32 soil-grown plants (41 days old), were randomly
placed into two groups (n = 16) and supplemented
with an additional 1-L nutrient equivalent of the
hydroponic solution. The next day, eight gravid female B. impatiens were caged on each of the plants
for infestation. Upon harvesting the plants for 20E
analysis, all infested plants contained at least four
live B. impatiens larvae. For data analysis, trial 1
utilized an Analysis of Variance (ANOVA) followed
by Tukey corrections for multiple comparisons
while trials 2 and 3 utilized simple t-tests.
Influence of 20E on B. impatiens Dietary Preference
Experiments 2A and B. Root 20E concentrations
were induced in spinach following B. impatiens attack. Thus, two experiments were designed to examine whether or not increased levels of dietary
20E influence larval feeding preference.
Experiment 2A. Test diets were constructed from
0.75 g of lyophilized and powdered mushroom
(Agaricus bisporus), 0.25 g agar, and 18 ml of distilled water. The mixture was briefly heated to a
boil, then 2-ml aliquots of either H20 or aqueous
solutions of 20E were stirred into the diets prior
to the agar solidifying in a glass petri dish. Dietary
20E concentrations of 0, 10, 25, and 50 mg/g (wet
mass) were constructed in this manner. Uniform
sections of diet were removed with a punch (64
mm2) and placed in the bottom of individual plasDecember 2002
207
tic soufflé cups (29.6 ml Solo-P100 Urbana, IL). A
25-ml drop of H20 was placed on the underside of
the plastic lids to elevate the humidity of the arena.
Sections of control and 20E-treated diets were placed
on opposite sides of the arena and slanted on preexisting circular ridges, which allowed larvae to crawl
under edges of the diet. Single 4th instar larvae were
placed into the center of the arena and given choice
of control and 20E-treated diets. Unless the experiment was being examined, the assay was maintained
in the dark. Observations of larval association with
diets were taken 24 and 44 h after the experiment
started. To be considered “associated” with the diet,
larvae needed to be within a 1-mm distance of the
diet. The small fraction of larvae not associated with
either diet was noted, but not included, in the chisquare analysis for associative preference of larvae
for paired diets that assumed equal larvae distribution between diets.
Experiment 2B. In order to estimate actual diet
consumption a tunneling bioassay was devised.
Diet was infused into sections of glass micropipettes; thus the removal of diet could be readily
visualized as B. impatiens larvae fed into the tubes.
Paired choice experiments were designed comparing spinach root diets made from uninduced control tissue with those of damaged roots, MJ-treated
roots, and control roots with added 20E.
Diets were constructed from lyophilized and
homogenized roots of 35-day-old plants that had
been either untreated, damaged (70% root mass
removal), or treated with MJ (200 mg/L hydroponic
solution) 3 days prior to harvesting (Schmelz et
al., 1999). Resulting root 20E concentrations were
48, 410, and 280 mg/g (dry mass) for the untreated,
damaged, and MJ-treated tissues, respectively. Agarbased diets were constructed from these roots using 50 mg powdered dry root plus 4.5 ml of molten
1.5% agar and 0.5 ml of either H20 or aqueous
20E solutions. Untreated roots were used to construct the 20E-treated diets, which resulted in additional 20E concentrations of 25 and 50 mg/g (wet
mass). Re-hydrated root fragments likely had 20E
concentrations one tenth of the reported dry mass
values. However, diffusion into the surrounding
agar matrix may have created even lower 20E con-
208
Schmelz et al.
centrations in the root fragments. The solidified
diets were loaded into 50 ml glass micropipettes
(VWR Scientifc Inc.) with a no. 22 syringe needle.
Paired sections of control and experimental diets
were placed into glass vials (8 ´ 40 mm, National
Scientific Company, GA) and laid horizontally. Individual 4th instar larvae were placed into the vials and could freely feed from either end of the
tubes. Within 24 h, most larvae had completely
tunneled into but not through one of the tubes,
to the point that they were no longer exposed. The
larvae could readily exit the tubes from the same
side they entered. After 48 h, the total distance tunneled into each control and experimental tube was
measured with an ocular micrometer. Based on the
a priori pairing design, paired t-tests were used to
statistically analyze this experiment.
Dietary 20E and B. impatiens Survivorship
Late instar B. impatiens larvae will feed on
healthy plant tissues when fungal food sources are
unavailable (Harris et al., 1996). However, B. impatiens species usually cannot complete their development on plant tissue alone, as fungi appear
to supply a required nutrient (Kennedy, 1974).
Thus, we address how PEs influence the ability of
larvae to use plant tissues as dietary supplements.
Three experiments (3A–C) were devised to examine larval survivorship curves on diets with and
without 20E. Survivorship among the different
treatments was examined using the SAS nonparametric Lifereg procedure for survival time analyses
(SAS Institute Inc). When significant differences
were detected, Tukey-type multiple pair-wise comparisons were performed.
Experiment 3A. We examined the effect of a high
20E concentration, equivalent to a root maximal
induction, on larval transition between the 3rd and
4th instars. Early 3rd instar larvae (N = 20) were
confined to individual 1.5-ml microcentrifuge
tubes containing either control or 20E-treated fungal diets (50 mg/g wet mass) identical to those used
in Experiment 2A. Larval survivorship and instar
were noted every 24 h for 4 days with the aid of a
dissecting microscope.
Experiment 3B. We investigated the differential
survivorship curves of individual 4th instar B. impatiens larvae confined on potato-based diets containing a range of 20E concentrations. Potato tuber
sections are highly attractive to B. impatiens larvae
and are used as lures in greenhouse monitoring
regimes (Evans et al., 1997). The diets consisted
of 100 mg lyophilized and powdered potato plus
4.5 ml of 1.5% molten agar. Before the diets solidified, 0.5 ml of either H20 or aqueous 20E solutions
were mixed in to create dietary 20E concentrations
of either 0, 5, 25, and 50 mg/g (wet mass). With a
syringe, approximately 100 ml of each of these diets was placed in the bottom of the microcentrifuge
tubes. Early 4th instar larvae (N = 50) were used
for each of the four diets. The larvae were examined approximately every other day for 3 weeks
while the diets were replaced every 4 days.
Experiment 3C. We investigated larval survivorship
on diets containing untreated and induced spinach roots. Three root diets were constructed of 50
mg of dry powdered roots plus 900 ml 1.5% molten agar and 100 ml of either H20 or aqueous 20E.
The final diets consisted of untreated roots, MJinduced roots, and untreated roots plus a 20E
concentration of 25 mg/g (wet mass). The untreated
and MJ-treated roots used in this study contained
20E concentrations of 48 and 280 mg/g (dry mass),
respectively. After hydration, the root fragments
likely contained one tenth of this 20E concentration. With a syringe, the solidified root diets were
injected into 22-mm sections of 50-ml glass micropipettes. The diet tubes were placed into glass vials
and anchored with 100–200 ml 1.5% agar, which
also served as a H20 reserve for maintaining humidity. Individual late 2nd instar larvae (N = 50) were
used for each diet type. Larval survivorship and instar were recorded every other day for 10 days.
Establishment of B. impatiens on Spinach
Experiment 4. In a more natural and complex soil
environment, we investigated the establishment of
B. impatiens on spinach roots previously untreated
or with induced 20E levels triggered by methyl
jasmonate (MJ) application. The word “establishArchives of Insect Biochemistry and Physiology
Induced Phytoecdysteroids and Plant Defense
ment” is used to include both oviposition and subsequent larval survivorship, as this experiment
combines these two processes. Fifty-eight soilgrown plants were separated into four groups of
twelve plants and two groups of five plants. To
understand both MJ and B. impatiens effects on
plant growth, the four plant groups consisted of
MJ-treated, MJ-treated with B. impatiens infestation,
untreated control, and untreated control with B.
impatiens infestation. At the start of the experiment,
the plants were 40 days old and had received a 1-L
nutrient equivalent of the hydroponic solution on
days 14 and 25 (see Schmelz et al., 1998). Plant
roots designated for induction received a 100-ml
drench of aqueous MJ (45 mM) while untreated
plants received 100 ml H20. One day later, five
gravid females were caged on each plant and allowed
to oviposit. The mesh cages were removed 5 days
later with no live adult B. impatiens remaining. At
this time, the two additional groups of five untreated
and five MJ-treated plants were harvested for root
20E analysis to confirm the 20E induction response.
All other plants were harvested 18 days after the
gravid females were introduced. The collection of
larvae and pupae followed from McConn et al.
(1997). The soil samples were stirred into a 60%
sucrose solution and allowed to settle. The larvae
floated to the surface and were collected with a forceps. The process of stirring, settling, and collecting
was repeated for each sample until no new larvae
were discovered between two separate searches. The
larvae were preserved in 70% ethanol for total
counts and instar analysis. Root and shoot dry mass
analysis utilized ANOVAs followed by Tukey corrections for multiple comparisons while t-tests were
used to examine root 20E concentration, Bradysia
establishment, and % of Bradysia at each stage.
Induction of 20E and Plant Protection
Experiment 5. We examined whether MJ-induced
roots with elevated 20E levels are better protected
from larval attack than previously uninjured control plants. Threefold differences in root 20E concentrations were achieved by treating the roots of
20-day-old hydroponic plants with aqueous MJ soDecember 2002
209
lutions (100 mg/plant). Fifteen untreated and MJtreated plants were harvested 3 days later and contained average root 20E concentrations of 104 and
312 mg/g (dry mass), respectively. At this time, 30
untreated and 30 MJ-treated plants were paired by
initial fresh mass (grand mean 404.5 ± 57.1 mg
wet mass) and potted in soil. From the 30 pots,
two groups of 15 pairs were either infested with
larvae or left uninfested. Fifty 4th instar larvae were
placed into the center of each of the 15 infested
pots and allowed to feed on the plants for 72 h,
after which all plants were harvested for biomass
analysis. Based on the initial a priori pairing of
plants by fresh mass, paired t-tests were utilized to
examine treatment effects on root, stem, and shoot
dry mass.
Statistics
Specific data analysis performed has been detailed in the individual experiments described
above, and was accomplished with the aid of the
JMP (SAS Institute Inc) and SAS (SAS Institute Inc).
RESULTS
Induction of Root 20E Levels by Larval Attack
Experiment 1. In all three trials, infestation of
plants with adult females resulted in the induction of root 20E concentrations. In trial one, significant increases (ANOVA F3,16 = 8.19, P = 0.002)
in root 20E concentrations of 5.0-fold or greater
were detected in plants infested with four or more
females (Fig. 1, Trial 1). However, significant root
loss by larval herbivory was not detected (ANOVA
F3,16 = 1.16, P = 0.354; Fig. 1, Trial 1). In trial two,
the high level of infestation resulted in a 6.6-fold
increase in root 20E concentrations (t-test t = 6.30,
df = 6, P < 0.001; Fig. 1, Trial 2). A decline in root
mass was suggested for the B. impatiens attacked
plants, but due to the variation in controls, was
not significant (t-test t = 1.96, df = 6, P = 0.098;
Fig. 1, Trial 2). In trial three, the infestation of
plants with 8 females resulted in a 4.0-fold increase
in root 20E levels (t-test t = 9.66, df = 30, P < 0.001;
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Schmelz et al.
Fig. 1. Mean (+S.E.) root 20E
concentrations and root dry mass
of soil-grown spinach plants, 21
days after infestation with gravid
female B. impatiens. In trial 1,
bars that share the same letter
are not significantly different (P
> 0.05, Tukey correction for multiple comparisons). *Significant
differences (t-tests, P < 0.05).
NSD, no significant difference.
Fig. 1, Trial 3). The large number of replicates enabled the detection of a significant 1.5-fold decrease in root mass for the infested group (t-test t
= 2.65, df = 30, P = 0.013; Fig. 1, Trial 3).
Influence of 20E on B. impatiens Dietary Preference
Experiments 2A and B. Larvae prefer control diets
over those containing induced plant roots or exogenous 20E concentrations of 25 mg/g (wet mass),
demonstrating that 20E acts as a feeding deterrent.
In experiment 2A, the 24-h examination of control and 20E-treated diets revealed that diets with
20E levels of 25 and 50 mg/g harbored significantly
fewer (chi-square, P < 0.05) larvae (Fig. 2). At 44
h, the preference difference for control versus 20E
diets of 25 mg/g was not significant (chi-square, P
> 0.05), while the 50 mg/g 20E diets were still
avoided (chi-square, P < 0.05; Fig. 2). Larvae did
not appear to discriminate between control and
20E diets containing 10 mg/g at either time point.
In experiment 2B, 4th instar larval diet consumption was estimated by measuring the distance tunneled into diets within 48 h. In each case, spinach
diets constructed from untreated control roots were
preferred over roots previously damaged, treated
with MJ, or untreated roots containing added 20E
at concentrations of 25 and 50 mg/g wet mass (all
paired t-tests t > 3.88, df = 19, P < 0.001; Fig. 3).
Dietary 20E Reduces B. impatiens Survivorship
Experiment 3A. Dietary 20E, responsible for feeding deterrency, also results in increased levels of
mortality. Third instar larvae confined to fungal diets containing 20E concentrations of 50 mg/g (wet
mass) displayed significantly reduced survivorship
compared to those on untreated control diets (Fig.
4). Untreated diets enabled 85% of the 3rd instar
larvae to successfully molt into 4th instars over a
Archives of Insect Biochemistry and Physiology
Induced Phytoecdysteroids and Plant Defense
211
Fig. 2. Paired-choice preferences of individual 4th instar B.
impatiens larvae on fungus-derived diets (Agaricus bisporous)
containing no PEs (control)
paired with treated diets containing 10, 25, or 50 mg 20E/g
wet mass. Larvae were considered “associated” with a diet if
they were within a 1-mm distance of it. Larvae associated
with control, 20E-treated, or neither diet are illustrated as white,
black, and gray bars, respectively. *Significant differences
in larvae distributions between
control and 20E-treated diets
(chi-square, P > 0.05).
4-day period (Fig. 4). Within this same period, 20Etreated diets resulted in significant mortality, with
only 15% of the individuals successfully molting
to 4th instars. One individual from the 20E-treated
diet formed a small yet complete pupa.
Experiment 3B. On potato-based diets, 20E concentrations as low as 5 mg/g (wet mass) were sufficient to cause decreased a survivorship of 4th instar
larvae compared to untreated control diets. After
3 days, 20E concentrations of 5, 25, and 50 mg/g
(wet mass) resulted in larval survivorship of 55,
28, and 5%, respectively (Fig. 5A). The mortality
on this same day was largely due to the formation
of non-viable “prepupae,” where 20E concentrations of 5, 25, and 50 mg/g (wet mass) resulted in
37, 61, and 73% formation of “prepupae” (Fig. 5B).
The non-viable “prepupae” were characterized by
an accelerated apolysis of the 4th instar cuticle with
a partial retraction of body segments and some cuticular tanning. The key character being incomplete
ecdysis of the head capsule, which likely impedes
proper head formation as the full pupal characteristics are not attained prior to death. By day 9, all
larvae on 20E diets suffered 100% mortality, while
73% of individuals reared on the untreated control diet remained alive. Over the next 2 weeks,
December 2002
Fig. 3. Mean (+S.E.) distances (mm) tunneled into spinach root-based diets after 48 h by individual 4th instar B.
impatiens larvae in paired choice arenas. Root-based diets
were constructed from uninduced controls or plants induced with damage or MJ treatments. 20E diets consisted
of control roots plus an additional amount of 20E increasing control root levels by 25 or 50 mg/g wet mass. *Significant differences (paired t-tests, P > 0.05) in this
estimation of diet consumption.
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Schmelz et al.
control survivorship steadily declined until the experiment was terminated on day 21 (Fig. 5A). During this time, larvae on untreated control diets also
formed non-viable “prepupae” to a lesser extent
with a final cumulative formation of 31% (Fig. 5B).
Experiment 3C. Second instar larvae fed on MJinduced root diets suffered significantly greater
mortality than those fed on untreated control root
diets (P = 0.022; Fig. 6). However, larval survivorship on diets constructed from untreated control
roots supplemented with a 20E concentration of
25 mg/g (wet mass) was intermediate and not significantly different from the other two treatments
(P > 0.138; Fig. 6). Larval death occurred at different stages between treatments. Of the larvae feeding on untreated root diets, the mortality that
occurred in the 2nd, 3rd, and 4th instars was 4,
29, and 2%, respectively. Only one death was attributed to molting disruption, all other individuals (33%) died without molting. Of those larvae
fed on diets made of MJ-induced roots, the percent larval death that occurred in the 2nd, 3rd and
4th instars was 4, 48, and 41%, respectively. Five
individuals (10%) died during a molt to the 4th
instar while three (6.5%) 4th instars formed nonviable “prepupae.” However, the majority of individuals (76%) died as larvae. Of those larvae fed
on diets made of untreated control roots supplemented with 20E, the percent larval death that occurred in the 2nd, 3rd, and 4th instars was 26, 22,
and 17%, respectively. Only two individuals (4%)
died during molting, all others (61%) died as larvae without molting. Based on observations, the
high percentage of deaths that occurred in the second instar were likely due to starvation caused by
feeding deterrency.
Fig. 4. Percent survivorship and development of 3rd instar B. impatiens larvae over 4 days on a fungal diets (Agaricus bisporous) containing either 0 (control) or 50 mg 20E/g
wet mass. Developmental stages of 3rd instar, 4th instar,
and pupae are denoted by white, black, and striped bars,
respectively. Percent total survivorship is indicated by the
line graph and is significantly different (P < 0.05) between
the control and 20E diets (Survival analysis using the SAS
Lifereg Procedure).
Induced Root 20E Levels Correspond With Reduced
Establishment of B. impatiens
Experiment 4. Spinach roots treated with MJ had
higher 20E levels and lower populations of B. impatiens larvae. On day five, MJ-treated roots demonstrated a 2.1-fold increase in 20E concentration
compared to untreated controls (t-test t = 3.78,
df = 8, P = 0.005; Fig. 7A). At the end of the exArchives of Insect Biochemistry and Physiology
Induced Phytoecdysteroids and Plant Defense
213
Fig. 6. Percent survivorship of B. impatiens larvae fed on
spinach diets constructed from either uninduced control
roots, control roots plus 20E (25 mg/g wet mass), or MJinduced roots. At time zero, all larvae were late 2nd instars and by day 10 all surviving individuals were 4th
instars. Larval survivorship curves sharing the same letter
are not significantly different (P > 0.05, Survival analysis
using the SAS Lifereg Procedure).
Fig. 5. A: Percent survivorship and (B) cumulative percent “prepupae” formation of 4th instar B. impatiens larvae
confined to potato-based diets containing 20E concentrations of either 0 (control), 5, 25, or 50 mg/g wet mass. The
larval survivorship on all 20E-treated diets is significantly
lower (P < 0.05) than the control diet (Survival analysis
using the SAS Lifereg Procedure).
periment, the number of B. impatiens present in
the plants pretreated with MJ was 2.0-fold lower
than the untreated controls (t-test t = 2.46, df =
22, P = 0.022; Fig. 7C). The percent of B. impaDecember 2002
tiens present in third instars, fourth instars, and
pupae did not differ between treatment groups
(all t-tests ts < 1.11, df = 22, P > 0.28; Fig. 7D),
suggesting that root induction did not alter the
rate of development in surviving larvae. Shoot
masses did not differ (ANOVA F3,44 = 1.15, P =
.341). However, significant differences in root dry
mass were detected (ANOVA F3,44 = 4.25, P =
0.010; Fig. 7B). MJ-treated plants not infested with
B. impatiens had larger root masses than all other
groups (Fig. 7B). Untreated control and MJ-treated
plants, which were infested, displayed identical
roots masses to the uninfested control plants (Fig.
7C). Thus, root herbivory may nullify a stimulatory effect of MJ on root growth.
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Schmelz et al.
Fig. 7. A: Mean (+S.E.) root
20E concentration (mg/g dry
mass) of control (Con) and MJtreated (45 mM root drench)
plants after 5 days. Five gravid
females were caged on individual control and MJ-treated
plants and analyzed for (B)
root dry mass remaining (mg),
(C) B. impatiens establishment
(total larvae present), and (D)
percent B. impatiens at each
stage, 18 days later. Additional
uninfested plants were analyzed for root mass only.
Plants With Induced 20E Levels Are Protected
Against B. impatiens Attack
Experiment 5. B. impatiens larvae preferentially attack untreated control roots over those pretreated
with MJ that contain induced levels of 20E. MJ
treatments resulted in a 3.0-fold increase (t-test t
= 11.74, df = 13, P < 0.001) in root 20E concentrations, with the untreated control and MJ-treated
roots exhibiting 104 and 311 mg/g (dry mass), respectively. For uninfested plants, the shoot, stem,
and root dry masses were not significantly different between the untreated control and MJ treatment pairs (all paired t-tests ts < 1.37, df = 14, P >
Archives of Insect Biochemistry and Physiology
Induced Phytoecdysteroids and Plant Defense
Fig. 8. Mean (+S.E.) shoot, stem, and root dry mass for
15 pairs of untreated control and MJ-treated plants that
were either uninfested (A-C) or infested (D-F) with fifty
December 2002
215
4th instar B. impatiens larvae for 3 days. Asterisks denote
significant dry mass differences (paired t-tests, P < 0.05).
216
Schmelz et al.
0.194; Fig. 8A–C). However, for infested plants, the
untreated controls suffered significant losses in
shoot (1.2-fold) and root (1.7-fold) mass compared to the MJ-induced plants (two paired t-tests
ts > 2.36, df = 14, P < 0.033; Fig. 8D,F). Stem dry
mass was not affected by infestation (t-tests t =
1.33, df = 14, P = 0.205; Fig. 8E). The root loss
experienced in the infested control plants caused
most shoots to become completely flaccid while
the leaves of MJ-induced plants remained turgid.
DISCUSSION
We examined the interaction of spinach roots,
PE concentrations, and B. impatiens larvae and conclude the following. First, root herbivory by B. impatiens larvae can result in 4.0- to 6.6-fold increases
in root 20E concentrations with levels reaching
over 300 mg/g dry mass. Second, in paired choice
tests, diets low or lacking 20E are preferred over
diets constructed from induced roots and also
those treated with 20E (25 to 50 mg/g wet mass).
Thus, dietary 20E can result in feeding deterrency.
Third, when B. impatiens larvae are confined to PEtreated diets, 20E concentrations as low as 5 mg/g
(wet mass) result in reduced survivorship compared
to controls. Elevated mortality on plant diets containing 20E was partially due to the accelerated
formation of non-viable “prepupae,” indicating
hormonal disruption. Fourth, plants with induced
levels of root 20E, stimulated by MJ treatments,
support the establishment of fewer larvae compared to uninduced control plants. Fifth, in paired
design, uninduced control roots were damaged significantly more than MJ-induced root systems.
Based on preference tests, elevated levels of 20E
present in the MJ-induced roots (28 mg/g wet mass)
are sufficient to account for a reduction in root
herbivory by B. impatiens larvae.
Root herbivory by B. impatiens larvae stimulated
increases in root 20E levels, in cases both with and
without detectable root loss. This result was predicted as stimulation of 20E biosynthesis can be easily triggered by mechanically wounding spinach
roots (Schmelz et al., 1998, 1999). In addition to
root herbivory, B. impatiens are also known to trans-
mit pathogenic fungi (Gardiner et al., 1990; Jarvis
et al., 1993); thus, changes in root 20E levels could
also be microbially mediated. However, using both
Pythium and Phytophthora spp. we previously demonstrated that pathogen attack does not result in
the induction of root 20E levels (Schmelz et al.,
1998). The interaction of spinach roots with insect
vectors of plant pathogens may have significant agronomic relevance, as fungal pathogens are responsible for major reductions in spinach production
(Larsson and Gerhardson, 1992).
PEs can function as insect feeding deterrents.
Ma (1969) first demonstrated that Pieris brassicae
feeding is reduced by 20E concentrations of 25 mg/
g (wet mass), with deterrency effects as low as 5
mg/g (wet mass) later established by Jones and Firn
(1978). Related investigations with the silk moth,
Bombyx mori, revealed that 20E concentrations of
100 mg/g (wet mass) stimulate a maxilla based R
receptor and reduce feeding (Tanaka et al., 1994).
Recently, dietary 20E at 40 mg/g (wet mass) has
been shown to be a potent feeding deterrent to
Inachis io, which does not normally encounter PEs
(Blackford and Dinan, 1997). We found that dietary 20E concentrations of 25 mg/g (wet mass)
were sufficient to reduce larval feeding preference
and association with treated diets. On average,
spinach tissue has a 91% water content (Schmelz,
unpublished data); thus, fresh mass 20E concentrations that larvae would encounter in roots are
approximately one tenth of our reported dry mass
values. Spinach roots with induced 20E concentrations of 250 mg/g (dry mass) or greater would
be expected to cause some feeding deterrency. Indeed, the damage and MJ-treated roots used in experiment 2B, which resulted in reduced feeding,
had 20E concentrations of 410 and 280 mg/g dry
mass, respectively.
Efficient PE detoxification is often associated
with phytophagous insects that are highly polyphagous. For example, many pest species of Noctuidae
(Lepidoptera) rapidly excrete apolar C-22 longchain fatty acids after the ingestion of 20E (Robinson et al, 1987; Kubo et al., 1987; Blackford et al.,
1997). An ecdysteroid-22-O-acyltransferase was
partly characterized from the midgut epithelial
Archives of Insect Biochemistry and Physiology
Induced Phytoecdysteroids and Plant Defense
membrane of Heliothis virescens (Zhang and Kubo,
1992), and is likely responsible for its high level
of PE tolerance (Kubo et al., 1981). The grasshopper, Locusta migratoria, is also unharmed by dietary
PEs (Carlisle and Ellis, 1968). In Locusta, rapid excretion of ingested PEs is important (Feyereisen et
al., 1976) but unlike the situation in Heliothis, 20E
is rapidly converted into a mixture of 20E-3-acetate
and 20E-3-acetate-2-phosphate (Modde et al.,
1984). Similarly, the enzyme activities were localized in the midgut and gastric ceca of Locusta
(Feyereisen et al., 1976), consistent with a role for
detoxifying dietary PEs. Monophagous and oligophagous insects, not known to include PE-containing plants in their host range, are often highly
susceptible to PEs (Kubo and Klocke, 1983; Blackford and Dinan, 1997). In non-adapted insects, ingested PEs are believed to cause mortality by
crossing the midgut, creating uncontrollable hemolymph ecdysteroid titers, and prematurely initiating developmental programs (Kubo et al., 1983).
Susceptibility to dietary PEs has been well studied
in the silk moth, Bombyx mori. Eight hours after
ingesting a dose of [3H] ecdysone, over 10% of
the radioactivity could still be found in the hemolymph (Zhang and Kubo, 1993). In this case, the
major inactivation product was 20-hydroxyecdysonic acid, which is believed to be a common inactivation product for endogenous ecdysteroid
metabolism (Lafont et al, 1983). Susceptible insects appear to lack specialized ecdysteroid conjugating enzymes to deal with dietary PEs .
Given the trend that insects with wide host
ranges often exhibit dietary PE immunity, the susceptibility of B. impatiens to 20E is somewhat curious. Harris et al. (1996) listed over 50 diverse
agronomic and horticultural plants known to be
attacked by B. impatiens larvae. While the PE status
of each listed plant is not known, the larvae clearly
encounter a broad range of plant secondary metabolites. While polyphagous with respect to the
plants attacked, fungi constitute the preferred diet
(Kennedy, 1974). Thus, even though a wide range
of plants are attacked, B. impatiens may have a narrower range of detoxification mechanisms similar
to some monophagous and oligophagous insects.
December 2002
217
Detoxification pathways for ingested 20E in the
Diptera have thus far not been examined. However, it is expected that 20E metabolites are similar to endogenous inactivation products, such as
20-hydroxyecdysonic acid, glucuronidases, b-glucosides, and sulfate conjugates (Lafont et al., 1983;
Briers et al., 1983).
When confined to 20E containing diets, larvae
exhibit greatly accelerated mortality compared to
the control diets. In Experiment 3A, control diets
allowed a normal developmental progression from
3rd to 4th instars, while diets containing 20E concentrations of 50 mg/g (wet mass) resulted in almost complete mortality within 4 days. With
potato-based diets, 20E concentrations as low as 5
ppm were found to drastically decrease larval survivorship compared to controls. Increased dietary
20E concentrations resulted in higher frequencies
of “prepupae” formation. Thus, when 20E-treated
diets are presented as the sole diet source, larvae
demonstrate extreme sensitivity. This suggests that
enough dietary 20E enters the hemolymph to cause
developmental disruption, with the mortality not
simply due to feeding deterrency induced starvation.
PE-containing diets had slightly different effects
on larvae depending on the design of the bioassay. Instead of a single dietary source, Experiment
3C also provided a small amount of 1.5% agar,
which was added to boost humidity levels in the
arena. Larvae were often seen crawling in and
around the agar and not feeding on the diet. In
this experiment, the larval survivorship on the 20E
diet containing 25 mg/g (wet mass) was intermediate between the control and MJ-treated root diets, and not significantly different from either. We
offer two possible explanations to account for this
difference in 20E sensitivity between bioassays.
First, the presence of the 1.5% agar substrate may
have allowed dietary mixing by the larvae, potentially diluting the concentration of 20E ingested.
This possibility has important implications for larvae attacking plants under natural conditions, as
dietary mixing would undoubtedly occur (Bernays
and Bright, 1993). Second, when larvae are confined to diets, 20E may be slowly absorbed across
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Schmelz et al.
the larval cuticle creating developmental disruption independent of ingestion. However, it is generally accepted that polar ecdysteroids cannot cross
the lipophilic cuticle of insects (Dinan, 1989).
Ohtaki et al. (1967) stated that topical applications
of PEs are completely inactive in the Calliphora assay, even with the aid of solvents like methanol
and dimethyl sulfoxide. In contrast, alcohols are
effective solvents for enabling PE penetration across
some lepidopteran cuticles (Sato et al., 1968;
Hasegawa and Ata, 1972). This suggests that not
all insect cuticles are impervious to PEs when in
contact with polar solvents. In a natural setting, B.
impatiens larvae are unlikely to encounter high levels of PEs in the soils surrounding roots as PEs
generally do not leach out of living plant tissues
(Matsumoto and Tanaka, 1991). However, the excretion of low levels of PE into water from fern
prothalli has been described (Reixach et al., 1997).
The possible absorption of PEs through the cuticle
of B. impatiens larvae was not specifically examined and is worthy of further investigation. Within
Experiment 3C, the contrast in larval survivorship
on root diets with exogenous 20E and diets made
with MJ-induced roots may be explained by the
complexity of induction. MJ is known to up-regulate a vast array of defense-related secondary metabolites (Karban and Baldwin, 1997). For example,
in solanaceous plants, jasmonic acid and its analogs stimulate the production of alkaloids, proteinase inhibitors, polyphenol oxidase, and volatile
terpenoids (Baldwin et al., 1997; Farmer and Ryan,
1992; Stout et al., 1998; Krumm et al., 1995). Numerous phenolases have been partially characterized in spinach roots (Sato, 1982). Thus, it is quite
possible that, in addition to 20E, MJ induces other
chemical changes that may have contributed to the
reduced survivorship observed.
B. impatiens establishment on roots induced
with MJ was significantly lower than on untreated
control plants. Establishment is a combination of
oviposition and larval survivorship; thus, this result needs to be cautiously interpreted. It is possible that B. impatiens oviposition was reduced on
MJ-treated soils. In a nearly identical experimental
design, McConn et al. (1997) found that Arabidopsis
plants sprayed daily with MJ (45 mM), spanning
the entire oviposition period, did not reduce the
number of larvae found in the soil 14 days later.
Additionally, their experimental design allowed
adult females to choose between treated and untreated plants for oviposition sites. Because we
treated the soil with MJ (45 mM) a full 24 h prior
to infestation and enclosed B. impatiens on individual plants, we feel that oviposition was unlikely
to be affected. Likewise, McConn et al. (1997)
demonstrated that direct spraying of larvae with
450 mM MJ did not increase mortality over a 48-h
period. Thus, we believe a direct effect of MJ on B.
impatiens is unlikely in our experiments. The decrease in B. impatiens larvae matched the MJ-induced increase in root 20E levels. A reduction in
root quality as a supplemental food source may
explain this result. Additionally, under sub-optimal conditions larvae can be cannibalistic (Wilkinson and Daughtery, 1970), and this may have
contributed also to the lower populations found
on previously induced plants. Based on the percentages of B. impatiens at each stage, changes in
plant chemistry did not significantly alter the rate
of larval development. A reduction in the concentration of 20E consumed through dietary mixing
and cannibalism is a possible factor that may explain why increased root 20E levels did not result
in altered rates of larval development.
MJ-induced roots are better protected against
larval herbivory than previously uninduced control roots. This result is important as it avoids the
artificial nature of constructed diets and relies on
the physiologically relevant differences between living root tissues. In the MJ-induced roots, 20E concentrations were estimated to be 28 mg/g (wet
mass). From our paired-choice preference tests, this
concentration is expected to result in feeding
deterrency. Thus, while a combination of plant
physiological responses may be responsible for the
MJ-induced decreases in root herbivory, the induced
20E levels alone are sufficient to afford spinach roots
increased protection against larval herbivory. Dietary 20E also reduces B. impatiens survivorship;
however, none of the plant-based diets were able
to support complete development. Regardless, durArchives of Insect Biochemistry and Physiology
Induced Phytoecdysteroids and Plant Defense
ing attack by B. impatiens larvae the presence of
induced levels of plant PEs increased both feeding
deterrency and reduced the nutritional quality of
the plant tissues. Interestingly, many PE-containing plants have the highest levels associated with
the root systems, some at concentrations near 1%
of the dry mass (Tomás et al., 1992; Corio-Costet
et al., 1993; Dinan 1995). At such high levels, complete protection against B. impatiens herbivory
would be expected. This defense may be particularly significant for some ferns, as cool damp places
provide ideal conditions for both fungi and sciarid
flies (Steffan, 1981; Jones, 1983). We believe that
the interaction between spinach roots and B. impatiens larvae is the first demonstration that PEs,
as inducible defenses, can provide protection
against insect herbivory.
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December 2002
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