Effect of generalist insect herbivores on introduced Lepidium draba (Brassicaceae): implications for...

Effect of generalist insect herbivores on introduced Lepidiumdraba (Brassicaceae): implications for the enemy releasehypothesisK. P. Puliafico1, M. Schwarzlander1, B. L. Harmon1 & H. L. Hinz2

1 Department of Plant Soil and Entomological Sciences, University of Idaho, Moscow, ID, USA

2 CABI Europe – Switzerland, 1 Rue des Grillons, Delemont, Switzerland

Introduction

Lepidium draba L. (=Cardaria draba (L.) Desv.), hoary

cress, is a perennial herbaceous crucifer (Brassicaceae)

indigenous to Eurasia. The plant was introduced to

North America in the mid 1800s (Mulligan and Find-

lay 1974), and is now listed as a noxious weed in 15

central and western US States and three Canadian

Provinces (Rice 2007). Hoary cress forms dense

patches in rangeland and natural areas, often exclud-

ing native flora (Mulligan and Findlay 1974; McKen-

ney 2005). Recent biogeographical comparisons of

hoary cress populations showed that plants grow

more vigorously and form denser patches in the USA

than in its native European range (McKenney

2005). Plant invasions have been explained with a

number of broad ecological hypotheses that assume

environmental and evolutionary changes following

Keywords

biological control, herbivore impact, herbivore

interaction, invasive species

Correspondence

Kenneth P. Puliafico (corresponding author),

Department of Plant Soil and Entomological

Sciences, University of Idaho, Moscow, ID

83844-2339, USA. E-mail: [email protected]

Received: September 17, 2007; accepted:

March 27, 2008.

doi: 10.1111/j.1439-0418.2008.01297.x

Abstract

The enemy release hypothesis (ERH) states that decreased regulation by

natural enemies allows plants to increase in distribution, abundance and

vigour following their introduction into an exotic range. Invasive plants

rarely escape herbivory entirely, and for hoary cress [Lepidium draba L.

(Brassicaceae)] it has been demonstrated that generalist insect

abundance is greater in its introduced North American range than in

the native European range. We assessed the role of increased generalist

herbivory on hoary cress using representatives of four important herbi-

vore niches commonly found in the introduced range. We experimen-

tally examined the density dependent impact of these herbivores

individually and in combination on hoary cress in a series of greenhouse

experiments. We found that defoliation of the oligophagous diamond-

back moth Plutella xylostella (L.) (Lep., Plutellidae) had the strongest and

most consistent impact, while damage by the stem-mining weevil

Ceutorhynchus americanus Buchanan (Col., Curculionidae) tended to have

the highest per capita effect. Plant response to feeding by the oligopha-

gous crucifer flea beetle Phyllotreta cruciferae (Goeze) (Col., Chrysomeli-

dae) was minor despite obvious feeding damage, and the impact of the

polyphagous tarnished plant bug Lygus hesperus Knight (Het., Miridae)

was negligible. In multiple-species experiments, herbivore impacts were

usually additive. In general, we found that hoary cress can tolerate high

densities of oligophagous insect herbivory and effectively resisted attack

by the polyphagous L. hesperus, but also the oligophagous C. americanus.

Our results indicate that a combination of plant resistance and tolerance

allows hoary cress to withstand increased generalist herbivore load in its

introduced range, consistent with the predictions of the ERH.

J. Appl. Entomol.

J. Appl. Entomol. 132 (2008) 519–529 ª 2008 The Authors

Journal compilation ª 2008 Blackwell Verlag, Berlin 519

the introduction of species (Alpert 2006). Exploring

specific mechanisms that cause a species such as hoary

cress to become invasive may greatly improve our

ability to understand and manage plant invasions

(Kolar and Lodge 2001).

The ‘enemy release hypothesis’ (ERH) is one of

the oldest (Elton 1958) and most commonly used

explanations for the invasiveness of introduced

species. The ERH states that non-indigenous plants

readily increase in distribution, abundance and

vigour in the introduced range because they experi-

ence a decrease in regulation by natural enemies

(reviewed by Keane and Crawley 2002; Wolfe 2002;

Colautti et al. 2004; Liu and Stiling 2006). The

ERH is based on evidence that herbivores, particu-

larly coevolved specialist insects, can impair plant

performance and fitness, exerting top down

regulation on plant populations (Keane and Crawley

2002). Consequently, release from specialist herbi-

vore pressure in the introduced range could provide

a competitive advantage over native neighbouring

plants still exposed to their own suite of coevolved

specialist herbivores. Although total escape is not

possible because generalist herbivores and pathogens

are present in both the native and introduced ranges

of a plant (Wolfe 2002), further competitive advan-

tage is gained if these insects cause less damage to

the exotic plants than their neighbours (Keane and

Crawley 2002). Because diversion of plant resources

to repair or replace tissues damaged by herbivory

can have direct fitness costs in a competitive

environment, changes in plant parameters, such as

biomass, are generally accepted as correlates of fit-

ness (Colautti et al. 2004). Several invasive plant

species have been shown to possess resistance traits

that reduce preference or performance of generalist

herbivores via chemical defences (Muller-Scharer

et al. 2004; Lankau 2007) or tolerance traits that

ameliorate damage caused by polyphagous and

oligophagous herbivores (Muller-Scharer et al.

2004).

Simultaneous surveys of the herbivore faunas

associated with hoary cress populations in both the

native European and introduced US range showed

that, in accordance with ERH assumptions, specialist

natural enemies were well represented in Europe,

but were almost absent in the USA (Cripps et al.

2006a,b). Contrary to ERH assumptions, generalist

herbivore abundance was greater in the introduced

than in the native range (Cripps et al. 2006b); how-

ever, it is unclear what effect oligophagous and

polyphagous herbivores have on hoary cress at the

densities observed. Any increased impairment of

plant performance caused by greater generalist her-

bivore pressure in the introduced range of the plant

may have implications for ERH, as it could counter-

act the release from specialist herbivores that hoary

cress is experiencing in the USA.

Plants are rarely attacked by a single insect species

in isolation. It is therefore necessary to assess the

impacts of individual herbivores and species combi-

nations to accurately predict herbivory pressure

experienced by the plant. Our null hypothesis was

that herbivore damage is additive; therefore, the

total impact is the sum of the individual species’

impacts (Fournier et al. 2006). Non-additive herbi-

vore impacts occur when interactions between spe-

cies improve or reduce their per capita effects on the

plant (Strauss 1991; Fournier et al. 2006). Synergism

can be the result of joint herbivore reduction of

plant defences, changes in plant tissues over time, or

other mechanisms (Ohgushi 2005). Reduced herbi-

vore effectiveness can result directly through

negative interactions between herbivore species,

such as resource competition, active interference or

aggression; or by indirect interactions mediated

through the host plant, such as reduction of food

quality, induction of plant defences or attraction of

mutual natural enemies (reviewed by Strauss 1991;

Denno et al. 1995; Vinson 1999; Ohgushi 2005).

Further, it is important to test combined effects of

herbivores at varying densities, as any of the above

outcomes and interactions may be density-depen-

dent (e.g. Briese et al. 2004; Schooler and McEvoy

2006; Gerber et al. 2007).

Here, we present data on the density-dependent

impacts of four oligophagous and polyphagous insect

herbivores commonly associated with hoary cress in

its introduced range. We compare the relative impact

of each individual species and discuss plant resis-

tance to herbivore attack and tolerance to injury

inflicted by insects in these specific feeding niches.

In addition, we combined species to explore their

interactions and herbivory pressures. We specifically

address which combinations and densities of herbi-

vores may impair hoary cress performance the most

and discuss implications for the enemy release status

of the plant in North America.

Materials and Methods

Study organisms

Hoary cress occurs in nearly all soil types and in a

wide range of habitats, but thrives in disturbed,

riparian or irrigated areas (Scurfield 1962). Hoary

Effect of generalist herbivory on hoary cress K. P. Puliafico et al.

520J. Appl. Entomol. 132 (2008) 519–529 ª 2008 The Authors

Journal compilation ª 2008 Blackwell Verlag, Berlin

cress reproduces vegetatively through rhizomes and

sexually by obligate out-crossing (Mulligan and

Findlay 1974). Vegetative growth allows the plant to

persist and increase in density once established at a

site, while seed production facilitates dispersal and

colonization of new sites (Larson et al. 2000). Seeds

usually germinate in autumn and early spring and

produce rosettes that bolt and flower the following

spring from April to June (McInnis et al. 2003).

Over 75% of hoary cress biomass is located below-

ground in established field sites (Miller et al. 1994)

and consists of vertical and lateral roots which pro-

duce adventitious buds, which in turn develop into

rhizomes and shoots (Mulligan and Findlay 1974;

Miller et al. 1994).

We used four different insect herbivores in our

experiments; the stem-mining weevil Ceutorhynchus

americanus Buchanan (Col., Curculionidae), the

crucifer flea beetle Phyllotreta cruciferae (Goeze) (Col.,

Chrysomelidae), the tarnished plant bug L. hesperus

Knight (Het., Miridae), and the diamondback moth

Plutella xylostella (L.) (Lep., Plutellidae).

Ceutorhynchus americanus is native to North America

and was the sole endophagous insect herbivore found

to feed on hoary cress during surveys in North Amer-

ica (Cripps et al. 2006b). Adult C. americanus have

been collected from several different Brassicaceae,

including the genus Lepidium (Buchanan 1937;

Scheibner 1963), but have only been reared from

L. virginicum (Buchanan 1937) and is considered an

oligophagous species. Adult C. americanus feed on

foliage and oviposit in the stems of hoary cress where

the larvae mine (K. P. Puliafico, unpublished data).

Phyllotreta cruciferae was introduced to North

America from Eurasia. Phyllotreta cruciferae is oligo-

phagous on species in the family Brassicaceae, and

can be a serious pest on several crucifer crop species

(Feeny et al. 1970). Plant injury occurs primarily

from adult flea beetle feeding on cotyledons and

slender stems of seedling plants, and continues as

shot holes or epidermal pit feeding on the leaves

(Feeny et al. 1970) and flowers of mature plants

(Leavitt and Robertson 2006).

Lygus elisus and L. hesperus are polyphagous plant

bugs native to North America, and are among the

most abundant insect species found to feed on hoary

cress (Cripps et al. 2006a,b). Hoary cress has been

found to be a reservoir for Lygus species that are

important economic pests in the USA (Fye 1980).

Although L. hesperus occurs on hoary cress less often

than L. elisus (Cripps et al. 2006a), they were more

widely available during the period in which we

conducted our study. Both Lygus species lacerate plant

tissue, inject salivary fluids which digest the tissue

extra-orally, and then ingest the liquefied tissue

(Butts and Lamb 1990). Lygus hesperus is also a very

capable predator that attacks a wide range of insect

prey (Zeng and Cohen 2001), allowing aggressive

competitive interactions.

Plutella xylostella is originally native to southern

Africa (Kfir 1998) but now has a cosmopolitan

distribution and occurs wherever crucifers are grown

(Talekar and Shelton 1993). It is considered the most

destructive insect pest of cruciferous crops through-

out the world (Talekar and Shelton 1993). Defoliat-

ing P. xylostella caterpillars are oligophagous within

the family Brassicaceae (Talekar and Shelton 1993)

and are abundant on hoary cress in North America

(Cripps et al. 2006b).

Plant and insect material

The study was conducted at the climate-controlled

Manis Entomological Greenhouse, University of

Idaho, Moscow, ID. Environmental conditions were

maintained with a 15L : 9D photoperiod at

24 � 2�C (day): 18 � 1�C (night) in the green-

house throughout all phases of the experiment

except for the seedling vernalization described

below.

Hoary cress seeds were collected from a population

in Latah County, ID (46�44¢N, 116�58¢W) in August

2003. This population comprised of individuals from

the four most common genetic clades found in 88%

of 26 North American populations of hoary cress in a

recent AFLP analysis (J. F. Gaskin, unpublished data).

Seeds were sown into Cornell ‘Peat-Lite A’ artificial

potting medium (Hartmann et al. 1990) in the green-

house. After formation of the first true leaves, seed-

lings were transplanted into plug trays using the same

potting medium. Seedlings were grown for 6 weeks in

the greenhouse and were then transferred to a

3 � 1�C cold room with 12L : 12D photoperiod for a

minimum of 60 days to initiate bolting and flower

production. During vernalization seedlings were ferti-

lized once a week with 0.33 ml of Miracle-Gro� per

litre of water (15-30-15 NPK; Scotts Miracle-Gro,

Marysville, OH). Following cold treatment, plants

were transplanted into 3 l plastic pots in an artificial

potting media (1 : 2 : 1 peat moss, vermiculite, perlite

mix; augmented with 5% sand, pH stabilizers and

trace elements), and fertilized with 2.8 g Osmocote�slow release fertilizer per litre soil (14 : 14 : 14 NPK,

Scotts Miracle-Gro). Plants were then grown in the

greenhouse for 6–9 weeks before they were used for

experimentation.

K. P. Puliafico et al. Effect of generalist herbivory on hoary cress



Ceutorhynchus americanus was collected from hoary

cress near Vale, OR (44�05¢N, 117�18¢W) 2 weeks

prior to the start of each experiment. Females were

tested individually for ovipositional status on cut

hoary cress stems prior to the start of the experiment

(Harmon and McCaffrey 1997), and only fecund

females were used in this study. Phyllotreta cruciferae

adults were collected 3 days before the start of each

experiment from Sinapis alba southeast of Genesee, ID

(46�33¢N, 116�55¢W). Adult and nymph L. hesperus

were collected from a mixed field of alfalfa and pas-

ture grasses at the University of Idaho Sheep Research

Farm, Moscow, ID (46�44¢N, 116�58¢W) 2 days prior

to the start of each experiment. Plutella xylostella eggs

from a strain without resistance to Bacillus thuringiensis

var. kurstaki (Bt) were obtained from Benzon

Research Inc. (Carlisle, PA). A laboratory colony was

reared on artificial diet in 473 ml Styrofoam cups with

plastic lids following the protocols of Shelton et al.

(1991). Adults of both sexes were caged together and

females were allowed to oviposit for 24–48 h on cab-

bage juice coated aluminium foil (Shelton et al.

1991). The foil was then cut into 1 cm2 pieces, eggs

were counted and combined into treatment densities,

then placed at the base of the plants.

Experimental design

For each experiment, plants were arranged in a

complete randomized block design with position on

greenhouse bench as the blocking factor. Stem

number and individual stem lengths were measured

for all plants prior to the start of experiments. Treat-

ment and control plants were caged with 80 cm long

mesh sleeve cages supported with internal wire

frames with the bottom of each sleeve held in place

with a metal hose clamp. Herbivores were released

according to treatments with 5–10 replicates (tables 1

and 2). All plants were caged for 40–44 days and

then destructively harvested by block. Plants were

clipped at the soil surface, the numbers of shoots

counted, and individual shoot lengths recorded to

the nearest 1 cm. Sub-samples of shoots from all C.

americanus treatment levels were dissected under a

binocular dissecting microscope to confirm oviposi-

tion. Roots were carefully washed to remove soil.

Root and shoot biomass was recorded to the nearest

0.1 g after drying for a minimum of 24 h at 80�C.

Experiments with P. xylostella were inspected daily

for pupae and adult moths, which were manually

killed to prevent a second generation. In addition,

all treatments were sprayed with Thuricide� Bacillus

thuringiensis var. kurstaki (Bonide Products, Inc.,

Oriskany, NY) at label rates every 5 days for 2 weeks

(total of three times) to control Bt susceptible P. xylo-

stella after the first pupae were found. No signs of a

second generation were detected within the cages at

harvest time. Survival of the other insect test species

was unaffected by the Bt spray.

Tolerance, as defined by Strauss and Agrawal

(1999), is the degree to which plant fitness is

affected by herbivore damage relative to closely

related but undamaged plants grown under the

same conditions, and was assessed by measuring

changes in plant growth parameters over a range of

herbivore densities. Plant resistance, in contrast,

reduces the performance (antibiosis) or preference

(antixenosis) of herbivores (Muller-Scharer et al.

2004). Visual observations of plant damage were

recorded to assess resistance through the antixeno-

sis effects of physical and chemical feeding deter-

rents. Adults of C. americanus, and P. cruciferae, and

nymphs of L. hesperus were recaptured from a mini-

mum of three cages in experiments 1 and 2 to

measure mortality and assess the antibiosis effects.

Observations of P. xylostella larvae and pupae were

recorded prior to application of pesticide to assess

plant related antibiosis.

The density dependent effects of each individual

herbivore species on plant biomass, shoot number

and length were tested in three experiments using

identical protocols. Insect densities (table 1) were

selected along an exponentially increasing scale and

chosen to encompass normal field densities from

survey data (Cripps et al. 2006b). Experiments 1–3

started on 29 April 2004, 12 August 2004 and

24 May 2005 respectively. To ensure continual

fertilization of eggs, C. americanus densities in experi-

ment 1 included 0, 1, 2, 4 and 8 ovipositing

females and 0, 1, 1, 2, and 4 males respectively.

Table 1 Design of single species experiments

Experiment Species Stage Density Replicates

1 Ceutorhynchus

americanus

Adult 0, 1, 2, 4, 8* 10

1 Phyllotreta

cruciferae

Adult 0, 10, 20, 40, 80 10

1 Plutella

xylostella

Egg 0, 75, 150, 300, 900 10

3 Egg 0, 15, 30, 70, 300 9

2 Lygus

hesperus

Adult 0, 10, 20, 40, 80 5

2 Nymph 0, 20, 40, 80, 160 7

*Number of ovipositing females, with 0, 1, 1, 2 and 4 males added

respectively.




Female-biased sex ratios were preferred because

reduced oviposition rates have been reported for

other Ceutorhynchus species because of interference

from males at high densities (Harmon and McCaf-

frey 1997). The sexes of the other species were not

determined.

The combined effect of paired herbivore species was

tested in another series of three experiments (table 2)

started on 2 August 2004, 12 August 2004 and 24

May 2005 respectively. Experiments 4 and 5 were set

up in a full factorial design, while experiment 6 tested

the impact of C. americanus (0, 1 sexed pair of weevils)

with each of the other three herbivores separately.

Data analysis

Data for each experiment were analysed using GLM

ancova models with herbivore treatments as fixed

factors and position within the greenhouse as a ran-

dom blocking factor. Pre-treatment plant parameters

were examined as covariates in preliminary experi-

ments and pre-treatment sum of shoot lengths was

found to be the best covariate for final dry weight

biomass (regression: shoots F1,28 = 11.72, P = 0.002,

r2 = 0.295; roots F1,28 = 9.99, P = 0.004, r2 = 0.263).

Maximum single shoot length of bolting hoary cress

at harvest was predicted by the pre-treatment maxi-

mum shoot length covariate (regression: F1,16 =

16.33, P = 0.001, r2 = 0.474). To meet underlying

assumptions for statistical analyses, maximum and

total shoot lengths were log10-transformed. In the

event of a significant herbivore effect, means were

compared using a Tukey HSD test and per capita

herbivore effects were examined using regression

analysis to assess tolerance to herbivory. Log-log

transformation of plant parameter responses and

herbivore treatment densities were examined using

linear regression (experiments 1–3) and multiple

regression (experiments 4–6). All analyses were

conducted using Minitab� v15 (Minitab Inc., State

College, PA, USA).

Results

Density dependent impact of single herbivore species

Plutella xylostella was the only herbivore that

caused significant decreases in hoary cress bio-

mass above (exp. 1: F4,35 = 40.09, P < 0.001, exp. 3:

F4,40 = 8.867, P = 0.009) and below ground (exp.

1: F4,35 = 47.17, P < 0.001, exp. 3: F4,40 = 3.21,

P = 0.022). Visible damage to plants was extensive at

all treatment levels of P. xylostella in experiment 1,

with total defoliation occurring at the highest egg

densities, and no larvae survived to pupation in the

900 egg treatment because of intra-specific competi-

tion. Remaining dead stem biomass and compensa-

tory re-growth of defoliated plants that occurred

before harvest resulted in no significant difference in

above-ground biomass between the three highest

egg densities (Tukey, P > 0.05). However, P. xylostella

feeding significantly decreased below-ground bio-

mass (Tukey, P < 0.02 for all means comparisons) at

rate of )0.193 (�0.026) log biomass/log density

(P < 0.001, r2 = 0.521).

Maximum shoot length was significantly reduced

by stem-mining of C. americanus larvae (F4,35 = 5.06,

P = 0.003), shot-hole feeding of P. cruciferae

adults (F4,35 = 3.32, P = 0.021), and defoliation of P.

xylostella (exp. 1: F4,35 = 5.14, P = 0.002; exp. 3:

F4,40 = 3.67, P = 0.012). In contrast, sap feeding by

L. hesperus adults (F4,20 = 0.89, P = 0.486) or nymphs

(F4,28 = 0.62, P = 0.654) had no impact on maxi-

mum shoot length. In experiment 1, C. americanus

caused the highest per capita decrease in maximum

shoot length [)13.13 (�0.39) cm/log density,

P = 0.001, r2 = 0.195], followed by P. xylostella

[)6.16 (�1.36) cm/log density, P < 0.001,

r2 = 0.300], and P. cruciferae [)4.39 (�1.77) cm/log

density, P = 0.017, r2 = 0.113]. On an absolute basis,

reductions in maximum shoot length at the highest

herbivore densities compared with the control were

greatest in P. xylostella (35.1% or 20.8 cm), followed

Table 2 Design of interaction experiments

Experiment Species 1 Stage Density Species 2 Stage Density Replicates

4 Phyllotreta cruciferae Adult 0, 20, 80, 160 Plutella xylostella Egg 0, 75, 300 8

5 0, 20, 80 Lygus hesperus Nymph 0, 20, 80 7

6 Ceutorhynchus americanus Adult 0, 1* Lygus hesperus Nymph 0, 10, 25 9

6 0, 1* Plutella xylostella Egg 0, 75, 300 9

6 0, 1* Phyllotreta cruciferae Adult 0, 20, 80 9

*One pair of sexed weevils.




by C. americanus (22.4% or, 12.3 cm) and P. cruciferae

(19.8% or 10.3 cm). Although there was little dam-

age attributable to adult feeding by C. americanus,

females laid eggs in more than 90% of the shoots

>2.5 cm regardless of weevil density. None of the

herbivore treatments in experiments 1–3 reduced

the number of vegetative shoots produced per plant.

Extensive feeding damage was observed by

P. xylostella larvae and P. cruciferae adults. No antibiosis

was observed for P. cruciferae adults or P. xylostella

larvae. This was confirmed by recapture of a mini-

mum of 70% of P. cruciferae adults and a high degree

of P. xylostella larval survival (as evident by numerous

pupal cases attached to cage walls). Ninety per cent of

adult C. americanus were recaptured across all

treatments and high oviposition rates suggested

that feeding deterrents were not effective against

C. americanus adults. However, upon dissection of

stems, we found undifferentiated callus tissue growth

around both C. americanus oviposition sites and larval

feeding trails, often resulting in larval mortality. No

L. hesperus were found alive and no visible feeding

damage was observed from any of the L. hesperus

treatments, although both adults and nymphs were

observed wandering within the cages the first 2 weeks

after introduction. Inspection of the vials used to

introduce L. hesperus revealed a low level of mortality

(<10%) because of handling and transfer into cages.

Density dependent impact of combined herbivore

species

In experiment 4 both P. xylostella (F2,76 = 13.25,

P < 0.001) and P. cruciferae (F2,76 = 2.95, P = 0.038)

decreased above-ground plant biomass (fig. 1a). The

per capita impact of P. xylostella [)0.031 (�0.011) log

biomass/log density, P = 0.005] was similar to that

of P. cruciferae [)0.028 (�0.013) log biomass/log den-

sity, P = 0.039, r2 = 0.120]. Below-ground biomass

was only decreased by P. xylostella (F2,76 = 6.01,

P = 0.004) (fig. 1b). Plants infested with P. xylostella

also tended to have shorter maximum shoot length

(F2,76 = 3.07, P = 0.052). There were no significant

interactions between the two herbivores, indicating

that their combined effect was additive. Shoot num-

bers were not affected by herbivores or herbivore

combinations.

In experiment 5, P. cruciferae significantly decreased

above-ground biomass (F2,47 = 17.08, P < 0.001)

(fig. 2a), however, only at the highest flea beetle den-

sity (80 adults) (Tukey, P = 0.001). Below-ground

biomass was only decreased by L. hesperus

(F2,47 = 7.70, P = 0.001) (fig. 2b). Total plant biomass

was significantly decreased by both herbivores (P. cru-

ciferae: F2,47 = 10.63, P < 0.001; L. hesperus:

F2,47 = 6.92, P = 0.002). However, the per capita

effect of P. cruciferae on total biomass [)0.089

(�0.025) log biomass/log density, P = 0.001] was one

and half times as much as that of L. hesperus [)0.060

(�0.026) log biomass/log density, P = 0.023,

r2 = 0.204]. None of the interaction terms were

significant, indicating that herbivore effects were

additive. Neither number of shoots nor maximum

shoot length was affected by the herbivores.

In experiment 6, Ceutorhynchus americanus decreased

total (F1,47 = 4.77, P = 0.034) and below-ground

biomass (F1,48 = 5.18, P = 0.027) when combined

with L. hesperus, but the impact of L. hesperus was not

significant (fig. 4a). The two herbivores did not affect

above-ground biomass.

Neither P. cruciferae nor C. americanus significantly

impacted plant parameters when combined,

although C. americanus feeding tended to decrease

total biomass as in the other experiments

(F1,48 = 3.70, P = 0.060). There was a significant

0 40 80 120 160

4

6

8

10

12

(a)

(b)

Ab

ove

-gro

un

d b

iom

ass

(g)

0 40 80 120 160

4

6

8

10

12

Bel

ow

-gro

un

d b

iom

ass

(g)

75 300 P. xylostella

P. cruciferae adults

0

Fig. 1 Effect of Phyllotreta cruciferae adult and Plutella xylostella

larval feeding (experiment 4) on hoary cress (a) above-ground and (b)

below-ground biomass after 40 days of combined herbivory at differ-

ent densities. Mean (�SE) shown.




negative interaction between these two herbivores

on total (F2,47 = 5.41, P = 0.008), and below-ground

biomass (F2,48 = 4.83, P = 0.012) (fig. 4b), indicating

that the impact of C. americanus was negated when

combined with P. cruciferae at high densities.

When C. americanus was combined with P. xylostella,

only P. xylostella decreased above-ground biomass

(F2,48 = 7.66, P = 0.001) (fig. 3c), while both

P. xylostella (F2,48 = 7.90, P = 0.001) and C. americanus

(F1,48 = 21.10, P < 0.001) decreased below-ground

biomass (fig. 4c). Total plant biomass was decreased

by both P. xylostella (F2,47 = 13.05, P = 0.001) and

C. americanus (F1,47 = 20.71, P < 0.001); however, the

per capita effect of C. americanus [)0.042 (�0.011) log

biomass/log density, P < 0.001, r2 = 0.227] was much

larger than that of P. xylostella [)0.012 (�0.006) log

biomass/log density, P = 0.072], which was only mar-

ginally significant. There were no significant interac-

tions between herbivores for any of the biomass

variables, indicating that effects were again additive.

Neither number of shoots nor maximum shoot length

was affected by any of the insect herbivores or herbi-

vore combinations.

Discussion

Our data suggest that hoary cress can be significantly

affected by representatives of the most abundant

generalist herbivore niches found in both the native

and introduced ranges (Cripps et al. 2006a,b). Defoli-

ation of P. xylostella had the strongest and most con-

sistent impact, while damage by C. americanus was

less visible but tended to have a higher per capita

0 20 40 60 80

1

3

5

7

9(a)

(b)

Ab

ove

-gro

un

d b

iom

ass

(g)

0 20 40 60 80

1

3

5

7

9

Bel

ow

-gro

un

d b

iom

ass

(g)

P. cruciferae adults20 80 L. hesperus0

Fig. 2 Effect of Phyllotreta cruciferae adult and Lygus hesperus

nymph feeding (experiment 5) on hoary cress (a) above-ground and

(b) below-ground biomass after 40 days of combined herbivory at dif-

ferent densities. Mean (�SE) shown.

0 5 10 15 20 25

8

10

12

14

16(a)

(b)

(c)

L. hesperus nymphs

0 20 40 60 80

8

10

12

14

16


0 75 150 225 300

8

10

12

14

16

P. xylostella eggs

Ab

ove

-gro

un

d b

iom

ass

(g)

Fig. 3 Effect of combined herbivory of Ceutorhynchus americanus

and either (a) Lygus hesperus, (b) Phyllotreta cruciferae or (c) Plutella

xylostella (experiment 6) on above-ground biomass of hoary

cress after 40 days of herbivory. Filled circle and solid line = 0

C. americanus. Open circle and dashed line = 1 sexed pair of

C. americanus. Mean (�SE) shown.




effect. Plant response to feeding by P. cruciferae was

minor despite obvious feeding damage, and the

impact of L. hesperus was negligible. Plutella xylostella

was the only herbivore that affected biomass accu-

mulation during single species experiments (but

see below). Herbivore combinations with the

defoliator, P. xylostella, significantly affected both the

above- and below-ground biomass accumulation

predominately because of the effects of caterpillar

feeding. The other two oligophagous herbivores

(C. americanus and P. cruciferae) affected plant archi-

tecture, i.e. maximum stem elongation and the sum

of shoot lengths (data not shown) in single species

experiments, but they had no effect on plant bio-

mass even at high densities.

The relatively greater impact of P. xylostella on

hoary cress in this study may not be representative

for the effect of the insect in the field because exper-

imental densities of P. xylostella eggs resulted in a

higher than expected number of larvae. We did not

surface sterilize eggs with chlorine bleach (as sug-

gested for artificial media culturing by Shelton et al.

1991), and egg mortality was greatly reduced as a

result. The lowest densities in all experiments

(except experiment 3) typically produced over 10

pupae per caged plant. This level of infestation is a

threefold increase over the average number of pupae

observed on individual crucifer plants (Haseeb et al.

2001). Field sampling by Cripps et al. (2006b) found,

on average, over 100 P. xylostella caterpillars per 30 s

sweep samples in North America, yet we believe it is

unlikely that individual ramets within a patch would

have a fraction of this amount because of the high

plant densities observed in established hoary cress

stands (McInnis et al. 2003; McKenney 2005). Iso-

lated hoary cress ramets near large patches have

been observed to have over 30 neonate larvae per

plant, but these are highly susceptible to predation

and parasitism and may rarely cause the impact

observed in our experiments (K. P. Puliafico, per-

sonal observation).

Combining insect species had largely additive

effects, with the exception of a negative interaction

between C. americanus and P. cruciferae. Combining

these herbivores reduced the impact of C. americanus

herbivory, particularly on below-ground biomass;

however, the mechanisms of interference are

unknown. The stem miner, C. americanus, impaired

total and below-ground biomass at low densities

when combined with both P. xylostella and L. hes-

perus, suggesting that the plant suffered cumulative

stress as the weevil impact was not significant in sin-

gle species experiments.

Negative interactions between herbivores can

reduce the impact on the plant, and insects within

the same niche tend to have the strongest compet-

itive interactions (Denno et al. 1995; but see Kap-

lan and Denno 2007). The vast majority of

haustellate species, sap feeders primarily in the

order Hemiptera, experience inter-specific competi-

tion (Denno et al. 1995; Kaplan and Denno 2007),

0 5 10 15 20 25

6

10

14

18

22(a)

(b)

(c)

L. hesperus nymphs

0 20 40 60 80

6

10

14

18

22


0 75 150 225 300

6

10

14

18

22

P. xylostella eggs

Bel

ow

-gro

un

d b

iom

ass

(g)

Fig. 4 Effect of combined herbivory of Ceutorhynchus americanus and

either (a) Lygus hesperus, (b) Phyllotreta cruciferae or (c) Plutella xylo-

stella (experiment 6) on below-ground biomass of hoary cress after

40 days of herbivory. Filled circle and solid line = 0 C. americanus. Open

circle and dashed line = 1 sexed pair of C. americanus. Mean (�SE)

shown.




potentially reducing the overall impacts of sap

feeders on hoary cress. Interestingly, haustellate

species do not interfere with mandibulate insect

herbivores as often (Denno et al. 1995), which

may explain the results presented in this study.

We found that combinations of mandibulate herbi-

vores (i.e. C. americanus and P. cruciferae) with the

sap feeding L. hesperus showed weak additive

effects but lacked evidence of negative interactions

that could have implications for future biologi-

cal control. Although other studies have found

that Mirid bugs, including L. hesperus, are capable

predators of soft bodied insect larvae (Zeng and

Cohen 2001), all of the agents currently being

considered for biological control release have

endophagous larvae (Cripps et al. 2006a) and are

therefore considered safe from potential interfer-

ence by Lygus species.

The clonal nature of L. draba in field populations

may help it tolerate feeding by above-ground herbi-

vores because its biomass is predominately located in

below-ground storage tissues (Miller et al. 1994),

which are not directly attacked by any herbivores in

North America (Cripps et al. 2006a,b). Carbon

reserves stored for above-ground reproduction in

these tissues can be mobilized for tolerance and

resistance responses (Strauss and Agrawal 1999).

Even with the small soil volumes used in our study

compensatory re-growth observed after defoliation

by P. xylostella resulted in significant decreases in

root biomass with increased herbivore densities even

though the above-ground biomass did not show a

density-dependent response.

Neither P. xylostella nor P. cruciferae suffered any

decrease in feeding preference or exhibited negative

physiological effects from prolonged feeding on

hoary cress. However, we found evidence of resis-

tance against the polyphagous insects, especially

L. hesperus but also the oligophagous C. americanus,

even though both species have previously been col-

lected from hoary cress (Cripps et al. 2006a).

Female C. americanus laid eggs in the majority of

shoots examined, and they were found actively ovi-

positing at the end of the single species experi-

ments. However, plant defence responses caused

high egg and larval mortality within the stems from

callus tissues found growing around oviposition

holes, eggs and larval mines. Oviposition sites and

eggs of the closely related C. napi Gyll., a stem-min-

ing pest of Brassica crop species, have been shown

to be encapsulated within callus tissues (Le Pape

and Bronner 1987), but it is unknown if larval

mines of this species are similarly affected. Like

other Brassicaceae, L. draba has qualitative chemical

defence mechanisms against generalist herbivores

(Rask et al. 2000). Levels of the most common

glucosinolate defence compound in hoary cress,

p-hydroxy-benzyl glucosinolate, were shown to

be significantly higher in populations from the

introduced range compared with native European

populations (Muller and Martens 2005). This could

explain the complete mortality and lack of repro-

duction of L. hesperus in our experiments. Although

Lygus species can occur at high densities on L. draba

in the USA (Cripps et al. 2006a), these insects may

utilize other host plants to moderate their intake of

glucosinolates.

Recent tests of ERH have used herbivore exclu-

sion field experiments to explore the impacts of

insects and other herbivores (Liu and Stiling 2006),

but this method does not allow quantitative assess-

ments of the specific impact of individual species or

feeding niches except when highly specific methods

(e.g. cages and pesticides) are used to exclude a

few susceptible species (e.g. Goolsby et al. 2004).

Greenhouse herbivore interaction experiments like

the one reported here are historically less common

than field experiments, but are useful for exploring

the mechanisms and symmetry of competition

(Denno et al. 1995). Assessing herbivore commu-

nity structure and plant response to the herbivore

niches present in North America could explain the

success of introduced L. draba populations and pro-

vide information on niches that may be better

exploited by specialists from the native range

(Puliafico et al. 2008). In summary, our results

show that hoary cress can tolerate high densities of

oligophagous insect herbivory and effectively resist

attack by not only the polyphagous L. hesperus, but

also the oligophagous C. americanus. Our data indi-

cate that L. draba is able to withstand this generalist

attack primarily because of a combination of plant

tolerance and resistance, but also because of weak

inter-specific herbivore competition.

Acknowledgements

We thank M. Cripps and J. McKenney for discus-

sions on experimental design, K. Schotzko, S. Gers-

dorf and M. Cole (all University of Idaho) for

technical assistance, and K. Marske (University of

Auckland) for reviewing an earlier draft. Funding

was provided by USDA NRI grant agreement

IDA00108-CG to MS, by the Idaho State Department

of Agriculture through its Cost Share Program, and

by the USDI Bureau of Indian Affairs.




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