Plant-mediated effects of drought stress on host preference and

performance of a grass miner

Jan Scheirs and Luc De Bruyn

Scheirs, J. and De Bruyn, L. 2005. Plant-mediated effects of drought stress on hostpreference and performance of a grass miner. �/ Oikos 108: 371�/385.

The plant stress hypothesis predicts that environmental stress increases the suitability ofplants as food for herbivores, especially for senescence feeders. Yet, performance isenhanced only at moderate stress intensities in several herbivores. Even moreparadoxically, a large number of insect species prefer and perform better onvigorously growing plants. In order to test plant stress theory, we conducted alaboratory experiment in which the influence of plant water stress on host preferenceand the performance of the grass miner Chromatomyia milii was studied. We imposed agradient of stress intensities, i.e. 25, 50, 75, 150, and 300-ml weekly-administered waterper grass pot, in order to study the full range of responses of C. milii to water stressedplants. Plant stress intensity was quantified by measuring individual plant mass, foliarwater content and the concentration of the photosynthetic pigments chlorophyll a and b.Plant mass had decreased from the 150 and 300-ml treatments to the lowest watertreatment at the end of our experiment, which was mainly a result of a reduction in leafarea and leaf number. Foliar water content was clearly negatively affected by watershortage. Chlorophyll a and b also decreased with water shortage. Finally, the stressintensity measurements showed that plants acclimated to water stress conditionsthroughout the experiment. Feeding and oviposition preference of C. milii waspositively related to water supply. No larvae survived on two lowest water treatmentsand only 38% survived on the 75-ml treatment, while more than 80% survived on the150 and the 300-ml treatments. Offspring development time was longer on the 75-mltreatment than on the 150 and the 300-ml treatments. We also evaluated themechanisms that could explain the response of C. milii to water stressed plants.Although no relationship between water treatment and foliar amino acid concentrationwas found, we observed significantly higher foliar protein concentrations in the 25 andthe 50-ml treatments. This supports the hypothesis that abiotic stress causes an increaseof nitrogenous compounds in plants. Leaf senescence following self-pruning, a processby which H. lanatus plants acclimate to drought conditions, was responsible for thedramatically high offspring mortality on the water stressed plants. The shape of theplant stress intensity�/herbivore response relationship showed strong variation anddepended both on the type of plant stress intensity measure and herbivore responsevariable involved. Yet, all relationships showed a monotonic increase of herbivorepreference and performance with decreasing plant stress intensity. This indicates thatC. milii prefers and performs better on vigorously growing plants. We found no supportfor an increased herbivore performance on moderately or severely stressed plants.

J. Scheirs, Max Planck Inst. for Chemical Ecology, Hans-Knoll-Strasse 8, DE-07745Jena (jscheirs@ice.mpg.de). �/ L. De Bruyn, Institute of Nature Conservation,Kliniekstraat 25, BE-1070 Brussels, Belgium.

Accepted 7 July 2004

Copyright # OIKOS 2005ISSN 0030-1299

OIKOS 108: 371�/385, 2005

OIKOS 108:2 (2005) 371

The plant stress hypothesis predicts that phytophagous

insects �/ especially those that feed on senescing rather

than flush tissue �/ will prefer and perform better on

stressed plants because abiotic stress causes an increase

of nitrogenous compounds in plants (White 1969,

1974, 1984, Mattson and Haack 1987a). Yet, perfor-

mance is enhanced only at moderate stress intensities

in several herbivores (Nelson et al. 1969, Oka and

Pimentel 1974, 1976, Perring et al. 1986, English-Loeb

1989, 1990a, b, Potter 1992, De Bruyn 1995, Schowalter

et al. 1999). Moreover, many phytophagous insects

prefer and perform better on vigorously growing

plants and plant modules (Price et al. 1987, Craig

et al. 1989, Kimberling et al. 1990, Inbar et al. 2001,

De Bruyn et al. 2002). These observations led to the

so called plant stress paradox (Larsson 1989, Mopper

and Whitham 1992) and the formulation of the

plant vigour hypothesis (Price 1991). The latter hypoth-

esis predicts that phytophagous insects, especially

those with a tight link between adult oviposition

and offspring feeding site and species in which

larval development is associated with plant growth

processes (Preszler and Price 1995), will prefer and

perform better on vigorously growing plants (Price

1991). Following Price (1991), the plant stress and

the plant vigor hypothesis should not be regarded as

strict alternatives. They are more likely to be relevant to

two ends of a spectrum with some herbivores favoured

by vigorously growing, unstressed plants, some by

moderately stressed plants and some by severely stressed

plants.

The mechanistic basis of the plant stress hypothesis

as proposed by White (1984) is straightforward. Plant

stress results in enhanced nitrogen concentrations, which

may benefit phytophagous insects as plant nitrogen

is of limiting availability to herbivores (Mattson 1980).

Despite the increase of nitrogen concentrations at

some level of stress in many plants (White 1984, Thomas

and Hodkinson 1991; but see Inbar et al. 2001),

a large group of phytophagous insects consistently avoid

and perform better on vigorously growing plants. Several

mechanistic explanations have been forwarded in order

to explain the latter pattern. First, high larval mortality

following early shoot (Craig et al. 1989) and leaf

abscission (Preszler and Price 1995) on stressed plants

has been related to a higher performance on and/or

preference for vigorously growing plants in endophagous

insects. Exophagous insects are probably less prone to

effects of leaf abscission. English-Loeb et al. (1997)

showed that the performance of the beet armyworm

larvae (Spodoptera exigua ) decreased on water stressed

plants despite an increase of plant N-concentrations

in the stressed plants. They measured a broad range

of plant chemical components and showed that seve-

ral mutually non-exclusive reasons could explain the

decrease in performance: i.e. an increase of plant defence

levels, a decrease of carbohydrates, and/or a decrease

in foliar water concentrations. Holtzer et al. (1988)

lists besides nutritional aspects a number of addi-

tional mechanisms by which water-stressed plants may

influence herbivores like plant availability and appa-

rency, microenvironment, and third trophic level inter-

actions. Hale et al. (2003) showed that performance

of the bird cherry-oat aphid (Rhopalosiphum padi )

decreased on drought stressed plants due to a decrease

in the feeding rates under drought-stressed conditions.

Yet, most studies provide only little insight into the

factors that underlie the response of insects to stressed

plants.

There is no universal agreement as to the definition of

stress in plants (Larsson 1989, Lichtenthaler 1996). We

define plant stress as any unfavourable condition or

substance that negatively affects plant metabolism,

growth or development (Lichtenthaler 1996). Yet, most

studies in plant�/insect research typically measure plant

growth as the only or primary indicator of stress (Willis

et al. 1993, De Bruyn 1995, Rehill and Schultz 2001).

Notably fewer studies use physiological measures like

water potential, stomatal conductance or plant photo-

synthetic capacity (but see Louda and Collinge 1992,

Preszler and Price 1995, Hanks et al. 1999). The latter

measures are frequently used by plant physiologists and

may therefore provide standardised measures of the

plant stress level (Larsson 1989). Moreover, different

plant stress endpoints do not necessarily co-vary and

may therefore provide different views of the plant stress

response (Hsiao 1973, Mattson and Haack 1987b,

Lichtenthaler 1996).

We investigated the relationship between plant stress

intensity and herbivore response with the grass

miner Chromatomyia milii (Kaltenbach) (Diptera, Agro-

myzidae) on the host plant Holcus lanatus L. We

subjected H. lanatus plants to a range of water treat-

ments, from very little to a presumed excess of water,

and studied the influence of stress intensity on the

response of C. milii . We applied a range of

stress intensities as recommend by Koricheva et al.

(1998) in order to be able to evaluate the existence

of non-linear relationships between plant stress intensity

and herbivore response. We quantified stress intensity

by measuring plant water status, plant growth and

plant photosynthetic capacity. Plant characteristics like

leaf area and the number of leaves along the stem

were monitored throughout the experiment in order to

evaluate the acclimation of the plants to stressed

conditions (English-Loeb et al. 1997). The response

of C. milii was established by recording host prefer-

ence and offspring performance. These data were then

used to study the shape of the between plant stress

intensity and herbivore response for the various plant

stress intensity measures. Finally, foliar protein and

amino acid concentrations were determined in order to

372 OIKOS 108:2 (2005)

evaluate whether stressed plants have enhanced concen-

trations of N-containing compounds and whether the

insect could benefit from it.

Methods

Life history of Chromatomyia milii

Chromatomyia milii is an oligophagous grass miner. It

can be found on individuals of four grass genera

(Griffiths 1980), but Holcus is its principal host in our

region. Females of C. milii insert their eggs directly in

the mesophyll layer between two veins of a grass leaf

(Scheirs et al. 2001a). Larvae emerge within one week

and immediately eat their way through the internal leaf

tissues. They mainly consume mesophyll cells, but also

parts of the sclerenchyma strands and bundle sheath

cells when crossing the numerous adjoining veins

(Scheirs et al. 2001b). Larvae complete four stadia

(3 larval and 1 prepupal) and then pupate within their

mines. Adults emerge through the leaf surface. Females

feed on leaf saps that exude from feeding punctures

made by their ovipositor in the leaf blades of host

grasses (Scheirs et al. 2001a). The number of feeding

punctures and eggs is positively related to feeding and

oviposition preference, respectively (Scheirs et al. 2003).

The C. milii females used in the experiments were

collected from a field population living on Holcus

lanatus L. at Hoboken, Belgium. We used only gravid

females of unknown age. Flies and grasses were only

used once in an experiment to avoid pseudoreplication.

Water stress

Holcus lanatus seeds (0.1 g pot�1) were sown in 10�/

10�/11 cm pots containing a soil mixture of 50% white

sand and 50% vermiculite. Grasses were grown in a

greenhouse under controlled conditions (16�/8 h L:D,

209/18C, 70 RH in the light �/ 100% RH in the dark) and

were watered weekly with 150 ml half-strength Hoagland

solution per pot (Hoagland and Arnon 1950). After ten

weeks, grass pots were randomly assigned to five water

treatments, i.e. 25, 50, 75, 150 and 300-ml water per pot.

Pots received these water treatments from week 11 till the

end of the experiment (week 18). The respective amount

of water was administered once a week. Nutrient levels

were kept equivalent to a 50% Hoagland solution in all

treatments. Three hundred grass pots were divided over

twenty five trays, which corresponds to twelve pots per

tray. Five trays were randomly assigned to each water

treatment. Plants in the 150 and 300-ml treatments had

always access to water. The water level of the 300-ml

treatment was equal to ground surface level (all below

ground plant parts submerged), while only the lower

half of the pot was submerged in the 150-ml treatment.

We applied the 300-ml treatment in order to investigate

whether an excess of water may also result in stressed

plants (Heinrichs 1988, p. 14), with potential effects on

the herbivore response as a consequence. We did not

consider higher water treatments than 300 ml week�1, as

this would submerge the aboveground plant parts and

flies would not have access to the leaves. The plants in

the 25, 50, and 75-ml had no access to water during one

or more days a week and suffered as a consequence from

a shortage of water (Results). A pilot experiment had

shown that plants in 100, 125 and 150-ml water

treatments had access to water during the entire period

of the experiment. Plants in the latter three treatments

showed no signs of water stress. We preferred to use the

150-ml treatments as this treatment was applied in most

previous experiments on host preference of C. milii

(Scheirs et al. 2001a, 2003) and because it would have

been too laborious to apply all treatments. The positions

of the trays were randomised weekly within the green-

house to avoid position effects.

Host preference and offspring performance

Five-way choice experiments were carried out when

plants were 13 weeks old. At this stage, leaves in the

25, 50 and 75-ml treatments showed signs of wilting. One

pot of each water treatment was offered simultaneously

to five females of C. milii during one trial. As individual

plant dry mass did not differ among treatments at this

point (mixed model Anova: F4,70�/0.76, P�/0.556),

there was no need to control for differences in plant

biomass among treatments. We conducted eight trials.

Pots were randomly positioned in a circle in a flight cage

(50 L�/75 B�/75 cm H). A trial lasted 48 hours, after

which the number of eggs and feeding punctures were

recorded as a measure of adult oviposition and feeding

preference respectively. Feeding punctures and eggs were

counted by examining each individual leaf under a

microscope with light projecting through the leaf.

Offspring performance was determined by offering

individual grass pots to single gravid females in a flight

cage. This was done for five pots per treatment when

grasses were 13 weeks old. Each pot came from

a different tray. Females were allowed to lay eggs for

48 hours. Afterwards, eggs were allowed to develop into

larvae, which were raised until they matured into

adult flies. The grasses were checked daily for pupated

and dead larvae. Pupated larvae were dissected from

the leaves and put into individual rearing containers,

which were checked daily for emerging adults. The

positions of the rearing containers were randomised

daily within the greenhouse to avoid position eff-

ects. Two days after pupation, the mass of the puparium

was assessed to the nearest 0.1 mg using a Cahn

microbalance. In this way we determined three measures

OIKOS 108:2 (2005) 373

of offspring performance: offspring survival, develop-

ment time, and pupal mass. Only larvae that completed

their development in the absence of competition

were considered in the analysis, because larval competi-

tion decreases offspring performance of Agromyzidae

(Quiring and McNeil 1984, Petitt and Wietlisbach 1992).

Finally, we also recorded the type and cause of offspring

mortality. We discriminated among three types of mor-

tality: i.e. larval mortality due to self-pruning, larval

mortality of unknown cause, and pupal mortality of

unknown cause. We considered the cause of dead to be

self-pruning if the larva was in a green leaf that wilted

and died. Leaves that die of age turn yellow and slowly

turn brown from the tip till the base of the leaf.

Plant stress intensity

All plant stress intensity measures were taken from

plants in pots that had not been exposed to the flies.

Individual plant growth was determined by mea-

suring total plant biomass at the end of the experiment

(week 18). All above- and below ground plant material

from 15 plants per treatment were collected. Above plant

parts (leaves and stem) and roots were dried at 1058Cand weighed after 48 hours in order to obtain total plant

dry mass. Relative root mass was determined in order to

evaluate whether water stressed plants invest relatively

more in root biomass than plants in the higher water

treatments (Chaves et al. 2003). Additional mechanisms

by which plants acclimate to water deficits are

the reduction of the number of leaves by self-pruning

and the reduction in size of newly developed leaves

(Sanderson et al. 1997, Chaves et al. 2003). In order to

study the effect of water treatment on the number of

leaves, three plants were sampled randomly from differ-

ent pots in a tray at weekly intervals from week 10 till 18.

The plants were immediately frozen at �/808C until

sample preparation for chlorophyll and nutrient analyses

(below). The number of leaves along the stem were

quantified just before samples were prepared for chlor-

ophyll and nutrient analyses. The leaf area of the fresh

predawn leaves collected for the foliar water measure-

ments (see below) was determined using a portable leaf

area meter (LI-COR 3000).

The influence of the water treatment on plant water

status was studied by analysing foliar water content

(Buckland et al. 1991, Hendry and Grime 1993). Five

leaves were sampled randomly from different pots in a

tray at weekly intervals during the light (noon) and the

end of the dark cycle (predawn). Leaves were immedi-

ately weighed after collection (FM), dried at 1058C, and

reweighed after 48 hours to obtain their dry mass (DM).

Water content is expressed as ([FM�/DM]/FM)�/100.

We collected leaves at noon and predawn because water

stress intensity is the highest during the light phase of the

day due to opening of the stomata, while plants

recuperate during the night when stomata are closed

(Holtzer et al. 1988, Prasertsak and Fukai 1997).

The concentration of the photosynthetic pigments

chlorophyll a and chlorophyll b were quantified as a

third plant stress intensity measure. The determination

of photosynthetic pigment concentrations has become a

classical method in plant stress research (Lichtenthaler

1996). Measuring photosynthetic pigments provides a

general indication of plant stress levels (Carter 1993,

Carter and Knapp 2001) as most stress factors, even if

they do not directly affect the composition of the

photosynthetic apparatus or its function, will affect the

photosynthetic process in the long run (Lichtenthaler

1996). Photosynthetic pigment concentrations decrease

as a general rule in water stressed plants (reviewed by

Chernyadev 1997, Yordanov et al. 2000). Three leaves

were sampled randomly for chlorophyll analysis from

different pots in a tray at weekly intervals during the

light cycle and were immediately frozen at �/808C until

sample preparation (see Plant growth). Weekly samples

were taken on the same hour and day of the week

(Wednesday at 12.00 h) in order to account for potential

confounding effects of sampling hour and day. The

middle 2-cm of the leaf was used for pigment extraction,

the remaining leaf parts for nutrient analyses (below).

The position of the leaf was recorded along the

stem axis. Pigments were extracted in the dark using

dimethylformamide. Absorbencies were recorded at 480,

646.8 and 663.8 nm. Chlorophyll a and b concentrations

were calculated using the extinction coefficients provided

by Wellburn (1994).

Chemical analyses

Proteins and amino acids are the principal N-containing

nutrients within a plant for insect herbivores (Mattson

1980). We assessed the protein and the amino acid

concentrations of H. lanatus leaves during the course of

the experiment. We used leaves that had not been

exposed to C. milii flies during the experiment. The

leaf samples (see chlorophyll analysis) were subsequently

lyophilised, ground, and extracted on ice for 30 min with

a Tris/EDTA buffer (pH�/7.6). Extracts were centri-

fuged at 5500 rpm and supernatants collected and stored

at �/808C until further analysis. We used whole leaf

samples for protein and amino acid analysis instead of

only the mesophyll as suggested for selective feeders

(Kimmerer and Potter 1987). Whole leaf samples are a

good estimate of the nutritional quality of the larval

food of C. milii (Scheirs et al. 2003) because (a) the

mesophyll layer of grasses consists of one large homo-

geneous layer (Vincent 1982), and (b) the larvae feed on

both the mesophyll and the veins (Scheirs et al. 2001b).

Foliar protein concentrations were determined using the

374 OIKOS 108:2 (2005)

Bradford assay with bovine serum albumin as a standard

(Sigma, B6916) (Jones et al. 1989). Prior to amino acid

analysis, proteins were precipitated using trichloroacetic

acid (Bollag et al. 1996) and supernatants collected after

centrifugation at 5500 rpm. Foliar amino acid concen-

trations were determined using the ninhydrin method

(Sigma, N1632) (Moore 1968, Allen 1989) with leucine

as a standard.

Statistical analyses

We used linear mixed model analysis (Littell et al. 1996)

in order to determine whether individual plant biomass

and relative root mass differed among the water treat-

ments. We included water treatment and the square of

the water treatment as continuous, independent variables

in our model. The square of the water treatment was

included in the model in order to account for potential

quadratic treatment effects. The logarithmic transformed

dry weight of plant biomass or the log-transformed

values of the relative root mass were used as dependant

variable. Tray, nested within water treatment, was

included in the analyses as a random effect.

We used a generalised estimating equations (GEE)

methodology (Liang and Zeger 1986, Diggle et al. 1994,

SAS Institute 1999) in order to compare the number of

leaves, leaf area, and the foliar concentration of water,

photosynthetic pigments, proteins and amino acids

among treatments. GEE methodology is a popular

technique for the modelling of correlated response data

that are either the result of repeated measurements on

the same subject (longitudinal data) or subjects that

share the same category that lead to correlation (cluster-

ing; Diggle et al. 1994, SAS Institute 1999). We do not

have longitudinal data as leaves or other subjects were

only measured once, but weekly measurements of

different leaves were most probably clustered within

trays. For the analyses of the leaf area, photosynthetic

pigments, proteins and amino acids, we used a compar-

able model as for the analysis of the individual plant

biomass data, but included water treatment, water

treatment2, week (continuous), week2, and the interac-

tions water treatment�/week, water treatment�/week2,

water treatment2�/week and water treatment2�/week2

as the independent variables. Tray, nested within water

treatment, was included in the analyses as a repeated

effect with an exchangeable correlation structure. The

foliar water, protein and amino acid concentrations were

log-transformed in order to meet the assumption of a

normally distributed error. The number of leaves along a

stem was also compared using GEE methodology, but

we used a Poisson error distribution in order to account

for the fact that we used count data in this analysis (SAS

Institute 1999). For the foliar water content analyses, the

model for the photosynthetic pigment concentrations

was used with the time of day (noon�/predawn) and all

additional interactions in the independent variable list.

We treated week as class variable in the latter analysis in

order to reduce the number of fixed effects to an

interpretable number.

The results of the choice experiment were analysed

using the Quade test (Quade 1979, Conover 1980). This

non-parametric test takes the lack of independence

among simultaneously offered food types into account

(Roa 1992). Quade-test statistics were calculated with

StatXact 3 (Mehta and Patel 1995).

Offspring survival was analysed using mixed model

logistic regression with logit link function and binomial

errors. Water treatment and water treatment2 were used

as independent, fixed variables, while tray nested within

water treatment was used as a random variable. We

compared the frequencies of the different classes of

offspring mortality with a Fisher exact test using

StatXact 3 (Mehta and Patel 1995). Total development

time and pupal mass were analysed with linear mixed

model analysis with normally distributed errors. The

dependant variables were log transformed. The indepen-

dent variables were water treatment, water treatment2,

gender and all possible interactions. Tray nested within

water treatment was treated as a random variable.

The nature of the relationship between plant stress

intensity and herbivore response was evaluated by fitting

linear and non-linear models through the data with

TableCurve (Jandel Scientific 1994). The best model fit

was established by using the best degrees of freedom

adjusted R-square as a criterion. Second, the model and

all parameter estimates had to be significant. The

number of feeding punctures and eggs per pot laid in

the choice trials were related to the mean foliar water

content per pot, and chlorophyll a and b per pot. We

used relative numbers of feeding punctures and eggs per

pot in a trial in order to account for the variation among

trials. We calculated mean foliar water content, chlor-

ophyll a and chlorophyll b from the measurements made

during week 13 and 14, which corresponds to the timing

of the preference experiments. We only evaluated the

stress intensity�/herbivore response relationship for

those offspring performance measures that differed

among water treatments, i.e. offspring survival and

mean development time per tray. For the plant stress

measures, we used per tray mean plant biomass, mean

chlorophyll a (or b) concentration, and the mean weekly

foliar water content. We only considered the plant stress

intensity measurements made from week 13 till 18 for the

calculation of the means as offspring development took

place during this period. As least squares curve fitting

assumes that the errors map to a Gaussian profile

(Jandel Scientific 1994), we checked for all equations

whether errors were normally distributed.

All previous GEE’s were modelled in the PROC

GENMOD module of SAS (SAS Institute 1999). All

OIKOS 108:2 (2005) 375

previous mixed model analyses were calculated using the

PROC MIXED module in SAS (Littell et al. 1996, SAS

Institute 1999) in the case of normal errors, and with the

GLIMMIX macro in the case of binomial errors (Littell

et al. 1996). The degrees of freedom of the fixed effects

F-test were adjusted for statistical dependence using the

Kenward�/Roger method (Kenward and Roger 1997).

Variance components were estimated by restricted max-

imum likelihood (REML, Littell et al. 1996). Model

reduction was performed using the method described by

Verbeke and Molenberghs (2000). We started with a ‘‘full

model’’ including all interactions. Model reduction was

performed by dropping non-significant (a�/0.05) fixed

effects in a backward hierarchical way, starting with the

least significant highest order interaction term.

Results

Plans stress intensity

Individual plant mass showed a convex relationship with

water treatment (Fig. 1; water treatment: F1,21.2�/27.61,

PB/0.001; water treatment2: F1,21.2�/16.60, PB/0.001).

Plant mass was the lowest in the 25-ml treatment and

increased over the 50 and the 75-ml treatment to the 150-

ml and 300-ml treatments. No relationship between

water treatment and relative root mass was observed

(water treatment: F1,22.5�/0.06, P�/0.807; Water treat-

ment2: F1,22.4�/0.25, P�/0.621). Leaf area was

clearly negatively affected by water availability in the

25, 50 and 75-ml treatments (Table 1; water treatment:

x2�/8.04, P�/0.005; water treatment2: x2�/8.64,

P�/0.003; week: x2�/7.16, P�/0.008; week2: x2�/7.34,

P�/0.007; water treatment�/week: x2�/8.20, P�/0.004;

water treatment2�/week: x2�/8.70, P�/0.009;

water treatment�/week2: x2�/8.38, P�/0.004; water

treatment2�/week2: x2�/8.78, P�/0.003). In the latter

cases, leaf area was halved from week 13 till week 17�/18,

Water treatment

25 ml 50 ml 75 ml 150 ml 300 ml

)g( ssam yrd tnal

P

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0.2

0.3

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10

15

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Fig. 1. Individual plant mass (black bars) and relative rootmass (white bars) for the five water treatments. Error barsrepresent 9/1 SE. T

able

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25

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75

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1.0

79

/1.2

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0.5

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2.5

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/1.1

41

1.8

29

/1.1

61

1.7

89

/1.0

53

.139

/0.1

33

.009

/0.1

42

.939

/0.1

53

.139

/0.2

43

.139

/0.1

91

11

3.5

29

/1.5

91

3.2

09

/1.3

21

3.4

49

/1.2

81

2.5

99

/1.0

21

1.2

69

/0.8

92

.809

/0.1

72

.939

/0.2

83

.939

/0.2

83

.339

/0.2

53

.009

/0.1

41

21

2.7

09

/1.2

51

2.2

49

/1.2

91

3.1

49

/1.3

41

3.5

99

/1.0

31

2.5

39

/1.2

23

.079

/0.1

23

.409

/0.1

92

.879

/0.1

72

.879

/0.2

73

.339

/0.2

91

31

0.5

79

/1.3

21

2.6

39

/1.0

31

2.3

89

/1.2

71

0.3

89

/0.8

11

1.2

29

/1.2

62

.809

/0.2

42

.939

/0.2

83

.279

/0.2

33

.339

/0.2

93

.879

/0.2

41

41

2.2

19

/1.5

61

2.4

49

/0.8

31

2.0

59

/1.0

41

1.7

09

/0.8

01

0.6

19

/1.0

82

.279

/0.2

32

.479

/0.3

23

.339

/0.1

63

.539

/0.1

74

.009

/0.3

51

51

0.7

69

/0.9

81

0.2

49

/1.0

11

0.4

09

/0.8

91

2.0

89

/0.8

71

1.3

49

/1.0

81

.939

/0.2

52

.809

/0.3

03

.279

/0.1

83

.479

/0.3

94

.139

/0.2

61

66

.089

/0.4

28

.799

/0.7

51

1.0

79

/1.3

91

1.2

29

/0.8

41

2.2

09

/1.0

32

.139

/0.2

22

.609

/0.1

63

.339

/0.1

64

.279

/0.3

63

.809

/0.2

01

75

.369

/0.5

16

.139

/0.9

66

.899

/0.6

29

.609

/0.9

51

1.1

89

/0.8

82

.209

/0.2

42

.739

/0.2

32

.739

/0.1

84

.209

/0.3

34

.409

/0.3

41

86

.229

/0.8

65

.739

/0.5

08

.069

/0.7

71

2.0

19

/0.9

21

1.7

29

/1.1

13

.009

/0.3

93

.139

/0.3

63

.609

/0.4

74

.439

/0.3

34

.009

/0.3

1

376 OIKOS 108:2 (2005)

while mean leaf area in the 150 and the 300-ml

treatments remained more or less constant. The mean

number of leaves along the stem increased in the 150 and

the 300 ml treatments from the start till the end of the

experiment or from around three till slightly more than

4 leaves (Table 1; water treatment: x2�/7.23, P�/0.007;

water treatment2: x2�/2.87, P�/0.090; week: x2�/5.83,

P�/0.016; week2: x2�/4.03, P�/0.045; water treatment�/

week: x2�/6.75, P�/0.009; water treatment2�/week:

x2�/4.33, P�/0.037; water treatment�/week2: x2�/3.94,

P�/0.047). Leaf number in the 25-ml, and to a lesser

extend in the 50-ml treatment, decreased from week 13

till 15, after which numbers increased back to levels

observed at the start of the experiment. Leaf numbers in

the 75-ml treatment remained more or less constant. The

reduction in plant growth and leaf number (Hale et al.

2003) and the reduction in leaf area (De Bruyn et al.

2002) is in line with previous work on drought-stressed

H. lanatus plants.

The kinetics of the foliar water content were very

complex for the different water treatments as indicated

by the many significant fixed effects in the generalised

linear model (Table 2; water treatment: x2�/7.98, P�/

0.005; water treatment2: x2�/9.15, P�/0.003; week: x2�/

18.50, P�/0.018; noon�/predawn: x2�/4.42, P�/0.036;

water treatment�/noon�/predawn: x2�/5.62, P�/0.018;

water treatment2�/noon�/predawn: x2�/6.37, P�/0.012).

The general pattern can be described as follows. Water

content was nearly equal for all treatments from week 10

till 12. Afterwards, water content dropped slowly for the

high water treatments (150 and 300-ml) and very rapidly

for the low water treatments. At the end of the

experiment, water content was the lowest in the 25�/ml

treatment and increased over the 50-ml and 75-ml

treatment to the 150-ml and 300-ml treatment. There

were large differences between predawn and noon water

content in the 25-ml water treatment from week 14 till 16

indicating severe water stress. There was still a noticeable

difference between predawn and noon water content in

the 50-ml treatment from week 14 till 16, but no or only

a very small difference in the other treatments. After

week 16, differences between predawn and noon water

content were greatly reduced in the 25 and the 50-ml

treatments.

The kinetics of the foliar chlorophyll a concen-

tration differed significantly among the water treatments

(Table 3; water treatment: x2�/5.89, P�/0.015; water

treatment2: x2�/3.47, P�/0.062; week: x2�/6.90, P�/

0.009; week2: x2�/6.57, P�/0.010; water treatment�/

week: x2�/5.89, P�/0.015; water treatment2�/week:

x2�/4.36, P�/0.037; water treatment�/week2: x2�/4.49,

P�/0.034). The chlorophyll a concentration of the 25-ml

treatment showed a decline from week 12 till week 16

and an increase from week 16 till 18. The kinetics of the

50-ml and the 75-ml were comparable. The concentra-

tions of the 50-ml treatment were in between those of the Tab

le2

.P

red

awn

an

dn

oo

nfo

liar

wat

erco

nte

nt

(%fr

esh

ma

ss)

fro

mw

eek

10

till

18

for

the

dif

fere

nt

wat

ertr

eatm

ents

.E

rro

rb

ars

rep

rese

nt9

/1S

E.

Pre

daw

nN

oo

n

25

-ml

50

-ml

75

-ml

15

0-m

l3

00

-ml

25

-ml

50

-ml

75

-ml

15

0-m

l3

00

-ml

Wee

k1

08

7.7

89

/0.5

78

8.0

49

/0.5

78

7.5

79

/0.3

58

7.9

99

/0.6

08

7.4

79

/0.4

58

8.7

69

/0.3

88

7.8

99

/0.5

48

8.6

29

/0.5

98

7.8

19

/0.6

98

8.0

79

/0.3

81

18

8.2

39

/0.5

68

7.7

29

/0.5

98

8.8

39

/0.7

08

6.6

69

/0.5

58

7.3

79

/0.4

58

7.9

89

/0.7

28

7.1

39

/0.5

48

7.9

49

/0.4

08

8.1

69

/0.4

78

7.0

89

/0.5

11

28

7.3

79

/0.5

08

6.4

99

/0.4

48

6.3

09

/0.6

08

7.4

79

/0.4

88

7.4

09

/0.4

58

8.1

69

/0.4

18

7.1

59

/0.4

78

8.4

39

/0.3

88

6.9

89

/0.3

08

7.4

49

/0.5

11

38

3.0

59

/1.0

18

2.6

89

/1.3

18

6.7

69

/0.4

98

7.4

69

/0.4

38

6.0

99

/0.6

38

1.5

49

/1.2

28

3.9

09

/0.7

28

5.7

89

/0.4

78

5.8

19

/0.5

78

5.8

69

/0.6

41

48

1.8

99

/0.7

38

4.5

49

/0.6

68

5.1

19

/0.8

48

6.2

89

/0.5

68

6.4

39

/0.3

87

2.7

89

/5.0

68

1.1

19

/2.0

38

4.4

59

/0.6

18

6.1

59

/0.7

88

6.0

59

/0.3

21

58

1.2

39

/1.5

68

2.6

29

/0.7

58

4.8

79

/0.5

78

6.1

39

/0.4

68

6.1

09

/0.4

47

1.2

39

/4.6

48

2.1

39

/0.9

18

4.1

69

/0.5

58

5.6

59

/0.4

58

4.9

89

/0.9

71

68

1.1

89

/0.6

88

3.4

99

/0.6

68

2.7

19

/0.8

28

5.5

09

/0.5

28

6.4

09

/0.4

57

6.2

89

/3.2

68

1.7

99

/0.9

28

1.8

99

/0.8

18

5.6

69

/0.4

78

7.3

49

/0.3

91

78

0.8

89

/0.9

17

9.2

69

/0.8

98

1.7

79

/0.6

78

5.6

39

/0.4

08

6.3

49

/0.4

28

0.2

69

/0.5

48

2.2

69

/0.7

18

2.7

09

/1.2

08

5.8

19

/0.5

48

6.2

69

/0.3

71

88

0.1

69

/0.6

88

2.4

99

/0.7

68

2.4

09

/0.7

28

6.0

29

/0.4

78

6.3

39

/0.4

17

7.2

69

/1.9

78

1.0

69

/0.7

68

0.9

89

/0.9

48

6.0

59

/0.6

18

5.2

59

/0.7

0

OIKOS 108:2 (2005) 377

25 and the 75-ml treatment from week 13 till 15.

The concentrations of chlorophyll a were higher in the

150-ml treatment than the lower water treatments during

weeks 14, 15 and 16. The chlorophyll a concentrations of

the 300-ml treatment were comparable to those of the

150-ml treatment till after week 15 when concentrations

in the 300-ml treatment decreased. There was less

variation in the kinetics of the foliar chlorophyll b

concentration. The statistical analyses showed that

chlorophyll b increased from the 25-ml, over the 50

and the 75-ml treatments to about equal levels in

the 150 and the 300-ml treatments (Table 3; water

treatment: x2�/5.20, P�/0.023; water treatment2: x2�/

4.40, P�/0.036).

Host preference and offspring performance

Host preference differed significantly among the differ-

ent treatments (feeding punctures: Q�/4.2, PB/0.01;

eggs: Q�/2.8, PB/0.05). The number of feeding punctu-

res and eggs clearly increased from the low water

treatments to the 150 and 300-ml treatments (Fig. 2).

There were very drastic effects of plant water stress on

offspring survival (F4,14.0�/76.99, PB/0.001) (Fig. 3). We

could not test for linear and/or quadratic stress effects as

the statistical program did not converge when water

treatment and water treatment2 were used as indepen-

dent variables. Offspring survival was higher than 80% in

the 150 and 300-ml treatments, while about 38% of the

larvae survived in the 75-ml treatment and no larvae

survived in the 25 and 50-ml treatments. There was also

considerable variation in the cause of offspring mortality

among treatments (Fig. 4; Fischer exact: FI�/132.5,

df�/8, PB/0.001). Most larvae in the 25, 50 and the

75 treatments died as a consequence of self-pruning by

H. lanatus. The mortality in the 150-ml and the 300-ml

treatments was not related to self-pruning, but the result

of unknown causes of larval and pupal mortality.

Offspring development time was longer in the 75-ml

treatment than in the 150 and the 300-ml treatments

(water treatment: F1,11.8�/35.60, PB/0.001; water treat-

ment2: F1,11�/28.61, PB/0.001). Pupal mass did not

differ significantly among treatments, but was higher

for females than males (F1,161�/192.61, PB/0.001). The

latter two performance measures could not be evaluated

for the 25 and the 50-ml treatment due to the 100%

offspring mortality rates on these water regimes (Fig. 3).

N-containing compounds

The generalised linear model analysis indicated that

the foliar protein kinetics was very complex (Table 4;

water treatment: x2�/4.53, P�/0.033; water treatment2:

x2�/4.69, P�/0.030; week: x2�/3.90, P�/0.048; week2:

x2�/3.14, P�/0.076; water treatment�/week: x2�/4.58,Tab

le3

.F

oli

ar

chlo

rop

hy

lla

an

db

con

cen

trat

ion

fro

mw

eek

10

till

18

for

the

wat

ertr

eatm

ents

.E

rro

rb

ars

rep

rese

nt9

/1S

E.

Ch

loro

phy

lla

(mm

ol/

gd

rym

ass

)C

hlo

rop

hy

llb

(mm

ol/

gd

rym

ass

)

25

-ml

50

-ml

75

-ml

15

0-m

l3

00

-ml

25

-ml

50

-ml

75

-ml

15

0-m

l3

00

-ml

Wee

k1

01

2.7

99

/1.1

11

1.9

19

/0.8

41

2.5

69

/0.8

81

2.2

79

/0.8

51

3.1

19

/0.7

04

.449

/0.4

93

.839

/0.3

54

.329

/0.4

24

.589

/0.4

44

.649

/0.4

11

11

3.1

49

/0.6

21

3.2

09

/0.6

51

1.6

79

/0.6

41

2.0

19

/0.5

91

3.5

19

/0.5

85

.009

/0.2

34

.869

/0.3

04

.299

/0.3

54

.759

/0.4

45

.179

/0.3

21

21

2.4

89

/0.6

01

0.8

89

/0.5

81

2.0

99

/0.7

21

2.1

19

/0.9

41

2.9

99

/0.6

54

.279

/0.2

33

.799

/0.2

94

.299

/0.2

84

.419

/0.4

34

.689

/0.2

81

31

0.5

79

/0.7

11

1.9

49

/0.8

21

3.3

79

/0.6

91

3.0

19

/0.8

71

2.0

29

/0.7

34

.169

/0.4

34

.319

/0.4

35

.149

/0.3

95

.119

/0.4

14

.569

/0.3

41

41

0.6

29

/0.7

61

2.0

59

/0.8

41

2.7

89

/0.5

71

3.9

19

/0.6

71

4.0

69

/0.6

63

.879

/0.3

64

.469

/0.4

04

.719

/0.3

75

.089

/0.3

55

.079

/0.2

51

51

0.6

59

/0.9

71

1.1

79

/0.8

61

2.3

69

/0.4

71

3.6

69

/0.6

91

4.5

59

/0.7

03

.629

/0.4

13

.839

/0.3

74

.579

/0.3

15

.329

/0.3

85

.769

/0.3

51

61

0.0

49

/0.7

09

.739

/0.8

91

0.4

29

/0.6

21

2.3

69

/1.0

41

0.4

99

/0.6

53

.259

/0.2

63

.089

/0.3

63

.469

/0.2

83

.989

/0.3

53

.909

/0.3

41

71

3.2

59

/0.5

61

2.4

29

/0.5

81

1.6

89

/0.8

21

4.6

49

/0.9

51

1.2

29

/0.6

64

.439

/0.2

74

.139

/0.2

33

.889

/0.3

45

.539

/0.4

33

.839

/0.3

01

81

3.1

99

/0.5

21

4.3

09

/0.7

01

3.2

29

/0.6

91

3.9

89

/0.5

61

2.1

59

/0.8

54

.319

/0.2

04

.859

/0.2

74

.329

/0.2

55

.109

/0.2

54

.239

/0.3

8

378 OIKOS 108:2 (2005)

P�/0.032; water treatment2�/week: x2�/4.80, P�/0.028;

water treatment�/week2: x2�/4.45, P�/0.035; water

treatment2�/week2: x2�/4.76, P�/0.029). The significant

interactions indicated that foliar protein concentrations

were higher in the 25-ml and 50-ml treatments in week

13, 15 and 16 than in the 150 and the 300-ml treatments.

This pattern is also confirmed by contrast analysis (25

and 50 against 75, 150 and 300-ml treatment: x2�/14.21,

df�/1, PB/0.001). We included the position of the leaf

along the stem as an additional fixed effect variable in

the previous analysis in order to investigate whether the

variation in foliar protein concentration among treat-

ments could be explained by the different age structure

of the leaves among treatments (above). This analysis

revealed a comparable model as the model in which leaf

position was not included with as main difference that

leaf position effect was also significant (water treatment:

x2�/4.10, P�/0.043; water treatment2: x2�/4.22, P�/

0.040; week: x2�/3.62, P�/0.057; Week2: x2�/3.02, P�/

0.082; water treatment�/week: x2�/4.23, P�/0.040;

water treatment2�/week: x2�/4.40, P�/0.036; water

treatment�/week2: x2�/4.22, P�/0.040; water

treatment2�/week2: x2�/4.46, P�/0.035; leaf age: x2�/

19.20, PB/0.001). This leaf position effect indicated that

protein concentration decreased with leaf age. This

suggests that a different age structure of the leaves as a

result of self-pruning in the water stressed plants only

partly explained the variation among treatments. The

foliar amino acid concentration did not differ signifi-

cantly among the treatments (Table 4). There was a

general decrease in foliar amino acids during the first

two weeks of the experiments after which levels remained

more or less constant (week: x2�/9.27, df�/1, P�/0.002;

week2: x2�/8.58, df�/1, P�/0.003). There was also

Water treatment

25 ml 50 ml 75 ml 150 ml 300 ml

serutcnup gnideeF #

0

50

100

150

200

250

sgg

E #

0

5

10

15

20

Fig. 2. Feeding (black bars) and oviposition preference (whitebars) of C. milii in the five-way choice experiments. Error barsrepresent �/1 SE.

)syad( emit tne

mpoleveD

0

10

20

30

40

MalesFemales

Water treatment

25 ml 50 ml 75 ml 150 ml 300 ml

)gm( ssa

m lapuP

0.0

0.1

0.2

0.3

0.4

0.5MalesFemales

)%( lavivru

S

0

20

40

60

80

100

Nosurvival

Nosurvival

Fig. 3. Offspring performance of C. milii on the different plantwater treatments. Error bars represent �/1 SE.

Water treatment

25 ml 50 ml 75 ml 150 ml 300 ml

)%( ytilatro

m fo esuaC

0

20

40

60

80

100

Larval mortality due to self-pruningLarval mortality not due to self-pruningPupal mortality

Fig. 4. Causes of mortality for larvae on the different watertreatments.

OIKOS 108:2 (2005) 379

a significant overall decrease in the foliar amino acid

concentration with leaf age (week: x2�/9.39, df�/1,

P�/0.002; week2: x2�/8.66, df�/1, P�/0.003; leaf age:

x2�/6.33, df�/1, P�/0.012).

Plant stress intensity�/herbivore response

relationship

The number of feeding punctures (FP) (FP (%)�/

�/670.40�/8.19�/WC, F1,17�/32.97, PB/0.001, DF adj-

R2�/0.62) and eggs (EGG (%)�/�/631.49�/7.74�/WC,

F1,17�/14.75, P�/0.001, DF adj-R2�/0.40) were posi-

tively related to foliar water content (WC) in a simple

linear way. We observed no significant relationship with

foliar chlorophyll a or b.

The best model fit of the relationship between plant

biomass per pot and offspring survival, and foliar

water content and offspring survival, was a sigmoid

curve (Fig. 5). A slightly convex or a linear curve

could best describe the chlorophyll a�/offspring survival

and the chlorophyll b�/offspring survival relationships.

The relationship between plant water content and

offspring development time was linear, while the rela-

tionship between plant biomass per pot and develop-

ment time was clearly non-linear. No significant model

described the relationship between chlorophyll a or b

and development time.

Discussion

Our observations indicate that there is no universal

answer as to the relationship between water stress

intensity and herbivore response. Both linear and non-

linear (from convex to sigmoid) relationships were

observed for the range of water treatments applied in

our experiments. Yet, all relationships showed a mono-

tonic increase of grass miner preference and perfor-

mance with increasing plant vigour. Chromatomyia milii

also satisfied one condition of the plant vigour hypoth-

esis (Price 1991, Preszler and Price 1995). It is namely an

endophagous insect with a tight link between adult

oviposition and offspring feeding site. We therefore

conclude that our study supports the plant vigour

hypothesis in all its aspects.

We found no evidence for an increased performance of

C. milii at moderate or severe stress levels, although

there was an increase of N-containing nutrients in the 25

and the 50-ml treatment (White 1969, 1974, 1984,

Mattson and Haack 1987a). According to White’s

original formulation of the plant stress hypothesis

(White 1984), senescence feeders �/ species that are

adapted to feed on senescing plant tissue �/ are

more likely to benefit from stressed plants than flush

feeders like C. milii (White 1993). Our findings areTab

le4

.F

oli

ar

pro

tein

an

da

min

oa

cid

con

cen

trat

ion

sfr

om

wee

k1

0ti

ll1

8fo

rth

ed

iffe

ren

tw

ate

rtr

eatm

ents

.E

rro

rb

ars

rep

rese

nt9

/1S

E.

Pro

tein

s(m

g/g

dry

ma

ss)

Am

ino

aci

ds

(mg

/gd

rym

ass

)

25

-ml

50

-ml

75

-ml

15

0-m

l3

00

-ml

25

-ml

50

-ml

75

-ml

15

0-m

l3

00

-ml

Wee

k1

01

4.3

09

/1.2

01

2.9

79

/1.2

91

4.1

79

/1.2

41

6.5

59

/1.7

31

3.7

09

/1.0

22

1.9

99

/1.5

51

9.4

59

/1.6

62

3.5

99

/1.4

62

1.3

69

/1.9

51

9.7

89

/2.3

51

11

6.2

99

/2.1

21

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52

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380 OIKOS 108:2 (2005)

therefore in line with most predictions of the plant stress

hypothesis (White 1984). Yet, many plant-insect re-

searchers do not take the senescence�/flush feeder

condition as formulated by White (1984) into account.

Consequently, they would have concluded that the

reduced performance on, and avoidance of, water

stressed plants in C. milii is not in support of the plant

stress hypothesis.

We like to stress that the observation of a monotonic

relationship between plant stress intensity and herbi-

vore response should not lead to the wrong conclusion

that a continuum view of plant stress is not essential

(Price 1991). Indeed, we were only able to exclude the

possibility that moderate plant stress increases the

performance of C. milii by studying the effects of plant

stress on herbivores at a broad range of stress intensities.

Mopper and Whitham (1992) hypothesised that only

long term sustained moderate plant stress (extended

stress that prevails when insects are both active and

inactive) can result in an increased herbivore response

at moderate stress levels. They argued that tempo-

rary simultaneous stress as applied in our experiments

(i.e. stress applied concurrent with insect feeding and

oviposition) has detrimental effects on insects with a

Fig. 5. Relationships betweenplant stress intensity andherbivore response. Theregression equations for thedifferent relationships are: plantbiomass (BM) �/ offspringsurvival (S): S�/88.77/(1�/

exp(�/(BM�/0.11)/0.019)),F2, 17�/35.87, PB/0.001, DFadj-R2�/0.77; foliar watercontent (WC) �/ offspringsurvival: S�/97.13/(1�/exp(�/(WC�/84.16)/0.92)),F2, 17�/101.62, PB/0.001,DF adj-R2�/0.91; Chlorophylla (CHLA) �/ offspringsurvival (convex�/solid line):S�/220.53�/(�/25646.71/CHLA2), F1, 18�/13.30, P�/

0.002, DF adj-R2�/0.36;chlorophyll a �/ offspringsurvival (linear�/dashed line):S�/�/264.13�/25.42�/CHLA),F1, 18�/12.58, P�/0.002, DFadj-R2�/0.34; chlorophyll b(CHLB) �/ offspring survival(convex�/solid line): S�/

297.18�/(�/1078.74/CHLB),F1, 18�/16.68, PB/0.001, DFadj-R2�/0.42; chlorophyllb �/ offspring survival (linear�/

dashed line): S�/�/192.94�/

55.04�/CHLB, F1, 18�/16.28,PB/0.001, DF adj-R2�/0.41;Plant biomass �/ developmenttime (DT): DT�/29.83�/

(0.059/BM2), F1, 12�/12.03,P�/0.005, DF adj-R2�/0.41;plant water content �/ offspringdevelopment time: DT�/

198.75�/(�/1.94�/WC), F1, 12�/

9.60, PB/0.01, DF adj-R2�/0.34.

Water (%)

76 78 80 82 84 86 88

)%( lavivru

S

0

20

40

60

80

100

Water (%)

76 78 80 82 84 86 88

)d( emit tne

mpoleveD

25

30

35

40

45

Chlorophyll b (µmol/g dry mass)

3.5 4.0 4.5 5.0 5.5

)%( lavivru

S

0

20

40

60

80

100

Chlorophyll a (µmol/g dry mass)

10 11 12 13 14 15 16

)%( lavivru

S

0

20

40

60

80

100

Biomass (g dry mass)

0.0 0.1 0.2 0.3 0.4 0.5 0.6

)%( lavivru

S

0

20

40

60

80

100

Biomass (g dry mass)

0.0 0.1 0.2 0.3 0.4 0.5 0.6

)d( emit tne

mpoleveD

25

30

35

40

45

OIKOS 108:2 (2005) 381

sigmoid relationship between stress intensity and herbi-

vore response as a result. Our observations support the

idea of Mopper and Whitham (1992) that the plant

stress hypothesis does not apply to insects under

simultaneous stress. Moreover, although many relation-

ships between stress intensity and herbivore response did

not follow a sigmoid pattern, the sigmoid relationships

between foliar water content and offspring survival, and

plant biomass and offspring survival are in accordance

with the simultaneous stress model of Mopper and

Whitham (1992).

Despite a significant increase of foliar protein, larval

performance was dramatically low (0%) in the 25 and the

50-ml treatments. Moreover, sixty two percent of the

larvae died in the 75-ml treatment. This high larval

mortality is mainly the result of self-pruning by the

H. lanatus plants. Indeed, 100% of the offspring

mortality in the 25 and 50-ml treatments and 94% in

the 75-ml treatments was caused by self-pruning. The

low offspring mortality in the 150 and the 300-ml

treatments was due to other, unknown causes of larval

and pupal mortality. Senescence of leaves and shoots are

an important, widespread resistance mechanism of water

stressed plants (Sanderson et al. 1997). We observed that

older H. lanatus leaves, which contain the larvae,

subsequently wilt and die. High larval mortality follow-

ing early shoot (Craig et al. 1989) or leaf abscission

(Preszler and Price 1995) on water stressed was also

observed in two other studies on endophagous insects.

We are not aware of examples on exophagous insects,

which is probably a consequence of the fact that

these insects can move freely among plant modules.

All together, these observations suggest that leaf or stem

senescence and resulting offspring mortality may be a

widespread cause for the reduced performance of

endophagous insects on water stressed plants.

The high offspring mortality on water stressed plants

seems to be a likely explanation for the fact that C. milii

avoided the plants of the 25, 50, 75-ml treatments. Yet,

there are still several other mutually non-exclusive

explanations. First, loss of leaf turgor may decrease the

interveinal distance. As a consequence, adult C. milii

females will have great difficulties to insert their ovipo-

sitor between two adjoining veins (Scheirs et al. 2001a).

Secondly, a loss of leaf turgor may decrease the amount

of plant saps that exude from feeding punctures made by

the females. Water stressed plants may therefore be a

poor resource for adults and larvae.

Plant water stress may result from a deficit or an

excess of water (Levitt 1972, Heinrichs 1988, p. 14). Yet,

only few studies have addressed the effect of an excess of

water on plant stress�/insect interactions (Heinrichs

1988). We assumed that the 300-ml treatment would

result in plant stress due to an excess of water, but there

were no differences in plant growth and foliar water

concentrations between the 300-ml and the 150-ml

treatments. However, the 300-ml plants turned yellow

at the end of the experiment (week 17 and 18) as

indicated by the decreased chlorophyll a and b concen-

trations. Chlorosis is a common effect of an excess of

water (Heinrichs 1988). Also protein levels started to

decrease in the 300-ml plants at week 17 and 18. These

observations indicate that stress effects were only

becoming apparent at the end of the experiment. Second,

we observed no effect of the 300-ml treatment on

herbivore preference and performance, which is logical

because the 300-ml plants were not stressed at the

moment that preference (week 13) and larval perfor-

mance (most larvae pupated before week 17) was

established. Nevertheless, more experiments are clearly

needed in order to investigate the influence of plants

stressed by an excess of water on herbivores. We did not

flood the plants because this would make them inacces-

sible to the herbivores, but supplied water to levels equal

to ground surface level. Flooding plants just before the

start of the trials with the herbivores may yield higher

stress levels and may be a better alternative to our

experiments.

Drought stress has been implicated as a causal factor

in outbreaks of phytophagous arthropods both in forest

and agricultural systems (White 1969, Mattson and

Haack 1987a,b). Our study gives no support to

this hypothesis. From our results, it is to be expected

that C. milii densities under natural conditions will be

the highest on vigorously growing plants. Two prelimin-

ary studies support this prediction. First, Rotheray

(1987) observed very high densities of C. milii on Holcus

plants in shaded areas and showed that shaded plants

grew more vigorously than plants from open habitats.

Holcus leaves from shade plants were also preferred for

adult feeding and oviposition over leaves from open

habitats in two-way choice trials. Second, a field study of

De Bruyn (1990) showed that larger leaves, or more

vigorously growing plant modules (Price 1991), had a

relatively higher chance of being mined by C. milii

larvae.

Several researchers believe that we may resolve the

plant stress paradox by comparing the plant stress

intensity�/herbivore response relationship in a wide

range of study systems (Larsson 1989, Waring and

Cobb 1992, Watt 1994, Koricheva et al. 1998). Yet, our

study shows that the shape of the relationship, and the

amount of variation explained by the models, depends

both on the type of plant stress intensity measure and

the herbivore fitness measure considered in the model.

Comparable observations were made in other studies.

For instance, English-Loeb (1989) studied the response

of the two-spotted spider mite Tetranychus urticae Koch

to water stressed plants. Water potential was related in a

complex non-linear way to population growth. He

observed a decline in mite abundance at slight stress,

an increase at moderate stress to densities comparable to

382 OIKOS 108:2 (2005)

unstressed conditions and a steep decline in mite

abundance at severe stress levels. The relationship

between water potential and offspring development

time of the two-spotted spider mite ranged from linear

to slightly convex. Second, Bjorkman (1998) studied the

response of the galling aphid Sacchiphantes abietis L. to

various stress types. He observed a linear relationship

between plant growth and gall size. Plant growth index

was however related to gall occurrence in a non-linear

way. An important implication of these observations is

that among studies comparisons of the plant stress

intensity�/herbivore response relationship should be

made with the same variables in the relationship.

Our analysis of the various plant stress intensity

measures and host plant chemistry showed that plants

acclimate to the stressed conditions. For instance,

chlorophyll a concentrations in the 25, 50 and 75-ml

treatments at the end of the experiment are restored to

the levels observed at the start of the experiment.

Second, water stress in the 25-ml treatment decreases

towards the end of the experiment as the difference

between noon and predawn water disappeared at

week 17 and 18. Third, foliar protein concentrations

differed between the two lower water treatments (25

and the 50-ml treatments) and the other treatments

at week 15 and 16, but they were similar at week 18

among the 25, 50, 75 and the 150-ml treatments.

As already mentioned above, self-pruning of old

leaves in combination with the reduction in leaf area of

newly developed leaves is probably an important water

stress resistance mechanism by which H. lanatus accli-

mated to the stress conditions. Both processes contribute

to water saving, but also allow the reallocation of

nutrients stored in older leaves (Chaves et al. 2003).

The reallocation of foliar proteins from old leaves that

died due to self-pruning to younger, smaller leaves may

be a complementary explanation to the younger leaf age

structure in water stressed plants for the enhanced foliar

protein concentrations in the 25 and the 50-ml treat-

ments. This is suggested by the fact that the younger age

structure of the water-stressed plants alone could not

explain the higher foliar protein concentrations in the 25

and the 50-ml treatments, although younger leaves

contained more proteins than older leaves. We found

no evidence that plants increased relative root mass in

order to acclimate to drought stress conditions. Finally,

an important consequence of plant acclimation are the

complex kinetics of the various plant characteristics that

were recorded during this study. This observation

reinforces the necessity (English-Loeb et al. 1997) to

monitor the plant response throughout the stress experi-

ment instead of taking one sampling point during the

course or at the end of the experiment as done by most

plant stress studies.

Acknowledgements �/ We thank Peggy Beers, Sofie Thys, InezVandevyvere and Natalie Van Houtte for technical assistance.

The comments of Stig Larsson and Peter Price greatly improvedthe manuscript. J.S. is a postdoctoral researcher of the FWO-Vlaanderen.

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