© 2005 The Netherlands Entomological Society

Entomologia Experimentalis et Applicata

115

: 179–186, 2005

179

Blackwell Publishing, Ltd.

Influence of leaf trichome density on the efficiency

of two polyphagous insect predators

Christer Björkman

1,

* & Karin Ahrné

2

1

Department of Entomology, Swedish University of Agricultural Sciences, PO Box 7044, SE-750 07 Uppsala, Sweden;

2

Department of Ecology and Plant Production Sciences, Swedish University of Agricultural Sciences, PO Box 7043, SE-750 07 Uppsala, Sweden

Accepted: 7 February 2005

Key words:

induced response, leaf hairiness, natural enemy behaviour, tritrophic level interactions,

Anthocoris nemorum

,

Ortothylus marginalis

,

Phratora vulgatissima

,

Salix cinerea

, Salicaceae, Coleoptera, Chrysomelidae, Heteroptera, Anthocoridae, Miridae

Abstract

Plant characteristics, such as leaf structure or hairiness, are important for the movement and attach-ment of insects. It has been suggested that increased trichome density on new

Salix cinerea L.

(Sali-caceae) leaves, produced after grazing by the willow leaf beetle

Phratora vulgatissima L.

(Coleoptera:Chrysomelidae), function as an inducible defence against the beetle and especially its larvae. Here westudied whether and how two of the main natural enemies of

P. vulgatissima

, viz.,

Anthocoris nemorum L.

(Heteroptera: Anthocoridae) and

Ortothylus marginalis L.

(Heteroptera: Miridae), were influencedby trichome density on

S. cinerea

leaves. The effect of trichome density on these two predators wasstudied on plants with different trichome densities, comparing natural enemy efficiency, measuredas number of

P. vulgatissima

eggs consumed or larvae missing and/or killed. To obtain differenttrichome densities, cuttings of several different clones of

S. cinerea

were used. In the experiment usingeggs as prey, an increase in trichome density was, in addition, induced through leaf beetle defoliationon half of the plants of each willow clone. Furthermore, a field study was performed to investigatewhether trichome density was correlated with natural enemy abundance. The results indicate thatneither the efficiency of these two natural enemies in the greenhouse, nor their abundance in the fieldwas influenced by trichome density. A well-known behavioural difference between the two predatorspecies could probably account for the higher disappearance of larvae after exposure to the moreactive predator. These findings are relevant for the development of pest management programs, notleast because the enemies are polyphagous predators. It is concluded that an induced increase in leafhairiness in willows in response to leaf beetle grazing could be a plant resistance trait worthy of further

study in this system, because no negative effects on the main natural enemies were observed.

Introduction

The surface of plants forms a framework within whichmany insects live. A plant surface covered with hair mightprevent insects from moving, whereas a slippery surfacemight make the insects lose their grip and fall off theplant. Insects are forced to adapt either behaviourallyor morphologically to overcome the problems posed byvarious plant surfaces (Southwood, 1986).

Several studies have shown that plant characteristics,such as leaf structure and trichome density, influence thenatural enemies of herbivorous insects (Hare, 2002). For

example, the walking speed of

Encarsia formosa

females

,

a parasitoid of whitefly, is three times greater on hairlesscucumber than on hairy varieties (Boethel & Eikenbary,1986). Walking speeds, rates of turning, and the flightpropensity of

Trichogramma exiguum

, an egg parasitoid of

Heliothis zea

, are influenced by differences in leaf structure,particularly trichome form and density (Keller, 1987).Kauffman & Kennedy (1989) found a significantly negativerelationship between the percentage parasitism of

Heliothis

spp. by

Trichogramma

spp. and the density of glandulartrichomes. A high trichome density usually hinders theinsects, but sometimes it results in a more thorough search,and thus in a higher encounter rate with the prey (Lauenstein,1980; Shah, 1982). These examples highlight the importance

*

Correspondence: E-mail: christer.bjorkman@entom.slu.se

180

Björkman & Ahrné

of considering more than two trophic levels when tryingto clarify the net effect of any plant trait on interactionsbetween insects and plants (Price et al., 1980; Karieva &Sahakian, 1990; Björkman et al., 1997).

The surface structure of plants may change after herbivoredamage. For example, trichome density increases on

Salix cinerea

L. (Salicaceae) leaves produced after grazing byadults of the leaf beetle

Phratora vulgatissima

L. (Coleoptera:Chrysomelidae) (Dalin & Björkman, 2003). The larvae of

P. vulgatissima

are negatively affected by increased trichomedensity because their feeding is reduced and becomes moredispersed on leaves with an induced high trichome density.The plant response has therefore been suggested to be aninducible defence mechanism (Dalin & Björkman, 2003).However, it is probable that increases in trichome densitynot only affect the next generation of

P. vulgatissima

, butalso influence its natural enemies.

In this study, we aimed to determine whether leaf trichomedensity affects the natural enemies of

P. vulgatissima

. Wechose to study nymphs of two of the most common naturalenemies, the heteropterans

Anthocoris nemorum

L. (Antho-coridae) and

Orthotylus marginalis

Reuter (Miridae)(Björkman et al., 2003)

.

These two predator species differin searching behaviour:

A. nemorum

has been characterisedas a ‘run and eat’ predator due to its habit of only eating oneor two food items before moving on, and

O. marginalis

asa ‘find and stay’ predator due to its habit of consuming allfood items before searching for a new feeding site (Björk-man et al., 2003). These two species therefore could bedifferently influenced by variation in trichome density. Wehypothesized that if an insect as

A. nemorum

moves con-siderably it would be more influenced by surface structurethan an insect such as

O. marginalis

, which is mostlystationary

.

Furthermore, it has been shown earlier thatsmaller insects are more affected by plant structure thanlarger ones (Obrycki & Tauber, 1984; Yang, 2000). Wetherefore hypothesized that smaller nymphs (of bothspecies) should be more negatively affected by hightrichome densities than larger nymphs.

To test these hypotheses we conducted two greenhouseexperiments; one with leaf beetle eggs as prey and one withlarvae as prey. The reason for also using larvae was to see iftheir movements, which supposedly increase with increasingleaf trichome density (Dalin & Björkman, 2003), couldaffect the effectiveness of the two predator species. To obtainplants with different trichome densities we used the cuttingsof several different clones of

S. cinerea.

In the experimentwith eggs as prey, we also used plants with an induced increasein trichome density, obtained by letting adult leaf beetles feedon half of the plants of each willow clone. The hypothesisthat smaller nymphs would be more affected by leaf trichomesthan larger nymphs was only tested using eggs as prey.

An earlier study on glandular trichomes on potatoplants and their influence on eight coccinellid species,natural enemies of aphids, showed that the results from agreenhouse are an unreliable predictor of the effects in thefield (Obrycki & Tauber, 1984). Therefore, we performed afield survey to obtain some indication of whether naturalenemy abundance was related to trichome density inthis system. It was assumed that natural enemy abundancewould reflect any possible effects of willow leaf trichomedensity on enemy preference or performance.

Materials and methods

Study organisms

Salix cinerea

is common in the Nordic countries, and isfound on moist, moderately nutritious soils along rivers,ditches, and pastures, as well as in forest glades. It growsinto bushes that are 1–4 m high.

Salix cinerea

is recognisedby its densely hairy current-year shoots and leaves that areinversely egg-shaped and covered with hairs (trichomes)(Mossberg et al., 1997; Dalin & Björkman, 2003). The hairson

S. cinerea

leaves are non-glandular and for simplicitywe have chosen to refer to them as trichomes (cf. Jeffree,1986).

Phratora vulgatissima

is commonly found on differentspecies of

Salix

, mainly on

S. aurita

and

S. cinerea

, and isalso found in short-rotation coppice crops such as

S. viminalis.

The insect is univoltine in Sweden and overwinters as anadult. The leaf beetle starts to feed in early May and lays itseggs soon thereafter, between late May and mid-June. Theeggs hatch after 1–2 weeks. The leaf beetle, and especiallyits larvae, may seriously damage

Salix

plants (Björkmanet al., 2000).

Anthocoris nemorum

is found on various species of

Salix

, the stinging nettle

Urtica dioica

L. (Urticaceae), andorchard trees. It is considered to be one of the most impor-tant predatory bugs in fruit orchards (Solomon, 1982).The species is known to switch between plant speciesduring its life cycle (Dwumfour, 1992). It has one generationper year, and overwinters as an adult. The first nymphscan be found in late May and adults are present until earlyOctober (Austreng & Sömme, 1980).

Ortothylus marginalis

is distributed throughout Europe, and is found on anumber of deciduous trees such as apple and different

Salix

species.

Ortothylus marginalis

also has one gener-ation per year (Austreng & Sömme, 1980). It overwintersas an egg and the first nymphs are found in early May, 1–2 weeks before

A. nemorum

nymphs and adults are present,until late August. Both

O. marginalis

and

A. nemorum

aregeneralist predators that feed on aphids, mites, mirids, andpsyllids, and they are important natural enemies of

P. vulgatissima

eggs and larvae (Dwumfour, 1992; Wheeler,

Influence of leaf trichomes on insect predators

181

2001; Björkman et al., 2003). Of these,

O. marginalis

atleast seems to play a significant role in the populationdynamics of

P. vulgatissima

(Björkman et al., 2004a).

Willow clones

Cuttings of

S. cinerea

used in the predation experimentswere collected from 24 and 20 different field locations inthe province of Uppland, respectively, for the eggs as preyand the larvae as prey experiments. The locations weresituated at least 3 km apart to minimise the risk of cuttingsbeing of the same clone. To further increase the probabilityof picking clones with different trichome densities, approxi-mately half of the locations were situated in open-fieldareas and the other half were situated in forest areas. Thehabitat origin was only tested for in the experiment witheggs as prey. Four and three cuttings from each clone wereplanted on 9 April and 29 April in the experimentsusing eggs and larvae as prey, respectively, and grown in agreenhouse. The plants were watered daily with watercontaining fertiliser (100 mg N l

−

1

).

Handling of natural enemies

Individuals of

A. nemorum

and

O. marginalis

were collectedfrom willow plantations near Uppsala 1 or 2 days beforeuse. They were kept in a refrigerator and starved for24 h before being put on plants to ensure they were equallyeager to feed. The 24 h starving period was chosen becausepreviously published experiments with

A. nemorum

haveshown that this time period is enough to make themhungry without making them inactive (Lauenstein, 1980).Because

O. marginalis

nymphs occur somewhat earlier thannymphs of

A. nemorum

, we did the experiment with eggs asprey and

O. marginalis

before we did those with

A. nemorum

.In the year the experiment with larvae was performed,nymphs of similar sizes were available at the same time.

Induction of increased trichome density

After 25 days, all the plants in the experiment with eggs asprey were placed in plastic cylinders (height 70 cm; 25 cmin diameter). To induce an increase in leaf trichomedensity, four leaf beetles (

P. vulgatissima

) were transferredto two randomly chosen plants from each clone. After9 days, the beetles were removed and the plants were left togrow for another 25 days. A detailed description of thismethod can be found in Dalin & Björkman (2003).

Measurement of trichome density and leaf areas

After the experiments, we counted the number of trichomeson the leaves of each plant. Trichome density was countedunder a stereomicroscope as the number of hairs crossinga 2 mm line. In the experiment with eggs, the trichomedensity was counted on five leaves at two levels on all

plants. The first level was the base, containing the leavesthat were present during the induction period. The secondlevel was the area where the induction should appear. Inthe experiment with larvae as prey, trichome density wascounted on five leaves from the middle of each plant. Aftera log10 transformation to reach normality, data wereanalysed with a two-sample t-test.

The leaf areas of all leaves in the experimental area weremeasured for two reasons: first, to estimate the area thatindividual predators could search when trying to find prey;and second, to analyse whether a possible increase intrichome density, on the beetle-defoliated plants, was dueto a decrease in leaf area or to an actual increase in absolutetrichome number. Leaf areas were measured with an imageanalysis program (cf. Dalin & Björkman, 2003). Data wereanalysed with Mann–Whitney U-tests.

Influence of trichome density on natural enemy efficiency

Two experiments were done: one with

P. vulgatissima

eggsas prey and the other with

P. vulgatissima

larvae as prey.Both experiments were carried out in a greenhouse, thewindows of which faced north to minimise temperaturefluctuations. The remaining variation in temperature wastaken into account in the ANCOVA by using the date ofexperiment as a covariate. Experiments with eggs as preyand

O. marginalis

were done between 5 and 15 June, andthose with

A. nemorum

were conducted between 16 and27 June in 2002. Experiments with larvae as prey andboth predators were conducted between 14 and 19 June in2004. Because the greenhouse was too small to study allreplicates simultaneously, we divided them into groups. Ifone or more insects escaped, the whole replicate was redone.

Experiments with eggs as prey.

To be able to measure whetherand how

A. nemorum

and

O. marginalis

were affected bytrichome density, we placed two clutches of

P. vulgatissima

eggs on separate leaves. The eggs were placed in an areawhere the induction would supposedly have appearedand in a corresponding position on control plants on thedamaged plants. Then we put the natural enemies onthe plants to let them search for and feed on the eggs. Thenumber of eggs eaten was used as a measurement ofthe insects’ efficiency on the different plants. To make theinsects stay in the desired area (with induced trichomedensity) we enclosed the experimental area using twoplastic cups turned with the inside towards each other. Theplastic cups were covered with Fluon (Fluortek TP50;Fluortek AB Kista, Sweden) which has a slippery surface, toprevent the insects from escaping. To compensate for anyvariations in leaf area among saplings, and to obtain anequal leaf area in all replicates, we adjusted the number ofenclosed leaves between the two cups. Mostly, about eight

182

Björkman & Ahrné

leaves (range 7–14 leaves) were enclosed between the cups.This was done on the beetle-defoliated plants as well as onthe control plants.

One individual predatory nymph was placed on eachplant. We used half of the

S. cinerea

clones for smallnymphs (2nd

−

3rd instar nymphs of

O. marginalis

and3rd

−

4th instar nymphs of

A. nemorum

) and half of theclones for large nymphs (5th instar nymphs of both species).Thus, 12 clones were used each for small and large nymphs.One control plant with

O. marginalis

(or

A. nemorum

)plus one beetle-defoliated plant with

O. marginalis

(or

A. nemorum

) was considered as one replicate. The fourplants originating from one clone were used as follows:(a) control plant with

O. marginalis

(small or large), (b)beetle-defoliated plant with

O. marginalis

(small or large),(c) control plant with

A. nemorum

(small or large), and(d) beetle-defoliated plant with

A. nemorum

(small or large).The nymphs were left on the plants for 2 days and thenremoved before the remaining eggs were counted.

The leaf beetle eggs used in the experiment were eithercollected from cages with

P. vulgatissima

in a greenhouseor from willow plantations. Before use, the number of eggsin each clutch was counted under a stereomicroscope andparasitised eggs were removed.

Experiments with larvae as prey.

A similar experimental set-up as that described above was used in the experimentwhere newly hatched larvae (younger than 24 h) were usedas prey. The only differences with the experiment with eggsas prey were that: (a) no plants with an induced increase intrichome density were used, (b) the number of leaveswithin the experimental area was fixed at seven, (c) theduration of the experiments was 24 h, and (d) only one sizeof predator was used, i.e., instar 4–5. Two groups with fiveleaf beetle larvae were placed on leaf nos. 2 and 6 (countedfrom the top), 2 h before placing the natural enemies onthe plants. Numbers of (A) dead and (B) missing + deadlarvae were used as measures of predator efficiency. Deadlarvae were easy to detect because the predators havesucking mouthparts and therefore leave corpses. In eachreplicate, three experimental shoots with 10 larvae wereused; one served as a control with no predators and theother two had one individual predator of each of the twospecies. The leaf beetle larvae used in the experimenthatched in the laboratory from eggs collected in the field,i.e., from nearby willow plantations.

Field study

To get some indication of whether natural enemy abundancewas related to trichome density, we carried out a field survey.We hypothesized that if the natural enemy specieswere affected by trichome density, their preference for, or

performance on, plants with different trichome densitiescould be influenced. This would, in turn, result in differencesin natural enemy densities on different clones. We revisited19 of the 24

S. cinerea

localities used in the experiment witheggs as prey and estimated the abundance of

O. marginalis

and

A. nemorum

. Of the 19 locations, 10 were situated inopen-field areas and the other nine were situated in forestareas. Twenty-five leaves were collected from each locality.The leaves were fully developed and were not damagedby beetles or any other defoliators. In the laboratory,we counted the number of trichomes on the leaves andmeasured leaf areas in the same way as before. Insects werecollected in a white plastic tub (23

×

35 cm and 12 cm deep).Forty branches on each plant were shaken over the tub andinsects sitting on the branches fell into the tub. We countedthe number of individuals of the two ‘enemy’ species.

Statistical analyses

When analysing the data we used parametric tests wherepossible, otherwise we used non-parametric tests. Data fromthe greenhouse experiments were analysed in ANCOVAs(GLM; Minitab). In the experiment with eggs as prey, fixedfactors were natural enemy species, instar, treatment, andhabitat, and covariates were trichome density, total leafarea for searching, and date. In the experiment withlarvae as prey, the fixed factor was natural enemy species,and covariates were trichome density, total leaf area forsearching, and date. In the experiment with larvae asprey, the control treatment was omitted from the statisticalanalyses because fewer than 0.25 larvae, on average,disappeared from the control plants. In the field study,differences between habitat types with respect to enemyabundance and leaf trichome density were tested with aMann–Whitney U-test and t-test, respectively.

Results

Induction of increased trichome density

Beetle-defoliated plants produced new leaves with a trichomedensity 92.5% higher than that of control plants (t = 5.94,P<0.001; n = 24). Trichome density on leaves at thebase did not differ between treatments (t = 0.46, P>0.5;n = 24). The increase in trichome density on previouslybeetle defoliated plants was not due to a decrease in leafarea on defoliated plants. Leaf areas did not differ betweentreatments, neither before nor after defoliation (base leaves:W = 504, P = 0.085; top leaves: W = 576, P = 0.82; n = 24).

Natural enemy efficiency

There was no evidence that trichome density influencedefficiency, measured as the number of eggs consumed orlarvae missing and/or dead, of either of the two natural enemy

Influence of leaf trichomes on insect predators

183

species (Figures 1 and 2; Tables 1 and 2). In the experimentwith eggs as prey, the only significant difference was thatlate instars, i.e., big nymphs, ate more eggs than earlyinstars, i.e., small nymphs (Table 1). In the experiment withlarvae as prey, the more active

A. nemorum

had a significantlystronger negative effect on the prey than

O. marginalis

(mean

±

SEM: 6.6

±

0.8 vs. 3.9

±

0.7) when both dead andmissing larvae were added together (Table 2). No such effectwas observed when only dead larvae were considered.None of the covariates explained any significant amountof variation in either the egg or the larval experiment. Inthe experiment with eggs as prey, no interactions at anylevel were significant. Similarly, there was no difference intotal leaf area in the larval experiment between plants offeredto the two enemy species (W = 389, P = 0.81; n = 19 and20), nor in leaf trichome density (t = 0.32, P = 0.75; n = 20and 20).

Field study

The results from the field study showed no evidence thatnatural enemy abundance correlated with leaf trichomedensity (Figure 3).

Ortothylus marginalis

was more abundanton open-field (of) plants than in the forest (f) plants (W =128, P = 0.026; n

of

= 10, n

f

= 9). There was no difference intrichome density between leaves picked from open-fieldplants and leaves picked from plants in the forest (t = 0.65,P = 0.53; n

of

= 10, n

f

= 9).

Discussion

The present study confirms the results of a previous studyshowing that Salix cinerea exposed to grazing by adults ofthe leaf beetle, P. vulgatissima, produces new leaves withan increased trichome density (Dalin & Björkman, 2003).It also confirms that the response is rapid, because the

Figure 1 Numbers of leaf beetle (Phratora vulgatissima) eggs consumed by two polyphagous insect predators in relation to density of leaf trichomes on Salix cinerea over 48 h. (A) Anthocoris nemorum nymphs in instar 3–4; (B) A. nemorum nymphs in instar 5; (C) Orthotylus marginalis nymphs in instar 2–3; (D) O. marginalis in instar 4–5. Two types of willow saplings were used: controls (�) and those exposed to feeding by adult leaf beetles (�) on which we observed a significantly induced increase in leaf trichome density. For statistics see Table 1.

184 Björkman & Ahrné

induction is expressed in new leaves within 25 days. Inaddition, this study shows that differences in behaviourbetween two predator species may affect their efficiency asnatural enemies: after exposure to the more active predatorA. nemorum, more prey larvae were missing than afterexposure to O. marginalis. However, we found no effect ofleaf trichome density on the efficiency of the predators.That is, our results gave no support to two of the hypo-theses we set out to test, viz.: (1) that more active predators(A. nemorum) should be more affected by leaf trichomedensity than less active predators (O. marginalis), and (2)that smaller predator individuals should be more affectedthan larger predators.

From our observations it appeared that trichomesprovided no obstacles to the predatory insects. However,in this study we did not examine whether their actual

Source d.f. Ms F P

Natural enemy species 1 112.5 2.10 0.15Instar 1 1673.3 31.22 <0.001Plant treatment 1 17.87 0.33 0.57Habitat 1 8.90 0.17 0.68Trichome density 1 4.85 0.09 0.76Total leaf area 1 69.11 1.29 0.26Date 1 7.42 0.15 0.70Natural enemy species × instar 1 1.73 0.03 0.86Natural enemy species × plant treatment 1 62.03 1.16 0.29Natural enemy species × habitat 1 37.95 0.71 0.40Instar × plant treatment 1 1.13 0.02 0.88Instar × habitat 1 15.86 0.30 0.59Plant treatment × habitat 1 1.11 0.02 0.89Error 70 53.60

Table 1 ANCOVA results of the effect of factors affecting the number of leaf beetle (Phratora vulgatissima) eggs consumed by two species of polyphagous insect predators Anthocoris nemorum and Orthotylus marginalis

Table 2 ANCOVA results of the effect of factors affecting the number of leaf beetle (Phratora vulgatissima) larvae: (A) dead, or (B) dead + missing, when exposed to two species of polyphagous insect predators Anthocoris nemorum and Orthotylus marginalis

Source d.f. Ms F P

(A) Dead larvaeNatural enemy species 1 5.91 1.42 0.24Trichome density 1 0.63 0.15 0.70Total leaf area 1 0.37 0.09 0.77Date 1 1.46 0.35 0.56Error 34 4.15

(B) Dead + missing larvaeNatural enemy species 1 59.35 4.72 <0.05Trichome density 1 6.59 0.52 0.47Total leaf area 1 0.06 0.00 0.95Date 1 0.74 0.06 0.81Error 34 12.56

Figure 2 Number of leaf beetle (Phratora vulgatissima) larvae that were: (A) dead; or (B) dead + missing when exposed to two polyphagous insect predators (Anthocoris nemorum and Orthotylus marginalis) on Salix cinerea saplings with varying in leaf trichome density during 24 h. For statistics see Table 2.

Influence of leaf trichomes on insect predators 185

behaviour differed depending on plant trichome density.It is possible that predatory insects move more slowly onplants with a high trichome density, but this is compensatedby a more thorough search, thus making their encounteringrate with their prey the same as on plants with a lowtrichome density. To test this, a behavioural study will beneeded where factors such as walking speeds and rates ofturning can be measured.

If larvae were to move more on leaves with highertrichome densities, as was found in a previous study (Dalin& Björkman, 2003), we would expect them to be morevulnerable to predators (cf. Boethel & Eikenbary, 1986).However, we found no evidence for this hypothesisedindirect effect of leaf hairiness on predator efficiencythrough increased larval movements on leaves with moretrichomes.

For practical reasons we chose not to use induced plantsin the experiment where larvae were used as prey. Oneconsequence of this was a reduced range of leaf trichomedensities. This could, in turn, have affected the possibilitiesof detecting any effects of leaf trichome density on theefficiency of the predators. In the experiment where eggswere used as prey, we were able to cover a larger range ofleaf trichome densities. However, in this experiment therewas no indication of any significant effect of leaf trichomedensity on the efficiency of the predators. One limitationwas that the number of observations at the upper part ofthe range was restricted. Thus, we cannot exclude thepossibility that leaf hairiness may have an effect at veryhigh trichome densities.

In the field study, the range of leaf trichome densities wasmore evenly represented. Even so, there was no indicationof any relationship between the density and the relativeabundance of the two predator species. Based on these

data, it is not possible to exclude that leaf trichome densityaffects the efficiency of one or both of the two predatorspecies, unless we assume that predator abundance insome way reflects the effect of leaf hairiness on predatorefficiency. However, the fact that O. marginalis was moreabundant in one habitat (open farmland) than another(forest), despite the similarity in leaf trichome densityin the two habitats, suggests that other factors are moreimportant in determining the density of these two poly-phagous predators. Most probably, enemy abundance ismore important from a biological control perspective thanany effect of trichomes on their efficiency because theeffect of trichomes seems to be small.

One significant difference between the two predatorspecies was the difference in the number of larvae thatdisappeared, although this was not connected to leaftrichome density. More larvae were missing at the end ofthe experimental period after exposure to the more activepredator A. nemorum than after exposure to the less activeO. marginalis. We observed that A. nemorum predators ranaround much more than O. marginalis predators, and thatthey often attacked larvae without consuming their con-tent. The attacked larvae sometimes seemed immobilisedby the attack. Thus, it is likely that the missing larvae wereactually dead or dying. In other words, the more activeA. nemorum was a more efficient predator than the lessmobile O. marginalis. However, if one takes into account thatO. marginalis is much more abundant than A. nemorum(Björkman et al., 2003), the total effect of O. marginalis asa mortality factor in the field is most probably higher(Björkman et al., 2004b).

The difference in behaviour between the two predatorspecies suggests that together they may constitute a ‘dynamicduo’ in biological control. That is, their effect may be com-plementary. An additional factor supporting the idea thatthey are a good combination is that O. marginalis seems toprefer eggs over larvae, whereas A. nemorum shows nosuch preference (Björkman & Liman, 2005). Furthermore,we observed no signs of negative interactions between thetwo species, in either laboratory or field studies (Björkman& Liman, 2005). The combination studied is thus one of anincreasing number of examples which suggest that the com-bined effect of several generalist predators may result in anoperative biological control of insect pests (Symondsonet al., 2002).

In conclusion, the effect of a change in plant surfacecharacteristics on an herbivore may be different dependingon whether natural enemies are present or not. It is there-fore of great relevance to consider more than two trophiclevels when studying insect–plant interactions, especiallywhen the aim is a development of pest managementprograms (Hare, 1992, 2002). Here, the inducible increase

Figure 3 Number of individuals of two polyphagous insect predators (Anthocoris nemorum and Orthotylus marginalis) in stands of Salix cinerea varying in leaf trichome density. For statistics see text.

186 Björkman & Ahrné

in leaf trichome density in willows should also functionas a plant resistance mechanism when natural enemies arepresent. That is, the effect of the predators seemed to beadditive.

Acknowledgements

We are grateful to Karin Eklund, Pettri Palkki, and MarcusTörnkvist for invaluable assistance and advice. We thankPeter Dalin, Barbara Ekbom, and one anonymous reviewerfor helpful comments on the manuscript. Steve Scott-Robson corrected the English. The study was financiallysupported by The Swedish National Energy Administrationand The Carl Trygger Foundation.

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