Ecology Letters, (2012) doi: 10.1111/j.1461-0248.2011.01724.x LETTER Cross-kingdom interactions matter: fungal-mediated interactions structure an community on oak

Abstract Ayco J. M. Tack,1* Sofia Although phytophagous and plant pathogens frequently share the same host plant, interactions among Gripenberg1,2 and Tomas Roslin3 such phylogenetically distant taxa have received limited attention. Here, we place pathogens and insects in the context of a multitrophic-level community. Focusing on the invasive powdery mildew Erysiphe alphitoides and the insect community on oak (), we demonstrate that mildew–insect interactions may be mediated by both the host plant and by natural enemies, and that the trait-specific outcome of individual interactions can range from negative to positive. Moreover, mildew affects resource selection by insects, thereby modifying the distribution of a specialist herbivore at two spatial scales (within and among trees). Finally, a long-term survey suggests that species-specific responses to mildew scale up to generate landscape-level variation in the insect community structure. Overall, our results show that frequently overlooked cross-kingdom interactions may play a major role in structuring terrestrial plant-based communities.

Keywords Erysiphe alphitoides, indirect defence, indirect interactions, invasive species, Microsphaera alphitoides, multi-trophic interactions, plant–fungus–insect interactions, trait-mediated interactions, tripartite interactions

Ecology Letters (2012)

whether or not different species within the same community respond INTRODUCTION in a similar way to the very same host pathogen (Stout et al. 2006). Phytophagous insects and plant pathogens are among the most Such differential effects of pathogens on herbivore performance may speciose groups worldwide (Strong et al. 1984; Agrios 2005), and be either direct, plant-mediated, or mediated by natural enemies several studies suggest that they frequently interact within local (Cardoza et al. 2003; Turlings & Wa¨ckers 2003; Stout et al. 2006). communities (Hatcher 1995; Johnson et al. 2003; Rosta´s et al. 2003; In addition to the effects of pathogens on herbivore performance, Simon & Hilker 2003; Stout et al. 2006). Indeed, in one of the earliest the presence of plant pathogens may also modify the strength with articles demonstrating pathogen– interactions, Karban et al. which insect herbivores are attracted to individual plant units. For (1987) asked ecologists to switch focus from interactions among example, herbivores may prefer to oviposit on either infected or non- closely related species – which were traditionally presumed to interact infected leaves (Simon & Hilker 2005) or host-plants (Hatcher et al. the strongest (Darwin 1859; Gause 1934) – to interactions between 1994; Biere et al. 2002; Ro¨der et al. 2007). Likewise, the rate of phylogenetically distant species. Despite this wake-up call, Stout et al. emigration may differ between infected and non-infected plants (2006) concluded in a recent review that pathogen–plant–insect (Ro¨der et al. 2007). Importantly, if insect taxa respond differently to interactions still receive limited attention, and identified several gaps in the presence of a plant pathogen, fungal-mediated interactions may our current knowledge. Most notably, few studies have examined generate variation in local community structure (Moran & Schultz interactions between plant-feeding insects and plant pathogens in the 1998; Kluth et al. 2001). field or placed such interactions in a wider community context (Stout In this article, we use a series of detailed experiments to pinpoint et al. 2006). A key question is how cross-kingdom interactions scale up the diversity of direct and indirect interactions between three plant- to affect the realised community structure at various spatial scales feeding guilds: leaf miners, free-feeding insects and the oak powdery (Hatcher 1995; Stout et al. 2006). mildew Erysiphe alphitoides. We then use two large field experiments to Pathogens have the potential to modify the structure of phytoph- understand whether fungal-induced changes in resource selection agous insect communities by differentially affecting the performance affect the distribution of a specialist herbivore at two spatial scales: of local community members, and by modifying the interactions among leaves within a single tree, and among trees within a landscape. between them. However, while laboratory experiments have convinc- Finally, we use long-term spatially explicit observations to assess ingly demonstrated that the impact of host plant pathogens on whether or not herbivore-specific responses to mildew infection individual insect species can range from negative to positive (Rosta´s translate into predictable patterns in insect community structure et al. 2003; Stout et al. 2006), few studies have experimentally assessed across the landscape. For comparison, we conducted a set of similar

1Metapopulation Research Group, Department of Biosciences, University of 3Spatial Foodweb Ecology Group, Department of Agricultural Sciences, PO Box 27 Helsinki, PO Box 65 (Viikinkaari 1), FI-00014 University of Helsinki, Helsinki, Finland (Latokartanonkaari 5), FI-00014 University of Helsinki, Helsinki, Finland 2Section of Biodiversity and Environmental Research, Department of Biology, *Correspondence: E-mail: [email protected] University of Turku, Turku FI-20014, Finland

2012 Blackwell Publishing Ltd/CNRS 2 A. J. M. Tack, S. Gripenberg and T. Roslin Letter experiments and observations to assess the impact of early-season experiment was conducted). As the bags were introduced before herbivory by free-feeding insects on our focal insect taxa. mildew could be detected, we distributed the bags across multiple trees (n = 12) in the hope of including both infected and non-infected leaves. Once mildew infection developed to a scorable level, MATERIALS we selected a set of focal mines on healthy (n = 52 on four trees) and infected (n = 52 on eight trees) leaves. As the presence of Study site and taxa conspecifics on the same leaf may affect larval performance (Tack The pedunculate oak Quercus robur is the only oak species in Finland et al. 2009), we focused on mines occurring singly on the leaf. and sustains a large community of specialist insect herbivores. In our To measure mine growth, we recorded the outline of the focal study area, four plant-feeding guilds are prominent: a biotrophic plant mines approximately every 11 days (27 July, 7, 18 and 30 August, 10 pathogen, free-feeding insects, leaf miners and gallers (for details of and 20 ⁄ 21 September 2007) using a fine marker and a transparent the study system, see Appendix S1). acetate sheet. The mine outlines were subsequently scanned and the The plant pathogen E. alphitoides (Griffon & Maublanc) U. Braun & areas measured in cm2 using ImageJ (Rasband 1997–2011). To detect S. Takamatsu 2000 (formerly Microsphaera alphitoides) attacks the young differences in larval weight among individuals on healthy oak leaves in early spring (Edwards & Ayres 1982). High densities of and mildew-infected leaves, we dissected the larvae from the leaves on the species in Europe were first observed in 1907 (Hariot 1907), as the last measuring date (coinciding with the end of the growing followed by a rapid epidemic spread across Europe (Foe¨x 1941; season). Larvae were then oven-dried at 80 C for 24 h and weighed Mougou et al. 2008). This study system then has the potential to reveal to the nearest microgram. the impact of an invasive fungal pathogen on the insect community As specific leaf area, leaf consumption per unit area and growth (Desprez-Loustau et al. 2007). efficiency [measured as the ratio (larval weight ⁄ (leaf weight consumed Tens of species feeding on oak are multivoltine. Their first per unit area · final mine area))] may differ among mildew-infected generations typically attack the expanding oak leaves at around the and healthy leaves, we also assessed leaf mass. For this purpose, we same time as the infection is initiated by powdery mildew (Feeny punched a single leaf disc (Ø 10 mm) from within the leaf mine and 1970). Subsequent generations can be found in the summer and two leaf discs from outside the mined area. These leaf discs were autumn. Hence, while the first generation feeds on the leaves when subsequently dried and weighed to the nearest microgram. The weight mildew infection is absent or just established, later generations may of the two leaf discs outside the leaf mine were averaged before face heavily infected leaves. analysis. In addition to free-feeders, more than twenty specialist late-season Experiment 2: The effect of mildew infection on the larval growth rate of leaf miner and galler species feed on the oak trees (see Tack et al. 2010 Acronicta psi: To assess the effect of E. alphitoides on late-season free- for more details). While some of the species are found in the early feeding insect herbivores (arrows 1a and 1b in Fig 1A), we used fifth- spring, their peak abundance is later in the season than that of free- instar larvae of A. psi for a feeding trial. Larvae were reared until the feeding insects. fifth instar in enclosures and fed oak leaves randomly collected across multiple host individuals. Offspring of three wild-caught female were randomly assigned to two treatments implemented in Experiments and observational data individual 10-cm diameter Petri dishes. In a no-choice setting, larvae To explore the effects of powdery mildew on the insect herbivore were fed with either two mildew-infected (n = 20 larvae) or two non- community, we combined a series of experiments with long-term infected (n = 23 larvae) leaves. Leaves were replaced every 24 h to observational data. The methods are subdivided to those addressing prevent them from drying. During a period of 96 h, we measured direct and indirect local interactions (Fig. 1A), those addressing larval weight gain (Ôweight at the end of the experimentÕ ) Ôinitial resource selection by adult insects (Fig 1B), and those examining weightÕ) as well as leaf consumption (Ôleaf weight beforeÕ ) Ôleaf realised effects on local community structure (Fig. 1C). All experi- weight afterÕ). Feeding treatments were continued until all larvae had ments, the interactions addressed, and the responses examined are pupated, after which we weighed and sexed the pupae. summarised in Table 1. Experiment 3: The effect of early-season herbivory on survival and weight of T. ekebladella: To investigate how leaf damage inflicted by free- Interactions within the local community feeding herbivores in the early spring directly and indirectly affect the To identify the types of interactions occurring locally, not only within performance of larvae of the leaf miner T. ekebladella feeding later in the same community but among members of different guilds (Fig 1A), the season (arrow 3 in Fig 1A), we used field-collected lepidopteran we performed a series of experiments aimed at establishing direct and larvae to establish damage treatments. We then allowed moths of T. plant-mediated interactions among fungus and insects (Experiments 1 ekebladella to oviposit on a single branch tip on each of 25 trees and 2; arrows 1a,1b and 2 in Fig 1A), at quantifying the impact of a free- representing three treatments: (1) control treatment, i.e. a tree without feeding herbivore on a late-season leaf miner (Experiment 3; arrow 3 in any experimental damage, (2) direct treatment, i.e. a tree where a free- Fig 1A), and at assessing parasitoid-mediated interactions among the feeder had been introduced to the T. ekebladella-branch earlier in the fungus and the leaf miner (Experiment 4; arrow 4 in Fig 1A). season and (3) indirect treatment, i.e. a tree where a free-feeder had Experiment 1: The effect of mildew infection on food consumption and utilisation been introduced on a branch tip next to the T. ekebladella-branch by ekebladella: To investigate how mildew infection on host earlier in the season. In the direct and indirect treatment, realised leaves affects the performance of T. ekebladella (arrow 2 in Fig 1A), we damage levels were more than twice as severe on experimentally enclosed a single pair (#$) in a muslin bag tied around a branch damaged branches as on control branches (50% vs. 21% of leaves tip. Moths were hatched from leaf mines collected in the early spring damaged respectively). A single pair of T. ekebladella was introduced in from various locations on the island of Wattkast (where the a muslin bag tied around each branch tip (see Tack et al. 2009 for

2012 Blackwell Publishing Ltd/CNRS Letter Fungus–plant–insect interactions 3

(a)

(b)

(c)

Figure 1 Ecological interactions and patterns addressed in this study. Panels A and B show potential interactions among members of three dominant plant-feeding guilds: free- feeding insects (represented by a larva of Acronicta psi; left in A), the oak powdery mildew (Erysiphe alphitoides; centre in A and shown as powdery structure in B and C), and leaf miners (represented by a mine and larva of Tischeria ekebladella right in A and moths in B). Panel A represents local interactions, where arrows illustrate specific interactions explored in this study. The performance of the free-feeder may be directly affected by the consumption of mildew mycelium, spores, or infected epidermal cells (arrow 1a). Alternatively, the free-feeder is affected by changes in the host plant induced by the mildew (arrow 1b). Since the leaf miner T. ekebladella and the oak powdery mildew do not feed upon the same leaf tissues, their interaction is mediated by changes in the host plant (arrow 2). Early-season herbivory by free-feeding insects may induce changes in the host plant that affect the late-season leaf miner (arrow 3), whereas mildew-induced changes in the plant may affect the natural enemies of the leaf miner (arrow 4). Panel B illustrates effects of mildew on resource selection by insects, including oviposition preference within (arrow 5) and among trees (left side of panel B). Panel C shows how interactions depicted in A and B scale up to affect the structure of local insect communities on oak trees. Galls and leaf mines on the leaves represent some of the target taxa identified in Appendix S1. Drawing by Ika O¨ sterblad.

2012 Blackwell Publishing Ltd/CNRS 4 A. J. M. Tack, S. Gripenberg and T. Roslin Letter

Table 1 A summary of the experimental and observational materials used in this study and of the models fitted for analyses. For each model, we specify the response examined, the fixed and random effects included, and the link function applied. For identity links, we assumed normally distributed errors, for logit links binomially distributed errors, and for log links Poisson distributed errors. Independent continuous variables are identified in italics

Interaction addressed Responses Scale Interaction targeted (Fig 1) examined Fixed effects Random effects Link

Local Exp1: the effect of Arrow 2 Mine area* Treatment + Time Tree + Bag(Tree)à Identity interactions mildew infection on Larval weight Treatment Tree + Bag(Tree) Identity food consumption and Specific leaf area* Treatment Tree + Bag(Tree) Identity utilisation by Leaf consumption Treatment Tree + Bag(Tree) Identity Tischeria ekebladella per unit area* Growth efficiency* Treatment Tree + Bag(Tree) Identity Exp2: the effect of Arrows Larval mass gain* Treatment + Initial larval weight + Sex Family Identity mildew infection on 1a & 1b Pupal mass* Treatment + Initial larval weight + Sex Family Identity the larval growth rate Leaf consumption Treatment + Initial larval weight + Sex Family Identity of Acronicta psi Growth efficiency Treatment + Initial larval weight + Sex Family Identity Exp3: the effect of Arrow 3 Larval survival Treatment + Conspecific density§ + Mildew Tree + Shoot(Tree) Logit early-season herbivory (0 ⁄ 1)§ + Leaf abscission (0 ⁄ 1)§ on survival and weight Larval weight Treatment + Conspecific density§ + Mildew Tree + Shoot(Tree) Identity of Tischeria ekebladella (0 ⁄ 1)§ + Leaf abscission (0 ⁄ 1)§ Exp4: rate of parasitism Arrow 4 Parasitism rate Mildew (0 ⁄ 1)§ + Herbivore damage Tree + Bag(Tree) Logit on the leaf miner Tischeria (0 ⁄ 1)§ + Conspecific density§ ekebladella on healthy, mildew-infected and insect-damaged leaves Resource Exp5: resource selection Arrow 5 Number of eggs Mildew (0 ⁄ 1)§ + Herbivore damage Tree + Bag(Tree) Log selection at the leaf level oviposited (0 ⁄ 1)§ Exp6: resource selection Panel 1B Number of leaf Mildew intensity + Herbivory + Year Log– at the tree level mines Realised Obs1: realised effects on Species-specific Species + Spatial connectivity + Mildew Log–** effects on insect community abundances intensity community structure structure

*ln-transformed. Two-way interaction(s) included. àAs mine area was measured several times during the growing season, we used a repeated measures design with an unstructured variance-covariance matrix. §Variables measured at the leaf level. –A quasi-Poisson distribution was used to account for overdispersion. **Separate models were built for each year. more details). At the end of the growing season, we scored the Resource selection survival of all leaf miner larvae (n = 1194) and dried and weighed all To investigate the impact of the fungus on the resource selection of live individuals (as described above). In addition, we recorded the insects across spatial scales, we examined whether or not moths of number of conspecific larvae, free-feeding damage (0 ⁄ 1), mildew T. ekebladella select healthy vs. infected leaves within trees (Experiment infection (0 ⁄ 1), and leaf abscission (0 ⁄ 1) for each leaf occupied by 5; arrow 5 in Fig. 1B), and how the infection status of the full tree T. ekebladella. affects the number of moths attracted to it (Experiment 6; left-hand Experiment 4: Rate of parasitism on the leaf miner T. ekebladella on healthy, part of Fig. 1B). In a similar vein, we also examined whether or not mildew-infected and insect-damaged leaves: To test whether parasitoids previous herbivory altered resource selection in T. ekebladella at both differentially affect leaf miner larvae on healthy, insect-damaged, or spatial scales. mildew-infected leaves (arrow 4 in Fig. 1A), we introduced a moth Experiment 5: Resource selection at the leaf level: To explore how the mildew pair (#$)ofT. ekebladella in a closed muslin bag (10 trees; three bags affects the spatial distribution of leaf mines within trees, we assessed per tree). The bags and adult moths were removed before the eggs whether moths of T. ekebladella actively avoid or prefer mildew-infected hatched. Hence, parasitoids had access to the leaf mines during the leaves (arrow 5 in Fig 1B) in a constrained-choice setting. To this end, entire developmental stage of the host larvae. (No egg parasitoids are we re-analysed a subset of the data from an experiment conducted in known for T. ekebladella.) In autumn, we recorded the number of 2004 (see Gripenberg et al. 2007). Here, we enclosed part of the foliage larvae, mildew infection (0 ⁄ 1) and damage by free-feeding herbivores on each of five oak trees in muslin bags (two shoots per bag, ten bags (0 ⁄ 1) for each leaf miner occupied leaf. To assess parasitism rate, we per tree; 729 leaves in total; this subset is referred to as the Ôwithin treeÕ reared adult insects from leaf mines by placing mined leaves treatment of the ÔTree pairsÕ material in Gripenberg et al. 2007). A pair individually in plastic cups and storing them in a cellar for hibernation of moths (#$) was later introduced in each bag for approximately (n = 895 leaf mines). A tissue paper was added to each cup to 4 days. After removing the moths, we counted the number of eggs laid decrease humidity. In early spring, the material was placed at room on each leaf. Three weeks later, we visually assessed the presence or temperature to stimulate the emergence of hosts and parasitoids. absence of mildew – and of herbivore damage – on each leaf.

2012 Blackwell Publishing Ltd/CNRS Letter Fungus–plant–insect interactions 5

Experiment 6: Resource selection at the tree level: At a scale larger than a normal distribution with an identity link. If necessary, data were leaves within trees, mildew may affect the preference of moth females log-transformed prior to analysis to achieve homoscedasticity and with respect to tree individuals. To examine the resulting effect of normality of residuals. For count data, we assumed a Poisson tree-level mildew infection intensity and herbivore damage on the distribution with a log link, and for binomial data, we assumed a number of moths attracted to previously unoccupied trees, we binomial distribution with a logit link. To derive degrees of freedom artificially removed all T. ekebladella from a set of 63 small trees in mixed models we used the Kenward–Roger adjustment (Littell (1–3 m) in the autumns of 2005, 2006 and 2007. These trees were et al. 2006). For models with multiple interactions, we used the located on the island Wattkast, Finland (see Gripenberg et al. 2008 for principle of backwards stepwise model simplification to arrive at a more detailed information and a map). We revisited each tree in each minimum adequate model, where variables were retained when subsequent autumn to establish the colonisation pressure of P < 0.1 (Crawley 2007). All model structures, their responses and T. ekebladella, as reflected by the abundance of leaf mines. At the link functions are summarised in Table 1. A more detailed same time, we assessed the intensity of mildew infection and of insect description of statistical analyses applied can be found in Appen- herbivory of the tree. For mildew, we used four categories: 0 = no dix S2. In reporting the results, we offer empirical means for factors mildew found on the tree; 1 = small amounts of mildew present on in simple models and least-squares means for factors in models the tree; 2 = parts of the tree with substantial amounts of mildew, including strong random effects, interactions and covariates. All other parts uninfected; 3 = the whole tree infected. For insect means and their standard errors are reported in the original scale of damage, we used four comparable categories: 0 = no herbivory; the response. 1 = light herbivory, only few leaves have been chewed upon; 2 = moderate herbivory, obvious that some leaves have been chewed RESULTS upon, but most leaves intact; 3 = more than 30% of the leaves have been chewed upon. Our experiments revealed a perplexing diversity of cross-kingdom interactions. The qualitative effects are summarised in Table 2, with Realised effects on insect community structure quantitative details given below. To assess whether or not the structure of the natural insect community varies with the mildew infection level of the oak tree The effect of mildew infection on the food consumption and (Fig. 1C), we conducted yearly surveys on an additional set of trees utilisation by T. ekebladella between 2003 and 2006. More specifically, we recorded the insect community on 88 small trees (1–3 m) on the island of Wattkast (Fig. 1 In Experiment 1, the difference in size among leaf mines on leaves in Appendix S1). For each tree, we recorded the level of mildew with and without mildew varied through time (interaction ÔTreat- infection (using the categories outlined in the previous section), and ment · TimeÕ:F5,96.74 = 7.21; P < 0.001). More specifically, the area the abundance of each of nineteen leaf mining and galling insect of the leaf mine was significantly larger on mildewed leaves during species on each of 20 shoots per tree. periods 3, 4 and 5 (Fig. 2). However, we detected no significant As the relative isolation of each tree will likely affect the number of difference in the mine area in the early development (periods 1 and 2; potential immigrants, the number of leaf mines observed on a tree was Fig. 2), in the final leaf mine area (period 6; Fig. 2), or in the final related both to its mildew infection status and to its connectivity to larval weight among leaves with and without mildew (mean ± SE: other trees (for a definition of connectivity, see Appendix S1). 1.25 ± 0.08 g vs. 1.09 ± 0.06 g respectively; F1,19.38 = 2.56; P = 0.13). The specific leaf area and the leaf mass consumed per unit area did Statistics not detectably differ among healthy and mildew-infected leaves To analyse the data, we used the framework of generalised linear (F1,7.96 = 0.03, P = 0.87; and F1,5.17 = 0.01, P = 0.94 respectively); mixed-effects models (Littell et al. 2006). All models were fitted with neither did growth efficiency differ among healthy and mildew- procedure GLIMMIX in SAS 9.2. For continuous data, we assumed infected leaves (F1,8.61 = 0.00; P = 0.96).

Table 2 A summary of effects uncovered with respect to individual interactions represented in Figure 1

Effect of: Interaction (Fig 1) Observed effect

Mildew on free-feeding herbivore 1a & b Growth rate of herbivore decreases***, pupal mass decreases*, growth efficiency decreases***; leaf consumption unaffectedNS Mildew on leaf miner 2 Mine size at dates 3*, 4** and 5** increases, mine size at date 1NS,2NS and 6NS, larval weightNS and growth efficiency unaffectedNS Free-feeder on leaf miner 3 Larval weight lower on herbivore-damaged leaves*, but not on systemic leavesNS; larval survival unaffectedNS Mildew on parasitism rate of leaf miner 4 Parasitism rate increases** Mildew on resource selection of leaf miner 5 Abundance of leaf mines at both leaf level** and tree level** decreases Mildew on insect community structure Panel 1C Insect abundances lower on mildew-infected trees in 2008***, species-specific responses to mildew in 2004** and 2006*

Asterisks denote significance levels, with interaction- and test-specific details given in the text: NS P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

2012 Blackwell Publishing Ltd/CNRS 6 A. J. M. Tack, S. Gripenberg and T. Roslin Letter

1.06 ± 0.04; mildew-infected leaves: 1.14 ± 0.04; F2,548.6 = 5.02; * * P = 0.03), whereas larval weight decreased with conspecific density * (F1,797.5 = 59.80, P < 0.0001) and with leaf abscission (least-squares means ± SE; attached leaves: 1.13 ± 0.03; abscised leaves:

2 0.98 ± 0.06;F1,797.7 = 9.36, P = 0.002 respectively). Larval weight was lower on leaves previously damaged by other herbivores than on undamaged leaves (least-squares means±SE: 1.08 ± 0.04 g vs. 1.14 ± 0.04 respectively; F1,835 = 4.67; P = 0.03).

Parasitism rate of the leaf miner T. ekebladella on healthy, mildew- infected and insect-damaged leaves Log(mine area) in cm In Experiment 4, parasitism rate was higher on mildew-infected than –3 –2 –1 0 1 2 Healthy leaves on healthy leaves (means ± SE: 0.34 ± 0.03 vs. 0.19 ± 0.02 respec- Infected leaves tively; F1,891 = 7.63; P = 0.006; Figure S1), whereas damage by free- 4

– feeding insects had no detectable effect (means ± SE: undamaged 27/7 7/8 18/8 30/8 10/9 20/9 leaves: 0.22 ± 0.02; herbivore-damaged leaves: 0.24 ± 0.03; 21/9 F1,891 = 0.34; P = 0.56). Parasitism rate per capita increased with an Date increase in the density of conspecific moth larvae (F1,891 = 4.01; P = 0.05). Figure 2 The performance of the leaf miner Tischeria ekebladella feeding on healthy and mildew-infected leaves. Shown are mine areas (log-transformed) as a function of time. Data points are jittered horizontally to reduce overlap, and horizontal bars Resource selection at the leaf level indicate treatment means for each time period. For each sampling date, a star In Experiment 5, females of the leaf miner T. ekebladella oviposited indicates a significant difference (P < 0.05) in mine area among healthy and mildew-infected leaves. more eggs on leaves without mildew infection than on those with mildew infection (a difference of 34%; least-squares means ± SE: 1.46 ± 0.44 and 1.09 ± 0.34; F1,726 = 9.57; P = 0.002). No signifi- cant difference in oviposition preference was detected between The effect of mildew infection on the larval growth rate of A. psi undamaged leaves vs. leaves damaged by insects (least-squares In Experiment 2, the free-feeding larvae gained mass faster when fed means ± SE: 1.41 ± 0.41 and 1.13 ± 0.37; F1,726 = 1.80; P = 0.18). control leaves than when fed mildew-infected leaves (means ± SE: 0.19 ± 0.01 g vs. 0.13 ± 0.01 g respectively; F = 15.15; 1,39 Resource selection at the tree level P = 0.0004; Figure S1). As leaf consumption did not detectably differ among treatments (F1,39 = 0.45; P = 0.51), the increased growth rate In Experiment 6, the number of T. ekebladella leaf mines encountered can be fully attributed to a higher growth efficiency (F1,39 = 20.42; on individual trees varied strongly among years (F2,184 = 8.10; P < 0.0001). Pupal weight was higher on the healthy leaves than on P = 0.0004; Fig. 3). Although we found a consistent and negative mildew-infected leaves (means ± SE: 0.188 ± 0.006 g vs. 0.165 ± 0.007 g respectively; F1,38 = 6.5; P = 0.02).

2006 The effect of early-season herbivory on survival and weight of 2007 T. ekebladella 60 2008 In Experiment 3, the majority of leaf miner larvae survived (ca. 80%), and no differences in survival were detected among larvae on branch tips exposed to direct earlier herbivory (least-squares mean ± SE: 40 T. ekebladella 0.79 ± 0.04), leaves on branch tips next to tips exposed to earlier herbivory (0.80 ± 0.04) and leaves on control branch tips 2006 (0.79 ± 0.05; F2,19.37 = 0.05; P = 0.95). Survival did not detectably 20 differ among leaves previously damaged by herbivores and undam- 20 aged leaves (least-squares means ± SE: 0.79 ± 0.03 and 0.81 ± 0.04 Abundance of 07 respectively; F = 0.00; P = 0.95). Whether a leaf was infected or 1,500 0 not by mildew had no significant effect on survival (least-squares mean ± SE: healthy leaves: 0.81 ± 0.04; mildew-infected leaves: 0123 Mildew infection level 0.79 ± 0.03; F1,237.4 = 0.39; P = 0.53), but survival decreased with conspecific density and with leaf abscission (F = 16.72, 1,430.9 Figure 3 Resource selection by Tischeria ekebladella at the level of trees with different P < 0.0001 and F1,501 = 7.16, P = 0.008 respectively). intensity of mildew infection. Each symbol represents the number of leaf mines The weight of larvae was not affected by the treatment applied encountered on a single tree experimentally cleared of previous leaf mines, and the (F2,19.06 = 0.70; P = 0.51). However, larval weight increased with curves refer to year-specific estimates from a generalised linear model. Dots for mildew infection (least-squares means ± SE; healthy leaves: different years are offset horizontally for visual clarity.

2012 Blackwell Publishing Ltd/CNRS Letter Fungus–plant–insect interactions 7

35 positive (see Table 2 for a summary of the effects uncovered). Teke n = 28 Sti Moreover, resource selection by insect herbivores was shown to be 30 Ppyg Phy modified by fungus infection of the host plant. Through these trait- Nque and species-specific effects, infection by mildew has the potential to 25 Mdry Hser shape the composition of local insect communities across the Ealb n = 37 Dsub landscape – as was empirically demonstrated by our survey of insect 20 Col Clon communities across 88 trees of variable infection levels. Calc 15 n = 18 Bulm Ainf Abundance Amit Interactions among fungus and plant-feeding insects structure the Acur 10 local insect community n = 11 5 Our experiments revealed a broad range of ecological interactions among individual community members on oak. Of these, the 0 interactions between the fungal pathogen and the insect herbivores 0123 were particularly versatile in character. Although early-season herbiv- Mildew infection score ory left little or no trace on different performance measures for late- Figure 4 Insect community structure on trees characterised by different levels of season insect herbivores, fungal infection had a detectable impact. mildew infection. As patterns vary among years, we show the results for 2006. Interestingly, our findings suggest that different insect taxa respond Stacked diagrams represent least-squares means for the abundance of 17 insect differently to the presence of the powdery mildew. More specifically, species on trees with different mildew infection score. Number of trees in each fungal infection reduced the growth rate, growth efficiency and pupal mildew category is stated above the vertical bars. For species abbreviations, mass of the free-feeding caterpillar A. psi (arrows 1a and 1b in Fig. 1). see Appendix S1. This will likely decrease its fitness, as body size has been shown to predict female fecundity in multiple lepidopteran species (Honeˇk 1993; Tammaru et al. 1996). In contrast, fungal infection increased the relationship between the number of mines and the strength of mildew growth rate, decreased the developmental time and increased the infection on the tree (F1,184 = 9.19; P = 0.003; Fig. 3), we did not parasitism rate of the leaf miner T. ekebladella (arrows 2 and 4 in detect any effect of herbivore damage (F1,184 = 0.33; P = 0.57). Year- Fig. 1). Taken together, these results support not only a significant specific regressions of leaf mine numbers on mildew infection level impact of a pathogenic fungus on the fitness of insect herbivores, but were significant for 2 of 3 years (P = 0.02 in both 2006 and 2007). also emphasise the wide range of ecological outcomes: mildew may improve individual performance measures for given insect taxa while impairing other performance measures. Realised effects on insect community structure The finer mechanisms behind the patterns detected remain to be In 2 of 6 years, individual herbivore taxa responded differently to the elucidated. In principle, the negative impact of the oak powdery presence of the powdery mildew (as suggested by a significant mildew on the free-feeder A. psi may be direct (as caused by the interaction between ÔSpeciesÕ and ÔMildewÕ; P = 0.001 and P = 0.02 in consumption of spores, mycelium and infected epidermal cells) or 2004 and 2006 respectively; Fig. 4). This finding indicates that the mediated by changes in plant chemistry (Hatcher 1995). However, a herbivore community structure differed significantly among trees with direct effect seems unlikely, as no toxic effect of spores or mycelium different mildew infection levels. In 2008, we detected no interaction, has been shown in powdery mildews – on the contrary, some but a significant main effect of the factor ÔMildewÕ (P < 0.001), with arthropods have specialised on, or increase growth when, consuming the lowest abundances on the most heavily infected trees. Herbivore this resource (e.g. Yarwood 1943; English-Loeb et al. 1999; Mondy & taxa were differentially affected by ÔSpatial connectivityÕ in 3 of 6 years Corio-Costet 2004). A more clear-cut picture then emerges for the (as revealed by a significant interaction between ÔSpeciesÕ and ÔSpatial interaction between the fungus and the leaf miner. Since T. ekebladella connectivityÕ (2003: P < 0.001, 2004: P < 0.0001, 2008: P = 0.003), only feeds upon the palisade parenchyma, it has no direct contact with whereas abundances were consistently lower (across species) on more the ectoparasitic powdery mildew, which only penetrates the isolated trees in 2 of 6 years (as suggested by the significant main epidermal cells with its feeding organs (Braun 1987). Hence, the effect ÔSpatial connectivityÕ; P = 0.01 and P < 0.0001 in 2005 and plant-mediated interaction may be caused by interactions between the 2007 respectively). Overall, these results show that both mildew and response pathways triggered by pathogens and herbivores (Bostock spatial context are important factors in structuring local insect 1999; Kessler & Baldwin 2002; Nimchuk et al. 2003; Taylor et al. communities across the landscape. 2004). Moreover, leaf miner performance may be affected by independent mildew-induced changes in oak leaf physiology, nutri- tional quality and chemistry (Hatcher 1995; Hajji et al. 2009). DISCUSSION Overall, plant-mediated indirect interactions between insect herbi- This study demonstrates that the ecological interactions among vores and pathogenic micro-organisms have recently received members of different kingdoms may be an important force in increased attention due to the realisation that in natural settings, any structuring local communities in nature. Here, we found that plant will have to deal with multiple enemies at the same time individual herbivore species were differently affected by the same (Hatcher 1995; Stout et al. 2006; Ro¨der et al. 2011). Yet few studies fungus, that interactions between the fungus and the insects may be have focused on enemy-mediated interactions among pathogenic mediated by both the plant and natural enemies, and that the trait- fungi and insect herbivores (Omacini et al. 2001; Biere et al. 2002; specific outcome of individual interactions ranged from negative to Turlings & Wa¨ckers 2003; Stout et al. 2006). Here, our findings

2012 Blackwell Publishing Ltd/CNRS 8 A. J. M. Tack, S. Gripenberg and T. Roslin Letter demonstrate that the leaf miner T. ekebladella suffered increased CONCLUSIONS parasitism on mildew-infected leaves in a natural setting (arrow 4 in Fig. 1). Possible explanations in our study system include that altered Overall, our multitrophic-level perspective on interactions within local volatile emissions from mildew-infected plants may affect herbivore oak communities demonstrates that effects among phylogenetically natural enemy responses (Cardoza et al. 2003; Turlings & Wa¨ckers distant taxa are abundant and diverse, and that they may be mediated 2003; Hare 2011), that differences in the larval phenology of the host by both the host plant and by natural enemies. Where previous on healthy and mildew-infected leaves may change the Ôwindow of research has demonstrated that insect species may respond to the opportunityÕ for parasitism (note differing patterns of growth in presence of a pathogenic fungus in a laboratory setting, our study Fig. 2A), or that the nutritional quality of larvae on healthy and places these findings in the context of more diverse natural mildew-infected leaves may result in selective parasitism. communities (see also Johnson et al. 2003; Rosta´s et al. 2003; Stout et al. 2006). In such communities, individual insect species react very differently to the presence of the same plant pathogen, and the Resource selection by insects ecological outcome can range from negative to positive depending on Our studies of insect oviposition revealed an effect of mildew the specific trait and species examined (see Table 2). As a likely result, presence on the behaviour of ovipositing female moths. This effect it seems that different species respond differently to plant pathogens was evident both in the number of leaf mines encountered at the level (e.g. Moran & Schultz 1998; Kluth et al. 2001; Ro¨der et al. 2007; of individual leaves within trees, and in the level of leaf miner Mouttet et al. 2011). As the literature reveals that insects vary widely in recolonisation of full trees experimentally cleared of leaf mines in the their response to host plant quality (Strong et al. 1984), induced previous year. responses (Karban & Baldwin 1997), and indirect defences (Turlings Within trees, the spatial distribution of mildew affects the fine-scale &Wa¨ckers 2003; Hare 2011), we expect that similar complexities are distribution of the herbivore, as T. ekebladella preferred to lay eggs on likely to emerge in future studies. These results emphasise that the leaves without mildew. Notably, these differences in oviposition traditional focus on herbivore–herbivore interactions or pathogen– choice at a small scale reflected spatial variation in the risk of pathogen interactions should be supplemented by work bridging both becoming parasitised, and may hence reflect an evolutionary fields in a natural setting. As herbivore–herbivore interactions appear adaptation to parasitism-mediated natural selection. As the difference to play a rather minor role in structuring our focal communities in parasitism rate among mildew-infected and healthy leaves was (see also Tack et al. 2009), the current study illustrates how relatively large (corresponding to a 1.8-fold increase in the risk of interactions across kingdoms may be crucial for our understanding becoming parasitised), it implies that mildew may have a marked of terrestrial plant-based communities. Strikingly, the epidemic impact on local insect performance. invasion of a fungal pathogen in northern Europe – now approxi- At a between-tree level, fewer leaf mines were found on trees mately 100 years ago – may have had a profound impact on the characterised by heavier mildew infection. This pattern may be caused structure, spatial ecology and evolutionary trajectory of the native by a shift in migration rates, with lower immigration and ⁄ or higher insect community. emigration from mildew-infected trees. Interestingly, our findings suggest that mildew-induced changes in movement and oviposition ACKNOWLEDGEMENTS decisions may contribute to shape the distribution of a specialist insect both within the local community (i.e. among leaves within a single This article could never have covered so many details across so many tree) and across the landscape. An effect of powdery mildew fungus spatial and temporal scales without the help of many fieldworkers. on spatial dynamics has also been demonstrated in the Glanville Among these, Liselotte Selter and Elena Blasco Martı´n deserve a large fritillary butterfly (Melitaea cinxia) system, where mildew infection was share of the gratitude. This study was financially supported by the shown to affect the local extinction risk of the herbivore (Laine 2004) Academy of Finland (grant numbers 126296 and 129142 to TR). and the colonisation rate of its parasitoid (van Nouhuys & Laine 2008). AUTHORSHIP AJMT conceived the idea of combining materials from studies Implications for herbivore community structure designed and implemented by AJMT, SG and TR; AJMT analysed the The most intriguing finding of our study is that the complex data, and all authors contributed to the manuscript. interactions among a pathogenic fungus and insect herbivores examined above actually scale up to affect the structure of the larger REFERENCES herbivore community. Our detailed experiments showed that individ- ual species responded differently to the presence of the same Agrios, G.N. (2005). Plant Pathology, 5th edn. Academic press, New York, NY. pathogen. They also revealed how mildew affected multitrophic Biere, A., Elzinga, J.A., Honders, S.C. & Harvey, J.A. (2002). A plant pathogen interactions among taxa. Together, they hence suggest that mildew reduces the enemy-free space of an insect herbivore on a shared host plant. Proc. R. Soc. Lond. B: Biol. Sci., 269, 2197–2204. may drive the relative abundances of different species in different Bostock, R.M. (1999). Signal conflicts and synergies in induced resistance to mul- directions, thereby moulding the structure of the full community. tiple attackers. Physiol. Mol. Plant Pathol., 55, 99–109. Indeed, the results from our long-term observational study support Braun, U. (1987). A monograph of the Erysiphales (powdery mildews). Beihefte zur this prediction: different insect species are differentially affected when Nova Hedwigia, 89, 1–700. species are confronted with the very same plant pathogen, and overall Cardoza, Y.J., Teal, P.E.A. & Tumlinson, J.H. (2003). Effect of peanut plant fungal community structure across the landscape is affected by mildew infection on oviposition preference by Spodoptera exigua and on host-searching infection level. behavior by Cotesia marginiventris. Environ. Entomol., 32, 970–976.

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Crawley, M.J. (2007). The R Book. John Wiley and Sons Ltd, London. Nimchuk, Z., Eulgem, T., Holt, B.F. III & Dangl, J.L. (2003). Recognition and Darwin, C. (1859). On the Origin of Species. John Murray, London, UK. response in the plant immune system. Annu. Rev. Genet., 37, 579–609. Desprez-Loustau, M.-L., Robin, C., Bue´e, M., Courtecuisse, R., Garbaye, J., Suffert, van Nouhuys, S. & Laine, A.-L. (2008). Population dynamics and sex ratio of a F. et al. (2007). The fungal dimension of biological invasions. Trends Ecol. Evol., parasitoid altered by fungal-infected diet of host butterfly. Proc. R. Soc. B: Biol. Sci., 22, 472–480. 275, 787–795. Edwards, M.C. & Ayres, P.G. (1982). Seasonal changes in resistance of Omacini, M., Chaneton, E.J., Ghersa, C.M. & Mu¨ller, C.B. (2001). Symbiotic fungal (sessile oak) leaves to Microsphaera alphitoides. Trans. Br. Mycol. Soc., 78, 569–571. endophytes control insect host–parasite interaction webs. Nature, 409, 78–81. English-Loeb, G., Norton, A.P., Gadoury, D.M., Seem, R.C. & Wilcox, W.F. Rasband, W.S. (1997–2011). ImageJ. U. S National Institutes of Health, Bethesda, (1999). Control of powdery mildew in wild and cultivated grapes by a tydeid mite. MD. http://rsb.info.nih.gov/ij/. Last accessed 6 February 2011. Biol. Control, 14, 97–103. Ro¨der, G., Rahier, M. & Naisbit, R.E. (2007). Coping with an antagonist: the impact Feeny, P. (1970). Seasonal changes in oak leaf tannins and nutrients as a cause of of a phytopathogenic fungus on the development and behaviour of two species spring feeding by winter moth caterpillars. Ecology, 51, 565–581. of alpine leaf beetle. Oikos, 116, 1514–1523. Foe¨x, E. (1941). LÕinvasion des cheˆnes dÕEurope par le blanc ou oı¨dium. Revue des Ro¨der, G., Rahier, M. & Naisbit, R.E. (2011). Do induced responses mediate the Eaux et Foreˆts, 79, 338–349. ecological interactions between the specialist herbivores and phytopathogens of Gause, G.F. (1934). The Struggle for Existence. Williams & Wilkins, Baltimore, MD. an alpine plant? PLoS ONE, 6, e19571. Gripenberg, S., Morrie¨n, E., Cudmore, A., Salminen, J.-P. & Roslin, T. (2007). Rosta´s, M., Simon, M. & Hilker, M. (2003). Ecological cross-effects of induced Resource selection by female moths in a heterogeneous environment: what is a plant responses towards herbivores and phytopathogenic fungi. Basic Appl. Ecol., poor girl to do? J. Anim. Ecol., 76, 854–865. 4, 43–62. Gripenberg, S., Ovaskainen, O., Morrie¨n, E. & Roslin, T. (2008). Spatial population Simon, M. & Hilker, M. (2003). Herbivores and pathogens on willow: do they affect structure of a specialist leaf-mining moth. J. Anim. Ecol., 77, 757–767. each other? Agric. For. Entomol., 5, 275–284. Hajji, M., Dreyer, E. & Marc¸ais, B. (2009). Impact of Erysiphe alphitoides on Simon, M. & Hilker, M. (2005). Does rust infection of willow affect feeding and transpiration and photosynthesis in Quercus robur leaves. Eur. J. Plant Pathol., 125, oviposition behavior of willow leaf beetles? J. Insect Behav., 18, 115–129. 63–72. Stout, M.J., Thaler, J.S. & Thomma, B.P.H.J. (2006). Plant-mediated interactions Hare, J.D. (2011). Ecological role of volatiles produced by plants in response to between pathogenic microorganisms and herbivorous . Annu. Rev. damage by herbivorous insects. Annu. Rev. Entomol., 56, 161–180. Entomol., 51, 663–689. Hariot, M.P. (1907). Note sur un oidium du cheˆne. Bulletin de la Socie´te´ Mycologique de Strong, D.R., Lawton, J.H. & Southwood, T.R.E. (1984). Insects on Plants. Harvard France, 23, 157–159. University Press, Cambridge, USA. Hatcher, P.E. (1995). Three-way interactions between plant pathogenic fungi, Tack, A.J.M., Ovaskainen, O., Harrison, P.J. & Roslin, T. (2009). Competition as a herbivorous insects and their host plants. Biological Reviews, 70, 639–694. structuring force in leaf miner communities. Oikos, 118, 809–818. Hatcher, P.E., Paul, N.D., Ayres, P.G. & Whittaker, J.B. (1994). The effect of Tack, A.J.M., Ovaskainen, O., Pulkkinen, P. & Roslin, T. (2010). Spatial location a foliar disease (rust) on the development of Gastrophysa viridula (Coleoptera: dominates over host plant genotype in structuring an herbivore community. Chrysomelidae). Ecol. Entomol., 19, 349–360. Ecology, 91, 2660–2672. Honeˇk, A. (1993). Intraspecific variation in body size and fecundity in insects – Tammaru, T., Kaitaniemi, P. & Ruohomaki, K. (1996). Realized fecundity in a general relationship. Oikos, 66, 483–492. Epirrita autumnata (: Geometridae): relation to body size and Johnson, S.N., Douglas, A.E., Woodward, S. & Hartley, S.E. (2003). Microbial consequences to population dynamics. Oikos, 77, 407–416. impacts on plant–herbivore interactions: the indirect effects of a birch pathogen Taylor, J.E., Hatcher, P.E. & Paul, N.D. (2004). Crosstalk between plant responses on a birch aphid. Oecologia, 134, 388–396. to pathogens and herbivores: a view from the outside in. J. Exp. Bot., 55, 159– Karban, R. & Baldwin, I.T. (1997). Induced Responses to Herbivory. University of 168. Chicago Press, Chicago, IL. Turlings, T.C.J. & Wa¨ckers, F. (2003). Recruitment of predators and parasitoids by Karban, R., Adamchak, R. & Schnathorst, W.C. (1987). Induced resistance and herbivore-injured plants. In: Advances in Insect Chemical Ecology (eds Carde´, R.T. & interspecific competition between spider mites and a vascular wilt fungus. Science, Millar, J.). Cambridge University Press, Cambridge, UK, pp. 21–75. 235, 678–680. Yarwood, C.E. (1943). Association of thrips with powdery mildews. Mycologia, 35, Kessler, A. & Baldwin, I.T. (2002). Plant responses to insect herbivory: the 189–191. emerging molecular analysis. Ann. Rev. Plant Biol., 53, 299–328. Kluth, S., Kruess, A. & Tscharntke, T. (2001). Interactions between the rust fungus SUPPORTING INFORMATION Puccinia punctiformis and ectophagous and endophagous insects on creeping thistle. J. Appl. Ecol., 38, 548–556. Additional Supporting Information may be downloaded via the online Laine, A.-L. (2004). A powdery mildew infection on a shared host plant affects version of this article at Wiley Online Library (www.ecologyletters.com). the dynamics of the Glanville fritillary butterfly populations. Oikos, 107, 329– 337. As a service to our authors and readers, this journal provides Littell, R.C., Milliken, G.A., Stroup, W.W., Wolfinger, R.D. & Schabenberger, O.. supporting information supplied by the authors. Such materials are (2006). SAS for Mixed Models, 2nd edn. SAS Institute Inc, Cary, NC. peer-reviewed and may be re-organised for online delivery, but are not Mondy, N. & Corio-Costet, M.-F. (2004). Feeding insects with a phytopathogenic fungus influences their diapause and population dynamics. Ecol. Entomol., 29, copy edited or typeset. Technical support issues arising from 711–717. supporting information (other than missing files) should be addressed Moran, P. & Schultz, J.C. (1998). Ecological and chemical associations among late- to the authors. season squash pests. Environ. Entomol., 27, 39–44. Mougou, A., Dutech, C. & Desprez-Loustau, M.-L. (2008). New insights into the Editor, Michael Bonsall identity and origin of the causal agent of oak powdery mildew in Europe. Forest Manuscript received 18 July 2011 Pathol., 38, 275–287. First decision made 19 August 2011 Mouttet, R., Bearez, P., Thomas, C. & Desneux, N. (2011). Phytophagous ar- Second decision made 17 November 2011 thropods and a pathogen sharing a host plant: evidence for indirect plant- Manuscript accepted 25 November 2011 mediated interactions. PLoS ONE, 6, e18840.

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