Performance of a leaf in a multitrophic context

Ylva Nordström

Ylva Nordström Degree Thesis in biology 60 ECTS Master‟s Level Report passed: 5 June 2016 Supervisors: Lars Ericson, Benedicte Albrectsen

Performance of a in a multitrophic context

Abstract The aim of my thesis was to assess the relative importance of bottom-up and top-down forces for a plant-herbivore system in a natural multitrophic context. The system consisted of the tall herb Meadowsweet, Filipendula ulmaria (Rosaceae), and the Strawberry leaf beetle tenella (Coleoptera: Chrysomelidae). I set up a field experiment with four clones of each of 16 genotypes varying in food quality. The four clones of each genet were assigned to one of four G. tenella egg density treatments (10, 20, 40 or 80 eggs). I hypothesized that (1) larval performance increases with plant edibility; (2) larval performance increases at higher larval densities (3) the parasitation rate by the hymenopteran parasitoid Asecodes parviclava increases with increasing larval density and decreasing food quality. The results showed significant relationships between larval density treatment and G. tenella survival (negative), pupal weight (positive), proportion of parasitoids (negative) and generalist predators (positive; for both functional groups “web- forming spiders” and “others”), and plant chewers. Parasitation rate was unaffected by treatments. Otherwise, complex interaction effects dominated and support for my hypotheses was low. Analysis of phenolic compounds, using liquid chromatography-mass spectrometry (LC-MS), showed positive correlations between plant damage and quercetin, and plant quality and catechin. Taken together my study indicates that larval performance is affected by both plant quality and larval density. It is likely that herbivore damage induces release of volatile organic compounds (HI-VOCs), attracting other . The strength and direction of those interactions will depend upon the composition of the local community.

Key Words Plant-herbivore interactions, parasitoids, generalist predators, multitrophic interactions, plant chemistry

Table of contents

1 Introduction...... 1 1.1 Background...... 1 1.2 Hypotheses...... 2 2 Materials and methods...... 3 2.1 Study system...... 3 2.2 Plant material...... 3 2.3 Study site……………...... 4 2.4 Experimental design………...... 4 2.5 Data collection...... 4 2.6 Chemical analyses...... 6 2.7 Statistical analyses...... 6 3 Results...... 8 3.1 Chemical analyses...... 8 3.2 Larval performance...... 9 3.3 Arthropod community…...... 13 3.4 Leaf damage……………………………………………...... 17 4 Discussion...... 18 4.1 Plant chemistry...... 18 4.2 Density treatment…………………………………………………...... 18 4.3 Plant quality…………………...... 19 4.4 Interaction effects………………………………………...... 19 4.5 Arthropod community...... 20 4.6 Conclusion…………………………...... 21 5 Acknowledgements...... 21 6 References...... 22 Appendix ...... 26

1. Introduction

1.1 Background

Which organism will, under natural conditions, benefit the most from the accessible information at hand in plant volatiles? – That is the still unanswered question raised in a number of recent detailed reviews on plant-arthropod interactions (Ode 2006, Poelman et al. 2008, Hare 2011, Gols 2014, Heil 2014). Plants may directly affect herbivore performance through stored metabolites that slow down larval development and deter feeding (Stenberg et al. 2006, Lehrman et al. 2012), or indirectly, in response to the level of herbivory experienced, by producing and releasing volatiles. Both these effects do to some extent shape their arthropod community (Vet and Dicke 1992, Abdala-Roberts and Mooney 2013). These volatiles are known as herbivore-induced volatile organic compounds (HI-VOCs), which can communicate to higher trophic (i.e. predatory) levels that prey is present (Hare 2011, Gols 2014, Heil 2014). HI-VOCs are emitted in part due to substances released orally by the herbivore when feeding (Paré and Tumlinson 1997) and are frequently herbivore-specific (Halitschke 2001), and detectable by parasitoids (Turlings et al. 1990, De Moraes 1998) and generalist predators (Johnson et al. 2011). Thus HI-VOCs constitute a valuable information source for both specialist and generalist parasitoids and/or predators in their search for hosts or prey (Castelo et al. 2010), and may potentially benefit the plant through top-down control of its herbivores (Hawkins et al. 1999, Kessler and Baldwin 2001).

However, the fitness gain for the plant has been widely debated, as HI-VOCs in theory are available to all arthropods in the community; herbivores, parasitoids and predators (Kessler and Baldwin 2002, Kessler and Heil 2011, Gols 2014, Heil 2014). For example, leaf damage has been shown to increase herbivore (Abdala-Roberts et al. 2012), parasitoid (Stenberg et al. 2007) and predator density (Abdala-Roberts et al. 2012). So, an intriguing issue is to which extent plant chemistry and volatile emissions will favour the host plant, herbivore, parasitoid, or generalist predator.

Direct and indirect defences can be constitutive and inducible, and the latter have been suggested to be favourable due to their lower cost (Kessler and Baldwin 2002). Studies also support that there is a trade-off between investment in direct and indirect defence due to resource limitations (Ballhorn et al. 2008, Wei et al. 2011). However, while indirect defence may influence herbivore behaviour (Bruce et al. 2005, Zakir et al. 2012), direct defence may have effects also on additional trophic levels (Ode 2006, Poelman et al. 2008). For example, HI-VOC-emitting plants have been found to reduce herbivory in neighbouring plants (Zakir et al. 2012). Therefore, studies of chemical defence, and its direct and indirect effect on the arthropod community, should be considered as a whole and not independently (Gols 2014).

Aggregative behaviour, a common phenomenon among many herbivores, has been interpreted as a strategy to overcome host plant defenses (e.g. Desurmont and Weston 2010, Rönnäs et al. 2010). Aggregation may shorten female oviposition time (Desurmont et al. 2014), increase larval/nymphal growth, pupation success and pupal/adult weight (Nahrung et al. 2001, Wise et al. 2006, Hambäck 2010, Desurmont et al. 2014), and furthermore also act as a dilution effect, increasing larval survival (Hunter 2000, Rönnäs et al. 2010). High larval densities may however attract more specialist or generalist parasitoids (Vamosi et al. 2011) and/or predators (Harwood et al. 2003, Horváth et al. 2005); raising the question under which conditions aggregative behaviour may be advantageous.

Several studies have shown that parasitation rate is affected by both herbivore and host plant quality (Ode 2006). Low plant food quality has been shown to slow down larval development, subsequently prolonging exposure time and increasing the risk of parasitation (Zvereva and Rank 2003, Kessler and Baldwin 2004). However, plant toxins have also been

1 interpreted as having a negative effect on parasitoid larval survival; parasitized herbivore larvae have been reported to show an increased chance of reaching adulthood when reared on plants with high levels of defensive chemicals, than on high food quality plants (Singer and Stireman 2003). Thus, direct defence chemicals may attenuate the bodyguard effect of volatiles (but see Gols et al. 2008).

Several studies have demonstrated that the outcome of tritrophic (i.e. plant-herbivore- parasitoid/predator) interactions may be strongly affected by generalist predators (Hawkins et al. 1999, Finke and Denno 2004), thus highlighting the importance to place these interactions in a broader, multitrophic context (McCormick et al. 2012, Moreira et al. 2012). High densities of generalist predators may suppress parasitoid pressure on herbivores, thus increasing herbivore density (Snyder and Ives 2001). Other studies have shown that the bodyguard effect of generalist spiders may vary depending on food plant quality (Denno et al. 2002). Recent studies have also stressed that high-quality food plants may provide non- prey food; satiating their omnivorous bodyguards and releasing the bodyguard effect (Lundgren 2009, Stenberg et al. 2011).

Many studies have shown that volatiles provide a valuable information source for herbivores as well as parasitoids and predators, thus demonstrating the key role of plant chemistry for bi-, tri- and multitrophic interactions under controlled conditions. However, due to variation in the abiotic and biotic environment, natural habitats constitute a complex world of volatiles. Hence, it can be asked whether this information in reality may become masked by background odours (Schroeder and Hilker 2008, Bruce and Pickett 2011, Büchel et al. 2014) and which trophic level benefits the most by the chemical information (Hare 2011, Kessler and Heil 2011, McCormick et al. 2012, Heil 2014). With this in mind, and to study the impact of the arthropod community on tritrophic interactions, further field studies have been encouraged (Hare 2011, McCormick et al. 2012).

For this study, I asked whether predictions based on controlled experiments may hold in a natural setting. I performed a field experiment on the tritrophic system: the tall herb Filipendula ulmaria (L.) Maxim. (Rosaceae), the chrysomelid herbivore Galerucella tenella (Coleoptera: Chrysomelidae) and its parasitoid Asecodes parviclava (Hymenoptera: Eulophidae). The male emit an aggregation pheromone (dimethylfuran-lactone) and the combination of the pheromone and the host plant attract females (Fors et al. 2015). Fors (2015) found no evidence that A. parviclava can exploit the adult pheromone for location of host larvae. However, parasitoids respond to leaf volatiles released from damaged host plants (Stenberg et al. 2007). In an earlier field experiment on the same study system (Nordström 2013), the major result was that spider densities were significantly higher both on less defended plants and at higher G. tenella larval densities. Hence, in this thesis, the occurrence of arthropods was recorded throughout the field experiment.

1.2 Hypotheses

The study was designed to address the following hypotheses: 1) larval performance (survival and pupal weight) is higher on less defended plants than on more defended plants, as larval development is faster, making larvae able to defend themselves against parasitoids and predators. An alternative hypothesis is that larval performance is higher on more defended plants, as low-defended plants may be attractive hunting grounds to generalist predators. 2) Larval performance will increase with increased larval density, provided that starvation does not occur. An alternative hypothesis is that parasitation rates will increase, resulting in decreased larval performance, with increased larval density. 3) Parasitation rates are higher on highly defended plants, as these hamper larval development (if hypothesis 1 is supported), making them easy targets for parasitoids. An alternative hypothesis is that parasitation rates are lower on these plants, if the toxins are detrimental to the parasitoid larvae.

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2 Material and methods

2.1 Study system

Meadowsweet, Filipendula ulmaria, (L.) Maxim. (Rosaceae) is a common tall herb that occurs in abundance on islands in the Skeppsvik archipelago (Stenberg et al. 2006). It has one major natural enemy in that archipelago, the Strawberry leaf beetle Galerucella tenella L. (Coleoptera: Chrysomelidae) (Stenberg et al. 2006), which in turn suffers high parasitation rates from a hymenopteran parasitoid, Asecodes parviclava (Thomson) (Hymenoptera: Eulophidae) (Stenberg et al. 2007, Hambäck et al. 2006, Hansson and Hambäck 2013). F. ulmaria shows marked spatial variation in traits, such as concentrations of individual phenolics and condensed tannins, in this small archipelago, which has been attributed to the duration of past coexistence between the host plant and its main herbivore G. tenella (Stenberg et al. 2006).

Adults of G. tenella over-winter in the soil and emerge in late May when they start to feed on their host plants. Oviposition starts in mid-June and eggs hatch in early July (Hambäck et al. 2006). At high population densities, higher egg densities are more often found on stems of the floral shoot than on leaves (own observation). Larvae feed for 2-3 weeks, preferably on younger leaves and flower buds, before pupating in the ground. After approximately eight days the pupae hatch and adults feed on leaves before over-wintering in mid-September (Stenberg et al. 2006). The major food source of G. tenella is F. ulmaria, but it also feeds on many other of Rosaceae. Three other host plants also occur in the archipelago; Potentilla anserina, P. palustris, and Rubus arcticus.

Food-choice experiments (with excised leaves) have shown that adults of G. tenella prefer to feed and oviposit on plants from younger populations of F. ulmaria, and that larval survival and development is higher on these plants (Stenberg et al. 2006). G. tenella shows an aggregative behaviour, and Fors et al. (2015) have recently demonstrated that G. tenella males produce the aggregation pheromone dimethylfuran-lactone.

A major natural enemy of G. tenella is the koinobiont endoparasitoid Asecodes parviclava (Hymenoptera: Eulophidae, earlier A. mento). This parasitoid has two other hosts, G. pusilla and G. calmariensis (Hansson and Hambäck 2013), the latter of which is also present in the archipelago, although monophagous on Lythrum salicaria. Parasitized G. tenella form black mummies when the parasitoid larvae pupate, and each mummy contains 1-14 parasitoids (Stenberg and Hambäck 2010). Parasitation rates vary between islands and populations; they are typically very high on G. tenella in populations where both F. ulmaria and L. salicaria co-occur (Hambäck et al. 2006). Parasitoids emerging from one host species have a higher success rate in future attacks on larvae of the same species (Fors 2015).

2.2 Plant material

Plants used in the experiment were raised from seeds sampled from islands in the Skeppsvik archipelago in fall 2006 (L. Ericson). Seeds were sown in 3x3x7-cm pots in an unheated greenhouse at the Umeå University campus, and germinating seedlings were later in 2007 transferred to plastic pots (7x7x10 cm) and kept for cultivation in the common garden. In 2009, 30 genets from mother plants that varied in resistance towards G. tenella were cloned by cutting the root stock in pieces and raised in pots (rose pots, diameter 15.5 cm, 1 l). These plants were in 2010 utilized in a bioassay experiment in order to determine the development time of G. tenella larvae (see below). Out of this cloned plant material of 30 genets, I selected plants from 16 genets in 2014 for the present experiment. Criteria for this selection were that the genets should represent a wide range in plant resistance, be in the flowering stage, and not vary too much in size. A garden soil (Hasselfors P jord) was used throughout the experiment.

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A proxy for plant resistance had previously been obtained in a bioassay experiment performed in an unheated greenhouse in 2010 (L. Ericson, unpublished). For each of 30 genets the survival, development time and pupal weight were determined for 20 G. tenella larvae. Newly hatched larvae were individually placed on leaves in Petri dishes and their development was followed daily or every second day, to obtain growth rates. Fresh new leaves were offered every second day. Survival of the larvae showed a broad variation between the different genets. For several genets no larvae survived to pupation. For that reason, the time when 50% of the larvae had died is used as a proxy for food quality, hereafter called „half-life‟ (data provided by Lars Ericson).

2.3 Study site

The field experiment was performed on the small island Antrevet in the Skeppsvik archipelago. We chose a small open moist depression on the middle part of the island. The depression was dominated by Purple loosestrife (Lythrum salicaria), Marsh cinquefoile (Potentilla palustris), and sedges (Carex nigra var. recta), and was surrounded by stands of F. ulmaria in front of deciduous stands of Gray alder () and Rowan (Sorbus aucuparia). The reason for the choice was that I both wanted to avoid the open shores with very high densities of hunting spiders (and subsequent very high predation risks) and ascertain the presence of high densities of A. parviclava. Sampling of Galerucella larvae in 2012 and 2013 had shown high parasitazion rates, for G. tenella 78 and 92 %, and for G. calmariensis 52 and 54 %, respectively (rearings of 50-60 larvae per species and year; data provided by Lars Ericson). A further reason for the sheltered location of the site was that exposure to strong winds may increase the risk of experimental plants coming in contact with each other.

2.4 Experimental design

For the experiment I used four clones from each of 16 genets, making a total of 64 plants. All plants were kept in rose pots throughout the experiment. On July 1, the four clones of each genotype were placed in trays with a distance of 40 cm between each pot to avoid interplant contact. The trays were filled with garden soil and placed about 1.5 m outside the nearby F. ulmaria stand bordering a small moist depression (Fig. 1), making sure that there would be no contact with experimental plants. The distance between two trays was 4-4.5 m. The trays were watered when needed.

The clones of each genet were assigned one of four different egg density treatments: 10, 20, 40 or 80 eggs per plant. Pieces of F. ulmaria stems with clusters of G. tenella eggs were harvested from another part of the island on July 2, carefully checked, and attached to the floral shoot of the experimental plants so that the newly hatched larvae would be able to crawl over to the experimental plant. As larvae pupate in the top soil close to the host plant, a top layer of sand was spread out in each pot. A loose soil structure is attractive to the larvae and will reduce the risk that the larvae will leave the pot before pupation (cf. Hambäck 2010).

2.5 Data collection

The plants were checked every third-fourth day (July 3, 6, 9, 13, 16, 20, 23, 27 and 31). Data was collected on number of G. tenella larvae and A. parviclava adults, as well as occurrence of herbivores, omnivores and generalist predators, and number of flowers in the inflorescence of F. ulmaria. In order to reduce the risk that the pupae should suffer high predation rates, large G. tenella larvae were collected before pupating. These larvae were individually placed in plastic jars, brought to the laboratory, where they were allowed to feed on leaves of the same genet. These larvae stopped feeding within 1-2 days, after which they pupated. The field experiment was terminated August 1 and the plants were brought back to

4 the experimental garden. The top soil in the pots was sifted in search for additional pupae that had been missed in the field. Number of fruits, seeds and percentage leaf damage of F. ulmaria were also estimated at the end of experiment. G. tenella pupae and A. parviclava mummies were weighed to nearest µg. Data on parasitoid adults are not given as most pupae had not hatched.

Egg batches were regularly checked for possible egg predation. Of 680 eggs scored, four were predated. The low predation may mirror the very low frequency of mirids and lady beetles during the experiment. The procedure adopted for data collection certainly resulted in an overestimation of survival. One limitation with this study is that small larvae that stopped feeding and later were found dead on the plant were not sampled. On some plants a large proportion of these larvae showed signs of parasitoid infection visible through a somewhat darker colour compared with uninfected larvae. About 30 of the sampled larvae died prior to or during pupation. The weather was rainy and chilly in the beginning of the experimental period. Temperature started to increase from day 7 and remained high from day 9; after day 20 the weather was very warm and dry and F. ulmaria inflorescenses surrounding the experimental site started to wither (day 20-24). Due to the dry weather plants were checked every 2nd day and regularly watered.

The arthropods were classified in the following broad functional groups: flower visitors (mainly pollen and nectar feeding Coleoptera), leaf chewers (mainly Symphyta larvae, some Lepidoptera larvae), sap suckers – phloem feeders (Homoptera: Aphidomorpha), sap suckers – xylem feeders (Homoptera: Cerceopidae and Cicadellidae), omnivores (Hemiptera: Miridae and Anthocoridae), generalist predators – spiders (mainly web-forming Aranidae, Linyphiidae and Tetragnatidae), generalist predators – others (a mixed group of Hemiptera: Nabidae and Pentatomidae, Coleoptera: Coccinellidae, Hymenoptera: Formicidae and Neuroptera). Three groups were scored but excluded from further tests due to their scarcity, namely hunting spiders, leaf miners (Microlepidoptera and Diptera), and gallers (Diptera). Flower-visiting diptera were not scored.

Figure 1. Design of the field experiment with 16 trays, each with four cloned plants of one of 16 genotypes of Filipendula ulmaria. For each genotype the four cloned plants were randomly attributed to one of four density treatments with 10, 20, 40 or 80 eggs of the chrysomelid beetle Galerucella tenella. Figures within trays denote half-life (days of G. tenella larval survival). Plant distance within trays 40 cm, distance between trays 4-4.5 m. Figure not drawn to scale.

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2.6 Chemical analyses

An earlier laboratory study by Stenberg et al. (2006) demonstrated a significant and negative correlation between G. tenella female behaviour (leaf consumption and oviposition) and larval development in response to increased leaf concentration of catechin and condensed tannins. For that reason I chose to screen for phenolic compounds in damaged, flowering plants and undamaged, flowering F. ulmaria leaves. I will here report data on catechin, which is considered to be a part of the constitutive defence in F. ulmaria (Stenberg et al. 2006), and quercetin which, based on my results, is considered an induced defence compound.

Chemical screening for phenolics was performed on material from leaves and flowers (data not reported) that were harvested from three different sets of plants. First, leaves were harvested from each plant that was used in the experiment on August 1. Flowers were not harvested from these plants, as the fruits had often suffered severe herbivore damage. Second, leaves and flowers were harvested on July 12 from control plants that had been kept in the experimental garden, safe from herbivores. However, in this plant material, seven of the genotypes used in the field experiment did not flower; thus, an additional nine genotypes were harvested, making a total of 20 genotypes. In order to obtain a chemical baseline, leaves from vegetative plants of 29 genotypes kept in the experimental garden were harvested (data not reported).

The leaves and flowers were dipped in liquid nitrogen directly after harvest and then stored with dry ice before placed in a -80 °C freezer. They were then ground with liquid nitrogen and weighed before extraction with a mixture of (MilliQ) water and organic solvents, MeOH:CHCl3:H2O 3:1:1. Labeled isotope D4 of salicylic acid was added as internal standard as a quality control. After extraction, solvents were removed through evaporation for three hours in a centrifugal evaporator. Samples were thereafter stored at -80 °C until diluted with MeOH and (MilliQ) H2O and analyzed with Agilent 6450 LC-qTOF-MS.

2.7 Statistical analyses

For an overview of statistical tests see Table 1. Statistical analyses on chemical data were only performed on undamaged leaves from floral shoots and damaged leaves from the experimental plants. When testing catechin concentrations in undamaged leaves I used a linear model, as there was no density treatment on these leaves. When testing for a significant effect of both half-life (genotype) and egg-density treatment, I initially used a multilevel linear model. If the multilevel linear model was not significant, and no interaction term was found, I proceeded with tests of normality (Shapiro Wilks), using linear models when allowed. If no normality was found, I used Kruskal-Wallis chi-squared test. Pearsons correlation test was used for testing the correlation between G. tenella leaf damage and the concentration of quercetin. Omnivores, sap suckers and flower visitors were excluded due to lack of statistical significance. All statistical analyses were performed in R (R version 3.1.2).

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Table 1. Transformations and tests of data assessed in the study. * = tests performed on cumulative data. G. t. = Galerucella tenella. A. p. = Asecodes parviclava. Data Transformation Test Catechin Continuous - Linear model Quercetin Continuous Log Multilevel linear model Total survival Proportion - Multilevel generalized linear model Proportion G. t. Proportion - Multilevel generalized linear model Proportion A. p. Proportion - Generalized linear model G. t. weight Continuous - Linear model A. p. weight Continuous Square-root Multilevel linear model Spider density Count - Kruskal-Wallis chi-squared Predator density Count * - Kruskal-Wallis chi-squared Omnivore density Count * - Kruskal-Wallis chi-squared Leaf chewer density Count * Square-root Linear model Sap suckers (phloem) Count * - Kruskal-Wallis chi-squared Sap suckers (xylem) Count * Square-root Linear model Flower visitors Count * - Kruskal-Wallis chi-squared G. t. leaf damage Proportion - Multilevel generalized linear model Total leaf damage Proportion - Multilevel generalized linear model Correlation - Pearsons correlation Quercetin/ G.t. leaf damage

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3. Results

3.1 Chemical analyses

Chemical screenings of undamaged F. ulmaria leaves showed a negative, although not significant (p = 0.173), correlation between catechin and increasing half-life (Table 2, Figure 2). This provides some support for the use of half-life as a proxy for plant food quality.

Chemical screenings of damaged leaves from plants in the field experiment showed that out of 35 tested compounds only one, quercetin, showed a significant response to the density treatment (Table 3, Figure 2). Interestingly, quercetin also showed a significant interaction effect to half-life (Table 3). Further, all genotypes showed an increased concentration of quercetin in response to increased density treatment, with the exception of one genotype with half-life 8.1 (Figure 3). A likely explanation for this deviating pattern is that the leaves of all clones of this genotype were severely damaged by sawfly larvae.

Table 2. Correlation between catechin concentration and half-life. Analysis performed on undamaged F. ulmaria leaves. Tested by using linear model in R. Estimate S. E. t value p-value Intercept 0.208 0.039 5.330 <0.001 HL -0.008 0.006 -1.421 0.173

Table 3. Model testing (ANOVA in R) for the effect of plant genotype (G) and egg density treatment (Ed, 10, 20, 40 and 80 eggs per plant) on the concentration of quercetin in leaves from the experimental plants. For test performed after removal of Hl = 8.1, p = 0.067. Model df AIC BIC logLik deviance χ² df p-value Quercetin G+Ed 4 108.14 116.72 -50.071 100.143 G*Ed 5 104.56 115.28 -47.280 94.561 5.582 1 0.018

Figure 2. Correlation of catechin concentration in undamaged F. ulmaria leaves to half-life.

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Figure 3. Correlation between quercetin concentration and egg density treatment (10, 20, 40 and 80 eggs per plant) for F. ulmaria plants with different half-lives (multilevel linear model). Explanations: mean concentration (thick, solid line), HL 4.0 - 6.5 (solid lines), HL 7.2 – 10.2 (dashed lines). Data from damaged F. ulmaria leaves from experimental plants.

3.2 Larval performance

Total survival (G. tenella eggs that survived to form either G. tenella pupae or A. parviclava mummies) was affected by the interactive effect of plant genotype and egg density treatment (Table 4). For less edible plants, survival was generally negatively affected by increased egg density, while survival was more often positively affected by increased egg density for more edible plants (Figure 4). A similar, though more marked, pattern was found for G. tenella survival (G. tenella eggs that survived to form G. tenella pupae) (Table 4, Figure 5). As densities increased, G. tenella survival decreased for less edible plants and increased on plants of higher food quality. There were some obvious deviations from the above-mentioned trends: regarding total survival for half-life 8.0 and 5.5, and regarding G. tenella survival for half-life 9.6 and 10.2. At least in the last mentioned case, this may be due to high numbers of Myrmica ruginodis.

The proportion of initial G. tenella eggs that survived to form A. parviclava mummies was negatively, although not significantly, correlated to increased density (Table 5), while no interaction effect was found in response to half-life (Table 4, Figure 6). Further, parasitation rate (defined as surviving A. parviclava/total survival) was neither affected by half-life nor density (p=0.275 and 0.571, respectively; Kruskal-Wallis chi-squared). Out of 358 surviving larvae, 177 (49.4 %) were parasitized by A. parviclava.

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Table 4. Model testing (ANOVA in R) of multilevel generalized linear model for the effect of plant genotype (G) and egg density treatment (Ed, 10, 20, 40 and 80 eggs per plant) on (i) total survival (expressed as proportion of eggs that survived to form either G. tenella pupae or A. parviclava mummies), (ii) G. tenella proportion (expressed as proportion of eggs that survived to form G. tenella pupae), (iii) A. parviclava proportion (expressed as proportion of eggs that survived to form A. parviclava mummies), (iv) weight of G. tenella pupae and (v) weight of A. parviclava mummies. Model df AIC BIC logLik Deviance χ² df p-value Total survival G + Ed 3 350.26 356.73 -172.13 344.26 G * Ed 4 339.66 348.29 -165.83 331.66 12.598 1 <0.001 Proportion G.t. G + Ed 3 285.9 292.38 -139.95 279.9 G * Ed 4 278.6 287.24 -135.30 270.6 9.2973 1 0.002 Proportion A.p. G + Ed 3 264.98 271.45 -129.49 258.98 G * Ed 4 263.97 272.61 -127.99 255.97 3.0035 1 0.083 Weight G.t. G + Ed 4 2010.1 2022.8 -1001.1 2002.1 G * Ed 5 2012.1 2028.0 -1001.0 2002.1 0.0056 1 0.940 Weight A.p. G + Ed 4 897.64 910.23 -444.82 889.64 G * Ed 5 888.70 904.44 -439.35 878.70 10.938 1 <0.001

Table 5. Effect of plant edibility (half-life, HL, in days) and density treatment (10, 20, 40 and 80 eggs per plant) on the proportion of added G. tenella eggs that survived to form A. parviclava mummies. Tested by using generalized linear model in R. A. parviclava survival Estimate S.E. z value p-value Intercept -2.243 0.174 -12.830 <0.001 Density -0.005 0.003 -1.781 0.075

Figure 4. Effect of plant edibility (half-life, HL, in days) and density treatment (10, 20, 40 and 80 eggs per plant) on the proportion of added G. tenella eggs that survived to form either G. tenella pupae or A. parviclava mummies; in the text referred to as total survival. Explanations: mean survival (thick, solid line), HL 4.0 - 6.5 (solid lines), HL 7.2 – 10.2 (dashed lines). Tested by using multilevel generalized linear model in R, with package lme4.

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Figure 5. Effect of plant edibility (half-life, HL, in days) and density treatment (10, 20, 40 and 80 eggs per plant) on (i) the proportion of added G. tenella eggs that survived to form G. tenella pupae (above), and (ii) the proportion of added G. tenella eggs that survived to form A. parviclava mummies (below). Explanations: mean survival (thick, solid line), HL 4.0 - 6.5 (solid lines), HL 7.2 – 10.2 (dashed lines). Tested by using multilevel generalized linear model in R, with package lme4.

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Figure 6. Effect of plant edibility (half-life, HL, in days) and density treatment (10, 20, 40 or 80 eggs per plant) on (i) the weight of G. tenella pupae (upper), and (ii) on the weight of A. parviclava mummies (lower). Explanations: mean survival (thick, solid line), HL 4.0 - 6.5 (solid lines), HL 7.2 – 10.2 (dashed lines). Tested by using multilevel generalized linear model in R, with package lme4.

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Table 6. Effect of plant edibility (Hl, half-life) on G. tenella pupa weight. Tested by using linear model in R. G. tenella pupa weight Estimate S.E. t-value p-value Intercept 321.840 19.931 16.147 < 0.001 Hl 7.542 2.641 2.856 0.005

G. tenella pupal weights were not affected by density treatment (Table 4), but were positively affected by increased plant edibility (Table 6, Figure 6). A. parviclava mummy weights were significantly affected both by plant edibility and density treatment, and the interaction between these two factors (Table 4). There seems to be a contrast in response to plant edibility. For more edible plants, mummy weights often increased with increasing density, while for less edible plants, mummy weights often decreased with increasing density (Figure 6). It should be noted that I do not have any data on number of parasitoids per mummy.

3.3 Arthropod community

The different arthropod groups varied strongly in abundance. The two most abundant groups were spiders and aphids (phloem suckers). Three groups showed significant and positive responses to increasing egg-density treatment: leaf chewers, spiders, and generalist predators (Tables 7-12).

Spiders started to colonize early during the field experiment, and at day 1 web-forming spiders were found on all plants except one (data not shown). Spider densities increased throughout the experiment on all treatment groups, but partly collapsed at day 27, which coincided with a very severe drought (Figure 7). Densities tended to be highest in density treatment 40.

Spiders showed a significant and positive response to increased egg-density during a long period during the experiment (day 13, 16, 20 and 23, Table 7 and 8, Figure 8 left panel); this positive response disappeared at day 27 following the severe drought (Table 7, Figure 8 right panel). However, spiders showed no significant response to half-life.

Table 7. Effect of plant edibility (Hl, half-life) and egg density (Ed, 10, 20, 40 and 80 eggs per plant) on spider density. Days that did not show statistically significant results were excluded (day 1, 3, 6, 27 and 31). Tested by using Kruskal-Wallis test in R. Day χ² df p-value 13 Hl 17.988 15 0.263 13 Ed 16.419 3 <0.001 16 Hl 19.789 15 0.180 16 Ed 10.749 3 0.013 20 Hl 23.558 15 0.073 20 Ed 8.0145 3 0.046 23 Hl 24.652 15 0.055 23 Ed 17.839 3 <0.001

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Table 8. Contrast analysis of spider density response to egg density treatment. Days that did not show statistically significant results were excluded (day 1, 3, 6, 27 and 31). Tested by using multiple comparison after Kruskal-Wallis test in R. Density Critical value Day 13 10-20 8.969 17.367 10-40 19.531 17.367 10-80 24.125 17.367 20-40 10.563 17.367 20-80 15.156 17.367 40-80 4.594 17.367 Day 16 10-20 13.531 17.367 10-40 16.250 17.367 10-80 19.844 17.367 20-40 2.719 17.367 20-80 6.313 17.367 40-80 3.594 17.367 Day 20 10-20 11.688 17.367 10-40 13.469 17.367 10-80 17.594 17.367 20-40 1.781 17.367 20-80 5.906 17.367 40-80 4.125 17.367 Day 23 10-20 10.719 17.367 10-40 23.063 17.367 10-80 23.594 17.367 20-40 12.344 17.367 20-80 12.875 17.367 40-80 0.531 17.367

Figure 7. Mean number of spiders for each density treatment (10, 20, 40 and 80 eggs per plant) over the course of the field experiment.

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Figure 8. Effect of egg density treatment (10, 20, 40 and 80 eggs per plant) on spider density on day 23 (left) and day 27 (right). Box plots show median, 25-75 percentiles (box), non-outlier range (bar), outliers and extremes.

Generalist predators (spiders excluded) were present throughout the experiment, but generally at low densities, and they, as well as leaf chewers showed a significant increase with increased density treatment (Table 9-12, Figure 9). Neither did however respond to half-life. Generalist predators were scarce, possibly owing to the unfavourable, cold weather at the start of the season.

Table 9. Effect of plant edibility (Hl, half-life) and egg density (Ed, 10, 20, 40 and 80 eggs per plant) on predator density. Tested by using Kruskal-Wallis test in R. χ² df p-value Predators (Hl) 16.332 15 0.360 Predators (Ed) 9.716 3 0.021

Table 10. Contrast analysis of spider density response to egg density treatment. Days that did not show statistically significant results were excluded (day 1, 3, 6, 27 and 31). Tested by using multiple comparison after Kruskal-Wallis test in R. Density Critical value Predators 10-20 3.563 17.367 10-40 12.813 17.367 10-80 18.000 17.367 20-40 9.250 17.367 20-80 14.438 17.367 40-80 5.188 17.367

Table 11. Effect of plant edibility (half-life) and egg density (10, 20, 40 and 80 eggs per plant) on the number of leaf chewers. Tested by using linear model in R. Leaf chewers Estimate S.E. t value p-value Intercept 1.990 0.262 7.585 <0.001 Egg density 0.014 0.006 2.432 0.018

Table 12. Leaf chewer density (mean ± s.d.) at four egg-density treatments (10, 20, 40 and 80 eggs per plant). Leaf chewers Mean 10 Mean 20 Mean 40 Mean 80 5.563 (±5.645) 6.250 (±4.359) 9.125 (±7.173) 10.563 (±9.252)

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Figure 9. Effect of egg density treatment (10, 20, 40 and 80 eggs per plant) on predator density ( left) and leaf chewers (right). Box plots show median, 25-75 percentiles (box), non-outlier range (bar), outliers and extremes.

3.4 Leaf damage

There was a strong positive correlation between G. tenella leaf damage and quercetin concentration (Pearson correlation = 0.58, p<0.001, see Appendix, Figure 1). Furthermore, both G. tenella leaf damage and total leaf damage showed a significantly positive correlation to density treatment and a significant interaction with half-life (see Appendix, Table 1, Figure 2 and 3).

4. Discussion

A general result of my study is that the arthropod community as a whole was affected by herbivore density, while plant genotype affected herbivore performance. One important outcome is the marked response of generalist predators (spiders in particular) to the density treatment. A second is the frequent occurrence of interaction effects between the density treatment and plant quality. A third is that my hypotheses that were based on constrained bi- and tritrophic studies were too simplistic. However, many of the observed patterns, although complicated, were logical in their response when considering shifts in the relative importance of bottom-up and top-down forces in play. This illustrates the need to assess plant–natural enemy interactions under natural multitrophic conditions.

4.1 Plant chemistry

The chemical screening, in spite of the rather small sample size for undamaged leaves from floral shoots, showed a negative, although not significant, correlation between catechin concentration and increasing half-life. Higher catechin concentrations have earlier been reported to be negatively correlated to both G. tenella female oviposition behaviour and larval development (Stenberg et al. 2006). For that reason, I am inclined to conclude that the half-life values can be used as proxies for constitutive defences in the plant genotypes; thus mirroring food quality in the experimental plants. Further, quercetin concentrations increased significantly with increased density treatment, suggesting that it may be part of the induced defence in F. ulmaria. While I do not know the actual effect of quercetin on G. tenella, other phenolics in the quercetin-group have been suggested to have negative effects

16 on the performance of the chrysomelid beetle Phratora vulgatissima (Lehrman et al. 2012). Both catechin and quercetin have been found to increase in response to abiotic stress in hawthorn, another species of the Rosaceae family (Kirakosyan et al. 2004). Towards the end of the experiment, a severe drought (an abiotic stress) occurred, which thus may have increased catechin and quercetin concentrations.

4.2 Density treatment

One marked pattern in this study is the significant, positive response of generalist predators (spiders in particular, but also other predators) and leaf chewers to an increased egg density. Further, there was an overall negative effect of egg density on total survival, G. tenella proportion and weight, as well as A. parviclava proportion (i.e. the proportion of total egg density that survived to form A. parviclava mummies) and weight. The negative egg-density effect on total survival and G. tenella performance is in contrast to my hypothesis that aggregative behaviour is beneficial for herbivore performance; a pattern that has been found frequently under controlled bi-and tritrophic studies (Nahrung et al. 2001, Wise et al. 2006, Costa et al. 2007, Hambäck 2010, Desurmont et al. 2014). There are two possible explanations as to why my results are in contrast to results from previous studies. First, a severe food shortage may have occurred, particularly on plants of lower food quality, e.g. resulting in lower pupae weights (Figure 6, upper panel). Second, high egg density treatment caused increased densities of generalist predators (Figure 8, left panel), which may have caused the decrease in larval survival (Figure 4). Thus, the challenge is to understand under which circumstances the generally assumed positive effect of herbivore gregariousness on G. tenella performance applies under natural conditions. In the light of the strong positive predator response to density, I am inclined to argue that generalist predation is more important than food limitation for the above patterns (but see further Interaction effects and Arthropod community).

I found no response of parasitation rates to the density treatment or plant edibility, possibly due to the small sample size of surviving larvae. Parasitized larvae may experience a higher mortality compared with unparasitized ones, attenuating the treatment effects. There are a number of possible explanations that are impossible to evaluate due to the experimental design. First, if exposed to multiple attacks of the parasitoid, host larvae may die from the injuries and this has been frequently observed in rearing experiments of collected larvae and particularly so when both the beetle and the parasitoid occur in high densities (own observations). Second, parasitized larvae are less mobile (own observation) and hence offer an easier prey for generalist predators. Third, the general negative response to the density treatment may also depend on an increased food shortage.

However, in contrast to my hypothesis, the proportion of A. parviclava mummies was negatively correlated to increasing egg density, although not significant. I had hypothesized an increased parasitation in response to increased egg density, as A. parviclava has been found to respond to leaf volatiles (Stenberg et al. 2007), and as leaf damage in the experiment also increased with increased egg density. A negative pattern can be interpreted as mirroring an increased food shortage resulting in smaller hosts and a subsequent, increased mortality among parasitized hosts (Jervis et al. 2008, Stenberg 2015). Another possibility is that immune responses in G. tenella are stronger when feeding on high-quality plants as found for other systems (Bukovinszky et al. 2009, Gols 2014). Faster growing larvae on high-quality plants also display a defensive behaviour by vigorously bending the abdomen when approached by A. parviclava (own observations). The last-mentioned interpretation is only partly supported by my data on A. parviclava mummy weights as they in some cases increased on more edible plants (see further Interaction effects). The above illustrates that although it is likely that the parasitoid is attracted by high larval densities, this does not necessarily imply an increased parasitation rate and parasitoid success.

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4.3 Plant quality

I hypothesized that herbivore performance should be positively affected by increased plant quality. In accordance with this, I found that G. tenella pupal weights were positively affected by higher plant quality. However, I did not find effects of plant quality alone on any other performance traits, which is in contrast to the frequent observations that performance of specialized herbivores and parasitoids are correlated to plant quality under controlled experimental conditions (Ode 2006, Kessler and Heil 2011, Gols 2014). Instead, a most striking result of this study is the frequent occurrence of interaction effects between egg density and plant quality. One conclusion is that bottom-up effects demonstrated in bi- and tritrophic studies may vary in strength under natural conditions where plants encounter a more diverse arthropod community (see further Interaction effects).

4.4 Interaction effects

My results show a number of complicated interaction effects, sometimes in support of, and sometimes in contrast to, my hypotheses that were based on results from previous from controlled, tritrophic studies. I argue that these shifting patterns can be understood by invoking the role of various generalist predators, and I will now discuss these aspects.

Although there was an overall negative response of G. tenella survival to increased egg density, five of the most edible genotypes (HL 9.9, 9.8, 8.2, 8.1 and 8.0, Figure 5) showed a strongly positive response to increasing egg density. This indicates that aggregative behaviour may sometimes be advantageous in the presence of high predator densities, albeit on high-quality food plants only. Aggregative behaviour has been shown to decrease larval development time (Nahrung et al. 2001, Reader and Hochuli 2003, Costa et al. 2007, Hambäck 2010). Accordingly, a possible interpretation of the above-mentioned pattern is that a shortened development time allows larvae to pupate earlier and escape predation on more nutritious plants. In contrast, judged on my data, aggregation rather seems to be a disadvantageous strategy on plants of lower food quality, possibly due to starvation or that a longer development time will result in an increased predation risk (e.g. Häggström and Larsson 1995). Furthermore, my results suggest that herbivores may experience enemy-free space on low-quality hosts, which has been demonstrated in other systems (Murphy 2004, Pöykkö 2011), although, in my case, at low densities only. This raises the question to which extent aggregative behaviour on low-quality plants will result in increased herbivore performance in a multitrophic context. One conclusion is that the fitness benefit from aggregative behaviour depends on a combination of food-plant quality and predation risk.

I hypothesized that G. tenella pupal weights would increase in response to increased egg density. However, G. tenella pupal weights were not affected by the density treatment. This lack of response can have several explanations. Mortality may have been skewed, as several generalist predators, for example stink bugs, have been shown to be attracted to larger prey (Gotthard 2000). An alternative explanation is that a smaller pupal size may be an effect of changed herbivore behaviour, as they have been found to feed less when predators are present (Stamp and Bowers 2000). So, the relative importance of these explanations remains elusive due to my experimental design.

The overall pattern for A. parviclava mummy weights was similar to that for G. tenella pupae weights, with a positive response to increased food plant quality. However, there are a few deviations. First, four of the seven least edible plants showed a strong, negative correlation to increased density treatment, with higher weights on the low-density treatments, which can be interpreted as an effect of enemy-free space. Second, two of the three most edible plant genotypes showed a strong positive response to increased density treatment. This provides the female parasitoid with two choices: to either parasitize hosts located on high-quality plants, which will likely deliver sufficient food, but possibly are more

18 exposed to predator attacks, or choose hosts at low densities located on plants of low quality, increasing progeny weight, but likely increasing host location time.

4.5 Arthropod community

I found marked differences between the arthropod groups in response to the egg-density treatment. While generalist predators, both spiders and “others”, as well as leaf chewers showed a positive response to increased egg density, the remaining groups (omnivores, sap suckers and flower visitors) did not. The marked positive response of generalist spiders, which almost entirely were represented by web-forming species, may seem odd in the light of them feeding mainly on small, flying species (aphids, diptera, sawflies, moths; e.g. Eggs and Sanders 2000). G. tenella is an unlikely primary food resource for web-forming spiders and a possible explanation for this positive response may be that the increased leaf damage that followed feeding by G. tenella larvae induced HI-VOC release by the plant attracting more suitable prey to the plant as well as to the web. This explanation has some support by the observation that other leaf-chewing herbivores (for example sawflies) showed a positive response to the density treatment which in turn enhanced the amount of leaf damage. A further support is the observed positive response of quercetin, the only compound that showed a positive response to the egg density treatment, suggesting that quercetin may play a role in the induced defence.

During recent years it has been increasingly acknowledged that spiders‟ choice of host plant is not only affected by prey but also by the availability of non-prey food. Pollen and nectar are important as supplementary food for web-forming spiders (Peterson et al. 2010, Schmidt et al. 2013), and Eggs and Sanders (2013) found that pollen trapped in the web amounted to 25% of the diet of juvenile orb-weaving spiders. This may also suggest a negative interaction between G. tenella and spider density. Increasing densities of G. tenella will result in depletion of flowers (and pollen) as G. tenella larvae feed on flower buds and flowers. During the experiment, I observed that the flowers in the high-density treatment were almost completely consumed by the larvae around day 23 (own observations). At the same time spider densities decreased on the highest egg-density treatment, and the positive response between egg-density treatment and spider density disappeared. Instead, an increasing effect of half-life became evident during the experimental period.

F. ulmaria is a dominant, apparent plant along the Bothnian coast. The large floral display offering plenty of pollen and nectar attracts high densities of flower visitors (own observations) and it is an important habitat for spiders (Kleemola 1969). The data from this experiment, as well as data from an earlier experiment on another island in the archipelago (Nordström 2013), suggests that larval survival is negatively correlated to spider density. However, rather than a direct influence, this may reflect that spiders respond to how other arthropods respond to HI-VOCs released by G. tenella-damaged plants. Hence, effects on selection pressures may depend on the local arthropod community composition and how its members respond to plant cues. As number of flowers in the F. ulmaria inflorescence varies between genotypes (Lars Ericson personal communication) future studies should also try to assess the role of floral display for the outcome of plant-herbivore-parasitoid interactions in a multitrophic context.

4.6 Conclusion

The major conclusion of this study is the complicated interaction effects between plant quality and egg density – most likely due to responses in the arthropod community. Thus, to understand the role of plant chemistry for reciprocal plant-herbivore-parasitoid interactions, such studies need to be performed in a multitrophic context. As the composition of the arthropod community (herbivores, parasitoids, omnivores and predators) varies in space and time, the outcomes of such studies are likely to be highly context-dependent. The role of

19 generalist predators need to be particularly assessed as their dependence on plant resources previously in general have been neglected. 5. Acknowledgements

I want to thank my supervisor Lars Ericson for providing plant material and excellent support. I also thank my supervisor Benedicte Albrectsen for support with the chemical part and Stefano Papazian for help with chemical analyses. Pär Ingvarsson is gratefully acknowledged for statistical support.

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Appendix

1.1 Leaf damage

Figure 1. Correlation between quercetin concentration and G. tenella leaf damage (%). Pearson correlation = 0.58, p<0.001.

Table 1. Model testing (ANOVA in R) of multilevel generalized linear model for the effect of plant genotype (G) and egg density treatment (Ed, 10, 20, 40 and 80 eggs per plant) on (i) G. tenella leaf damage and (ii) total leaf damage. Model df AIC BIC logLik deviance χ² df p-value G. t. leaf G+Ed 3 1247.41 1253.89 -620.71 1241.41 damage G*Ed 4 916.03 924.66 -454.01 908.03 333.39 1 <0.001 Total leaf G+Ed 3 2331.9 2338.4 -1162.98 2325.9 damage G*Ed 4 1583.8 1592.4 -787.88 1575.8 750.2 1 <0.001

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Figure 2. Effect of egg density treatment (10, 20, 40 and 80 eggs) on G. tenella leaf damage (‰). Explanations: mean survival (thick, solid line), HL 4.0 - 6.5 (solid lines), HL 7.2 – 10.2 (dashed lines). Figure made using multilevel generalized linear model in R, with package lme4.

Figure 3. Effect of egg density treatment (10, 20, 40 and 80 eggs per plant) on the total leaf damage (‰). Explanations: mean survival (thick, solid line), HL 4.0 - 6.5 (solid lines), HL 7.2 – 10.2 (dashed lines). Figure made using multilevel generalized linear model in R, with package lme4.

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