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Modification of plant-induced responses by an insect ecosystem engineer influences the
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colonization behaviour of subsequent shelter-users
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* Akane Uesugi (corresponding author)
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Department of Ecology & Evolutionary Biology, Cornell University, Corson Hall,
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Ithaca, NY 14853, USA
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Current address:
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School of Biological Sciences, Monash University. Building 18, Victoria 3800
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Australia.
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Kimberly Morrell
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Department of Ecology & Evolutionary Biology, Cornell University, Corson Hall,
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Ithaca, NY 14853, USA
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Erik H. Poelman
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Laboratory of Entomology, Wageningen University, P.O. Box 16, 6700 AA
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Wageningen, The Netherlands
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Ciska E. Raaijmakers
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Department of Terrestrial Ecology, Netherlands Institute of Ecology,
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Droevendaalsesteeg 10, 6700 EH Wageningen, The Netherlands
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André Kessler
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Department of Ecology & Evolutionary Biology, Cornell University, Corson Hall,
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Ithaca, NY 14853, USA
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Running headline: modification of induced responses
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*Correspondence author. Email: [email protected]
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SUMMARY
1. Herbivores that modify plant morphology, such as gall forming insects, can
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disproportionately impact arthropod community on their host plants by providing
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novel habitats and shelters from biotic and abiotic stresses. These ecosystem engineers
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could also modify plant chemical properties, but how such changes in plant quality
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affect the behaviour of subsequent colonizers has rarely been investigated.
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2. We explored how an initial infestation of the tall goldenrod (Solidago altissima) by an
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ecosystem engineer, the rosette gall-midge (Rhopalomyia solidaginis), affects
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colonization behaviour of a shelter-using beetle (Microrhopala vittata) through plant-
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induced responses in the field.
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3. Beetles preferentially colonized plants with galls and exhibited a clumped distribution
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on those plants, which suggested a possible advantage for aggregating on galled
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plants. Accordingly, we found that beetles remained longer on galled plants with
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previous beetle damage than those without beetle damage. No such effect of beetle
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damage was found on plants without a gall. Similar interactions between galler-
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infestation and beetle damage were found in beetle’s feeding choice, leaf diterpene and
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serine protease inhibitor production, and volatile organic compound (VOC) emission.
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These plant metabolic induction and herbivore response patterns indicated that the
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gall-midge can alter how plants respond to the beetle damage, and that gall presence
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coupled with beetle damage improves leaf palatability for the beetle. Finally, we found
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reciprocal effects of beetles on gall-midge performance to be neutral to slightly
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positive, suggesting that the observed field association of the two herbivores could be
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formed by plant-mediated facilitation.
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4. Synthesis: Our study suggests that an ecosystem engineer could have significant
impact on herbivore community not only by changing plant morphology, but also by
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altering host quality and modifying plant induced responses to subsequent herbivory.
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As such, R. solidaginis also functions as a keystone herbivore that has
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disproportionate effects on community dynamics and composition meditated by
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induced plant growth and metabolic responses.
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Key-words: induced defense, herbivore community, mutualism, plant–herbivore interactions,
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secondary metabolites, volatile signalling
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INTRODUCTION
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Insect herbivores that modify plant morphology (e.g. gall-makers, leaf-miners and rollers) can
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disproportionately impact arthropod community composition and dynamics on plants through
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non-trophic interactions (Jones et al 1994; Lill & Marquis 2007). These ecosystem engineers
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are thought to alter herbivore diversity and abundance (Fukui 2001; Lill & Marquis 2007;
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Cornelissen et al 2015; Wetzel et al. 2015) by providing novel habitats and shelters from
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natural enemies and abiotic stresses (Kudo 1994; Larsson et al 1997; Fukui 2001; Danks
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2002; Marquis & Lill 2007; Crawford et al. 2007). Insect ecosystem engineers are also known
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to manipulate plant metabolism for their own benefit (Sagers 1992; Sandberg & Berenbaum
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1989; Marini-filho & Fernandes 2012). But how such alteration of plant metabolic quality
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affects the behaviour and performance of subsequent shelter-users has rarely been
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investigated (Cornelissen et al 2015; but see Crawford et al 2007).
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Plant induced responses to herbivore damage can drastically alter the pattern of
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subsequent colonization (Kessler & Baldwin 2004; Viswanathan, Narwani & Thaler 2005;
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Ohgushi 2008; Poelman et al. 2010) by inducing or suppressing plant resistance traits,
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including trichomes, latex, secondary metabolites, and volatile compounds that attract natural
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enemies (Van Zandt & Agrawal 2004; Kessler & Baldwin 2004; Viswanathan et al. 2005;
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Tooker et al. 2008). Recent studies have recognized that early colonizers could further modify
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plants’ physiological abilities to respond to the secondary colonizers by inducing changes in
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phytohormone levels (Tooker et al. 2008; Ali & Agrawal 2014). For example, an initial aphid
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infestation that induced salicylic acid (SA) can suppress plant’s ability to respond to the
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subsequent caterpillar damage by interfering with the jasmonic acid (JA)-induced defense
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pathways (Ali & Agrawal 2014).
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Such modifications of plant metabolic plasticity could influence the field distribution
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pattern of subsequently colonising mobile herbivores. These herbivores may use induced
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secondary metabolite changes as information cue to disperse away from, or aggregate on, the
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damaged plants (Hoy, Head & Hall 1998; Roslin et al. 2008). Thus, we hypothesize that
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ecosystem engineers would not only provide novel habitats, but also alter the chemical
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information landscape by inducing plant chemical changes and affecting how new
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information is generated by altering plant endogenous signalling pathways (Kessler 2015). In
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this way, herbivore ecosystem engineers could also function as keystone herbivores that affect
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dynamics and composition of the entire arthropod community beyond the effect expected
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form their mere biomass alone (Hunter 1992, Poelman & Kessler 2016).
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Here, we examine an interaction between an ecosystem engineer, the rosette gall-
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midge (Rhopalomyia solidaginis: Diptera), and the shelter-using beetle (Microrhopala vittata:
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Coleoptera) on the tall goldenrod (Solidago altissima). Larvae of R. solidaginis induce
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rosette-shaped galls on a lateral bud of the host plant, which provides microhabitats for a
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diverse array of arthropods (Crawford et al. 2007) including adult M. vittata (Maddox & Root
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1990). Crawford et al. (2007) found greater arthropod abundance and diversity on galled than
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ungalled S. altissima plants, suggesting that R. solidaginis is an ecosystem engineer that
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disproportionately impact arthropod community structure.
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Gall infestation often triggers changes in nutritional quality of plant tissue (Takei et al.
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2015). Infestation by another gall-midge, Rhopalomyia sp. was found to increase nutrient
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levels within galled compared to non-galled tissues (Marini-Filho & Fernandes 2012). Other
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galling insects are also known to reduce defensive secondary metabolites (e.g. phenolics and
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peroxidase) in the internal tissues they feed on, while increase these compounds in outer
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layers, presumably to defend themselves against their natural enemies (Nyman & Julkunen-
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Tiitto 2000, Allison & Schultz 2004, Takei et al. 2015). Consequently, such gall-induced plant
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metabolic changes are expected to directly or indirectly affect interactions with other
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herbivores on the plant. Crawford et al. (2007) found that resistance to a non-native,
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generalist herbivore, Spodoptera exigua, did not differ between the R. solidaginis-galled and
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ungalled plants (Crawford et al. 2007). However, whether and how R. solidaginis changes
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plant metabolic quality and the interactions with native specialist herbivores that are directly
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associated with the gall, has not been explored. Moreover, whether R. solidaginis modifies
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plant responses to the subsequent herbivory by gall-inhabiting beetles is unknown. Such
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modification of metabolic plasticity is predicted, as S. altissima was shown to respond to
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another galling insect, Eurosta solidaginis, by altering phytohormonal levels (Mapes &
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Davies 2001; Tooker et al. 2008).
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To investigate how plant chemical changes induced by R. solidaginis affect the beetle
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colonization pattern, we first quantified the field distribution pattern of the beetle on galled
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and ungalled plants. We then conducted a series of field and laboratory bioassays and
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chemical analyses to test whether the gall infestation induces changes that affect plant
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attractiveness to the beetles, and how it modifies plant induced response to the beetle damage.
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Finally, we asked whether the interaction between the shelter-inducer and the shelter-user is
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asymmetric (Fukui 2001), i.e. whether the feeding damage by the beetle negatively influences
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the gall-midge performance.
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MATERIALS AND METHODS
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Study system
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The tall goldenrod, Solidago altissima (Asteraceae), is a perennial forb that dominates old-
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fields throughout eastern North America. The plant is attacked by a diverse array of insect
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herbivores (~ 100 species), including stem- and leaf-gallers, leaf-miners, leaf rollers, sap-
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feeders and external feeders, most of which are Solidago specialists (Root 1996). Upon
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herbivory, S. altissima induces various biochemical responses that are specific to the attacking
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herbivore species, including alteration in secondary metabolites (Uesugi, Poelman & Kessler
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2013), anti-nutritive protease inhibitors (Bode, Halitschke & Kessler 2013), and volatile
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organic compounds (VOCs; Tooker et al. 2008; Kessler & Morrell 2010).
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Rhopalomyia solidaginis (Diptera: Cecidomyiideae) is a specialist gall-forming midge
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on S. altissima that is known for its role as an ecosystem engineer (Crawford et al. 2007). The
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larvae induce rosette growth of leaves on lateral buds. The resulting galls contain 1-12 larval
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chambers (Raman & Abrahamson 1995). The galls attract a suite of herbivores and predators,
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thus altering the community structure on S. altissima (Crawford et al. 2007). A chrysomelid
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beetle Microrhopala vittata (Coleoptera) is one of the herbivores that are often associated
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with the rosette galls (Maddox & Root 1990). The beetle is a Solidago specialist and spends
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its larval stage as a leaf-miner, but feeds externally as an adult. During the fall, adults are
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often found feeding on rosette leaves. Overwintering M. vittata adults that emerge in early
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spring exhibit aggregation behaviour when they feed, mate, and oviposit on their host plants
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(Cappuccino 1991), as a high egg density per leaf increases the probability of mine
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establishment and larval survival (Damman 1994). All of the experiments in this study were
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conducted in July-August 2013, and used the second generation of R. solidaginis and the adult
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M. vittata that eclosed during the late summer.
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Field survey of M. vittata beetle colonization of galled plants
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To examine if the beetle colonization of S. altissima is determined by the presence of the
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rosette galls, we conducted field surveys in late August, 2013, in four old-fields within
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Tompkins Co., NY: Durland Bird Preserve (42°26’17”N, 76°23’54”W), Cornell Lab of
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Ornithology (42°28’55”N, 76°26’58”W), Liddell Field Station of Cornell University
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(42°27’39”N, 76°26’40”W), and Whipple Farm (42°29’20”N, 76°25’50”W). In each site, we
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haphazardly sampled 75-100 galled plants and the equal number of ungalled plants, and
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counted the number of M. vittata and other herbivorous and predatory arthropods on the
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whole plant, and identified to species, or to family when species identification was not
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possible in the field.
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Arthropod richness, abundance and Shannon's diversity index on each plant was
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calculated using the above classification, and compared between plant type (galled or
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ungalled) using an ANOVA. The abundance of M. vittata and predatory arthropods
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(combination of spiders, predatory bugs and ants) were tested specifically. Due to a large
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number of zeros in the data set, we were unable to transform data to achieve normality, thus
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the count numbers were converted into presence/absence (“colonization”) of each
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species/group. First, we tested whether the relationship between colonization and plant type
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depends on survey sites by examining a three-way interaction using log linear analyses (loglm
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function in R). If no such interaction was found, we collapsed the data ignoring sites to
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examine overall pattern of colonization. When we found a significant interaction, we
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conducted contingency table analyses for each site.
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Using the data from galled plants across the four fields, we tested if the beetles show
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clumped or uniform dispersion (Ludwig & Reynolds 1988). A clumped dispersion would
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indicate that beetles aggregate within galled plants, potentially suggesting an advantage of
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being with conspecific individuals (Denno & Benrey 1997; Aukema & Raffa 2004). A
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uniform distribution, in contrast, would suggest that beetles avoid each other, perhaps due to
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reductions in plant quality with increased beetle damage (Kouki 1991). Initially, the observed
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distribution was tested against a random Poisson distribution. Because a Chi-squared test
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showed divergence from the Poisson expectation (see Results), we then tested for an
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agreement with negative binomial distribution, which indicates clumped distribution. Finally,
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we calculated the degree of clumping as Green's Index, which has the maximum value of 1
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(Ludwig & Reynolds 1988).
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Beetle preferences and plant induced responses
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Three field experiments were conducted to examine whether the field distribution patterns of
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the beetles reflect their preference for suitable habitats. First, we examined the likelihood of
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beetles remaining on plants on galled vs. ungalled plants ('plant type') that were previously
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damaged or undamaged by beetles ('damage treatment'). The study was conducted in an area
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of Whipple Farm where galls were abundant, but beetles were rare. We haphazardly selected
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20 pairs of galled and ungalled plants that were growing next to each other, and considered a
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pair as a block. Because S. altissima grows clonally, two plants within a pair is likely to be
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genetically identical. All plants were bagged individually near the tip (~20 cm) with fine mesh
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sleeves (Breather mesh sleeves, Palm Tree Packaging, Apopka, FL), and 10 blocks were
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randomly chosen to receive the damage treatment where we released 3 beetles per bag for 4
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days. The remaining blocks were undamaged controls. After the initial damage treatment, we
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removed the bags and initial beetles, and carefully placed five new beetles on each plant. The
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number of remaining beetles was counted after 20, 44, 68, 87, 183, and 129 hours. The count
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data were analyzed using a generalized linear model with plant type and damage treatment as
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fixed effects and hours since release as a random effect.
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The second experiment tested if the re-colonization probability of the beetles on galled
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plants depended on prior beetle damage. The experiment was conducted in an area of Whipple
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Farm where both galls and beetles were relatively abundant. We randomly marked 96 galled
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plants with previous beetle damage, and the equal number of galled plants without beetle
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damage. We removed beetles from galls when present, and counted the number of beetles that
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colonized the plants after one week.
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The third experiment tested if the beetles are attracted to the presence of a beetle itself
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rather than the damaged plants. The experiment was conducted at the Brown Road site
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(42°28'58"N 76°27'19"W) using an outbreaking beetle population. We marked 100 galled
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plants, removed all beetles, and added one beetle back in half of the galled plants, leaving the
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other half of the plants without beetles. The recolonization (i.e. final number - initial number
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of beetles) was examined after a week. Galls that initially received a beetle but had no beetles
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after a week were removed from the analysis. Re-colonization probability in the second and
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third experiments was analyzed using a contingency table analysis.
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Feeding choice assays
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To test the hypothesis that beetles’ habitat selection is driven by plant quality, we conducted
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two types of feeding choice assays. We focused on local effects of gall midge because
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herbivore interactions occur on the rosette leaves rather than systemic leaves. First, we
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examined in situ choice—we selected 50 pairs of galled and ungalled plants that were
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growing next to each other at Whipple Farm, enclosed each pair together in a mesh sleeve
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bag, and allowed 3 beetles to feed freely for 4 days. At the end of the experiment, we removed
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the beetles, and counted the number of characteristic feeding holes on each plant.
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Because the in situ choice assays can be influenced by the architectural differences
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between galled and ungalled plants, we conducted a subsequent experiment in Petri dishes
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using detached leaves from the galled and ungalled plants. Initially, 20 pairs of galled and
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ungalled plants were bagged individually, and half of the pairs were damaged by adding 3
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beetles per bag for 4 days. The other half of the pairs without beetles served as controls. We
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collected 4 leaves per plant, placed the petioles in moist floral foam to keep the leaves fresh,
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and randomly paired leaves from galled and ungalled plants within each damage treatment in
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Petri dishes. One beetle was released per dish and allowed to feed for 36 hrs. The leaf area
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eaten was measured by scanning the leaf area before and after the beetle damage, and
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analysing leaf tissue loss with ImageJ (Schneider, Rasband & Eliceiri 2012).
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The number of holes in the in situ choice assay was analyzed using a generalized
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linear mixed model with plant type as a fixed effect, and bag as a random effect, with a
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Poisson distribution. In the Petri-dish experiment, leaf area eaten was log transformed to
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improve normality, and analyzed using a linear mixed model with plant type as a fixed effect,
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and a Petri dish within a plant as random effects. Here, the data for beetle-damaged and
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undamaged treatments were analysed separately because beetles were not given a choice
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between damaged and undamaged leaves.
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Plant chemical analyses
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To understand potential chemical mechanisms that explain beetle’s colonization behavior and
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host plant choice, we first measured leaf tissue chemistry of galled and ungalled leaves under
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the beetle damage treatments. We focused on two defence-related compound classes in S.
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altissima: Serine protease inhibitors (SPIs), and diterpene acids. SPIs are anti-digestive
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compounds that inhibit gut protease activity (Green & Ryan 1972). Solidago altissima is
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known to induce SPI activities in response to herbivory (Bode et al. 2013). Diterpene acids
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are secondary metabolites that are often correlated with plant resistance to multiple herbivores
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(Hull-Sanders et al. 2007; Uesugi et al. 2013). Leaf samples (one leaf per plant) were
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collected from the same plants used in the Petri-dish feeding assays. Leaves were flash frozen
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in liquid nitrogen, and stored at - 80°C.
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SPI analysis was conducted following methods described in Bode et al. (2013) using
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Synergy HT multi-detection microplate reader (Bio-Tek, Winooski, VT, USA). Approximately
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200 mg of fresh leaf samples were extracted in 1 mL of extraction buffer. Leaf protein content
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was quantified using Bradford assays (Sigma-Aldrich, St. Louis, MO, USA), and SPI activity
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was calculated by contrasting each sample’s inhibition of trypsin with a standard curve
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generated from Soybean trypsin inhibitor standards (Sigma-Aldrich, St. Louis, MO, USA).
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The SPI activity was expressed as mg SPI per mg total protein.
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Analysis for diterpene acids followed methods used in Uesugi et al. (2013). Leaf
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samples were extracted in 1 mL 90 % methanol, analyzed with high-performance liquid
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chromatography (HPLC; Keinanen, Oldham & Baldwin 2001) on an Agilent® 1100 series
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HPLC equipped with a Gemini C18 reverse-phase column (3μm, 150×4.6 mm, Phenomenex,
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Torrance, CA, U.S.A.). Three diterpene acids (acetoxykolavenic acid, unknown labdane
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diterpene acid, and kolavenic acid) were quantified at 230 nm, and the relative concentration
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of each compound was expressed as peak intensity relative to tissue fresh mass of each
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sample.
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Finally, plant emission of volatile organic compounds (VOCs) was measured because
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some specialist herbivores are known to use plant VOCs as cues for locating suitable plants
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(Loughrin, Potter & Hamilton-Kemp 1995; Halitschke et al. 2008). At Whipple Farm, we
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haphazardly selected seven pairs of galled and ungalled plants with no prior beetle
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colonization, and bagged individually with a fine mesh. Four pairs of plants received 3
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beetles, and the rest were kept untouched. VOCs were collected after 4 days from the
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headspace of leaves by enclosing the upper 15 cm of the plants in 500 mL polyethylene cups
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and pulling air through ORBO-32 charcoal adsorbent tubes (Supelco, Bellefonte, PA, USA),
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using a 12 V vacuum pump (GAST®, Gast Manufacturing Inc., Benton Harbor, MI, USA).
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Compounds bound to the ORBO-32 traps were desorbed with dichloromethane and samples
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were analysed by GC-MS (Kessler & Baldwin 2001) on a Varian Saturn 2200 GC/MS/MS
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with a CP-8400 Autosampler, using an Agilent J&W GC Column (DB-WAX FAME, 30m x
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0.25mm ID, DF=0.25). We integrated peak areas of individual compounds using selected ion
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chromatograms, and normalized by the area of the internal standard (tetraline, Sigma-Aldrich,
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St. Louis, MO, USA).
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We generated dissimilarity indices for VOCs and diterpene acids using the vegdist
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function in R composition, and compared them between plant types and damage treatments
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using PERMANOVAs (adonis function in R, Oksanen et al. 2011). This method is robust
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even when multiple variables are highly correlated, which is likely to be the case for these
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secondary metabolites. The above analysis showed significant plant type effects for both
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VOCs and diterpene acids (see Results). Thus, we further conducted a Random Forests
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analysis with package VarSelRF in R to extract a minimum set of compounds that best
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distinguish between the plant types (Ranganathan & Borges 2010). We calculated the
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percentage that the same set of predictor compounds appeared in 200 iterations (model
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frequency), the mean decrease in accuracy for removal of each compound from the model
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(MDA), and the prediction error of the best model (Ranganathan & Borges 2010). Finally, the
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total amount of VOCs, diterpene acids and SPIs were compared between plant types and
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damage treatment using ANOVA.
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Effects of beetle damage on the gall-midge performance
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To test how beetle feeding affects the performance of gall-midges, we manipulated levels of
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beetle damage on the galls at Whipple Farm. Fifty galled plants without prior beetle damage
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were bagged in July when gall midges were still at early larval stages, and half of those bags
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received six beetles each. After 8 weeks, we harvested galls and counted the number of larval
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chambers per gall, the number of aborted galls (galls with no larval chamber), and the number
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of parasitized larvae. The effect of beetle damage on the larval chamber count was analysed
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using a general linear model with a Poisson distribution; the probability of aborted galls was
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analysed with a contingency table; and the probability of parasitism was analysed using a
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general linear model with a binomial distribution. All statistical analyses were done with R (R
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v.3.1.1: R Foundation for Statistical Computing, Vienna).
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RESULTS:
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Field survey of beetle distribution pattern
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Across the four fields surveyed, both species richness (galled = 1.72 ± 0.92, ungalled = 1.17 ±
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0.87, F1,981 = 92.1, P < 0.0001) and arthropod abundance (galled = 2.55 ± 2.04, ungalled =
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1.54 ± 1.89, F1,981 = 63.9, P < 0.0001) were higher on galled than on ungalled plants.
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Accordingly, the Shannon’s diversity index was greater on galled than ungalled plants (F1,981
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= 67.6, P < 0.0001). Microrhopala vittata was almost exclusively found on galled plants (Χ2 =
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354.8, P < 0.0001, Fig 1a), with 40 - 80 % of the galled plants being colonized across the
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survey sites (colonization x plant type x site: Χ2 = 6.69, P = 0.08). Galled plants also
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harboured a higher density of predatory arthropods (i.e. spiders, predatory bugs, ants and
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lady-beetles) than ungalled plants in all (Χ2 > 20.2, P < 0.0001) but the Lab of Ornithology
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site (Χ2 = 0.6, P = 0.42).
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In total, 489 beetles were observed on 494 galled plants examined across the four
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sites. On those galled plants, we observed more plants with high numbers of beetles (> 4
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beetles per plant), as well as plants with no beetles, than expected from a random Poisson
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distribution (across sites: Χ2 = 39.4, P < 0.0001, Fig. 1b, Ludwig & Reynolds 1988). The test
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against negative binomial distribution revealed no difference between observed distribution
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(Χ2 = 13.4, df = 7, P = 0.063), suggesting that the beetle population exhibits clumped
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dispersion, but the level of clumping was relatively low (Green's Index = 0.001).
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Beetle preferences and plant induced responses
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We measured the residence time of beetles on galled and ungalled plants that had been
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damaged or undamaged by previous beetle colonizers. The number of beetles declined with
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time in all plants (time effect: z = -4.13, P < 0.0001), but beetles were likely to stay longer on
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galled than ungalled plants (plant type effect: z = 7.58, P < 0.0001, Fig. 2). Damage treatment
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alone did not affect the number of beetles remaining (damage treatment effect: z = -0.46, P =
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0.65), but beetle damage increased their residence time on galled plants but not on ungalled
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plants (plant type x damage treatment: z = 2.6, P = 0.01).
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To test if beetles are attracted to conspecific damage on galls, we estimated the re-
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colonization probability on damaged vs. undamaged galls. Only 19 out of 193 plants were re-
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colonized, but re-colonization occurred predominantly on previously damaged galls (Χ2 =
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4.13, P = 0.042). On beetle-damaged galls, however, an addition of a conspecific individual
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did not influence re-colonization rate (Χ2 = 0.16, P = 0.69), suggesting that beetles are
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attracted to the leaf damage rather than the presence of conspecific individuals.
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To test the hypothesis that galled plants are of higher quality for the beetle than
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ungalled plants, we conducted in situ feeding choice assays in the field. Here, we found that
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beetles preferentially feed on galled over ungalled plants (z = 7.6, P < 0.0001, Fig. 3a). In the
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Petri-dish assays with no prior damage, however, beetles tended to prefer ungalled over galled
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leaves (F1,30 = 3.9, P =0.057), but did not discriminate between the plant type when leaves
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were previously damaged (F1,30 = 0.14, P =0.70, Fig. 3b).
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Plant chemical analyses
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Serine protease inhibitor (SPI) activities did not differ between plant type (F1,35 = 0.6, P =
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0.44) or beetle damage treatment (F1,35 = 3.7, P = 0.062), but showed a significant interaction
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(gall x damage: F1,35 = 9.8, P = 0.0036, Fig 4a). In particular, SPI activity did not differ
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between plant types when plants were not damaged, but when damaged, ungalled plants
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induced SPI activities whereas galled plants did not, resulting in higher SPI activity in
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ungalled plants.
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Leaf diterpene composition differed between galled and ungalled plants (F1,31 = 3.4, P
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= 0.023), but beetle damage did not affect the composition (F1,31 = 0.9, P = 0.43) nor did it
357
interact with gall presence (F1,31 = 0.75, P = 0.52). The Random Forests analysis identified
358
unknown labdane diterpene acid and kolavenic acid as minimum set of predictor compounds
359
(model frequency = 61 %, MDA = 0.55 and 0.99, respectively). The bootstrap prediction error
360
for the model was 0.23. The total amount of leaf diterpenes tended to be higher in ungalled
361
than galled plants (F1,28 = 3.7, P = 0.064), but the beetle damage and its interaction with galls
362
did not affect total diterpenes (F1,28 < 0.9, P > 0.4, Fig. 4b).
363
Plant volatile analysis shows that galled and ungalled plants produced different blends
364
of VOCs (PERMANOVA: F1,13 = 6.79. P = 0.005), but the beetle damage did not affect the
365
volatile composition (F1,13 = 0.84. P = 0.47) nor did it interact with plant type (F1,13 = 1.6. P =
366
0.17). The Random Forests analysis identified a combination of three compounds, copaene,
367
benzophenone and β-pinene, as minimum VOCs that are needed to distinguish between the
368
plant type (model frequency of 63 %). The mean decrease in accuracy for removal of each
369
compound from the model (MDA) for these compounds was 0.57, 0.47 and 0.39, respectively
370
(Table S1). The bootstrap prediction error was 0.103. Interestingly, emission of both copaene
371
and β-pinene were higher in galled than in ungalled plants (F1,12 = >19.8, P < 0.0008), but
372
benzophenone was present only in ungalled plants.
373
Total VOCs was greater in galled than in ungalled plants (F1,10 = 10.2, P = 0.0095), but
374
beetle damage did not affect the total VOCs (F1,10 = 0.13, P = 0.7, Fig. 4a). Although the
375
interaction between plant type and beetle damage was not significant for total VOCs (F1,10 =
376
2.9, P = 0.11), a post hoc tests showed that difference in total VOCs between galled and
377
ungalled plants was significant only under damaged treatment (Post hoc test: Padj = 0.023, Fig
378
4c).
379
380
Effects of beetle damage on the gall-midge
381
Finally, we examined the reciprocal effect of beetle damage on gall-midge performance by
382
manipulating beetle damage on galled plants. We found that beetle damage had no effect on
383
the number of larval chambers within a gall (z = 0.11, P = 0.9), or on the proportion of galls
384
aborted (i.e. no larval chambers: Χ2 = 1.5, P = 0.22). Interestingly, beetle damage tended to
385
reduce the probability of larval parasitism (z = -1.8, P = 0.067).
386
387
DISCUSSION
388
Insect herbivores that function as ecosystem engineers can disproportionately alter the
389
arthropod community structure on their host plants by providing novel habitats and shelters
390
(Jones et al. 1994, Lill & Marquis 2007). Here, we explored whether the rosette gall-midge, a
391
microhabitat engineer, would further alter the chemical environment of the host plant, thereby
392
influencing the colonization behaviour of a common shelter-user, the beetle M. vittata.
393
394
Colonization pattern of beetles in field
395
Consistent with the previous study that suggested R. solidaginis as an ecosystem engineer
396
(Crawford et al 2007), we found that arthropod diversity was significantly greater on galled
397
than on ungalled plants. In particular, M. vittata beetles almost exclusively colonized galled
398
plants. Because we found that the abundance of potential predators was also greater on galled
399
than ungalled plants, the beetle’s preference for galled plants was not likely to be explained by
400
the presence of an enemy-free space (Price et al. 1980; Crawford et al. 2007). Although
401
weakly, beetles also showed clumped distributions among galled plants, suggesting a potential
402
additional advantage of being with conspecific beetles on a galled plant. Adult beetles have
403
relatively limited mobility (Zu Dohna 2006), but the observed colonization pattern suggests
404
that beetles are actively searching for galled plants, especially ones that have been previously
405
damaged by conspecific beetles.
406
407
Does the colonization pattern reflect beetle foraging preference?
408
We found that beetles are likely to stay longer on galled plants than on ungalled plants.
409
Furthermore, prior damage by conspecifics increased beetles’ residence time on galled plants,
410
but not on ungalled plants. This interaction between plant type and beetle damage suggests
411
that the gall-midge could modify plant’s response to beetle damage, thereby increasing its
412
attractiveness to the beetle. In support for this hypothesis, re-colonization experiments
413
suggested that beetles are attracted to the damage itself, rather than the presence of
414
conspecific beetles on galled plants.
415
Because adult beetles need to feed in the autumn until they hibernate, their habitat
416
selection should be, at least partially, driven by the quality of the plants that they colonize. In
417
the field assays where beetles chose between galled and ungalled plants, we found that beetles
418
fed more on galled plants. While the results support the hypothesis that galled plants are
419
higher in quality than ungalled plants, one alternative explanation may be that beetles prefer
420
to feed on plants with greater architectural complexity (Lawton 1983).
421
422
To eliminate the architectural differences between galled and ungalled plants, we
tested beetle choice using detached leaves in Petri dish. In the absence of prior beetle damage,
423
beetles tended to prefer ungalled over galled leaves—the opposite of what we observed in the
424
field experiment. Interestingly, such preference for ungalled leaves disappeared when leaves
425
were previously induced by beetle damage, suggesting that previous beetle damage could
426
improve the leaf quality on galled plants. Such induction of susceptibility in galled plants may
427
also explain the field assay results: plants in the field experiment are likely to be induced
428
strongly by the beetles as they fed for 4 days on intact test plants. Our results differed from
429
that of Crawford et al (2007), who showed that Spodoptera exigua did not discriminate
430
between galled and ungalled leaves when plants were not previously damaged. The lack of
431
response may suggest that non-native generalists, such as S. exigua that rarely occurs on S.
432
altissima, may not have evolved ability to detect differences in plant quality between galled
433
and ungalled plants (Bode & Kessler 2012). The discrepancy between studies also emphasizes
434
the fact that plant induced responses and the effect of induction can be herbivore species-
435
specific (Agrawal 2000; Uesugi et al. 2013).
436
437
Secondary metabolite changes underlying observed variation in beetle behaviour
438
Induced changes in the levels of defense-related leaf compounds partially explained the
439
feeding choice of the beetles. While SPI activities did not differ between galled and ungalled
440
plants, the plant type and beetle damage had an interacting effect on SPI activities, where
441
ungalled plants induced SPI activities while galled plants attenuated the induction in response
442
to the beetle damage. Thus, under beetle attack, galled plants have significantly lower SPI
443
activity than ungalled plants, presumably making galled plants better quality for the beetles
444
(Bode et al. 2013). These results are consistent with the pattern of SPI activity observed under
445
interactions between another galling insect (Eurosta solidaginis) and specialist external feeder
446
(Trirhabda virgata) on S. altissima (A. Hogan & A. Kessler, unpublished data).
447
Similar to SPIs, we found a trend that diterpene acid concentration was lower on
448
galled plants than ungalled plants when damaged by beetles (Fig. 4b), suggesting a possible
449
attenuation of the secondary metabolite induction in response to beetle damage. Together,
450
these chemical analyses support the idea that initial colonization by galled insects modifies
451
plant induced response to the secondary herbivores, making galled plants more attractive to
452
the subsequent colonizers than ungalled plants.
453
An interaction between the plant type and damage treatment was also observed in
454
VOC emission, suggesting that beetles may use VOCs as reliable cues when they forage
455
(Loughrin et al. 1995). We found that galled plants emitted distinctive blends of VOCs from
456
ungalled plants, with overall higher levels of total VOCs. Furthermore, there was an
457
interaction effect on total VOCs between beetle damage and plant type, suggesting that the
458
initial gall-midge colonization not only alters the constitutive levels of plant VOC emission,
459
but further increases VOC inducibility in response to beetle damage. While such interactive
460
effects of gall-makers and external feeders on volatile induction have been demonstrated
461
previously on S. altissima, its direction was inconsistent (Tooker et al. 2008): VOC induction
462
due to Heliothis virescens damage was suppressed in plants galled by Eurosta solidaginis, but
463
not in plants galled by Gnorimoschema gallaesolidaginis (Tooker et al. 2008).
464
Because we did not experimentally manipulate the presence of galls, but used
465
naturally galled and ungalled plants, it is possible that these plants are intrinsically different in
466
chemical properties, as gall-midges are found to preferentially colonize certain plant
467
genotypes (Crawford et al. 2007). However, we used pairs of galled and ungalled stems that
468
are growing in close proximity, which are most likely to be genetically identical. Moreover,
469
manipulative infestation by E. solidaginis strongly induced metabolic change in S. altissima
470
(Tooker et al. 2008). Together, we argue that the differences we observed between galled and
471
ungalled plants are likely to be the results of plant-induced responses to the gall-midge attack
472
rather than constitutive variation among plant genotypes.
473
How does the gall infestation modify plant responses to the beetle? Previous studies
474
have suggested that antagonistic interactions between phytohormones, salicylic acid (SA) and
475
jasmonic acid (JA), explain plant responses to sequential herbivory (Tooker et al. 2008;
476
Thaler, Humphrey & Whiteman 2012; Ali & Agrawal 2014). For example, the initial plant
477
colonization by an aphid induced SA in the common milkweed, Asclepius syriaca, which in
478
turn, suppressed JA induction when damaged by monarch caterpillars. This resulted in an
479
attenuation of cardenolides induction by the caterpillar in the presence of aphids (Ali &
480
Agrawal 2014). Similar SA suppression of JA responses has been found to underlie aphid
481
facilitation of caterpillar performance in other plant species (Rodriguez-Saona et al. 2010;
482
Soler et al. 2012; Stam et al. 2014). Infestation of S. altissima by E. solidaginis also lead to an
483
induction of SA, which seemed to suppress the emission of JA-dependent volatiles (Tooker et
484
al. 2008). Similarly, gall formation by an aphid Tetraneura nigriabdominalis was shown to
485
suppress responsiveness of galled tissue to JA, which then suppressed VOC emission from
486
galled leaves (Takei et al. 2015).
487
Our results, in contrast, show conflicting support for SA-JA antagonism hypothesis.
488
On one hand, we found that galled plants tended to suppress induction of SPIs and diterpene
489
acids upon beetle damage, supporting the hypothesis that JA responsiveness has been
490
compromised. On the other hand, galled plants had enhanced induction of total VOCs, which
491
goes against the hypothesis. Future analyses on JA and SA on galled vs. ungalled plants may
492
reveal whether an alteration in endogenous signalling could differentially affect defense
493
compound classes.
494
495
Alternative explanation may be that R. solidaginis induce production of “younger
leaves” (Larson & Whitham 1991). A congeneric gall-midge, Rhopalomyia yomogicola, is
496
known to secrete auxin and cytokinins into its host plants, which maintains fresh and nutritive
497
cell tissues as seen in younger leaves (Tanaka et al. 2013). These younger leaves induced by
498
the gall-midges are likely to be more plastic and responsive to herbivory than older leaves on
499
the ungalled stems (Van Dam et al. 2001). A previous study with larval M. vittata showed that
500
the beetles preferred and performed better on plants previously damaged by conspecifics than
501
heterospecifics, and attenuated induction of secondary metabolites, suggesting that the beetles
502
can manipulate plant response to its own benefit (Uesugi et al. 2013). If this is also the case
503
for the adult stage, it is possible that beetles may be better able to manipulate younger leaves
504
induced by R. solidaginis than older leaves on ungalled plants. Further investigation is
505
required to elucidate the mechanisms underlying the modification of induced responses by the
506
gall-midge.
507
While we presented evidence suggesting that gall-midge infestation coupled with
508
conspecific damage may increase plant quality for the beetles, a variation in leaf quality itself
509
only partially explains nearly exclusive use of galled plants over ungalled plants by the beetle.
510
Additional benefit of colonizing galled plants may be that they provide protection from harsh
511
environmental conditions (Kudo 1994; Larsson et al 1997; Fukui 2001; Danks 2002;
512
Crawford et al. 2007; Marquis & Lill 2007). The aggregation behavior in the fall may also
513
provide the ground for effectively locating mates when they emerge from hibernation (Zu
514
Dohna 2006). Overall, the plant phenotypic changes induced by the gall-midge seem to have
515
positive effect on the beetles, and may facilitate colonization and subsequent aggregation of
516
the beetles in the field.
517
518
Beetle damage affects performance of gall-midges
519
What is the consequence of the intense leaf damage by the beetles on the performance of gall-
520
midge itself? Previous studies on non-trophic interactions via ecosystem engineering suggest
521
that the benefit is unidirectional, i.e. engineers do not benefit from colonization of other
522
arthropods (Fukui 2001). We found that beetle damage had no effect on the number of larval
523
chambers within a gall or the probability that galls are aborted (i.e. galls with no midge
524
larvae). Thus, the beetle damage does not seem to negatively affect gall-midge growth and
525
development. Interestingly, however, we found that beetle damage tended to reduce parasitism
526
by Platygaster variabilis on gall-midges, suggesting that beetle damage could have a positive
527
effect on the galls. Because the parasitoid attack R. solidaginis eggs prior to gall formation
528
(Stireman et al. 2006), the probability that parasitoids attack the midge should not depend on
529
beetle damage. But the ability of the midge to encapsulate parasitoids may be affected by
530
beetle-induced changes in plant chemistry (Bukovinszky et al. 2009). Although mechanisms
531
behind such positive interactions need further investigations, our study suggests that the field
532
association of the two herbivores could be formed by plant-mediated facilitation.
533
534
Conclusion
535
By combining field observations, behavioural assays, and chemical analyses, we
536
demonstrated that R. solidaginis presence could strongly impact the colonization behaviour
537
and field distribution pattern of M. vittata, by not only altering host plant morphology, but
538
also by altering host quality and modifying plant induced responses to the beetle damage.
539
Although the current study focused on a paired interaction between the gall-midge and the
540
beetle, our field surveys and a previous work by Crawford et al. (2007) suggested that the
541
gall-midge, in addition of being an ecosystem engineer, is a keystone herbivore (Hunter 1992)
542
that has a disproportionate effect on the abundance of other members of the arthropod
543
community on S. altissima. Thus, we further suggest that plant-mediated interactions that
544
involve keystone herbivores, such as R. solidaginis, could have even stronger impact on
545
driving herbivore community dynamics and composition than previously considered
546
(Poelman & Kessler 2016).
547
548
ACKNOWLEDGEMENT
549
We thank Rayko Halitschke for his assistance with chemical analysis. This work was
550
supported with funds from the National Science Foundation (USA, NSF-IOS-0950225), the
551
National Institute of Food and Agriculture, U.S. Department of Agriculture (Hatch, 1004266),
552
and the School of Biological Sciences at Monash University.
553
554
DATA ACCESSIBILIT
555
Data deposited in the Dryad repository:
556
http://datadryad.org/resource/doi:10.5061/dryad.440n9
557
558
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FIGURE LEGENDS
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Figure 1: Colonization pattern of Microrhopala vittata in fields: a) Percent colonization of
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ungalled (gray bars) and galled (black bars) plants at Durland Bird Preserve (Durland),
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Cornell Lab of Ornithology (LabO), Liddell Field Station of Cornell University (Liddell), and
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Whipple Farm (Whipple). Asterisks indicate significance at > 0.0001 by χ2 tests. b) The
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frequency distribution of observed (black bars) and expected (hatched bars) number of M.
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vittata on a galled plant across the survey fields. We observed more plants with high number
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of beetles (> 4 beetles per plant), as well as those with no beetles, than expected from random
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distribution, suggesting that beetles show clumped dispersion (Χ2 = 39.4, P < 0.0001).
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Figure 2: The residence time of beetles released on ungalled (gray) and galled (black) plants
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under beetle damage treatments: undamaged (solid lines) and damaged (dashed lines).
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Number of beetles remaining was counted at 20, 44, 68, 87, 183, and 129 hours after beetles
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were released onto test plants (time: z = -4.13, P < 0.0001, plant type: z = 7.58, P < 0.0001,
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plant type x damage treatment: z = 2.6, P = 0.01).
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Figure 3: Beetle feeding choice between ungalled (gray bars) and galled (black bars) plants in
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a) in situ field assays measured as number of feeding holes, and b) Petri dish assays measured
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as leaf area eaten (cm2). Petri dish assays were conducted separately for leaves with and
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without previous damage. Asterisks indicate significant difference in feeding between
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ungalled and galled plants (*** < 0.0001).
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Figure 4: Variation in a) leaf SPI activity, b) total diterpene acids and c) total VOC emission in
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ungalled (gray bars) and galled (black bars) plants under undamaged control and beetle
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damage treatments. Letters above bars indicate significance in post hoc tests with Tukey’s
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HSD.