Virus infection influences host plant interactions with non-vector herbivores and predators

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1 Functional Ecology 2015, 29, doi: / Virus infection influences host plant interactions with non-vector herbivores and predators Kerry E. Mauck 1,2, Erica Smyers 1, Consuelo M. De Moraes 1,2 and Mark C. Mescher*,1,2 1 Department of Entomology, The Pennsylvania State University, University Park, Pennsylvania, USA; and 2 Department of Environmental Systems Science, ETH Z urich, Z urich 8092 Switzerland Summary 1. Viruses are widespread in both natural and agricultural plant communities and can significantly alter diverse traits of their host plants that mediate key interactions with other organisms. Yet, the impacts of plant viruses on broader community dynamics remain little studied. 2. Here, we explore the effects of Cucumber mosaic virus, a common non-persistently transmitted plant virus, on short- and long-term interactions of herbivorous and predatory insects with squash (Cucurbita pepo) plants in a weedy field setting, as well as virus-induced changes in plant phenotypes that mediate these interactions. Cucumber mosaic virus has previously been shown to have numerous effects on host plants that likely influence interactions with arthropods, including reduced plant size, increased volatile emissions, and diminished plant quality and palatability for aphid vectors. 3. Infection reduced the likelihood of many herbivorous insects arresting and feeding on plants, as well as the apparency of plants to herbivores that base in-flight foraging on visual cues. In particular, infection drastically reduced numbers of a specialist squash herbivore (Anasa tristis) on plants in the field a pattern likely driven by a reduction of phagostimulatory sugar levels in leaf tissue and concurrent increase in amino acid levels, as nymphal development was not obviously impacted by infection status. Relative to effects on herbivores, virus infection had little impact on the ability of predatory insects to locate aphid prey, although an experiment examining plant visitation in the absence of aphids revealed reduced numbers of foraging Syrphidae (Diptera) and Coccinellidae (Coleoptera) on infected plants but increased visitation and oviposition by Chrysopidae (Neuroptera). 4. CMV infection may reduce overall herbivore pressure on infected plants through effects on palatability and apparency, yet predators appear to locate herbivorous prey that do occur on infected plants as efficiently as those on healthy plants. 5. Virus infection can significantly influence plant interactions with the insect community (including non-vector as well as vector insects) with potential implications both for disease spread and for broader community dynamics. Key-words: Anasa tristis, Cucumber mosaic virus, Cucurbita pepo, herbivore preference, nonpersistent transmission, plant community ecology, plant viruses, predator prey interactions Introduction Plant viruses are near-ubiquitous components of natural and managed ecosystems (Jeger et al. 2004; Wisler & Norris 2005; Ng & Falk 2006; Power 2008; Roossinck 2010; Malmstrom, Melcher & Bosque-Perez 2011) that frequently modify host plant phenotypes, including plant traits that mediate interactions with insects and other organisms. For example, viruses can alter plant nutritional *Correspondence author. mescher@usys.ethz.ch quality (Blua, Perring & Madore 1994; Mauck, De Moraes & Mescher 2010; McMenemy et al. 2012), as well as defence responses against insect herbivores or other pathogens (Lewsey et al. 2010; Zhang et al. 2012; Zhou & Zhou 2012). Virus infection can also alter plant-derived olfactory and visual cues that mediate important ecological interactions among plants and insects (Ajayi & Dewar 1983; Eigenbrode et al. 2002; Jimenez-Martınez et al. 2004a; Srinivasan et al. 2006; Medina-Ortega et al. 2009; Werner et al. 2009; Mauck, De Moraes & Mescher 2010; McMenemy et al. 2012; Shapiro et al. 2012). Because of their 2014 The Authors. Functional Ecology 2014 British Ecological Society

2 Virus infection changes plant insect interactions 663 prevalence and often significant impacts on plant phenotypes, viruses may be expected to strongly influence broader community interactions, with significant implications for the population dynamics and fitness of interacting organisms (Castle & Berger 1993; Christiansen-Weniger, Powell & Hardie 1998; Eigenbrode et al. 2002; Jimenez- Martınez et al. 2004b; Culver & Padmanabhan 2007; Mauck, De Moraes & Mescher 2010; Alexander et al. 2013). Yet, to date, research examining virus effects on plant insect interactions has focused almost exclusively on implications for virus transmission by insect vectors (reviewed in Mauck et al. 2012), while viruses have received little attention in the broader literature addressing plant community ecology and plant herbivore natural enemy interactions. For example, a recent review exploring plant pathogen effects on arthropod communities included only 11 references explicitly focused on plant viruses (out of a total of 134), with the remainder focused largely on fungal or bacterial pathogens (Tack & Dicke 2013). Consequently, there is a clear need for more work addressing the broader effects of plant virus infections on multi-trophic interactions among plants, herbivores and predators in a community context (Malmstrom, Melcher & Bosque-Perez 2011). Plant viruses may be expected to influence plant insect interactions through effects on both host plant quality for (vector and non-vector) insect herbivores and via effects on the visual and olfactory cues that guide foraging by herbivores and their natural enemies. Some viruses have been reported to enhance host plant quality for insect vectors, so that they more readily settle and feed on infected plants and reproduce at higher rates (Montllor & Gildow 1986; Castle & Berger 1993; Musser et al. 2003; Jimenez- Martınez et al. 2004b; Alvarez et al. 2007; reviewed in Mauck et al. 2012; Mauck, De Moraes & Mescher 2014b), while others decrease host quality, favouring rapid vector dispersal from infected plants (Donaldson & Gratton 2007; Mauck, De Moraes & Mescher 2010, 2014a,b). Either effect can potentially be conducive to virus spread, depending on the mode of transmission (Mauck et al. 2012). A number of studies have also demonstrated vector attraction to virus-induced changes in plant visual cues (e.g. leaf yellowing) (Ajayi & Dewar 1983; D oring & Chittka 2007) or to quantitative and/or qualitative changes in plant volatile emissions (Eigenbrode et al. 2002; Jimenez- Martınez et al. 2004a; Srinivasan et al. 2006; Medina-Ortega et al. 2009; Werner et al. 2009; Mauck, De Moraes & Mescher 2010; McMenemy et al. 2012). The latter are known to serve as key foraging cues for both insect herbivores and their natural enemies (Turlings, Tumlinson & Lewis 1990; De Moraes et al. 1998; De Moraes, Mescher & Tumlinson 2001; Kariyat et al. 2012, 2013). But, little work has yet explored the implications of virus-induced changes in such plant traits for interactions with nonvector insects. Some research efforts focused on vectorborne transmission have considered multi-tropic interactions involving predators. For example, natural enemies of aphids the primary vectors of many plant viruses can potentially influence virus transmission to new hosts through impacts on aphid behaviour and dispersal (Chau & Mackauer 1997; Dader et al. 2012). Several studies have reported increased rates of virus transmission by vectors when predators or parasitoids were introduced into mesocosms containing infected and susceptible host plants and aphids (Christiansen-Weniger, Powell & Hardie 1998; Smyrnioudis et al. 2001; Hodge & Powell 2008; Hodge, Hardie & Powell 2011; Jeger et al. 2011; Dader et al. 2012). And other studies have found virus effects on parasitoid fitness, mediated by direct effects of virus circulation within vectors (Christiansen-Weniger, Powell & Hardie 1998), which could potentially feed back to influence both virus transmission and top-down regulation of vector populations. The current study extends our limited knowledge of virus effects on the ecology of host plant interactions with the broader insect community by examining the influence of a widespread plant virus, Cucumber mosaic virus (CMV), on the interactions of its host plant, squash (Cucurbita pepo L.), with the insect community including non-vector herbivores and predators in a semi-natural field setting. In previous work, we have shown that CMV has significant impacts on host plant quality for and attractiveness to aphid vectors (Mauck, De Moraes & Mescher 2010, 2014a,b). Specifically, we observed that CMV elicits an overall elevation of host plant volatile emissions that enhances aphid attraction to infected plants, but decreases host plant quality for aphids, leading to rapid aphid dispersal following virus acquisition (Mauck, De Moraes & Mescher 2010) an overall pattern that appears conducive to the transmission of this non-persistently transmitted virus (Mauck, De Moraes & Mescher 2010; Mauck et al. 2012). Here, we report new findings that address the following community-level questions: (i) How do the combined effects of CMV infection and aphid damage influence the attraction of herbivorous and predatory insects to hosts in the field? (ii) What are the effects of CMV infection on colonization (settling, feeding or oviposition) of plants by naturally occurring non-vector herbivores and predators? (iii) How does CMV infection influence colonization and survival of an important specialist herbivore: the squash bug Anasa tristis De Geer? and (iv) How does CMV infection alter plant-derived cues mediating interactions with generalist and specialist herbivores? Materials and methods CULTURE OF PLANTS AND VIRUS INOCULATIONS Squash plants (Cucurbita pepo cv. Dixie, Willhite Seeds Inc.) were grown in square pots (12 cm 3 ) in autoclaved ProMix potting soil containing 5 g of slow-release fertilizer (Osmocote N-P-K) and trace micronutrients (Scott s Micromax) in an insect-free walk-in growth chamber with a 16 : 8 light : dark

3 664 K. E. Mauck et al. photoperiod (23 C day and 21 C night). Plants at the cotyledon stage were mechanically inoculated with the equivalent of 5 cm 2 of frozen stock tissue infected with CMV-FNY (stored at 80 C) using established protocols (Walkey 1991) (details in Appendix S1, Supporting information). Plants used in the trapping field experiment were transplanted to 35 L pots and given an additional 3 g of Osmocote fertilizer one week before use (when plants were approximately 2 weeks old). Plants used in the observation field experiment were transplanted from the 12 cm 3 square pots directly into field plots. TRAPPING FIELD EXPERIMENT This experiment was designed to trap insects visiting CMVinfected and healthy plants with and without aphid colonies under semi-natural field conditions (infection treatment x aphid damage treatment). CMV is endemic in the area where this experiment was performed, but to avoid additional virus spread, the experiment was kept small and performed within a field planted primarily with alfalfa, which is not readily infected by CMV. In early spring 2009, a 60 m 9 30 m plot was marked out in a field at the Russell E. Larson Agricultural Research Farm in Rock Springs, PA. Alfalfa was seeded in the area around this plot, and weeds (grasses and forbs) from the seed bank and field edge were allowed to colonize among the alfalfa plants. In the first week of July, 35- week-old squash plants in pots (20 CMV-infected plants and 20 healthy plants) were randomly placed into the plot. Plants were placed 1 m apart to isolate them from neighbours and deter interplant movement of aphids. Plants were watered every other day, except following rainfall. Three days after establishment of the plot, ~150 apterous Aphis gossypii per plant (mix of instars) were placed on half of the infected plants and half of the healthy plants, yielding 10 plants per virus x aphid treatment (aphid rearing protocol presented in Appendix S1). Aphids were allowed to establish for several days prior to the start of sampling. Insects were sampled from plants using clear plastic water pan traps (20 cm long 9 12 cm wide 9 10 cm deep) (Fig. S1, Supporting information illustrates the trapping design). These traps are effective at sampling very small herbivores that are weak flyers (e.g. generalist winged aphids) based largely on visual cues and for sampling a wide range of stronger flyers responding to both visual and odour-based cues (other hemipterans, parasitoids and smallto medium-sized predators) (Duelli, Obrist & Schmatz 1999). The first three-day trapping period began on July 11 and the second on July 21. Plants and their associated insects were harvested after the second round of trapping by pulling a previously positioned mesh bag from the base of the stem up over the canopy and freezing the entire plant in the bag to allow later identification of colonizing insects. Healthy plants were monitored visually for symptoms throughout the experiment to ensure that they did not become infected with CMV or other viruses. Insects were identified to order and to family where possible. Insects that could be identified as predators and herbivores were further classified into functional groups based on feeding style and phylogeny (details in Appendix S1). OBSERVATION FIELD EXPERIMENT A second field experiment was performed in 2010 to monitor insect interactions with healthy and infected plants over a longer period of time (without insect removal due to trapping). For this experiment, two transects were set up in a field of second-year alfalfa mixed with annual and perennial forbs (in a different area of the same field used for the 2009 trapping experiment). Every 3 m along each transect, we cleared a 025-m plot and planted a 25-week-old squash plant (June 15 16) that was either healthy (mock inoculated), or infected with CMV (40 plants per treatment). Plants were assigned randomly to positions along the transect and watered during establishment. Starting on June 26, all observable insects present on each plant were counted between 9:00am and 2:00 pm once every 7 10 days, with the last census on August 8 (the time between censuses varied slightly to ensure that observations were always made on clear, sunny days with little wind). Insects were identified in the field as predators, herbivores or others and further placed into phylogeny-based feeding guilds where possible (details in Appendix S1). Squash bugs (Anasa tristis) occurred only towards the end of the season (in very large numbers, but with a patchy distribution) and were analysed separately, as this species is a key specialist herbivore of squash responsible for most of the insect-caused squash plant mortality in the field. SQUASH BUG LABORATORY EXPERIMENTS To further explore how CMV influences interactions between squash and its specialist herbivore Anasa tristis, we performed oviposition choice tests and nymph growth experiments in the laboratory (squash bug rearing protocol presented in Appendix S1). Oviposition choice tests were conducted with paired healthy and infected 35-week-old plants (nine pairs). Equal amounts of leaf tissue from each plant were enclosed at opposite ends of a fine mesh sleeve with drawstring ends (25-cm long). Two similarly aged gravid female bugs were released in each sleeve, and the ends were drawn closed. Two males were also included in each sleeve to ensure further mating opportunities were available. Bugs were allowed to oviposit for 5 days after which time the total number of eggs laid on the enclosed leaves of each treatment were counted. One test was excluded because no eggs were laid. Nymph growth experiments were performed by establishing populations of first-instar squash bug nymphs on 35-weekold plants (three infected plants and three healthy plants). Experiments were conducted in this way because bugs are naturally gregarious as nymphs (usually occurring as groups of 15 30) and are also highly mobile, feeding on many parts of the plant during development. In pilot experiments, placing single bugs in small clip cages resulted in near 100% mortality regardless of host plant infection treatment. Nymphs used were all from the same generation and came from a pool of eggs laid by a group of ~15 female bugs collected from the main colony. Each infested plant was placed in its own pop-up mesh cage (30 cm 9 30 cm 9 30 cm Bioquip), and the cages were housed in a walk-in Conviron growth chamber (16L:8D, 24 C day, 22 C night, 50 60% RH). In previous work with A. tristis nymphs repeated handling increased bug mortality, so we assessed survival only at the end of the experiment (5 weeks), when most bugs had progressed to the final instars but not yet matured. Surviving bugs were weighed at 5 weeks and placed into one of five possible instar categories based on morphology. The number of bugs surviving the experiment was calculated, and the distribution of surviving bugs was compared between the two treatments. MEASUREMENT OF VISUAL AND NUTRITIONAL CUES Visual cues presented to flying insects were assessed for CMVinfected and healthy plants using top-down photography under controlled lighting conditions in a growth chamber with incandescent and fluorescent lighting (Fig. S2, Supporting information). Plants received either CMV inoculation or mock inoculation at the cotyledon stage as described above (10 plants per treatment). They were photographed when they were 35 weeks old, using a Canon EOS 5D Mark III positioned on a tripod approximately 125 m above the floor with the lens facing directly down and

4 Virus infection changes plant insect interactions 665 fixed, so that each image had an identical field of view (details in Appendix S1). ADOBE PHOTOSHOP CS5 software was used to quantify the total exposed leaf surface area and the mean red, green and blue (RGB) colour components of the leaf surface on a scale of (as in Salvaudon, De Moraes & Mescher 2013). To examine virus and herbivore-induced changes in plant nutrients, we performed a factorial laboratory experiment examining the combined effects of CMV infection and squash bug damage on levels of free amino acids and simple carbohydrates (sugars) in C. pepo. Plants were inoculated with CMV, or mock inoculated, as described above. After two weeks, the two lower leaves of each plant were enclosed in a mesh cage with draw-string closures on either end (following Biernacki & Lovett-Doust 2002). Half the plants in each infection treatment received three late-instar squash bugs (3rd or 4th instar); the other half received empty cages only. Sample sizes were 10 plants per infection x squash bug treatment. Bugs were allowed to feed for 20 days, a period found to yield host plant developmental shifts in another Cucurbit host (Biernacki & Lovett-Doust 2002). At the conclusion of this period, we harvested tissue discs from the most recent fully expanded leaf of each plant using a cork borer, weighed the discs, and immediately placed them into 2-mL Eppendorf tubes that were then flash-frozen in liquid nitrogen. All discs were harvested between 10am and 2 pm. Samples were stored for a short time at 80 C then ground to a fine powder under cryogenic conditions using a Genogrinder (SPEX Sample Prep). One sample was lost from each treatment due to cap failure during grinding, resulting in a final sample size of nine per infection 9 squash bug treatment. Free amino acids and simple sugars (glucose, fructose and sucrose) were analysed in each sample using the methods of Lisec et al. (2006). Blanks (without leaf tissue) were also taken through the procedure. All chemicals used were obtained from Sigma-Aldrich (HPLC or derivatization grade or higher), and all water used was distilled, deionized and filtered through a Millipore water sterilization system. Details of the gas chromatography and mass spectrometry instrument configurations used to quantify the compounds of interest can be found in Appendix S1. Chromatograms were analysed using CHEMSTATION software Ó 2003, Agilent Technologies (Santa Clara, CA, USA). Peak areas for leaf samples were converted to microgram amounts relative to the known amount of internal standard added to each sample, then corrected for the weight of leaf tissue collected. If derivatization produced more than one peak for a compound (verified by standards), the amounts for the two products were summed. treatment x infection status interaction (statistical results in Table S1). Infected plants attracted fewer weak flying herbivores (e.g. aphids) via water pan traps (Fig. 1a). Other herbivores that were both trapped in pan traps and harvested from whole plant samples (providing access to contact cues that further mediate the insect s decision to remain on the plant or disperse) showed few differences based on infection status (Fig. 1a). Most predator groups did not show a clear pattern of response to either infection status or aphid treatment, but predatory Diptera that preferentially feed on aphids (Aphidoletes spp. and Leucopis spp.) responded strongly to the presence or the absence of aphids across infection treatment (Fig. 1b). Coccinellid beetles also showed a marginally insignificant attraction to aphid-infested plants (Fig. 1b). Results TRAPPING FIELD EXPERIMENT Data were analysed by two separate MANOVAs with infection treatment, aphid treatment and the interaction as terms and the herbivore or predator groups described in the Appendix S1 as the variables (rank transformed) (Minitab v. 14). Univariate tests (Mann Whitney) were performed on the untransformed values of each variable using a reduced model including only significant terms in the MANOVA (Minitab v. 14). Infection treatment influenced overall herbivore abundance, while the presence of aphids did not influence visitation by subsequent herbivores, and there was no significant interaction between aphid treatment and infection status (statistical results in Table S1, Supporting information). Conversely, predator abundance was not influenced by infection status, but was influenced by the presence of aphids, again with no significant aphid Fig. 1. Insect group abundances in the trapping experiment. Means SE for visual reference of herbivore groups trapped or collected from whole plant samples (due to the presence of an aphid treatment on some plants, winged aphids were only counted from pan traps). * indicates a significant difference in the mean ranks of herbivores per individual plant between the two infection treatments [for aphids, W(37) = 2635, P = 0001] (aphid prey treatment had no effect on herbivore abundance, see Table S1). Mean SE for visual reference of predator groups trapped or collected from whole plant samples. * indicates significant difference in the mean ranks of herbivores per individual plant between the two aphid prey treatments [for predatory dipterans, W (37) = 237, P = 0000] (infection had no effect on predator abundance, see Table S1).

5 666 K. E. Mauck et al. OBSERVATION FIELD EXPERIMENT AND SQUASH BUG LABORATORY EXPERIMENTS Per-plant totals for herbivores and predators at each census point were rank transformed and analysed using repeated measures ANOVAs with infection status as a fixed factor, census date as a random factor (six dates) and their interaction. Additionally, the total number of visitors from each insect group was summed across all of the censuses (as in Gange 1995) and analysed with infection status as the factor using Mann Whitney tests. Squash bugs were analysed only for plants that became infested (Mann Whitney tests for eggs laid and nymphs present with infection as the factor) (Minitab v. 14). Herbivores were responsive to infection status across the experiment, with higher numbers observed on healthy plants relative to virus-infected plants (statistical results in Table S2, Supporting information, Fig. 2a). In contrast, predators did not respond strongly to infection status (statistical results in Table S2, Fig. 2b). Both groups varied in abundance by date (Table S2), and the only time that herbivore abundance on infected plants approached that on healthy plants was near the conclusion of the experiment possibly due to healthy (but not infected) plants succumbing to squash bug damage or contracting powdery mildew disease (August 8 census) (Fig. 2a). All herbivore groups preferred to settle or feed on healthy plants over infected plants (Figs 3a and S3, Supporting information). Coccinellids, syrphids and the mixed other predatory insect group also showed a slight preference for healthy plants over infected plants (Figs 3b and S4, Supporting information). However, Chrysopidae preferred infected plants as settling and oviposition sites over healthy plants, especially during the early censuses (Figs 3b and S4). Squash bugs appeared on plants in substantial numbers only during the final two weeks of the experiment. While the overall proportion of plants infested with eggs or nymphs for each treatment was the same (11 infected and 11 healthy plants), adults laid more eggs on healthy than infected plants ( on healthy vs on a single infected plant) and nymphs also preferred to Fig. 2. Total insects observed on plants by census and infection status. Mean total herbivores SE on infected and healthy plants at each census. Mean total predators SE on infected and healthy plants at each census. Dates of each census are indicated on the x-axis. N = 39 plants per treatment after accounting for initial plant mortality. Statistics are in Table S2. Fig. 3. Major insect community groups observed on plants across all censuses. Mean SE for visual reference of herbivore groups observed on plants across all censuses. * indicates significant difference in the mean ranks of herbivores per individual plant between the two infection treatments (for Cicadellidae, W (76) = 76, P = 0000; for Miridae, W(76) = 1714, P = 0033; for other herbivores, W(76) = 1726, P = 0015). Mean SE for visual reference of predator groups observed on plants across all censuses. * indicates significant difference in the mean ranks of predators per individual plant between the two infection treatments [for Chrysopidae, W(76) = 13075, P = 005; for Syrphidae, W(76) = 1651, P = 0029; for Coccinellidae, W(76) = 1703, P = 0014; for other predators, W(76), P = 0015].

6 Virus infection changes plant insect interactions 667 reside and feed on healthy plants ( on healthy vs on infected, W (17) = 1355, P = 0004). A laboratory experiment with controlled oviposition tests revealed the same pattern, with gravid females depositing more eggs on healthy plants (307 total eggs) and generally avoiding oviposition on infected plants (20 total eggs deposited only on one plant) (analysis by chi-squared test, d.f. = 1, v 2 = 25189, P = 00001). Despite these clear preferences, experiments conducted with squash bug nymphs under controlled conditions in a growth chamber produced little evidence of detrimental effects of host plant infection on squash bug development. Bugs did not exhibit lower rates of survival on infected plants (43 of 71 bugs surviving across all infected plants and 44 of 71 bugs surviving across all healthy plants) (d.f. = 1, v 2 = 0011, P = 0915). There were slightly fewer bugs in the 4th and 5th instar categories on infected plants by 5 weeks, while a number of bugs on the healthy plants appeared to be arrested in the 2nd instar stage, with only one 2nd instar occurring on an infected plant (accounting for the marginal chi-squared value in a multi-way chi-squared test, Fig. 4a). Bugs in the 3rd and 5th instars did not weigh less on the infected relative to healthy treatments, but those in the 4th instar did weigh significantly less on the infected treatment (Fig. 4b). Repetition of this experiment using slightly modified methods (described in Appendix S1) yielded similar results, with no differences in survival, instar progression or weights within instars (Fig. S5, Supporting information). MEASUREMENT OF VISUAL AND NUTRITIONAL CUES Surface area data were analysed using a 2-sample t-test, and RGB components were log-transformed and used as variables in a MANOVA with infection status as a fixed effect (as in Salvaudon, De Moraes & Mescher 2013) and post hoc univariate tests. Glucose, fructose, sucrose and carbohydrate to amino acid ratios were log-transformed and analysed by ANOVA with infection status, damage treatment and their interaction term in the model. Amino acid composition was analysed using principal component analysis (PCA; with each amino acid as a variable), followed by ANOVA (with terms as described above) using rank-transformed scores (newly generated orthogonal data values) for components 1 and 2 (MINITAB v. 14). The MANOVA was significant for infection treatment (Wilks k = 0113, F = 4205, d.f. = 0188, P = 0000), with infected plants having significantly more of the red component (F = 786, d.f. = 1, P = 0012), and a marginally increased level of the green component (F = 318, d.f. = 1, P = 009) (Fig. 5a). These two components are responsible for the characteristic yellowing observed in virus-infected plants. Healthy plants had significantly more exposed surface area in top-down images relative to infected plants (Fig. 5b). Although we did not quantify plant size in the field, uniform discrepancies in size between infected and healthy plants were observed throughout all experiments. Infection reduced fructose and sucrose levels, and bug Fig. 4. Patterns of growth among different squash bug instars by infection treatment. Number of bugs in each instar at 5 weeks post-infestation pooled across host plants within each treatment (overall distributions did not differ significantly by multi-way chisquare, d.f. = 3, v 2 = 7210, P = 0065). Mean bug weight within each instar SE. Since only one bug was in the second instar for the infected treatment, statistical analysis was not possible for this group. Among instar categories, only 4th instars on infected plants weighed significantly less than 4th instars on healthy plants [W(19) = 89, P = 0003, indicated by *], while 3rd and 5th instars did not differ (P > 005). Sample sizes for each instar weight comparison are displayed in. damage significantly increased levels of glucose in leaves regardless of infection status (statistical results in Table S3, Supporting information, Fig. 6a). The ANOVA for ratios of total sugars to total amino acids was significant for all terms, with infected plants having lower ratios relative to healthy plants regardless of bug damage, and healthy plants having higher ratios for bug-damaged plants relative to undamaged plants (statistical results in Table S3, Fig. 6b). Amino acid compositions differed between infected plants and healthy plants along the main axis, PC1 (significant infection term in the ANOVA, F = 9534, d.f. = 1, P = 0000), and between bug-damaged and undamaged plants along PC2 (significant damage term in the ANOVA, F = 638, d.f. = 1, P = 0017) (Fig. 7). However, PC2 only explains 122% of the variation in amino acid composition, so bug effects are slight compared to the effects of virus infection (PC1 explains 648% of variation).

7 668 K. E. Mauck et al. This pattern is likely driven by much higher concentrations of most amino acids in virus-infected plants (Fig. S6). Discussion Our results indicate that CMV infection alters the host plant phenotype both visually and chemically and that these changes influence plant interactions with vector and non-vector insects. In the trapping experiment, aphids exhibited reduced attraction to CMV-infected plants (Fig. 1a), a preference likely based on the reduced visual apparency of CMV-infected hosts (Fig. 5). In the observation experiment, some insects were attracted to infected and healthy plants at relatively equal rates (Fig. 1a) but tended not to settle on virus-infected plants when permitted unimpeded plant contact (Table S2, Figs 2a and 3a), a preference likely driven by altered ratios of carbohydrates to amino acids in CMV-infected leaf tissue (Fig. 6). In particular, the specialist herbivore A. tristis exhibited a strong preference for oviposition and feeding on healthy vs. virusinfected plants even though squash bug offspring performed as well on CMV-infected plants as on healthy hosts in laboratory experiments (Figs 4 and S5). In experiments in which aphid colonies were placed on infected and healthy plants, predators and parasitoids appeared largely indifferent to infection status and responded primarily to the presence of aphid prey (particularly predators which rely heavily on aphids as part of their diet) (Table S1, Fig. 1b). In the absence of aphid prey, the observation experiment indicated that Chrysopidae prefer to visit and oviposit on CMV-infected plants, while several other predators showed reduced foraging on CMV-infected plants (Fig. 3b). These patterns of insect visitation and colonization, along with virus- and herbivore-induced changes in plant phenotype, have implications for the fitness of the pathogen and its host plant, as well as the abundance and behaviour of generalist and specialist insects. IMPLICATIONS OF HERBIVORE INTERACTIONS WITH INFECTED HOSTS Non-vector herbivores were less likely to visit or colonize CMV-infected hosts (Figs 1a, 2a and 3a). In particular, squash bugs did not seem to recognize CMV-infected plants as suitable oviposition sites in either the laboratory or the field. In line with these observed preferences, phagostimulatory sugars (fructose and sucrose) and sugar to amino acid ratios were significantly reduced in infected plants (Fig. 6). Sugars are universal feeding stimulants (Chapman 2003), and squash bugs engage in extensive tasting of cell sap with the stylet prior to feeding in order to identify suitable hosts (Cook & Neal 1999). The ratio of sugars to amino acids is also a major determinant of feeding propensity for piercing sucking insects, with ratios above 5 : 1 being preferred (Mittler 1967; Abisgold, Simpson & Douglas 1994), and this ratio was also significantly lower in virus-infected plants (Fig. 6). Squash bug feeding did little to alter nutrient levels or ratios in infected plants (although effects were observed in healthy plants), which suggests that bug feeding does not induce shifts in nutrient production or partitioning that would improve the palatability or quality of infected hosts. However, infected plants did have higher levels of free amino acids (Figs 7 and S6, Supporting information). Insect herbivores must obtain adequate levels of amino acids from host plants, either in a free form or from breakdown of plant proteins (Behmer 2008); thus, for insects that obtain nutrients from non-vascular leaf tissue, infected plants may actually provide sufficient nutrition if insects can overcome the unpalatability of reduced sugar levels. This is consistent with what we observed in our no-choice performance assays, where infection status had no effect on A. tristis nymph development or survival. In addition to effects on nutritional cues, we recently reported that CMV infection of C. pepo causes the upregulation of certain plant defence hormones (salicylic acid, ethylene), while reducing the responsiveness of induced defence pathways regulated by jasmonic acid (Mauck, De Moraes & Mescher 2014a). Both salicylic acid and jasmonic acid regulate defence responses to herbivorous insects. For instance, herbivores that are sensitive to jasmonic acidinduced defences (many chewing Lepidoptera) may perform better on CMV-infected plants, while those that are sensitive to ethylene- or salicylic acid-induced defences may find CMV-infected plants less suitable (some piercing sucking insects) (Anstead et al. 2010; Avila et al. 2012; Thaler, Fig. 5. Colour and leaf area analysis of infected and healthy squash. Mean SE of average red, green and blue components in infected and healthy plants (N = 10 per treatment). A MANOVA including all three colour variables (log-transformed) is significant for infection status (Wilks k = , F = 42053, d.f. = 01875, P = 0000), with a significant ANOVA (indicated by *) for the red component (F = 786, d.f. = 1, P = 0012). Mean SE of leaf surface area of infected and healthy plants from top-down view. *indicates significant difference by t-test (T = 1606, d.f. = 17, P = 0000).

8 Virus infection changes plant insect interactions 669 Fig. 6. Carbohydrate levels and carbohydrate to amino acid ratios. Mean SE for fructose, glucose and sucrose levels (N = 18 plants per infection treatment, 18 plants per bug damage treatment). * indicates significant difference at P < 005 and treatments on the X-axis correspond to significant terms in the overall ANOVA (see Table S3 for statistics). Comparisons not displayed were not significantly different. Mean SE for total sugar to total amino acid ratios for the significant interaction term in the ANOVA (Table S3) (N = 9 plants per infection x bug damage treatment). Different letters indicate significant differences among treatments at P < 0001 according to post hoc Tukey s tests. Humphrey & Whiteman 2012). Thus, patterns of herbivore colonization in our field and laboratory experiments may be influenced in part by the perception of and response to plant defences that are altered by CMV infection. The rejection of infected C. pepo plants by generalist and specialist herbivores might be expected to have significant implications for plant fitness. For instance, squash bug feeding often results in plant mortality, as these insects inject salivary proteins into plant tissues that disrupt cellular osmotic balance, causing the expulsion of nutrients and water (Neal 1993; Miles & Taylor 1994). In our observation experiment, during the period between the July 30 and August 8 censuses (when squash bugs appeared in the plot), 10 healthy plants died due to what appeared to be squash bug damage or interactive effects of squash bug feeding and powdery mildew infection. Only one CMVinfected plant died during the same time period, and CMV-infected plants also exhibited reduced severity of powdery mildew infection (personal observation). Thus, even though plants infected with CMV may experience reduced fruit output and pathological effects on seed development, they may escape a number of additional stressors that can remove reproductive output entirely by killing the plant (Xu et al. 2008). If infected host plants escape mortality (or extensive tissue damage) due to herbivory, they will persist longer in the landscape, increasing their likelihood of serving as inoculum sources for new infections. At the community level, under herbivore pressure and other stresses, such as drought (e.g. Xu et al. 2008), infected plants might be expected to increase in frequency over time, so that vectors would be forced to visit infected hosts more often, which we have observed for CMV-infected C. pepo in previous field experiments (Mauck, De Moraes & Mescher 2010). An increase in the proportion of infected hosts in a given area may be particularly important for CMV spread, since our data suggest that CMV-infected plants are less visually apparent than healthy plants (Fig. 1a, Fig. 5b). Infected plants are slightly more yellow than healthy plants (Fig. 5a), which may counteract some of the effects of reduced plant size on the attraction of aphids (D oring & Chittka 2007) and Cicadellidae (Chu et al. 2000; Rodriguez-Saona, Byers & Schiffhauer 2012) both of which are attracted to yellow. The water pan traps we used are effective at catching aphids descending from the air column above a plant via dropping behaviour elicited in response to the visual stimulus of green foliage against a soil background (D oring et al. 2004). However, aphids are generally not attracted to plants via volatile cues at this range due to an inability to orient while in flight (Nottingham & Hardie 1993; Goldansaz & McNeil 2006; reviewed in Powell, Tosh & Hardie 2006; Webster 2012). In contrast, both alate and apterous aphids make extensive use of volatile foraging cues when walking (e.g. Nottingham et al. 1991; Goldansaz & McNeil 2006; Webster et al. 2008; Medina- Ortega et al. 2009; reviewed in Webster 2012), and we have previously documented the attraction of walking aphid vectors to the elevated odours of CMV-infected plants (Mauck, De Moraes & Mescher 2010, 2014b). Since infection by CMV makes the host plant less visually apparent (Fig. 5b), the virus-induced elevation of odour cues that are attractive to walking aphids may have even more significance than previously thought (Mauck, De Moraes & Mescher 2010; reviewed in Mauck et al. 2012). Changes in host plant size and appearance are often unavoidable consequences of virus effects on host physiology or the host defence response (Wang & Metzlaff 2005). However, the negative effects of virus infection on plant size may be

9 670 K. E. Mauck et al. Fig. 7. Principal component analysis of amino acids in infected and healthy plants. PCA output showing a scatterplot of component 1 (x) and component 2 (y) scores for each replicate plant coded according to infection treatment x bug damage treatment (N = 9 plants per infection x bug damage treatment). PC1 explained 648% of the variation in amino acid composition, and PC2 explained 122% of the variation. Loading plots of the different compounds (variables in the PCA) composing the amino acid blend (grey lines). A longer line distance from the origin along a given axis indicates a larger contribution of that compound to the variation explained by the component associated with that axis. Letters correspond to standard single-letter abbreviations for coding amino acids. Symbols correspond to non-coding amino acids and are as follows: inverted triangle = ornithine, square = hydroxyproline, diamond = beta-alanine and hexagon = homoserine. offset (in terms of viral fitness) by subtle changes in volatile emissions that render plants more attractive to walking vectors in combination with drought protection for the host (Xu et al. 2008; Westwood et al. 2013) and drastic changes in nutrient levels that both encourage dispersal of viruliferous vectors after probing and deter non-vector herbivore feeding. IMPLICATIONS OF PREDATOR INTERACTIONS WITH INFECTED HOSTS While CMV-infected plants appear to experience reduced herbivore pressure, our data suggest that predators are less influenced by plants infection status. Importantly, our results indicate that even though plants infected with CMV are less visually apparent due to reduced size (Fig. 5b), major predators of aphid vectors are still able to effectively locate prey on these plants (Table S1, Fig. 1b), possibly due to the fact that volatile cues are enhanced by CMV infection under both laboratory and field conditions (Mauck, De Moraes & Mescher 2010). Some predators in the Chrysopidae, Coccinellidae and Syrphidae, as well as parasitic wasps, also respond more strongly to yellow vs. green traps in sampling experiments, so the enhanced yellow colour of CMV-infected plants may help counteract reductions in apparency due to smaller size (Figs 3b, 5a) (Maredia et al. 1992; Rodriguez-Saona, Byers & Schiffhauer 2012). Conversely, for predators that also utilize floral resources (e.g. Syrphidae), we found a reduction in visitation to CMVinfected plants in the absence of aphid prey (Figs 3b and S5), which correlates with a reduction in floral output due to CMV infection (Fig. S7, Supporting information). Recent empirical and theoretical work suggests that the spread of viruses, and particularly of non-persistently transmitted viruses like CMV, is enhanced by natural enemy foraging within aphid-infested plant patches (Smyrnioudis et al. 2001; Hodge, Hardie & Powell 2011; Jeger et al. 2011; Dader et al. 2012). Natural enemies may even be indirectly responsible for a significant proportion of new infections, though they also reduce the overall number of vectors through consumption (Hodge, Hardie & Powell 2011). Thus, the observation that most predators are indifferent to infection status in the presence of aphid prey suggests that changes to plant phenotype in response to CMV infection are not likely to result in a loss of any fitness benefits conferred to a virus by predator disturbance of aphid vectors. Our data also indicate that an infected plant that is successfully colonized by an herbivore is likely to experience overall similar rates of predator visitation (relative to healthy plants), which will increase host survival (and potentially fitness) as well as the likelihood of that host serving as a source for new infections. Conclusions and future directions Our results demonstrate that virus infection influences the distribution of herbivores in a plant community by altering the apparency and palatability of potential host plants an effect that will impact herbivore population dynamics across time and space, and which certainly will have effects on host plant survival and fitness. While we observed few effects of virus infection on predator visitation, we did not explore whether virus infection alters the quality of prey for predators, or whether predators are able to learn and respond to such alterations in prey quality. There is limited evidence from laboratory studies that such effects do occur (e.g. Christiansen-Weniger, Powell & Hardie 1998), but their community-level impacts remain unexplored. More broadly, our findings suggest that the direct and indirect impacts of plant viruses on arthropod community dynamics

10 Virus infection changes plant insect interactions 671 and selection for or against different plant genotypes are important to consider even though plant virus infection has not figured prominently to date in the exploration of broader ecological patterns (Malmstrom, Melcher & Bosque-Perez 2011). Future work should examine virus effects in truly natural (unmanaged) plant communities (Alexander et al. 2013), where viruses are ubiquitous (Roossinck et al. 2010) and often unapparent in terms of visual symptoms (Roossinck 2010; Prendeville et al. 2012), but may still have significant effects on aspects of the plant phenotype that mediate interactions with beneficial and antagonistic organisms. Acknowledgements The CMV-FNY pathogen was kindly provided by Dr. John Murphy (Auburn University). 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