Specificity in Induced Plant Responses Shapes Patterns of Herbivore Occurrence on Solanum Dulcamara Author(S): Danush V

Specificity in Induced Plant Responses Shapes Patterns of Herbivore Occurrence on Solanum dulcamara Author(s): Danush V. Viswanathan, Anita J. T. Narwani, Jennifer S. Thaler Source: Ecology, Vol. 86, No. 4 (Apr., 2005), pp. 886-896 Published by: Ecological Society of America Stable URL: http://www.jstor.org/stable/3450842 Accessed: 05/11/2008 19:17

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http://www.jstor.org Ecology, 86(4), 2005, pp. 886-896 ? 2005 by the Ecological Society of America

SPECIFICITY IN INDUCED PLANT RESPONSES SHAPES PATTERNS OF HERBIVORE OCCURRENCE ON SOLANUM DULCAMARA

DANUSH V. VISWANATHAN,' ANITA J. T. NARWANI, AND JENNIFER S. THALER2 Departmentof Botany, Universityof Toronto,25 WillcocksStreet, TorontoM5S 3B2, Canada

Abstract. Interspecific competition between phytophagous insects can occur when plant responses induced by an early-season herbivore alter host quality for later colonizers. Recent evidence for specificity in the elicitation of induced plant responses by different attackers suggests that dynamics of host use in the field may be more complex than previously anticipated, because host suitability for colonizing herbivores may depend on which herbivore species has initially damaged a plant. In each of two years, we manipulated the first herbivore to attack Solanum dulcamara plants in an experimental population using several different arthropod species and subsequently monitored colonization by natural herbivores over the course of the growing season. We additionally performed weekly herbivore counts in wild S. dulcamara populations following natural variation in herbivore arrival. Plant- mediated interactions occurred primarily between two leaf-feeding beetles, Psylliodes affinis and Plagiometriona clavata. In both manipulative and observational experiments, P. clavata oviposition was reduced on plants initially damaged by P. affinis (or a third leaf-feeding beetle, Lema trilinea) relative to plants that were initially undamaged. Lowered P. clavata occurrence continued through subsequent life-history stages, resulting in decreased emergence of second-generation P. clavata adults on these plants. The occurrence of P. affinis was also lowered on plants damaged by conspecifics in both manipulative and observational experiments. Resistance against P. affinis also followed applications of jasmonic acid, an elicitor of plant defensive responses. Conversely, early-season damage by P. clavata did not influence plant quality for either later conspecifics or P. affinis. Initial herbivory by the spittlebug Aphrophora saratogensis or generalist taildropper slugs (Prophysaon sp.) likewise had no influence on P. clavata and P. affinis colonization, whereas L. trilinea damage did not affect later arriving P. affinis. Hence, only a subset of early-season damagers influenced herbivore occurrence on S. dulcamara. Preference tests examining P. affinis feeding and P. clavata oviposition confirmed that specificity in elicitation of induced plant responses produced the divergent herbivore occurrence patterns observed in the field. Over- all, the existence of plant-mediated competitive asymmetry between herbivore species on S. dulcamara highlights the dynamic nature of plant resistance and its potential role in organizing herbivore communities. Key words: bittersweetnightshade; competition; herbivory; induced responses;phytophagous insect communities;Plagiometriona clavata; plant-mediated interactions; Psylliodes affinis; Solanum dulcamara;specificity.

INTRODUCTION work has begun to extend these results to other feeding et al. and further that Increasingly widespread documentation of induced guilds (Denno 2000), suggests induced a in her- plant responses to herbivory has fostered interest in the responses may play part structuring bivore communities Gonzalez-Me- possibility of plant-mediated interactions between dif- (Tscharntke 1999, Gomez Van Zandt and ferent herbivores (Faeth 1986, Denno et al. 1995). Such gias and 2003, Agrawal 2004). Plant-mediated interactions between herbivore indirect effects may occur when physical or chemical species are therefore as an mechanism plant responses induced by a given herbivore affect emerging important of in To resource quality for subsequent foragers (Karban and competition many systems. date, however, most field studies have examined Baldwin 1997). Indeed, a review by Denno et al. (1995) experimental only whether a can in- found that induced responses were involved in over single early-season inducing agent fluence later and Niemela half of the published studies in which competition oc- foragers (e.g., Haukioja curred between mandibulate herbivores. More recent 1979, Faeth 1986, Hanhimaki 1989, Karban 1993, Tha- ler et al. 2001, Wise and Weinberg 2002). This ap- Manuscriptreceived 13 February2004; revised 15 June2004; proach mimics natural conditions in systems where a 23 accepted August2004. CorrespondingEditor: K. C. Larson. is the exclusive or early- I E-mail: danush@ particular species predominant botany.utoronto.ca season In however, several 2 Present address: Departmentof Entomology, Cornell damager. many systems, University, 3128 Comstock Hall, Ithaca, New York 14853 different herbivore taxa feed abundantly on a common USA. host plant species during its early growth phase. The 886 April2005 SPECIFICITYOF PLANT RESPONSES 887 dynamics of host use over a growing season may be substantially more complex than previously expected if the host plant exhibits specificity in induced responses to different early-season attackers. Specificity in plant response has two components: effect and elicitation. Specificity of effect occurs when herbivore species are differently affected by a given induced response (Stout et al. 1998, Agrawal 2000; see ? '4 also Hartley and Lawton 1987). Experiments assessing colonization of treated and untreated plants by natural assemblages of herbivores following early-season in- PLATE. 1. Plagiometriona clavata (tortoise beetle) adult duction frequently find some degree of specificity of feeding on Solanumdulcamara (bittersweet nightshade) leaf. effect (e.g., Hunter 1987, Inbar et al. 1998, Agrawal Photo credit: D. Viswanathan. 1999, Agrawal and Sherriffs 2001, Thaler et al. 2001, Riihimaki et al. 2003). Conversely, specificity of elicitation occurs when herbivore species differentially in- to induce pathogenesis-related proteins, peroxidases, duce phenotypic responses in a particular host plant polyphenol oxidases, and cell necrosis in response to (Stout et al. 1994, 1998, Agrawal 2000, Traw and Daw- herbivory by gall mites (Bronner et al. 199 la, b). These son 2002). In the field, specificity of elicitation is ev- responses reduce intraspecific gall mite damage to the ident when a given attacker varies in abundance across plant (Westphal et al. 1991), exert negative interspecific plants initially damaged by different herbivore species. effects on rust mite fecundity (Westphal et al. 1991), Using lab preference tests, Hartley and Lawton (1987) and positive interspecific effects on spider mite fecun- revealed specificity of elicitation in field-collected dity (Westphal et al. 1992). Additionally, herbivory by birch leaves damaged by different herbivores. Addi- Lema trilinea (three-lined potato beetle) induces tionally, Inbar et al. (1998) showed that different chem- changes in polyphenol oxidase and proteinase inhibitor ical elicitors of induced plant responses varied in their activity, which can influence the growth rate of L. tri- ability to influence the colonization of tomato plants linea larvae (Viswanathan and Thaler 2004). by specific herbivores. Otherwise, however, field col- Natural populations of S. dulcamara occur at the onization of plants treated with different damage types Koffler Scientific Reserve at Jokers Hill (hereafter Jok- has been explicitly compared only between plants with ers Hill), King City, Ontario, Canada, where all field herbivore vs. mechanical damage (e.g., Agrawal 1999, experiments were conducted. Several herbivore species Agrawal and Sherriffs 2001; but see Van Zandt and feed on S. dulcamara at Jokers Hill, five of which were Agrawal 2004). examined in this study: two univoltine native special- In this we two of field data from paper, present years ists on solanaceous plants, Lema trilinea White and whether the iden- manipulative experiments examining Plagiometriona clavata Fab. (tortoise beetle; see Plate of the initial attacker on Solanum dulcamara tity plants 1); an introduced univoltine solanaceous specialist, can influence of colonization patterns by subsequent Psylliodes affinis Paykull (European potato flea beetle); counts of herbivore abundance on foragers. Regular Aphrophora saratogensis Fitch (Saratoga spittlebug), over the season enabled us to assess the plants growing a native that feeds on understory herbs as a nymph in duration and of in- strength specific, plant-mediated the spring and later specializes on pine trees as an adult teractions between different herbivores. We also used froghopper; and native generalist taildropper slugs early- through late-season damage estimates to distin- (Prophysaon sp.). guish between the direct effects of initial damage per se on later colonization and the indirect effects of early Manipulative experiments damage type mediated through differences in subse- Manipulative experiments of S. quent levels of herbivory. We additionally performed using transplants dulcamara were conducted in of both weekly herbivore counts in wild S. dulcamara popu- June-August 2001 and 2002 = 400 and 500, Seeds lations following natural variation in herbivore arrival (n respectively). had been collected from wild S. dulcamara to determine whether similar patterns occur in natural previously at Jokers and were conditions. Finally, we conducted preference tests us- populations Hill, seedlings grown in ProMixBX soil Riviere-du- ing a subset of treatments from manipulative experi- (Premier Horticulture, contained in 131-mL un- ments to expressly investigate specificity in the elici- Loup, Qu6bec, Canada) pots der a 16:8 L:D for three weeks tation of induced plant responses as the mechanism greenhouse cycle prior to Both were underlying results from the field. transplantation. manipulative experiments conducted in the semi-shaded understory of a young METHODS forest where S. dulcamara occurs naturally, with trans- Study system plants spaced -30 cm apart in a square grid. Solanum dulcamara L. (bittersweet nightshade) is a Following transplantation, each plant was protected perennial vine in the Solanaceae that has been shown from ambient herbivores with a spun polyester sleeve 888 DANUSH V. VISWANATHANET AL. Ecology, Vol. 86, No. 4

(Rockingham Opportunities Corporation, Reidsville, camara population occurring in a pine stand, where North Carolina, USA) and was allowed to acclimate to 100 ramets were randomly selected for use in the study. field conditions for a week. In 2001, eight different Ramets occurred in clumps spaced 0.5-3 m apart, with treatments were then randomly imposed, with 50 rep- clumps varying in size from 10 to 50 ramets. Only one licates of each treatment. Five treatments consisted of ramet was selected per clump to reduce the probability inoculations with either adult L. trilinea, P. clavata, P. of sampling within a genet. Selected ramets varied in affinis, Prophysaon sp., or A. saratogenis nymphs. The size from 20 to 100 cm, measured from their point of remaining three treatments included a I-mL spray of connection with other ramets, and were sampled over a 0.5 mmol jasmonic acid (an elicitor of plant defensive this entire length. responses) solubilized in 1% acetone solution (see Tha- At the end of May, when S. dulcamara herbivores ler et al. [1996] for dosage), and two controls (plants were first appearing, the species that had first attacked undamaged and sprayed with a control solution of wa- each seedling or ramet was assessed; S. dulcamara her- ter and 1% acetone, or plants mechanically damaged bivores generally have a characteristic pattern of dam- with a hole punch to produce leaf loss without specific age that can be used to identify early-season attackers induction cues). In 2002, the experiment was repeated when levels of damage are relatively low. A number at an adjacent site without the A. saratogensis and me- of plants were still undamaged at this point. Plant chanical damage treatments, resulting in -92 replicates height, number of leaves, and visual estimates of perfor each of the remaining six treatments. centage tissue removed were also recorded on the initial Over a one-week initial damaging phase, the amount sampling date. At weekly intervals for the next two of tissue consumed was equalized at 15-20% across months, these three variables, as well as herbivore leaf-feeding herbivore treatments using herbivore ad- abundance and identity, were measured on each plant. ditions and removals. ANOVA results compared dam- Preference tests age measurements using an acetate grid on a subset of 20 plants from each treatment (for 2001, F495 = 0.71, In 2003, P. affinis feeding and P. clavata oviposition P = 0.56; for 2002, F376 = 0.08, P = 0.97). Equal- preference tests were conducted using potted plants, ization of tissue removal was used to prevent dose- because these two herbivores discriminated between dependent differences between treatments in induced treatments in field experiments (see Results). For these responses. On average, 2-4 herbivores per plant were tests, plants were grown in the greenhouse for three required to produce this level of herbivory. For the A. weeks as previously described, after which experimen- saratogensis treatment, four individuals were placed tal treatments were imposed. A no-choice feeding assay on each plant, which was sufficient to cause wilting in was used to examine the absolute preference of P. af- 10% of replicates. After one week, -15% leaf tissue finis for different induced plant phenotypes. For this was removed per plant in the mechanical damage treat- assay, four treatments were randomly assigned across ment, and the jasmonic acid treatment was applied. 44 plants: control, or herbivory by adult L. trilinea, P. After the treatments had been imposed, all sleeves clavata, or P. affinis. Each plant was covered with a and treatment herbivores were removed. Colonization spun polyester sleeve, and the amount of tissue con- by naturally occurring herbivores was then assessed sumed across herbivore treatments was equalized at through regular counts of the abundance and identity 15-20% using herbivore additions and removals, as per of all aboveground herbivores on each plant. Because the manipulative experiments. Following a one-week herbivore colonization may be influenced by plant size, damaging phase, all herbivores and sleeves were re- plant height was recorded at each sampling interval in moved. After a subsequent week of growth, a sleeve both years, along with the number of leaves in 2002. was used to enclose the bottom (damaged) portion of In both years, visual estimates of total percentage dam- each plant, while a second larger sleeve was used to age were also recorded, because colonizing herbivores contain the entire plant. Single P. affinis adults were may be sensitive to differences between treatments in placed in each large sleeve, and the leaf area consumed levels of herbivory following initial damage. In 2001, was recorded after two days, along with available leaf plants were sampled weekly throughout June, and sub- area and total plant height. Because assay herbivores sequently every second week until mid-August, for a did not have access to damaged portions of the plant, total of seven sampling dates. In 2002, plants were we interpret differences in feeding between treatments sampled every 10 days, with three sampling dates in to indicate specificity in the elicitation of induced plant each of June, July, and August. responses. A choice assay was used to examine the oviposition Observational study preference of P. clavata females for different induced In May-August of 2002, observational studies were phenotypes relative to uninduced plants. A choice assay conducted at Jokers Hill in two populations of S. dul- was used instead of a no-choice assay because P. cla- camara. Site 1 consisted of 200 second-year seedlings, vata females seem to oviposit on low-quality plants transplanted into an open forest understory in the pre- when higher quality resources are not available (D. V. vious year. Site 2 consisted of a large natural S. dul- Viswanathan and J. S. Thaler, unpublished data). Two April2005 SPECIFICITYOF PLANT RESPONSES 889 trials of the P. clavata oviposition choice assay were In the current study, repeated-measures Poisson re- conducted, using 82 and 64 plants, respectively. For gressions were used to compare the relative odds of each trial, half of the plants were left as undamaged herbivore occurrence across treatments over several controls. Every control plant was paired with another weeks. If there was a significant week by treatment plant assigned to one of three treatments: adult L. tri- interaction in the repeated-measures analysis, individ- linea, P. clavata, or P. affinis herbivory. All four treat- ual weeks were also examined separately. When the ments were imposed as previously described. After a effect across individual weeks was similar, only the week of growth following the initial damage phase, the repeated-measures analysis were reported. In the ob- bottom (damaged) portion of each plant was covered servational study, treatments were classified by which with a sleeve. Each pair of damaged-control plants was herbivore species had first damaged a plant, with un- then enclosed together in a larger sleeve. A single grav- damaged plants as "controls." At each observational id P. clavata female was placed in every large sleeve, site, the number of plants damaged by different her- and the differences between the two plants in eggs laid, bivore species, or initially undamaged, was similar. available leaf area, and total plant height were calcu- Plant height and leaf number were used as covariates, lated after four days. Each damaged-control pair because these variables may additionally influence her- served as a replicate, with 41 and 32 in each trial, bivore foraging choice. In the observational study, the respectively. As with the P. affinis feeding test, bio- amount of damage imposed by initial damagers was assay P. clavata did not have access to damaged por- used as a covariate for similar reasons. Covariates that tions of the plant. Hence, we likewise interpret differ- were not significant were removed from the analysis. ences in relative oviposition across the different treat- Initial damage type may directly influence later her- ment-control combinations to indicate differences in bivore occurrence, or may act indirectly through dif- plant quality induced by initial herbivory. ferences in subsequent levels of herbivory. Hence, we also used damage estimates from each previous sam- Data and statistical analyses pling interval as a covariate to distinguish between Across all field experiments, L. trilinea, P. clavata, direct and indirect effects of initial damage type. Pois- P. affinis, and several slug species colonized the plants. son regressions were performed with and without the However, only P. clavata and P. affinis occurred at high damage estimate covariate. If inclusion of damage lev- enough frequencies to perform statistical analyses. For els from the previous week eliminated a significant P. clavata, overwintering adults oviposit on S. dul- main effect of treatment, we conclude that effects on camara leaves over 3-4 weeks in early summer, with herbivore occurrence resulted from differences be- the entire juvenile stage subsequently being completed tween treatments in levels of damage following initial on leaf surfaces. Hence, we were able to record all life herbivory, and not directly from lasting effects of initial stages for this species: adults, eggs, larvae, pupae, and damage type per se (cf. Van Zandt and Agrawal 2004). exuviae (the presence of a pupal molt can be used to For the P. affinis no-choice preference test, leaf area infer successful eclosion; D. V. Viswanathan, personal consumed was analyzed using a one-factor ANOVA, observation). P. affinis larvae feed on roots, so only with damage type as the main effect and available leaf the occurrence of adults could be scored for this spe- area and total plant height as covariates. For the P. cies. clavata choice preference test, the difference in number Herbivore occurrence data were not normally distrib- of eggs laid between damaged and control plants was uted; there were many zero and one values. Dichoto- analyzed using a two-factor ANOVA, with damage mous, binomially distributed data are analyzed using type and trial as the main effects and differences in logistic regression, which can be generalized for poly- both available leaf area and total plant height as co- chotomous dependent variables with a multinomial dis- variates. Significant ANOVA results were further ex- tribution (Trexler and Travis 1993). Hence, generalized amined using Tukey post hoc comparisons. estimating equations were used to perform repeated- measures poisson regressions on herbivore counts RESULTS (GEE-PROC GENMOD in SAS System Version 8; Manipulative experiments Allison 1999). Poisson regression calculates likelihood- ratio chi-square statistics, with degrees of freedom equal For both 2001 and 2002, the repeated-measures anal- to one minus the number of levels of each factor. It also ysis revealed a significant week by treatment interac- produces an odds ratio for each level of every factor tion on the odds of Psylliodes affinis occurrence (Table adjusted for other terms in the model, indicating the odds 1). For the 2001 data, examination of individual sam- (or likelihood) that the response will occur. The odds pling dates revealed that initial treatment had a sig- ratio for each level is calculated relative to a reference nificant effect only in the second week after initial dam- level, with a value of one indicating an equal likelihood age (X2 = 47.7, df = 7, P < 0.001). Similarly, treatment of the event taking place. Values above or below one in 2002 had a significant effect only one week follow- indicate greater or lesser odds, respectively, when com- ing initial damage (X2 = 68.7, df = 5, P < 0.001). The pared to the reference level. effect of each specific treatment was consistent across 890 DANUSH V. VISWANATHANET AL. Ecology, Vol. 86, No. 4

TABLE 1. Results from repeated-measuresPoisson regres- stages. There were strong effects of initial treatment sions examiningthe occurrenceof Psylliodes affinisadults in 2002 number and clavata on Solanumdulcamara on P. clavata oviposition (Table 1; egg Plagiometriona eggs trilinea and P. plants following differentearly-season damage types. was not recorded in 2001). Both Lema affinis damage decreased the odds of P. clavata ovi- Effect X2 df P position by -50% relative to undamaged controls, with a) Psylliodes affinis adults no influence of Prophysaon or mechanical damage Manipulativeexperiment 2001 (Fig. lb; see the Appendix for raw means). Addition- Treatment 8.6 7 0.28 ally, the occurrence of P. clavata larvae was signifi- Week 88.7 5 <0.001 affected initial treatment in both (for Treatmentx Week 75.8 35 <0.001 cantly by years = = P = for = Percentagedamage 12.4 1 <0.001 2001, x2 16.1, df 7, 0.02; 2002, x2 df = 5, P < 0.001). As with numbers, odds Manipulativeexperiment 2002 28.45, egg Treatment 17.3 5 0.004 of occurrence for P. clavata larvae were -50% lower Week 159.9 6 <0.001 on L. trilinea and P. affinis damaged plants relative to Treatmentx Week 74 30 <0.001 undamaged controls (Fig. 1). Plant height 28.3 1 <0.001 Leaf number 4.92 1 0.027 We further examined whether decreased larval oc- of lower relative Observationalsite 2 currence was a consequence ovipo- Initial damager 23.8 2 <0.001 sition on L. trilinea and P. affinis damaged plants rather Week 4 7 0.78 than lasting effects of initial treatment. Counts of lar- Initial x Week 20.2 14 0.12 damager vae in the week when they were maximally abundant b) Plagiometrionaclavata eggs in 2002 were analyzed using Poisson regression, with 2002 Manipulativeexperiment egg number from the last week of oviposition as an Treatment 17.4 5 0.004 ovi- Week 64.8 2 <0.001 additional covariate. The 2-3 weeks between final Treatmentx Week 8.9 10 0.59 position date and maximal larval abundance is long Percentagedamage 14.9 1 <0.001 enough for all P. clavata eggs to have hatched, yet too Observationalsite 1 soon for pupation to have occurred. Egg number sig- Initial damager 8.7 3 0.034 influenced larval occurrence, with initial Week 11 1 <0.001 nificantly treatment (Table 2). Initial damagerx Week 1.1 3 0.78 remaining marginally significant Percentagedamage 5.4 1 0.02 In 2002, similar effects of treatment on the occurrence Observationalsite 2 of P. clavata pupae (X2 = 30.2, df = 5, P < 0.001) Initial damager 14.4 2 <0.001 and exuviae (X2 = 32.6, df = 5, P < 0.001) were Week 69.1 5 <0.001 driven lower occurrences in previous life Initial x Week 19.4 8 0.013 entirely by damager adult P. clavata Plant height 7.5 1 0.006 stages (Table 2). Hence, oviposition determined occurrence in all other life stag- Notes: In total dam- preference manipulativeexperiments, percentage es. and exuviae were too few to Poisson age in the week previousto each samplinginterval and plant Pupae perform height and leaf number in the current week were used as regressions in 2001. covariates. Percentageof leaf area consumed on each plant by initial herbivoreswas used as an additionalcovariate in Observational study the observationalstudy. Covariates not significantat an alpha value of 0.05 were removed from the analysis and are not At site 1, damage levels on the initial sampling date reported. ranged from 0% to 15%, with P. affinis and Prophysaon sp. as the main initial damagers (plants damaged by both species are not included in this study). Both of years: P. affinis damage and jasmonic acid decreased these herbivores were subsequently found at extremely the odds of P. affinis occurrence relative to undamaged low frequencies, while later arriving P. clavata were controls by >50% and 75%, respectively, whereas Pla- more abundant. Initial damage type significantly af- giometriona clavata damage increased the odds of P. fected the number of P. clavata eggs (Table 1), with affinis occurrence by >50% relative to controls, al- the odds of oviposition being 47% lower on plants though this comparison was not statistically significant damaged by P. affinis relative to plants that were ini- in 2001 (Fig. 1; see the Appendix for raw means). tially undamaged. However, no such influence of Pro- Conversely, Aphrophora saratogensis, Prophysaon sp., physaon damage was apparent (Fig. 2a; see the Ap- or mechanical damage did not influence P. affinis oc- pendix for raw means). Significant overall effects of currence (Fig. 1). Effects of initial treatment were not early-season damager on the occurrence of larvae (X2 = = significant in subsequent weeks, despite the continued = 9.57, df 3, P 0.02; see Fig. 3), pupae (X2 = = = presence of P. affinis adults throughout each season. 16.2, df = 3, P 0.001), and exuviae (X2 17.1, df A similar consistency was apparent across years for = 3, P < 0.001) were due to the previous distribution P. clavata. Although adult occurrence was not influ- of eggs (Table 2). Adult P. clavata occurred at too low enced by initial treatment (for 2001, x2 = 12.1, df =7, a frequency to allow Poisson regression. P = 0.10; for 2002, X2 = 2.7, df = 5, P = 0.75), Poisson At site 2, damage levels on the initial sampling date regressions revealed significant effects on other life also ranged from 0% to 15%, with P. clavata and P. April 2005 SPECIFICITYOF PLANT RESPONSES 891

2.5 a) 2001 O Plagiometriona clavata larvae A Psylliodesaffinis Z.un

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u Psylliodes PlagionmetionaPryphasaon Lema Jasmonic affinis clavata sp. trilinea acid FIG. 1. Results from repeated-measures Poisson regression showing relative odds of herbivore occurrence on Solanum dulcamara plants following several different early-season manipulations in 2001 and 2002, calculated relative to undamaged controls (dotted line). Psylliodes affinis data are from week 2 only. Symbols and bars show the mean + 1 SE for each treatment. Values above and below 1 indicate greater and lesser odds, respectively, of herbivore occurrence when compared to the control. An asterisk indicates a significant difference at an alpha value of 0.05. Note that odds of occurrence for Plagiometriona clavata larvae in panel (b) result from a similar distribution of P. clavata eggs, with continuing effects on P. clavata pupation and eclosion (see Results). For treatment details, see Methods: Manipulative experiments. affinis as the main initial damagers. Subsequently, these were again due to the previous distribution of eggs same two species were the main colonizers. Initial dam- (Table 2). The occurrence of P. clavata adults was not age type significantly affected colonization by P. affinis affected by differences in early-season attacker (X2 = (Table 1), with 60% lower odds of occurrence on plants 1.7, df = 2, P = 0.43). receiving conspecific herbivory relative to plants that Direct vs. indirect were initially undamaged (Fig. 2b; see the Appendix effects for raw means). There was no such effect of P. clavata For the results just reported, significant effects of herbivory (Fig. 2b). P. clavata oviposition was also initial damage type on herbivore occurrence were never significantly influenced by initial damage type (Table eliminated when damage estimates from the previous 1), with 27% lower odds of oviposition on P. affinis week were included in Poisson regressions. Neverthe- damaged plants relative to controls. However, there was less, percentage damage was a significant covariate in no influence of P. clavata herbivory on oviposition by one-third of the analyses, whereas plant height and conspecifics (Fig. 2b). Similarly, significant effects of number of leaves only occasionally influenced foraging initial damage type on larvae (X2 = 15.5, df = 2, P < herbivores (Tables 1 and 2). Percentage damage by 0.001; see Fig. 2b), pupae (X2 = 9.01, df = 2, P = initial herbivores never significantly affected later her- 0.011), and exuviae (X2 = 15.4, df = 2, P < 0.001) bivore occurrence in the observational study (Tables 1 892 DANUSH V. VISWANATHANET AL. Ecology, Vol. 86, No. 4

TABLE 2. Results from Poisson regressionsexamining the ber of between and control occurrenceof clavata and eggs damaged plants (F265 Plagiometriona larvae, pupae, = P = with P. clavata females exuviae on Solanumdulcamara plants following different 4.44, 0.016), strongly early-seasondamage types. disfavouring plants damaged by P. affinis (Fig. 3b). No effect of trial was apparent (F, < 0.001, P = 0.99), Effect X2 df P nor was there a trial x treatment interaction (F2,65 = a) Larvae 0.52, P = 0.598). Manipulativeexperiment 2002 Treatment 11 5 0.052 DISCUSSION Egg number 183.9 1 <0.001 Percentagedamage 5.7 1 0.017 Experimental demonstrations of induced plant re- Observationalsite 1 sponses influencing herbivore success are one of the Initial 3.7 3 0.3 damager main reasons for renewed interest in competition be- Egg number 126.1 1 <0.001 tween insects (Denno et al. 1995). A number of field Observationalsite 2 studies in Initial damager 1.9 2 0.39 examining plant-mediated competition have, Egg number 97.1 1 <0.001 fact, shown that early-season herbivory can decrease b) Pupae the population size of subsequent colonizing species, Manipulativeexperiment 2002 including phytophagous mites (English-Loeb et al. Treatment 9.1 5 0.11 1993), microlepidoptera (Karban 1993), and sap-feed- Larval number 414.5 1 <0.001 ing planthoppers (Denno et al. 2000). In this study, we Observationalsite 1 similarly show strong influences of early-season in- Initial damager 3.7 3 0.29 Larval number 21 1 <0.001 duction on occurrence across herbivore generations. In both and natural indi- Observationalsite 2 addition, experimental patterns Initial damager 1.9 2 0.39 cate that such lasting effects of initial herbivory may Larval number 97.1 1 <0.001 depend on the order of herbivore arrival when plants c) Exuviae exhibit specificity in the elicitation of induced plant Manipulativeexperiment 2002 responses. Treatment 8.9 5 0.11 Across both manipulative and observational exper- Pupal number 479.2 1 <0.001 iments, heterospecific herbivory by P. affinis consis- Observationalsite 1 decreased P. clavata when Initial 5 3 0.17 tently oviposition compared damager that received no Decreased Pupal number 46.5 1 <0.001 to plants initially damage. Percentagedamage 6.8 1 0.009 egg number resulted in decreased occurrence of larvae Observationalsite 2 and pupae, with subsequently fewer second-generation Initial damager 4.5 2 0.1 adults emerging on P. affinis damaged plants. The same number 44.2 1 <0.001 Pupal effect was observed following L. trilinea damage in Notes: Analyses were performedon data from the week the manipulative experiments, whereas conspecific her- when each life was with numbers stage maximallyabundant, bivory never influenced P. clavata occurrence. Inter- from the final week of the previous life stage as a covariate. In manipulativeexperiments, total percentagedamage in the estingly, adult P. clavata occurrence was never affected week previousto the samplinginterval and plant height and by the identity of early-season attackers, suggesting leaf numberin the currentweek were also used as covariates. that adults forage independently of initial damage type, Percentage of leaf area consumed on each plant by initial herbivoreswas used as an additionalcovariate in the obser- but nevertheless assess host plant quality when making vational study. Covariatesnot significant at an alpha value oviposition decisions. of 0.05 were removedfrom the analysis and are not reported. Early-season damage by conspecifics consistently decreased P. affinis occurrence relative to plants that initially received no herbivory. However, heterospe- and 2), whereas in the manipulative experiments, her- cific damage did not have a similar effect. In the ma- bivore occurrence was never different between me- nipulative experiments, only P. affinis herbivory had chanically damaged plants and undamaged controls negative effects on conspecifics 1-2 weeks after initial (Fig. 1). damage (with weak evidence for a short-term positive effect of P. clavata herbivory on P. affinis occurrence). tests Preference At site 2 of the observational study, negative effects In the no-choice assay, P. affinis feeding rate was of conspecific damage on P. affinis lasted the entire significantly affected by initial damage type (F339 = season (with neither short- nor long-term effects of P. 4.64, P = 0.007), with 47% less tissue consumed on clavata herbivory). Because the length of the sampling plants damaged by conspecifics relative to controls interval exceeded the recorded generation time for this (Fig. 3a). A similar reduction did not occur following species by several weeks (Wheeler and Hoebeke 1983), P. clavata or L. trilinea herbivory (Fig. 3a). In the P. P. affinis adults at the end of the season most likely clavata oviposition choice assay, there was a significant included progeny of early-season colonizers. Hence, effect of initial damage type on the difference in num- effects of initial P. affinis herbivory on conspecifics April 2005 SPECIFICITYOF PLANT RESPONSES 893

1.0 a) Site 1 0.8

-- 0.6 0 c o 0 0.4 0 Plagiometrionacavata a) 0.2 0? Plagiometrionaclavataclavata larvaelarvae a)(- I (D Psylliodes affinis Prophysaon sp.

1.6 a)0 o Site 2 > 1.4 b) ._ ) 1.2

U) 1.0 ...... v 0 0.8

0.6

0.4 ~* Ay/2\ * Plagiometrionaclavata eggs 0 Plagiometrionaclavata larvae 0.2 A Psylliodes affinis

Psylliodes affinis Plagiometrionaclavata FIG. 2. Results from repeated-measures Poisson regression showing relative odds of herbivore occurrence on Solanum dulcamara plants following natural early-season damage by different herbivore species at site 1 and site 2, calculated relative to plants that initially received no herbivory(dotted line). Symbols and bars show the mean + 1 SE for each damagetype. Values above and below 1 indicategreater and lesser odds, respectively,of herbivoreoccurrence when comparedto initially undamagedplants. An asteriskindicates a significantdifference at an alpha value of 0.05. Note that odds of occurrencefor P. clavata larvae result from a similar distributionof P. clavata eggs, with continuingeffects on P. clavata pupationand eclosion (see Results).For treatmentdetails, see Methods:Observational study. may have persisted across generations in the obser- that induction in the absence of any external cues left vational study. by herbivores is sufficient to cause changes in the suit- Several lines of evidence together suggest that spec- ability of S. dulcamara. ificity in the elicitation of induced plant responses The pattern of induced resistance in this study may caused the divergent herbivore occurrence patterns ob- have resulted from specific changes in the quality of served in the field. Most importantly, findings from the plant tissues (Stout et al. 1994, 1998). In addition, spe- preference assays match those from field experiments. cific induction of volatile compounds may have influ- P. affinis had lower feeding rates on undamaged tissue enced either host use by herbivores (e.g., Pallini et al. from conspecifically, but not heterospecifically, dam- 1997) or the attraction of predators and parasitoids aged plants when compared to controls. P. clavata fe- (e.g., De Moraes et al. 1998, Meiners et al. 2000). males also showed a relative oviposition bias against Preliminary analysis of tissue collected from the ma- undamaged tissue from P. affinis damaged plants, al- nipulative experiments following initial damage has though the expected bias against L. trilinea damaged shown that P. affinis did, in fact, induce qualitatively plants was not found. Secondly, herbivore occurrence different chemical responses in S. dulcamara leaves never differed between mechanically damaged plants than did P. clavata (D. V. Viswanathan, O. A. Lifchits, and undamaged controls, indicating that removal of and J. S. Thaler, unpublished data). The responses in- tissue in the absence of herbivore-specific induction duced by P. clavata seemed simply not to have influ- cues did not influence plant quality for subsequent for- enced later colonizers. Although it is possible that her- agers. Finally, treatment with jasmonic acid consis- bivory by subsequent attackers further altered the tently lowered the occurrence of P. affinis, showing strength or quality of initial plant responses, the sim- 894 DANUSH V. VISWANATHANET AL. Ecology, Vol. 86, No. 4

cm E E 1 * * ab 35- 4-a) 0a) 30- 0) a) 25 0c b 20- 0) -J 15

10 Control Psylliodes Plagiometriona Lema affinis clavata trilinea

t0 4 )

C 2 0) E I o

CO-2 a) c -4

C 0) -6

0) -8 ]Q Psylliodes Plagiometriona Lema affinis clavata trilinea FIG. 3. (a) Amount of Solanum dulcamara leaf area consumed by a single Psylliodes affinis adult over two days of feeding on systemic tissue producedfollowing one of four initial treatmentsin a no-choice feeding experiment.(b) Differencein the numberof eggs laid by single gravid Plagiometrionaclavata females over four days between pairs of damagedand control Solanumdulcamara plants. Damage was imposedusing one of threedifferent herbivore species, andoviposition was restricted to tissue producedafter the damagingphase. In both panels (a) and (b), boxes and bars show the mean + 1 SE for each treatment.Treatments that do not share a common letter above boxes are significantlydifferent at an alpha value of 0.05, calculatedwith Tukey'spost hoc comparisonsfollowing ANOVA. For treatmentdetails, see Methods:Preference tests.

ilarity of results between preference tests and field ex- Conversely, results for P. affinis occurrence were more periments suggests that responses to initial damagers conclusive, although slightly inconsistent: definite sea- were, by themselves, sufficient to determine host plant son-long effects in the observational study contrasted quality. with solely short-term effects in both manipulative ex- Specificity in induced responses produced lasting periments. This difference may have been caused by differences in herbivore occurrence in the field, but transplantation shock preventing the induction of whether this stemmed from equivalently long-term al- strong responses with lasting effects in the manipula- terations in plant phenotype was not always clear. De- tive experiments. Additionally, relatively close spacing spite an early-season difference between treatments in of individuals in manipulative experiments may have oviposition quality for P. clavata, subsequent effects promoted competition as plants grew, which can at- of initial damage type on the relative survival of larvae tenuate the expression of plant responses (Cipollini and and pupae were not apparent. Although these results Bergelson 2001). might suggest a mid-season diminution in the strength Because induced plant responses are a product of the of induced responses or lowered sensitivity of later life interaction between plant and herbivore, it is not clear stages, several unmeasured life-history characteristics from the current data whether the observed pattern of could have in fact been influenced by damage type, specificity is adaptive for either S. dulcamara or its including time to pupation, pupal mass, or emergence. attackers. Alternatively, it may be solely a functional April 2005 SPECIFICITYOF PLANT RESPONSES 895 consequence of different herbivore feeding styles or wild radish. Annals of the Entomological Society of Amer- ica 94:71-75. salivary elicitors. Whether or not there is a clear adap- Allison, P. D. 1999. Logistic regressionusing the SAS sys- tive however, interactions explanation, plant-mediated tem: theory and application. SAS Institute, Cary, North on S. dulcamara took place largely between its most Carolina,USA. abundant colonizers, P. affinis and P. clavata. These Bronner,R., E. Westphal,and E Dreger. 1991a. Enhanced effects were unidirectional: P. affinis feeding had neg- peroxidase activity associated with the hypersensitive re- of Solanum dulcamara to the mite Aceria cla- ative effects on both P. clavata and conspecifics, sponse gall dophthirus(Acaria, CanadianJournal of whereas P. clavata did not influence either Eriophyoidea). damage Botany 69:2192-2196. itself or P. affinis. This result is not unusual; interac- Bronner,R., E. Westphal,and F Dreger.1991b. Pathogenesis- tions between insects are often asymmetric, particularly related proteins in Solanumdulcamara L resistant to the among mandibulate herbivores (Denno et al. 1995). gall mite Aceria cladophthirus (Acaria, Eriophyoidea). and MolecularPlant 38:93-104. However, asymmetric competition between herbivores Physiological Pathology Cipollini, D. E, and J. 2001. Plant density and caused in the elicitation of induced Bergelson. by specificity plant nutrient availability constrain constitutive and wound-in- responses is a novel possibility in the study of plant- duced expression of trypsin inhibitors in Brassica napus. insect interactions. Similar specificity in prey responses Journalof ChemicalEcology 27:593-610. to multiple predator species has recently received much De Moraes, C. M., W. J. Lewis, P. W. Pare, H. T. Albron, and J. H. Tumlinson. 1998. Herbivore-infested selec- experimental attention from behavioral and evolution- plants tively attract parasitoids. Nature 393:570-573. et al. ary ecologists (Sih 1998, Relyea 2003), following Denno, R. F, M. S. McClure, and J. R. Ott. 1995. Interspecific earlier theoretical work showing that trade-offs among interactions in phytophagous insects: competition reex- predator-specific defenses in prey can promote the co- aminedand resurrected.Annual Review of Entomology40: 297-331. existence of different predator species (Matsuda et al. Denno, R. F, M. A. Peterson, C. Gratton, J. G. A. 1993) and overall et Cheng, community complexity (Matsuda Langellotto,A. F Huberty,and D. L. Finke. 2000. Feeding al. 1994). Hence, our results (and similar findings by inducedchanges in plantquality mediate interspecific com- Van Zandt and Agrawal 2004) suggest that comparable petition between sap-feeding insects. Ecology 81:1814- attention to specificity in induced plant responses may 1827. G. R. and D. provide related into the of herbiv- English-Loeb, M., Karban, Hougen-Eitzman. insights dynamics 1993. Directand indirect between mites orous insect communities. competition spider feeding on grapes. Ecological Applications 3:699-707. In sum, there was a strong similarity of results across Faeth, S. H. 1986. Indirectinteractions between temporally experiments in this study. Manipulative experiments separated herbivores mediated by the host plant. Ecology indicated that initial damage type could affect herbi- 67:479-494. and J. M. Gomez. 2003. vore occurrence in the field, and observational studies Gonzalez-Megias, A., Consequences of a herbivore for the abundance and verified that herbivore distribution in natural removing keystone popula- diversity of arthropods associated with a cruciferous shrub. tions of S. dulcamara is indeed influenced by the order Ecological Entomology 28:299-308. of arrival of herbivore species. Subsequent preference Hanhimaki,S. 1989. Inducedresistance in mountainbirch: tests showed that specificity in induced plant responses defence against leaf-chewing insect guild and herbivore 81:242-248. underlay these patterns. Overall, we therefore found competition.Oecologia Hartley, S. E., and J. H. Lawton. 1987. Effects of different evidence for in the elicitation of induced specificity types of damage on the chemistry of birch foliage, and the plant responses that was consistent across time and responses of birch feeding insects. Oecologia 74:432-437. space, with lasting effects on herbivores. Haukioja, E., and P. Niemela. 1979. Birch leaves as a resource for herbivores: seasonal occurrence of increased resistance ACKNOWLEDGMENTS in foliage after mechanical damage of adjacent leaves. Oec- We thankJennifer Chalmers, Graham Cox, AbbyDeshman, ologia 39:151-159. Joey Dodgson, Celine Griffin,Ray Holberger,Lisa Plane,and Hunter, M. D. 1987. Opposing effects of spring defoliation Pete Van Zandt for help with field work, AnuragAgrawal, on late-season oak caterpillars. Ecological Entomology 12: Peter Abrams,Don Jackson, and Pete Van Zandt for devel- 373-382. opmentof ideas, and A. Agrawaland P. VanZandt for helpful Inbar,M., H. Doostdar,R. M. Sonoda, G. L. Leibee, and R. commentson the manuscript.This researchwas supportedby T. Mayer.1998. Elicitorsof plantdefensive systemsreduce the CanadianFoundation for Innovationand a NaturalSci- insect densities and disease incidence. Journal of Chemical ences and EngineeringResearch Council (NSERC) of Canada Ecology 24:135-149. operating grant to J. S. Thaler, and NSERC graduateand Karban, R. 1993. Induced resistance and plant density of a undergraduatescholarships to D. V. Viswanathanand A. J. T. native shrub, Gossypium thurberi, affect its herbivores. Narwani, respectively. Ecology 74:1-8. Karban, R., and I. T. Baldwin. 1997. Induced responses to LITERATURE CITED herbivory. University of Chicago Press, Chicago, Illinois, Agrawal, A. A. 1999. Induced responses to herbivory in wild USA. radish: effects on several herbivores and plant fitness. Ecol- Matsuda, H., P. A. Abrams, and M. Hori. 1993. The effects ogy 80:1713-1723. of adaptive anti-predator behavior on exploitative com- Agrawal, A. A. 2000. Specificity of induced resistance in petition and mutualismbetween predators.Oikos 68:549- wild radish: causes and consequences for two specialist and 559. two generalistherbivores. Oikos 89:493-500. Matsuda, H., M. Hori, and P. A. Abrams. 1994. Effects of Agrawal, A. A., and M. F Sherriffs. 2001. Induced plant predator-specificdefence on communitycomplexity. Evo- resistance and susceptibility to late-season herbivores of lutionaryEcology 8:628-638. 896 DANUSH V. VISWANATHAN ET AL. Ecology, Vol. 86, No. 4

Meiners, T., C. Westerhaus, and M. Hilker. 2000. Specificity Trexler, J. C., and J. Travis. 1993. Nontraditional regression of chemical cues used by a specialist egg parasitoid during analyses. Ecology 74:1629-1637. host location. Entomologia Experimentalis et Applicata 95: Tscharntke, T. 1999. Insects on common reed (Phragmites 151-159. australis): community structure and the impact of herbivory Pallini, A., A. Janssen, and M. W. Sabelis. 1997. Odour- on shoot growth. Aquatic Botany 64:399-410. mediated of mites to responses phytophagous conspecific Van Zandt, P. A., and A. A. Agrawal. 2004. Community-wide and 110:179-185. heterospecific competitors. Oecologia of herbivore- induced plant responses in milkweed R. A. 2003. How to combined impacts Relyea, prey respond preda- syriaca). 85:2616-2629. tors: a review and test. 84:1827-1839. (Asclepias Ecology empirical Ecology D. and J. S. Thaler. 2004. Plant vascular P. and K. Ruohomaki. 2003. Viswanathan, V., Riihimaki, J., Kaitaniemi, Spa- in resource tial of two herbivore to a larva architecture and within-plant spatial patterns responses groups geometrid Journal of Chemical on mountain birch. Oecologia 134:203-209. quality following herbivory. Ecology Sih, A., G. Englund, and D. Wooster. 1998. Emergent impacts 30:521-533. 1991. Induced re- of multiple predators on prey. Trends in Ecology and Evo- Westphal, E., E Dreder, and R. Bronner. lution 13:350-355. sistance in Solanum dulcamara triggered by the gall mite Stout, M. J., K. V. Workman, R. M. Bostock, and S. S. Duffey. Aceria cladophthirus (Acaria, Eriophyoidea). Experimental 1998. Specificity of induced resistance in the tomato, Ly- and Applied Acarology 12:1.11-118. copersicon esculentum. Oecologia 113:74-81. Westphal, E., M. J. Perrotminnot, S. Kreiter, and J. Guitierrez. Stout, M. J., K. V. Workman, and S. S. Duffey. 1994. Dif- 1992. Hypersensitive reaction of Solanum dulcamara to ferential induction of tomato foliar proteins by arthropod the gall mite Aceria cladophthirus causes an increased sus- herbivores. Journal of Chemical 20:2575-2594. Ecology ceptibility to Tetranvchus urcticae. Experimental and Ap- J. M. J. R. and S. S. 1996. Thaler, S., Stout, Karban, Duffey. Acarology 15:15-26. simulate insect in tomato plied Exogenous jasmonates wounding Wheeler, A. G., and E. R. Hoebeke. 1983. New records of a in the and plants (Lycopersicon esculentum) laboratory flea beetle, in eastern North field. Journal of Chemical 22:1767-1781. palearctic Psylliodes affinis, Ecology America of the Thaler, J. S., M. J. Stout, R. Karban, and S. S. 2001. (Coleoptera: Chrysomelidae). Proceedings Duffey. of 85:594-597. Jasmonate-mediated induced plant resistance affects a com- Entomological Society Washington and A. M. 2002. Prior flea beetle munity of herbivores. Ecological Entomology 26:312-324. Wise, M. J., Weinberg. Traw, M. B., and T. E. Dawson. 2002. Differential induction herbivory affects oviposition preference and larval perfor- of trichomes by three herbivores of black mustard. Oec- mance of a potato beetle on their shared host plant. Eco- ologia 131:526-532. logical Entomology 27:115-122.

APPENDIX A table showing raw means for (A) Psylliodes affinis adult and (B) Plagiomatriona clavata egg abundance on Solanum dulcamara plants following different early-season damage types is available in ESA's Electronic Data Archive: Ecological Archives E086-047-A 1.