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Mediation of herbivore attack and induced resistance by plant vigor and ontogeny Jean Carlos Santos a , * , G. Wilson Fernandes b , c a Instituto de Biologia, Universidade Federal de Uberlândia, CP 593, Campus Umuarama, Bloco 2D, Rua Ceará s/n, 38400-902 Uberlândia, Minas Gerais, Brazil b Ecologia Evolutiva & Biodiversidade/DBG, CP 486, ICB/Universidade Federal de Minas Gerais, CP 486, 30161 970 Belo Horizonte, MG, Brazil c Planta Ltda, Rua Martim de Carvalho 549, 30190-010 Belo Horizonte, MG, Brazil a r t i c l e i n f o Article history: Received 4 November 2008Accepted 14 September 2010Available online 8 October 2010 Keywords: Amazonian rain forest Anacardium occidentaleContarinia HerbivoryHypersensitivityInsect galls a b s t r a c t A large number of insect galls induced by Contarinia sp. (Cecidomyiidae) on cashew plants, Anacardiumoccidentale L. (Anacardiaceae), and induced resistance (hypersensitivity) against galling were observed in ve restored different-aged stands in the Amazonian tropical rain forest. We tested three hypotheses: (1)the effect of age-dependent changes on the attack by Contarinia sp. and on induced resistance of A.occidentale to herbivory (plant ontogeny e herbivory hypothesis); (2) the effect of leaf size on theoviposition preference by the gall-midge (plant vigor hypothesis), and (3) whether past attack couldin uence future attack and induced resistance (attack prediction hypothesis). Tree age positively in u-enced attack levels and gall density. The leaves of older trees experienced four-fold greater attack andsupported two-fold more galls. Hypersensitive response was also positively affected by tree age. Thisinduced resistance was six-fold higher on older trees. Therefore, we suggest that induced resistance in A.occidentale was age-dependent, hence supporting the plant ontogeny e herbivory hypothesis. Higherpreference of Contarinia sp. on larger sized leaves of A. occidentale was only observed in old stands, henceproviding support for the plant vigor hypothesis. The same trend was observed in hypersensitiveresponse. Only two older plots (5 e 7-year-old) were better predictors of current attack and resistance of A. occidentale , hence supporting the attack prediction hypothesis. Our results suggest that plant devel-opment is an important factor that contributes to the structuring of interactions between host plant andinsect herbivores. However, more information about ontogenetic changes and regeneration processes isneeded to understand plant e herbivore interactions in the Amazonian forest. 2010 Elsevier Masson SAS. All rights reserved. 1. Introduction Interactionsamongplantsandherbivorousinsectsconstitute animportant component of most terrestrial and aquatic ecosystems(e.g. Dorn et al., 2001; Cebrian and Lartigue, 2004). These interac- tions occur at low trophic levels and consequently result ina cascade of effects that often in uence entire food webs (Poliset al., 2000). Species interactions have a crucial role in the recy-cling of matter, energy and nutrient ows in an ecosystem (Price,1997, 2002). Insect herbivory has shown to be in uenced bydisturbance (e.g., Ayres and Lombardero, 2000), but may have a positive or negative in uence on plant succession (e.g., Turneret al., 1998; Schädler et al., 2004), plant distribution and speciesdiversity (e.g., Olff and Ritchie, 1998).Herbivory in natural communities, as well as in man-madeecosystems such as in agriculture and forestry, can be high,reducing growth and reproduction of individual plants (Price,1997). Nevertheless, to diminish the impact of herbivores, plantspresent a wide spectrum of defensive mechanisms (Fernandes,1994; Karban and Baldwin, 1997). Among them, inducedresponses by the host may reduce the performance and/or prefer-ence of herbivores. Previous studies have demonstrated that initialherbivore attack decreases the nutritional quality of the attackedplant tissue and/or increases levels of chemical and physicaldefenses in a wide variety of plants (reviewed by Karban andBaldwin, 1997). Among the many hypotheses raised to explainthe patterns of attack by herbivorous insects the plant vigorhypothesis (PVH; Price, 1991) has gained strong support (seeCornelissen et al., 2008). This hypothesis predicts that insectherbivores will choose preferentially large, more vigorouslygrowing plants or plant modules and that offspring performancewillbegreateronthesemorevigorousplantsorplantmodules(seealso Thompson,1988; Thompson and Pellmyr, 1991). Hypersensitivity is an induced defense mechanism wherebyplants locate and kill the gall-inducing larva (Fernandes, 1990;Fernandes et al., 2000; Harris et al., 2003). The occurrence of galls * Corresponding author. Tel.: þ 55 34 32182243. E-mail address:
[email protected] (J.C. Santos). Contents lists available at ScienceDirect Acta Oecologica journal homepage: www.elsevier.com/locate/actoec 1146-609X/$ e see front matter 2010 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.actao.2010.09.007 Acta Oecologica 36 (2010) 617 e 625 and plant-induced defense to galling offered a unique opportunityto study the interactions in a system involving the host plant, itscecidomyiid galling herbivore and plants of different ages. Plantscanbeexposedtodifferentlevelsofherbivoryastheydevelopfromseedstomaturestages.Hence,aplant ’ sdefensescanchangeduringdevelopment (see Boege and Marquis, 2005; Korndörfer and Del-Claro, 2006). In several systems, induced resistance has beenfoundtobestronglyaffectedbyplantontogeny.AccordingtoKarbanand Baldwin (1997) plantontogeny should produce age-dependentpatterns in induced resistance to herbivores. Moreover, the onto-genic stage, can also positively or negatively, affect the damage,abundance and preference of herbivores, as well as plant defenses(see Boege and Marquis, 2005), and contribute tothe structuring of insect communities (e.g., Waltz and Whitham,1997). In this study, we tested the plant vigor hypothesis (Price, 1991)which predicts that galling insects prefer and achieve higherperformance on larger plant modules compared to smaller plantmodules. If leaves or plants are preferentially attacked, they shouldpresent moreresourcesthat would enhance offspringsurvivorship.Also, we expected that plants should be more responsive to attackon the preferentially attacked host organs or plants as a directattempttodiminishherbivoreperformance.Consequently,ahigherfrequency of hypersensitive reactions against the galling herbivoreshould also be found on the most vigorous or attacked hosts andhost organs. We also tested the plant ontogeny e herbivoryhypothesis (Boege and Marquis, 2005) which predicts that plantontogeny in the restored forest causes age-dependent changes inherbivore attack and induced resistance to herbivory. In addition,we test the attack prediction hypothesis that predicts that pastattackandresistancetothegallingherbivorein uencefutureattackand induced resistance. Little is known of the effects of herbivoreattackandplantdefenseonfutureattacksonplants(seeKarbanandAdler,1996; Prado and Vieira,1999; Cornelissen et al., 2002). During an investigation of insect galls on a restored Amazonianrain forest we observed the occurrence of a large number of insectgalls induced by a still unidenti ed species of Contarinia sp.( ¼ Stenodiplosis sp.) (Diptera: Cecidomyiidae) on the cashew plant, Anacardium occidentale L. (Anacardiaceae), in several restoredforest stands of different ages. Casual eld observations also indi-cated that A. occidentale plants responded to attack by elicitinga hypersensitive reaction to the galling larva. Then, we speci callyaddressed the following questions: 1) What is the effect of leaf sizeof A. occidentale in the oviposition preference and Contarinia larvalsurvival?;2) Doesthefrequencyofattackedplantsof A. occidentale ,abundance of successfully induced galls, and the plant hypersen-sitive response to Contarinia increase with stand age?; and, 3) Doespast attack and A. occidentale resistance to Contarinia in uencefuture attack and induced resistance? 2. Materials and methods 2.1. Organisms studied The cashew, A. occidentale L. (Anacardiaceae) is an evergreentree that reaches 5 e 20 m height, with glabrous leaves, reddishwhen young, 8 e 14 cm long and 6 e 8 cm wide (see Lorenzi, 2000). The commercial cashew nut, native to elds and dunes of the northcoast of Brazil, is grown as a plantation crop and has becomenaturalized in many tropical countries throughout the world.Bleicher and Melo (1996) related the occurrence of 99 insectspecies and 7 mite species associated with the culture of thecashewindifferentcultivatedareasinBrazil(seealsoBleicheretal.,1993a,b, 2002, Bleicher and Melo, 1996; Mesquita et al., 2003 unpublished report). Rickson and Rickson (1998) reported on antspecies attracted to a large number of extra oral nectaries presenton the plant. One of the most important herbivore on A. occidentale is the galling cecidomyiid (Diptera) Contarinia sp. ( ¼ Stenodiplosis sp.)thathasanaveragedegreeofinfestationof25%(Mesquitaetal.,2002 unpublished report). Otherwise, we were unable to nd anypublished information on the interaction of this important herbi-vore and the host plant. 2.2. Study location The study was done in a bauxite mine operated by MineraçãoRio do Norte SA in the Saracá-Taquera National Foreston an uplandmesa at an elevation of 180 m, 65 km northwest of the town of Oriximiná and 30 km south of the Trombetas River inwestern ParáState,Brazil(1 40 0 S,56 27 0 W).Theregionalvegetationisevergreenequatorial moist forest, within which the forests occupying theupland mesas and surrounding slopes have average canopy heightsof 20 e 35 m, with emergent trees up to 45 m tall (Parrotta et al.,1997). Since 1979 the mining company has implementedapioneerreforestationprogram,restoringforestcoverdestroyedatarateofapproximately100hayear 1 duringbauxiteoreextraction.At the mine site the reforestation method employed the inclusionof a wide variety of forest species, 80 e 100 species of native forestspecies, one of which was A. occidentale (for details see Parrottaet al., 1997; Parrotta and Knowles, 1999, 2001).GallsamplingwascarriedoutinNovember2003in verestoredstands, with plantations started in the years 1997,1998,1999, 2000and 2001. Trees on each stand were seven, six, ve, four, and threeyears old respectively at sampling periods. Prior to planting, plantspecies were grown from seeds collected in the eld. Seeds weregerminated under ambient temperature, light, and humidity inopen-airgreenhousesandthentheyweretransplantedinthe eld.Plants did not receive any further fertilization or irrigation. On allstand plots, A. occidentale individuals grew under similar environ-mental conditions (light, water, and nutrients). 2.3. Sampling To address how plants responded to gall induction and how theinsect herbivore responded to module vigor and the in uence of past generation on new generation of galls, 15 individuals of approximately 4 e 5 m high were randomly marked in each agestand, except for the 2001 stand, where we sampled only 14 indi-viduals. From each tree, 25 young and 25 old leaves were randomlycollected from the canopy up to 2.5 m high. Old leaves representedherbivore attack of the previous generation while young leavesrepresentedtheherbivoreattackofthecurrentgeneration.Thetwoleafagetypeswerereadilyseparatedinthe eldduetotheirclearlydifferent color pattern and toughness. Sampled leaves were takento the laboratory in marked plastic bags for measurements of length, number of galls successfully developed and those killed byhypersensitiveresponse(HR).Thetotalattackby Contarinia wastheresult of the sum of the number of galls developed (successfulattack) plus HR (unsuccessful attack). Galls killed by the plant ’ shypersensitiveresponsewereinferredbytheoccurrenceofaroundnecrotic spot around the attempted site of penetration by thegalling larva ( sensu Fernandes, 1990; Fernandes and Negreiros,2001). Mortality factors caused by parasitism, predation, or path-ogens were not addressed in this study. 2.4. Statistical analyses To determine how leaf traits, Contarinia attack, and inducedresistance change with age of A. occidentale ( ¼ stand age) we usedPearson ’ s correlations. At the stand level, we tested PVH using thetree ’ sagethroughtheKruskal e Wallistestbecausedatawereneither J.C. Santos, G.W. Fernandes / Acta Oecologica 36 (2010) 617 e 625 618 normally distributed nor had homogenous variance. Afterward, weused a post-hoc pairwise comparison of means. To test for any rela-tionship between galling insect preference and plant vigor, leaveswere divided into 2 cm classes and analyzed by simple linearregression. Preference was de ned as non-random oviposition onplant resource offered simultaneously (see Singer, 1986; Thompson,1988) and estimated by quantifying number of galled leaves,numberofgallsandtotalattack(sumofgallssurvivedandgallskilledbyhypersensitiveresponse).Finally,todeterminethepredictabilityof galling attack and induced resistance, between past (old leaves) andrecent (new leaves) attack, we used simple regression linear (Zar,1996). All statistical analyses were performed using Statistica 6.0StatSoft (2001). 3. Results 3.1. Patterns of Contarinia attack and plant-induced resistance We recorded 3926 galls successfully induced by Contarinia and21,904 galls that failed to develop due to hypersensitive responseon 3693 leaves of A. occidentale . Hence, there were 25,830 attacksby the galling cecidomyiid on the 74 plant individuals sampled inthe ve age-classes studied. Number of hypersensitive responses(HRs), galls, attacks (attempt to induce galls by individual larva),and attacked leaves showed strong and signi cant positive corre-lations with stand age ( r values range from approximately 0.30 to0.57; Table 1). The other measured leaf traits weresigni cantlyandpositivelycorrelatedsuchasleaflengthwithwidth,numberofgallswith number of attacks, HR with number of attacks, HR withnumber of attacked leaves, galls with number of attacked leaves,and attack with number of attacked leaves ( r values range fromapproximately 0.56 to 0.99; Table 1). Leaf size did not vary amongthe A. occidentale trees studied. Furthermore, there was no associ-ation between a tree ’ s average leaf size and attack or inducedresistance (Table 1 and Fig.1), indicating that plant traits were not in uenced by stand age.Induced resistance in both Contarinia attack and A. occidentale varied with stand age. Attack by Contarinia (Kruskal e Wallis test:H 4,74 ¼ 38.671, p < 0.001, Fig. 2a); galls successfully developed(Kruskal e Wallis test: H 4,74 ¼ 29.183, p < 0.001, Fig. 2b), and A.occidentale- induced resistance (Kruskal e Wallis test:H 4,74 ¼ 33.624, p < 0.001, Fig. 2c) were correlated positively withstand age. Comparison within treatments among tree age revealedthat attack was higher on A. occidentale found in the older stands(6- and 7-year-old). In these older stands, A. occidentale supportedmore galls but had a higher incidence of induced resistancecompared with individuals in the other younger stands, thereforecorroborating the plant ontogeny e herbivory hypothesis (Figs. 1and 2a e c). 3.2. Plant vigor: preference and induced resistance at leaf level Intermediate leaf length classes were always very abundant,while smaller and longer leaves were rare (Fig. 3a,b). In old stands(5 e 7-year-old), Contarinia showed higher oviposition preferencerates [number of galled leaves (7-year-old e r 2 ¼ 0.78, y ¼ 51.107 þ 2.726 x , p < 0.01; 6-year-old e r 2 ¼ 0.71, y ¼ 60.217 þ 2.104 x , p < 0.01; 5-year-old e r 2 ¼ 0.77, y ¼ 10.899 þ 2.272 x , p < 0.001), gall successfully developed (7-year-old e r 2 ¼ 0.50, y ¼ 0.926 þ 0.144 x , p < 0.05; 5-year-old e r 2 ¼ 0.83, y ¼ 0.217 þ 0.063 x , p < 0.001), and total attack (7-year-old e r 2 ¼ 0.82, y ¼ 1.326 þ 0.644 x , p < 0.001; 6-year-old e r 2 ¼ 0.78, y ¼ 2.432 þ 0.463 x , p < 0.0013; 5-year-old e r 2 ¼ 0.63, y ¼ 0.385 þ 0.115 x , p < 0.01)] for larger leaves of A. occidentale (Fig. 3a,b), while in younger stands (3- and 4-year-old) thehypothesis was not supported (Fig. 3a,b).The pattern for plant-induced resistance (hypersensitive reac-tions) followed that observed for successfully developed galls; i.e.,thenumberofHRswashigheronlargerleavesinoldstands(6-and7-year-old, r 2 ¼ 0.87, y ¼ 0.309 þ 0.485 x , p < 0.0001 and r 2 ¼ 0.85, y ¼ 0.465 þ 0.499 x , p < 0.001 respectively; Fig. 3b) but not inyounger stands, suggesting that in spite of supporting more galls,larger leaves were also more defended. 3.3. Predicting current attack and plant-induced defense from past attack and defense Past attack rates in uenced current attack rates only in olderstands. Variation in past attack rates by Contarinia explained a largeproportion of the current attack in 5-year-old ( r 2 ¼ 0.40, y ¼ 0.0335 þ 0.3318 x , p < 0.05), 6-year-old ( r 2 ¼ 0.53, y ¼ 3.3773 þ 0.2979 x , p < 0.01), and 7-year-old ( r 2 ¼ 0.39, y ¼ 2.6046 þ 0.5862 x , p < 0.05) stands (Fig. 4). Otherwise, nostatistically signi cant relationship was observed in 3-year-old( r 2 ¼ 0.10, p > 0.05),and4-year-old( r 2 ¼ 0.02, p > 0.05)stands(Fig.4).The same trend was observed for plant-induced resistance.Variationinpast attack explained approximately28%, 34%, and 29%of the variation in current resistance of A. occidentale , in 5-year-old( r 2 ¼ 0.28, y ¼ 0.1799 þ 0.2332 x , p < 0.05), 6-year-old ( r 2 ¼ 0.34, y ¼ 2.0415 þ 0.2041 x , p < 0.05), and 7-year-old ( r 2 ¼ 0.29, y ¼ 1.9201 þ 0.3925 x , p < 0.05) stands (Fig. 4). No statisticallysigni cant relationship was found between past and currentinduced resistance in stands of 3-year-old ( r 2 ¼ 0.07, p > 0.05), and4-year-old ( r 2 ¼ 0.04, p > 0.05) stands (Fig. 4). Table 1 Pearson ’ s correlation coef cient ( r values) among leaf traits (length and width),induced resistance (hypersensitivity) and galling attack of Contarinia sp. (galls,attack, and attacked leaves) on young leaves of Anacardium occidentale (Ana-cardiaceae) in 3 e 7-year-old reforestation area study plots at the Trombetas bauxitemine. Note the positive correlations of variables (hypersensitivity, galls, attack andattacked leaves) with age.Length Width Hypersensitivity Galls Attack Attacked leavesStand age 0.01 0.04 0.41 b 0.30 a 0.41 b 0.57 b Length 0.85 b 0.02 0.03 0.02 0.09Width 0.00 0.04 0.02 0.05HR 0.56 b 0.93 b 0.75 b Galls 0.83 b 0.62 b Attack 0.78 ba p < 0.05; b p < 0.001. Fig.1. Leaf length average and proportion of galled leaves ( x SE) by Contarinia sp. onyoung leaves of Anacardium occidentale (Anacardiaceae) in 3 e 7-year-old reforestationarea study plots at the Trombetas bauxite mine. J.C. Santos, G.W. Fernandes / Acta Oecologica 36 (2010) 617 e 625 619 4. Discussion 4.1. Herbivore attack and plant-induced resistance through plant ontogeny Plant individuals change their structure, physiology, anddefenses during ontogeny (see Boege and Marquis, 2005). Corre- spondingly, herbivores should also be able to respond to theontogenetic changes of their host plants. For example, herbivoresshould exhibit differential female preference for and larval perfor-mance on different ontogenetic stages of their host plants. Thepresent study reveals an ontogenetic pattern of Contarinia gallpopulation throughout the development of its host, A. occidentale .Among trees, stand age positively in uenced the number of attacked leaves, attack rates (female preference) and abundance of galls (larval performance). Leaf attack was approximately 60%higher on older trees compared to younger trees (Fig. 1). Themedian value for female attack was 11-fold higher on older treescompared to younger trees and older trees supported two-foldmore galls than young trees (Fig. 2a,b).Plant responses to damage may depend on the plant type,ontogenetic stage and growth rate, and on the timing, amount andtypeofdamage(KarbanandBaldwin,1997;NykänenandKoricheva,2004). Our results also showed that A. occidentale hypersensitiveresponse (induced resistance) was in uenced by plant ontogeny.Themedianvalueofinducedresistancewasapproximatelysixtimeshigher on older trees (Fig. 2c). Therefore, we suggest that inducedresistancein A.occidentale wasage-dependent;thussupportingtheplant ontogeny e herbivory hypothesis.Several studies have indicated that differences in the develop-mentalphaseofplantscanstronglyin uenceresistancetoherbivory(Kearsley and Whitham, 1989; Karban and Thaler, 1999; Campos et al., 2003; Boege, 2005; Boege and Marquis, 2005; Fonseca et al., 2006) as also found in this study. For instance, Kearsley andWhitham (1989) found that the performance of two species of insects transferred onto different-aged trees of the same naturallyoccurring clones of narrowleaf cottonwood, Populus angustifolia ,showed opposing and signi cant changes in host resistance asa function of tree age. The gall-inducing aphid Pemphigus betae was70 times as common on mature trees as on juvenile trees andsurvivorship on mature trees was 50% higher than on juvenile trees.Nevertheless, the leaf feeding beetle Chrysomela con uens exhibitedopposite distribution on hosts, with densities 400 times higher on juvenile than onmature trees andsurvivorshipon mature trees was50%lowerthanonjuveniletrees.Infact,themagnitudeanddirectionof plant-induced responses and their effects on herbivore perfor-mance depend on the planttype (deciduous vs evergreen), inherentgrowth rate and ontogenetic stage (Nykänen and Koricheva, 2004). Other factors, such as habitat structural complexity, may in u-ence the attack by Contarinia and plant resistance. Barrett andAgrawal (2004) argued that interactions among genotype, envi-ronment, and ontogeny are strong determinants of herbivory.Although no measurement was taken on the structural complexityof restored stands in this study, habitat structural complexity andplantrichnessincreasewithtimeinstudiedstands(see Parotta andKnowles, 2001). Further studies will focus on the in uence of thesevariables on herbivore attack and plant ontogeny, as well as theeffect of tritrophic interactions, such as that between ants, insectgalls, and extra oral nectaries, on the dynamic interactioninvolving Contarinia and A. occidentale . 4.2. Plant vigor and induced resistance Thesizeofplantmodulesin uencesovipositionchoice,survival,andpupaloradultbiomassofmanyguildsofsedentaryherbivorousinsects, such as leaf miners and gall formers (Price, 1991; Clancyet al., 1993; Cornelissen et al., 2008), because size represents a high-quality indicator of resource availability to herbivores. Thegalling herbivore Contarinia oviposited and successfully inducedmoregallsonlargerleavesof A.occidentale ,aspredictedbytheplantvigor hypothesis (Price, 1991). Otherwise, our study corroborates the vigor hypothesis only in the older age stands. We propose thatchangesinplanttraitsrelatedtoplantontogenymayin uenceplant Fig. 2. Median number of attacks (all developed galls and killed by hypersensitivity)(a), developed galls (b) and hypersensitive reactions (HR) (c) in the cashew Anacardiumoccidentale (Anacardiaceae) in 3 e 7-year-old reforestation area study plots at theTrombetas bauxite mine. Letters represent comparisons between tree age treatments(post-hoc comparisons, p < 0.005). J.C. Santos, G.W. Fernandes / Acta Oecologica 36 (2010) 617 e 625 620 Fig. 3. Resource availability, preference of Contarinia sp. (Cecidomyiidae) and induced resistance of the cashew Anacardium occidentale (Anacardiaceae) in 3 e 7-year-old refores-tation area study plots at the Trombetas bauxite mine. The frequency distribution resource expressed as the percentage of total leaves in each leaf length class. Preference wasrepresented by percentage of galled leaves (a), number of galls/leaf (a) and number of attacks/leaf (b), while induced resistance (hypersensitive reaction e HR) was represented bynumber of HR lesions/leaf (b). J.C. Santos, G.W. Fernandes / Acta Oecologica 36 (2010) 617 e 625 621
