Mechanisms of Resistance to Insect Herbivores in Isolated BreedingLineages of Cucurbita pepo

Lauren J. Brzozowski1 & Michael Mazourek1 & Anurag A. Agrawal2

Received: 20 August 2018 /Revised: 5 November 2018 /Accepted: 15 January 2019 /Published online: 1 February 2019# Springer Science+Business Media, LLC, part of Springer Nature 2019

AbstractAlthough crop wild ancestors are often reservoirs of resistance traits lost during domestication, examining diverse cultivatedgermplasm may also reveal novel resistance traits due to distinct breeding histories. Using ten cultivars from two independentdomestication events of Cucurbita pepo (ssp. pepo and texana), we identified divergences in constitutive and induced resistancemeasured by growth of generalist caterpillars and leaf traits. C. p. texana cultivars were consistently more resistant toTrichoplusia ni and Spodoptera exigua, and this was not due to expected mechanisms including cucurbitacins, nitrogen, stickyphloem sap, or toxicity. Although more susceptible on average, C. p. pepo cultivars showed stronger induced resistance,suggesting a trade-off between constitutive and induced resistance. To test the hypothesis that leaf volatiles accounted fordifferences in resistance to caterpillars, we devised a novel method to evaluate resistance on artificial diet while larvae areexposed to leaf volatiles. In both subspecies, cultivar-specific induced volatiles that reduced T. ni growth were present in highlyinducible cultivars, but absent in those that showed no induction. These results have important agricultural implications ascultivar-specific resistance to caterpillars mirrored that of specialist beetles from field trials. Overall, the eponymous cucurbitacindefenses of the Cucurbitaceae are not the mechanistic basis of differences in constitutive or induced resistance between C. peposubspecies or cultivars. Instead, deterrent cultivar-specific volatiles appear to provide general resistance to insect herbivores.Divergence during breeding history within and between subspecies revealed this pattern and novel resistance mechanism,defining new targets for plant breeding.

Keywords Cucurbita pepo . Cucurbitacins . Herbivore induced plant volatiles . Plant-herbivore interactions . Trichoplusia ni

Introduction

Crop plants are often less resistant to herbivores than theirwild ancestors (Chen et al. 2015a; Whitehead et al. 2017),yet variation for resistance persists within crop germplasm.During breeding history, traits impacting herbivore resistancewere altered by direct (natural or human-directed) selection,indirect consequences of selection on other traits, and genetic

drift (Ladizinsky 2012). The effect of breeding history onherbivore resistance is amplified when crop germplasm is iso-lated in distinct breeding pools, and comparisons betweenthese pools provide opportunities to elucidate mechanismsof resistance. Indeed, tracking plant resistance through severalgenetic lineages revealed a decline in resistance from wildrelatives to landraces to modern cultivars (Dávila-Floreset al. 2013; Rodriguez-Saona et al. 2011; Rosenthal andDirzo 1997), and occasionally uncovered qualitative lossesof major resistance traits between isolated breeding lineages(Rasmann et al. 2005). Studying distinct lineages providesinsight not only on losses, but other possible outcomes, suchas novel resistance traits.

Elucidating resistance mechanisms through comparisonsof cultivated plants with distinct breeding histories is alreadysituated within the context of crop lineages, and thus may beadvantageous for further agricultural applications. First, dur-ing domestication, human consumers typically selected forpalatability and against toxicity (Meyer et al. 2012), presum-ably eliminating sources of resistance that would be

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s10886-019-01046-8) contains supplementarymaterial, which is available to authorized users.

* Lauren J. Brzozowskiljb279@cornell.edu

1 Section of Plant Breeding, School of Integrative Plant Science,Cornell University, Ithaca, NY, USA

2 Department of Ecology and Evolutionary Biology, and Departmentof Entomology, Cornell University, Ithaca, NY, USA

Journal of Chemical Ecology (2019) 45:313–325https://doi.org/10.1007/s10886-019-01046-8

unacceptable for consumption. Additionally, mechanisms ofeffective resistance in crop plants may differ from resistancetraits in their wild relatives. Indeed, some resistance traits areimportant in wild plant populations, but neutral or detrimentalunder cultivation (Gaillard et al. 2018; Turcotte et al. 2014). Aseparate suite of resistance traits may be more relevant inagricultural contexts because the community and intensity ofpests also differ (Chen et al. 2017). For instance, potential pestpressure from specialist insects at the onset of domesticationof maize may have disrupted balancing selective forces fromgeneralist and specialist herbivores (Gaillard et al. 2018).

Evaluation of isolated breeding pools that arose post-domestication have also provided insight into novel resistancemechanisms, including plant volatiles. Volatile compoundsserve as information to herbivores, for instance, a warning ofcompetition (Bruce and Pickett 2011), or as feeding deterrents(Shiojiri et al. 2006; Veyrat et al. 2016). A notable example isthe discovery that independent maize breeding programs inNorth America and Europe diverged in the production of thevolatile compound, (E)-β-caryophyllene (Degen et al. 2004).This volatile is induced in maize roots by the corn rootworm(Diabrotica virgifera virgifera) and provides critical indirectresistance through recruitment of entomopathogenic nema-todes (Rasmann et al. 2005). Across plant species, substantialvariation in plant volatiles exists in cultivated germplasm, andseveral domesticated plants have greater volatile induction ascompared to wild relatives (Rowen and Kaplan 2016). Thus,use of multiple cultivars with independent breeding historiesprovides a structure to identify novel resistance mechanisms,like plant volatiles, some of which could become the target ofbreeding programs.

Cucurbita pepo provides an excellent system to test howdivergent breeding histories in independent lineages may haveled to altered defensive traits. Within C. pepo, there are twocultivated subspecies, C. pepo ssp. pepo (hereafterC. p. pepo)and C. pepo ssp. texana (syn. C. pepo ssp. ovifera; hereafterC. p. texana), that have been bred to include multiple marketclasses, including pumpkin and zucchini in C. p. pepo, andacorn squash and summer squash inC. p. texana (Paris 2000).These subspecies arose from two separate domesticationevents (Decker 1988; Nee 1990; Sanjur et al. 2002), and crossbreeding is uncommon in modern breeding programs (Gonget al. 2012).C. p. pepo is thought to have been domesticated incentral Mexico from a yet unknown wild progenitor (Sanjuret al. 2002), but cultivar development predominately occurredin Europe starting in the sixteenth century (Paris 2000). Incontrast, C. p. texana was domesticated and developed inthe eastern United States and northern Mexico (Decker1988; Sanjur et al. 2002; Smith 2006). Because C. p. texanahas been developed in its center of origin, it exclusively hasbeen exposed to specialist beetle pests endemic to NorthAmerica (Metcalf 1985) throughout its breeding history.North American specialist beetles in the Diabroticina subtribe

(Coleoptera: Chyrsomelidae), including Acalymma vittatumand Diabrotica spp., strongly prefer C. p. pepo over C. p.texana cultivars (Ferguson et al. 1983; Hoffmann et al.1996; Brzozowski et al. 2016). Accordingly, these breedinglineages provide an opportunity to examine how independentdomestication events and breeding histories shaped the mech-anistic basis of plant resistance to these, and other, herbivores.

Cucurbitacins, intensely bitter and toxic triterpenoids, arean important form of resistance across the Cucurbitaceae togeneralist herbivores (Da Costa and Jones 1971; Metcalf1985), yet are tolerated and sequestered as a defense againstpredators by some specialist beetles (Ferguson and Metcalf1985; Metcalf et al. 1980; Metcalf 1986; Tallamy andKrischik 1989). Fruits of wild Cucurbita spp. containcucurbitacins, but today, Cucurbita spp. food crops lackcucurbitacins due to the identification of bitter-free mutantsduring the domestication process (Nee 1990). The implica-tions of loss of fruit bitterness for leaf-chewing herbivores ofC. pepo are unknown. While the full molecular pathway ofcucurbitacin production is yet to be elucidated in C. pepo, lossof fruit bitterness does not preclude cucurbitacin production inleaves in other Cucurbitaceae, like cucumber (Cucumissativus) (Shang et al. 2014; Zhou et al. 2016). Indeed,cucurbitacins are present in cotyledons of some domesticatedC. pepo (Ferguson et al. 1983), but have been reported to benil or at low constitutive concentrations in true leaves (Metcalfet al. 1980, 1982; Theis et al. 2014). Herbivory was previouslyshown to induce a substantial increase of a leaf cucurbitacin inone cultivar ofC. pepo (Tallamy 1985), but cucurbitacins haveonly been evaluated in a small number of cultivars, and theirpotential role in resistance and breeding requires additionalinvestigation. Additional defenses of C. pepo have also beenevaluated in some contexts: deterrent plant volatiles have beenmeasured in one cultivar (Peterson et al. 1994), and mucilag-inous sap has been studied in other Cucurbitaceae (McCloudet al. 1995; Dussourd 1997), but not C. pepo. Apart fromdefenses, leaf nutrient content is important for herbivore pref-erence and performance (Awmack and Leather 2002; Mattson1980). Nitrogen content was measured in a survey ofCucurbitaceae species but was not associated with specialistbeetle (A. vittatum) abundance (Theis et al. 2014).

In this study, we determine how plant resistance mecha-nisms diverged between the two cultivated C. pepo subspe-cies, representing two independent domestication eventswhere isolated breeding pools have been maintained. Onesubspecies, C. p. texana, has had continuous interaction withspecialist beetle pests, while the other (C. p. pepo) was geo-graphically separated.We thus hypothesized that these distinctbreeding histories would lead to disparate defense strategies,with different effects for generalists and specialists. We usedfive cultivars each of C. p. pepo and C. p. texana to test 1) fordivergence in constitutive and induced resistance to the twoleaf-feeding generalist caterpillars, Trichoplusia ni and

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Spodoptera exigua, as measured by larval performance, and 2)if differences in resistance were associated with secondarymetabolites or a suite of other foliar traits. Plant chemical traitsincluding cucurbitacins and nitrogen were measured andcomplemented by insect growth and behavioral assays. Todistinguish between deterrence and toxicity, we examinedgrowth efficiency of caterpillars as well as the impact of con-stitutive and induced volatiles on caterpillar growth. Inductionby caterpillar feeding was also compared to elicitation byjasmonic acid, the plant hormone primarily responsible fororchestrating induced resistance. And finally, given that spe-cialist beetle associations differentially affected C. pepobreeding histories, 3) we sought to test how findings fromgeneralist herbivores related to specialist beetle preference.Specifically, we tested the generality of resistance by relatingresistance to caterpillars to field preference of a major agricul-tural leaf-feeding specialist beetle pest (A. vittatum). In sum-mary, we used the independent domestication events ofC. pepo as a means to identify mechanisms of resistance tomultiple herbivores that may be useful in plant breeding.

Methods and Materials

Plant Material Five Cucurbita pepo cultivars (Table 1) eachwere used from two cultivated subspecies,C. p. pepo andC. p.texana (Gong et al. 2012; Paris et al. 2003). Plants were startedfrom untreated seed (source, Table 1) in the Cornell UniversityAgricultural Experiment Station greenhouses (Ithaca, NY,USA). In the greenhouses, a 14 hr photoperiod was main-tained, and the day and night temperatures, were 27 C and21 C, respectively. Plants were watered daily, treated with

standard greenhouse fertilizing practices (150 ppm 21–5-20NPK fertilizer, (Peters Company, Allentown PA, USA) fivetimes a week), and non-chemical pest control (bio-control)was used as necessary. Lambert LM-111 potting mix(Rivière-Ouelle, Québec, Canada) was used in all assays ex-cept the volatile assay, in which McEnroe Organic LiteGrowing mix (Millerton, NY, USA) was used to be in com-pliance with requirements of a certified organic greenhouse.Seeds were sown in individual 10 cm diameter pots for assaysthat required whole plants (whole plant feeding assays), and72-cell flats for assays that used excised plant tissue (mass perunit area consumed of leaf discs, volatile assay).

Insects Trichoplusia ni is polyphagous on plants in at least 36botanical families, including C. pepo and multipleCucurbitaceae (Sutherland and Greene 1984). Spodopteraexigua is likewise a generalist herbivore feeding on more than20 plant families, also including Cucurbitaceae (Tietz 1972).Prior to conducting the experiments described here, we con-ducted a feeding trial (in February 2015) to assess degree offeeding on C. pepo, and we observed substantial feeding byboth species (data not presented). Spodoptera exigua eggswere sourced from Benzon Research (Carlisle, PA, USA),and Trichoplusia ni eggs were supplied from a colony atCornell University (Dr. Ping Wang, Cornell AgriTech,Geneva, NY, USA). Due to similarity of results between cat-erpillar species we found at the subspecies level, and highS. exigua mortality in the induced resistance assay, T. ni wasused in all subsequent assays. For assays where caterpillarmass was measured, two unfed neonate caterpillars were ap-plied to each plant or diet. At the conclusion of each assay, allliving caterpillars were placed in Eppendorf tubes and frozen

Table 1 Cultivar listSubspecies Type Cultivar name Abbreviation Seed source

C. p. pepo pumpkin Charisma PMRa,f CH Johnny’s Selected Seeds

C. p. pepo zucchini Dunjaa,b,d DU Johnny’s Selected Seeds

C. p. pepo pumpkin Magic Lanterna,c,e ML Harris Seeds

C. p. pepo zucchini Costataa,c,f CO Cornell University

C. p. pepo zucchini Reward F1a,c RE Osborne Seed Company

C. p. texana acorn Honey Beara,c HB Johnny’s Selected Seeds

C. p. texana delicata PMR Bush Delicataa BD Cornell University

C. p. texana straightneck Success PMa,b,c,e SP Cornell University

C. p. texana crookneck Sungloa,f SU Osborne Seed Company

C. p. texana acorn Sweet Rebaa,b,c,d SR High Mowing Organic Seeds

a Included in induced resistance trialb Included in jasmonic acid assaysc Included in leaf trenching assayd Included in mass per unit leaf area assays, and volatile assays as most inducible cultivare Included in mass per unit leaf area assays, and volatile assays as least inducible cultivarf Not included in beetle correlation estimates because defoliation data was not available

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at −20 C for individual weighing later (AT21 ComparatorMicrobalance, Mettler-Toledo, Columbus, OH, USA). Inevaluating leaf trenching, T. ni were raised to second instaron high wheat germ diet (Bell et al. 1981) in a 26 C growthchamber before the assay. The same diet was also used in thevolatile assay.

Induced Resistance and Plant Chemistry in the TwoSubspecies – Induced Resistance Assay All cultivars weregrown to assess constitutive and induced leaf chemistry traitsand plant resistance to T. ni and S. exigua. This experimentwas conducted in two iterations in a randomized completeblock design with three blocks per iteration. In each block,there were seven plants of each cultivar and each plant wassubjected to one of seven treatments (Bt^): (t1) T. ni and (t2)S. exigua induction for chemical analyses, (t3) no herbivorycontrol for chemical analyses, (t4) T. ni and (t5) S. eixguainduction to measure the effect on subsequent conspecificherbivory, and finally untreated controls for induction by (t6)T. ni and (t7) S. exigua. Plants induction was achieved by fivedays (days 1–5) of herbivory by neonates immediately prior tochemical analyses or measuring effect on subsequent conspe-cific herbivory. The effect of induction was measured by cat-erpillar mass after five days (days 6–10) of feeding.

Seeds were sown in February 2015, approximately twoweeks prior to treatment to allow for plants to reach the 1–2leaf stage, and then plants were enclosed in mesh sleeves(30 cm × 18 cm). On the first day of the experiment(March 2015), plants were infested with T. ni (t1, t4),S. exigua (t2, t5), or left as is (t3, t6, t7). On day five, leaftissue was collected from plants with treatments for chemicalanalyses (t1-t3; i.e. cv. Dunja with T. ni feeding, S. exiguafeeding, and no herbivore control). The caterpillars were alsoweighed from those plants, and the plants were discarded.Also on day five, caterpillars were removed from the remain-ing T. ni and S. exigua feeding treatments (t4, t5), andweighed. Those plants (t4, t5) were then infested with newneonate conspecifics to test the effect of induction by conspe-cific prior herbivory. At the same time, the no herbivore con-trol plants were infested with T. ni (t6), or S. exigua (t7) tocompare to the effect of prior herbivory. The caterpillars ap-plied on day five were allowed to feed until day 10 when theywere removed and weighed. All caterpillars collected fromplants on day five were analyzed to examine constitutive re-sistance in all cultivars (n = 12 per cultivar). Caterpillars col-lected from plants on day 10 were used to examine the degreeof induced resistance (n = 6 per cultivar-treatmentcombination).

Induced Resistance and Plant Chemistry in the TwoSubspecies – Cucurbitacin Analysis Cucurbitacins were ex-tracted in a method similar to Theis et al. (2014), and a de-tailed description of the extraction, quantification and analysis

protocol is included in Supporting File S1. Briefly,cucurbitacins were extracted from freeze-dried tissue withmethanol, and were then purified with solid phase extraction.Cucurbitacins were quantified in a triple-quadrupole LC-MS/MS system (Accela-Quantum Access; Thermo Scientific)equipped with a C18 reversed-phase column (Kinetex2.6 μm EVO C18, 150 × 2.1 mm; Phenomenex).

Induced Resistance and Plant Chemistry in the TwoSubspecies – Nitrogen Analysis Tissue for nitrogen analysiswas sourced from the same freeze dried tissue used forcucurbitacin analysis. Tissue was finely ground (120 s at27 Hz, MM300, Retsch, Haan, Germany) and submitted tothe Cornell University Stable Isotope Laboratory (Ithaca, NY,USA) for continuous flow analysis of percent nitrogen andcarbon with an elemental analyzer. Per cultivar, five to sixsamples of controls and each treatment (T. ni and S. exiguaprior herbivory) were measured, with the following excep-tions: n = 4 for cv. Success PM – S. exigua; n = 3 for cv.Sweet Reba – S. exigua, T. ni.

Induced Resistance and Plant Chemistry in the TwoSubspecies – Jasmonic Acid Treatment Three cultivars wereused to test if jasmonic acid (JA) treatment had similar induc-tion effects to prior herbivory. We included two cultivars wehad found to be highly inducible, C. p. pepo (cv. Dunja) andC. p. texana cultivar (cv. Sweet Reba), by T. ni in previousassays, and a less inducible C. p. texana cultivar (cv. SuccessPM). Seed were sown in September 2015, and appropriateplants were sprayed with jasmonic acid (0.5 mM JA, dis-solved in ethanol) ten days after sowing. In one test, JA treatedplants were compared to plants sprayed with solvent (ethanol)alone with two to six replicates per treatment-cultivar. A sep-arate set of JA treated plants were compared to plants thatreceived two neonate T. ni, or nothing (control) with 2 itera-tions of 6 blocks (where three blocks were complete, and threewere nearly so, typically missing a single treatment-cultivarcombination) per cultivar and treatment. For both tests, fivedays after the treatments commenced (for JA treatments,there was one spray at the beginning of the five dayperiod), initial T. ni were removed from the appropriatetreatments, and then all treatments and control wereinfested with two neonate T. ni, which were allowedto feed for five days before weighing.

Feeding and Growth Bioassays – Qualitative Leaf TrenchingAsubset of six cultivars (three per subspecies, see Table 1) weresown on January 2017, and were grown to the three leaf stagebefore second instar T. niwere placed on the plants. The plantswere observed daily for eight days for evidence of trenching,and qualitative notes and photographs were taken. Leaftrenching was observed in Epilachna borealis in response tomucilaginous plant sap in C. maxima, wild C. p. texana, and

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C. okeechobeensis (McCloud et al. 1995), and also from T. niin C. sativus and C. moschata (Dussourd 1997). Observationof T. ni trenching behavior between C. pepo subspecies waschosen to test if T. ni exhibited a differential response that maybe indicative of differences in plant sap defenses.

Feeding and Growth Bioassays – Mass Gained per Unit LeafArea To address how larval growth was associated with leafconsumption, which can provide an indication of deterrenceversus toxicity, we conducted a bioassay of T. ni on four cul-tivars. Using the most and least T.ni - inducible cultivars weidentified in the full cultivar panel from each subspecies(Table 1), we presented T. ni with a single 10 cm2 leaf discon moistened filter paper in a plastic petri dish. Seeds weresown in March 2016, discs were removed from the newestfully expanded true leaf with a cork borer after two weeks ofgrowth, and T. ni fed on the discs for five days. Cultivars cv.Dunja and cv. Success PM had 20 replicates, cv. MagicLantern had 19 replicates, and cv. Sweet Reba had nine repli-cates. Leaf discs were imaged, and area damaged was mea-sured in imageJ (Schneider et al. 2012).

Later, the cultivars were grown to measure 10cm2 leaf discfresh and dry weight. Seeds were sown in November 2017,and nine samples per cultivar from separate plants were re-moved and weighed two weeks after sowing (HR-120, A&DCompany, Tokyo, Japan). The discs were lyophilized(FreeZone 2.5, Labconco, Kansas City, MO, USA) until dryand weighed immediately.

Feeding and Growth Bioassays – Volatile DeterrenceWe test-ed if foliar volatiles influenced T. ni feeding on artificial dietusing leaf tissue from cultivars we previously found to be themost and least inducible by T. ni from each subspecies(Table 1). This experiment was conducted in two iterationsof a randomized block design. Iteration 1 (August 2017) hadthree complete blocks, and iteration 2 (January 2018) hadseven blocks (where three blocks were complete, and fourwere nearly so, typically missing a single treatment-cultivarcombination). Each cultivar had an induction treatment (T. nifeeding on leaf tissue), or control treatment (leaf alone) perblock. The seeds were sown approximately 14 days before theassay commenced. Photographs and a detailed description ofthe experimental arena are shown in Fig. S4, and describedbriefly here. In each arena, neonate T. ni were placed on anexcess of diet in a small plastic cup covered by cheesecloth.Excised leaves were placed in 9.5 mL floral tubes (FloralSupply, Fruit Heights, UT, USA) filled with water, and refilledas necessary. An organza mesh bag (SumDirect manufactur-ing, Dongguan, China) was secured around the leaf, and twoneonate T. niwere added to leaves of the induction treatments.The diet cup and excised leaf were placed together in a 1 Lplastic container (Clear Lake Enterprises, Port Richey, FL,USA), and closed with a lid with small holes. The diet cup

was on the bottom of the container, and the leaf wassuspended 10 cm above the diet cup, and was kept in placeby the floral tube. As a check of the effect of the non-plantmaterials, a control treatment with no leaf but all accessorieswas also used. After three days, leaves with the T. ni inductiontreatment were scouted to confirm T. ni presence and feeding,and T. ni feeding on diet were recovered and weighed.

Resistance Comparison to A. vittatum To address the gener-ality of resistance mechanisms, the mass of T. ni caterpillarswas compared to previously obtained preference data of aspecialist herbivore of cucurbit crops, Acalymma vittatum(Brzozowski et al. 2016). The experiment is detailed inBrzozowski et al. (2016), and the objective was to assessA. vittatum preference for cultivars in the two subspecies ofC. pepo used in this experiment. Briefly, a field choice testwith n = 27 cultivars (n = 17 C. p. texana, and n = 10 C. p.pepo) (Brzozowski et al. 2016, Table 2) was conducted in2015 under naturally occurring A. vittatum infestation inFreeville, NY. Cultivars were grown with five replicates inthree-plant plots in a randomized complete block design, andA. vittatum preference was measured as estimated percent leafdefoliation of plants with one to three leaves (not flowering).Seven of the cultivars used in the field experiment were alsoused in experiments with T. ni (see Table 1). Importantly, inboth experiments, plants were at the same growth stage (1–3leaves, non-flowering). To test for C. pepo cross-resistance tothese herbivores, we determined the correlation betweenA. vittatum preference and T. ni performance on thesecultivars.

Table 2 Anova table from linear mixed effects model of herbivore massin cultivar panel induced resistance assay

Insect Effecta,b DF F-value P value

T. ni Subspecies 1 8.241 0.004

Treatment 1 6.133 0.005

Subspecies X Treatment 1 2.344 0.130

Iteration 1 4.834 0.015

Residuals 97

S. exigua Subspecies 1 3.482 0.066

Treatment 1 0.239 0.626

Subspecies X Treatment 1 0.200 0.657

Iteration 1 0.135 0.714

Residuals 70

Numbers in bold indicate significant differences at α = 0.05a Treatment refers to induction by conspecific feedingb There were two nested random effects: randomized complete blocknested within iterations of the experiment (Biteration^) (T. ni modelσ2 = 0; S. exigua model σ2 = 0.0002) and cultivar nested within subspe-cies (T. ni model σ2 = 0.0104; S. exigua model σ2 = 0.0009)

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Statistical Analysis Linear mixed models were used to modelthe response variables of caterpillar performance or chemicalconcentration as a function of plant cultivar and experimentalparameters. For each caterpillar sample, the mass of the twocaterpillars was averaged, and the average was used in furtheranalysis. If only one caterpillar was recovered, that mass wasused.

For the induced resistance and chemical assays, caterpillarmass or chemical concentration, respectively, were modeledwith subspecies, treatment, subspecies by treatment interac-tion, and iteration as fixed effects, and cultivar nested withinsubspecies and block nested within iteration were included asrandom effects. For assays with a subset of cultivars (jasmonicacid, mass per unit leaf area consumed, volatiles), caterpillarmass was modeled with cultivar, treatment, and cultivar bytreatment interaction as fixed effects. In the jasmonic acidand volatile assay, iteration was treated as a fixed effect, andblock nested within iteration was included as a random effectfor the volatile assay.

All statistical analyses were performed in R (R Core Team2016). Linear mixed models were calculated with the ‘lmer’function in the ‘lme4’R package (Bates et al. 2015), and linearmodels with the ‘lm’ function. In all cases, analysis of

variance was used to test significance of fixed effects, exceptfor in the mass per unit leaf area consumed assay where anal-ysis of covariance was used. Tukey’s honest significant differ-ence test was used to separate effect levels with the‘TukeyHSD’ function in ‘agricolae’ R package (deMendiburu 2016). Finally, correlation between A. vittatumpreference and T. ni mass was calculated using the ‘cor.test’R function.

Results

Induced Resistance and Plant Chemistry in the TwoSubspecies Both generalist caterpillars, T. ni and S. exigua,showed >40% lower mass after five days of feeding on C. p.texana cultivars compared to C. p. pepo cultivars (Fig. 1a,c;T. ni: F1,93 = 20.00, P < 0.001; S. exigua, F1,89 = 10.08, P =0.002). Induced resistance following herbivory reducedgrowth of both species, although the effects were most pro-nounced in C. p. pepo with T. ni. Induced resistance reducedT. ni mass by 21% in C. p. pepo, and 9% in C. p. texana(Table 2; Fig. 1b). For S. exigua, induced resistance reducedmass by 7% in C. p. pepo, but had no effect in C. p. texana

Fig. 1 Mass of generalistcaterpillars after feeding onC. pepo subspecies with andwithout induction by priorconspecific herbivory.Differences in constitutiveresistance by subspecies andcultivar are shown for (a) T. ni and(c) S. exigua. The effect ofinduction is summarized acrossvarieties by subspecies (b, d).Shown are means ±1 SE andasterisks indicate P < 0.05 for theeffect of subspecies in ANOVAsdescribed in the text. Cultivarabbreviations are listed in Table 1

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(Table 2; Fig. 1d). Based on these results, we selected the mostand least inducible cultivars from each subspecies for furtherassays (Table 1; Fig. S1). Mean caterpillar mass reductionacross both subspecies after induction by prior herbivorywas greatest in cv. Dunja (C. p. pepo; 28% decrease; F1,17 =5.08, P = 0.038), and cv. Sweet Reba (C. p. texana; 26% de-crease; F1,10 = 5.49, P = 0.041). In contrast, cv. Magic Lantern(C. p. pepo) and cv. Success PM (C. p. texana) showed notrends of induction, and were chosen as the least inducible(Fig. S1).

Cucurbitacins B, D, and E were detected in leaf tissue butonly in trace concentrations; in the majority of samples, noneof the cucurbitacins reached detectable levels (greater than0.05 ng g−1 dry weight for cucurbitacins B and E, and above1.0 ng g−1 dry weight for cucurbitacin D), and there was nopattern of cucurbitacins by subspecies or induction treatment(Table 3). In control plants, mean leaf nitrogen was nearly 9%higher in C. p. texana as compared to C. p. pepo cultivars(F1,51 = 11.60, P = 0.001, Table S1). However, following in-duction by T. ni herbivory, nitrogen remained constant inC. p.texana, but dropped 5% in C. p. pepo as compared to controls(Fig. 2a; Table S2). Leaf nitrogen also slightly decreased inC. p. pepo with induction by S. exigua, and had a smallerchange in C. p. texana as compared to controls (Fig. 2b;Table S2).

We tested whether the effects of induced resistanceby prior herbivory could be reproduced by applicationof jasmonic acid (JA) using a subset of cultivars (oneinducible C. p. pepo, one inducible C. p. texana, and

one non-inducible C. p. texana; Table 1; Fig. S1).Effects of induction by JA were similar to inductionvia prior conspecific herbivory (Fig S2; Table S4), and dis-tinct from controls (Table S3).

Feeding and Growth Bioassays T. ni exhibited trenching (toavoid sticky phloem sap) on all C. pepo cultivars(Fig. 3), but appeared to avoid feeding on C. p. texanacultivars for a longer period of time before trenching(LB, personal observation).

To address the nutritional quality or potential toxicity ofC. pepo,we conducted an assay tomeasure larval mass gainedby T. ni per unit leaf area consumed using four cultivars (twoof each subspecies, representing extremes of inductionresponse Table 1; Fig. S1). T. ni mass on C. p. pepo cultivarleaf discs was 32% higher than those feeding on C. p. texanacultivars (F3,64 = 6.77, P < 0.001; Table S5). Similarly, T. nicaterpillars consumed 58% more leaf area of C. p. pepo discs(F3,64 = 7.75, P < 0.001, Table S5). However, there was nodifference between cultivars in T. nimass attained per unit leafarea consumed (Fig. S3; Table 4). Separately, leaf disc freshand dry mass was measured, and fresh mass was significantlydifferent between cultivars (F3,31 = 14.50, P < 0.001;Table S5); nonetheless dry mass per area was consistent(F3,31 = 1.55, P = 0.211; Table S5). The results did not changewhen we converted area consumed to fresh mass consumed:T. ni caterpillars consumed 65% more fresh leaf mass of C. p.pepo discs (F3,64 = 13.37, P < 0.001; Table S5), and T. nimassgained per fresh mass consumed did not differ between culti-vars (F3,64 = 0.98, P = 0.41). In summary, these feeding assaysstrongly indicate that caterpillar growth was proportional tofeeding, and did not appear to be due to leaf toxicity.

In our volatile bioassay, neonate T. niwere fed artificial dietwith exposure to volatiles from an excised leaf of one of fourcultivars, representing extremes of induction response in eachsubspecies (Table 1; Fig. S1; Fig. S4).Mass of T. ni feeding ondiet was compared between those exposed to constitutiveplant leaf volatiles and those exposed to plant volatiles in-duced by active T. ni feeding. The mass of T. ni feeding onartificial diet was significantly affected by cultivar of the ex-cised leaf and induction treatment (F3,57 = 3.82, P = 0.015;Table 5; Fig. 4). Volatiles from leaves with active conspecificfeeding significantly loweredmass of T. ni feeding on artificialdiet in the two cultivars previously found to show the stron-gest induction response (C. p. pepo cv. Dunja: F1,16 = 5.59,P = 0.031; C. p. texana cv. Sweet Reba: F1,13 = 7.44, P =0.017; Fig. 4), but not in the two cultivars with weak inductionresponses (C. p. pepo cv. Magic Lantern: F1,17 = 0, P = 0.99;C. p. texana cv. Success PM: F1,8 = 0.80, P = 0.40; Fig. 4).

Resistance Comparison to A. vittatum Correlation betweenadult A. vittatum defoliation previously measured in the field(Brzozowski et al. 2016) and T. ni mass gain on the same

Table 3 Leaf cucurbitacin measurements

Cultivar Subspecies Cucurbitacin (ng g−1 dry tissue)a,b

Control / T. ni Inducedc,d

D B E

Charisma PMR C. p. pepo - / - - / - - / -

Dunja C. p. pepo - / - - / - - / -

Magic Lantern C. p. pepo - / - - / - - / -

PMR Costata C. p. pepo - / - - / 0.06 0.47 / 1.13

Reward F1 C. p. pepo - / - - / - - / -

Honey Bear C. p. texana - / - - / - - / -

PMR Bush Delicata C. p. texana - / - 0.07 / - - / -

Success PM C. p. texana - / - - / 0.10 - / 0.06

Sweet REBA C. p. texana 5.30 / - - / - - / -

a B-^ indicates that the particular cucurbitacin was not detected inthe sampleb Cucurbitacin I was not detected in any samples, and not presentedin this tablec BT. ni Induced^ refers to five days of T. ni damage prior to samplingd Induced cucurbitacins were additionally measured after S. exigua incultivars C. p. pepo cv. Charisma PMR, C. p. pepo cv. Dunja, and C. p.texana cv. Success PM, but none were detected

J Chem Ecol (2019) 45:313–325 319

cultivars measured here (Table 1) was strongly positive(Pearson’s r = 0.893, df = 5, P = 0.007; Spearman’s rank cor-relation, rho = 0.964, P = 0.003) (Fig. 5).

Discussion

Evaluating defensive traits in multiple cultivars from two paralleldomestication events revealed contrasting levels of resistanceand a novel mechanism of plant resistance to insects in

C. pepo. While C. p. pepo had lower constitutive resistance, itwas more strongly inducible when assayed bymeasuring growthof two generalist, leaf-chewing herbivores (T. ni and S. exigua).Lower resistance to these generalists in C. pepomirrored greaterpreference of an important specialist beetle pest, A. vittatum.Specific analyses of multiple metabolites, including leafcucurbitacins, did not explain these differences, and a growthassay demonstrated that caterpillars gained equal mass per unitleaf consumed in cultivars from both subspecies (consistent withthe lack of a toxic principle between cultivars). These results

Fig. 3 Examples of semi-circulartrenching by second instar T. ni onleaf edge when feeding on (a)Success PM (C. p. texana), (b)Reward (C. p. pepo), and exam-ples of major vein cutting on (c)Honey Bear (C. p. texana), (d)Magic Lantern (C. p. pepo). Allcultivars included in this assay areindicated in Table 1

Fig. 2 Leaf nitrogen content intwo C. pepo subspecies andinduction by prior herbivory by(a) T. ni and (b) S. exigua. Shownare means ±1 SE and the asteriskindicates P < 0.05 for subspeciesby induction treatment term inANOVAs described in Table S2

320 J Chem Ecol (2019) 45:313–325

echo Theis et al. (2014), where they likewise found thatneither leaf nitrogen nor cucurbitacins (nor other mea-sured leaf traits) predicted leaf damage by A. vittatumon 20 varieties from 12 diverse Cucurbitaceae species.

An important result of our work is that volatiles induced byactive T. ni damage in some varieties had a deterrenteffect on T. ni growth when feeding on a standard diet.As discussed below, the impact of plant volatiles onlarval feeding has been little studied, and is an area inneed of further investigation. The volatile deterrent ef-fect we found was not specific to either C. pepo sub-species, but instead was detected in cultivars that wefound to have the strongest inducible resistance. Whilefloral volatile composition in Cucurbita spp. and theimplications for specific herbivores and pollinators are well-characterized (Andersen and Metcalf 1986; Andrews et al.2007; Theis et al. 2009, 2014), knowledge of leaf volatilecomponents is limited. Volatiles from leaf trichomes ofone C. p. texana cultivar not used in this study wereshown to have attractant and repellent compounds to thepickleworm moth (Diaphania nitidalis) (Peterson et al.1994). Volatiles from induced by bacterial wilt infection(Erwinia tracheiphila) in wild C. pepo are also knownto attract more A. vittatum than healthy plants (Shapiroet al. 2012). Finally, in other Cucurbitaceae, inducedvolatiles have been implicated in the attraction of naturalenemies of herbivores (Agrawal et al. 2002; Kappers et al.2011; Takabayashi et al. 1994).

Plant domestication and breeding trajectory has a range ofeffects on herbivore-induced plant volatiles. Overall, recentmeta-analyses across systems showed that domesticatedplants have greater induced volatile production than wildplants (Rowen and Kaplan 2016), although parasitoid andpredator attraction are not consistently greater in cultivatedplants (Chen et al. 2015b). In maize, there was considerablegenetic variability in induced volatile production in a diversegroup of cultivars and wild accessions, and this was not pre-dicted by domestication status or subsequent breeding(Gouinguené et al. 2001). Nonetheless, by examining finer-scaled contrasts inmaize germplasm, it was revealed that plantbreeding history shaped herbivore-induced root volatilescritical for indirect defense: isolated North Americanand European breeding programs diverge in productionof (E)-ß-caryophyllene (Rasmann et al. 2005), and mod-ern hybrids lack oviposition induced volatiles present inlandraces (Tamiru et al. 2011). In a more recently do-mesticated crop with a known pedigree, cranberry(Vaccinium macrocarpon), induced volatiles measuredfollowing treatment with exogenous jasmonic acid wereconsistent across pedigree and state of cultivar improve-ment (Rodriguez-Saona et al. 2011). These results indi-cate that while varietal-specific induced volatiles arecommon, we may be best able to elucidate changes toherbivore-induced plant volatile production by evaluat-ing clearly defined contrasts of plant breeding lineages.

Mechanistically, volatile repellents can affect herbivoresthrough signaling status as a non-host with criterion as subtleas one component of a volatile blend, or warning herbivores ofplant defenses, potential competition, or natural enemies (Bruceand Pickett 2011). The impact of plant volatiles on larval feedingbehavior has thus far not received substantial attention, althoughit may have potential as a resistance mechanism. Inmaize, indoleis an herbivore induced plant volatile and was recently shown toreduce feeding and increase mortality of Spodoptera littoralis(Veyrat et al. 2016). Deterrent volatiles produced during theday also drive the nocturnal feeding behavior of Mythimnaseparate on maize (Shiojiri et al. 2006). Overall, deterrent plantvolatiles can impact caterpillar feeding behavior, but more workis needed to understand the mechanistic basis and ecologicalrelevance of such signals.

While we found cultivar-specific induced volatile deter-rence in both subspecies, there are remaining questions aboutwhat mechanisms support the large and repeatable differencesin overall resistance between C. pepo subspecies. It is possiblethat induced volatiles are more ubiquitous in one subspecies,and that could be addressed by testing induced volatile deter-rence in a broader C. pepo panel. We observed no evidence ofleaf toxicity, but instead less consumption of C. p. texanacultivars in no-choice assays, which indicates that there maybe differences in constitutive deterrent volatiles between sub-species that our volatile assay did not detect. Moving beyond

Table 5 Anova table from linear mixed effects model of T. nimass afterfeeding on artificial diet while exposed to plant volatiles

Effecta,b DF F-value P value

Cultivar 3 5.276 0.003

Treatment 1 6.939 0.011

Cultivar X treatment 3 3.818 0.015

Iteration 1 2.136 0.149

Residuals 57

Numbers in bold indicate significant differences at α = 0.05a Treatment refers to conspecific leaf feedingb Effect of block was treated as a nested random effect within experimen-tal iteration (σ2 = 0.008)

Table 4 Ancova table for the leaf area consumed covariate for T. nimass in the mass gained per unit leaf area bioassay

Covariate Effect DF F-value P value

Leaf area consumed Cultivar 3 2.289 0.088

Leaf area 1 37.563 <0.001

Cultivar X Leaf area 3 0.692 0.561

Residuals 60

Numbers in bold indicate significant differences at α = 0.05

J Chem Ecol (2019) 45:313–325 321

secondary metabolites, there are morphological differences inplant traits between subspecies, including leaf color andshape, but their relation to herbivore resistance is unknown(Paris et al. 2012). Perhaps examination of these traits, or othermore multi-functional traits, like leaf water content (Theiset al. 2014), or leaf turgor pressure (McCloud et al. 1995),would provide insight into the differences in plant resistancein C. pepo.

Additional work on mechanisms of preference in C. peposhould also examine causes of divergence in constitutive andinduced resistance between the independent domesticationevents. Induction is overall stronger in susceptible C. p. pepo,but there is greater constitutive resistance in the resistant C. p.texana. Induced resistance may be favored when herbivorepressure is intermittent and cost of defense is high (Karbanand Baldwin 1997), although tradeoffs between constitutive

and induced resistance strategies are apparently less commonin domesticated than wild plants (Kempel et al. 2011). Anexploration of costs of endemic herbivore damage in the re-gion of domestication and subsequent breeding may provideinsight into whether natural or human-mediated selection mayhave favored one type of resistance over another in eachsubspecies.

Because C. p. pepo appears to be most palatable forboth generalists assayed and specialist beetles, whatevermechanism increases C. p. pepo susceptibility to thetwo generalist herbivores assayed here could be thesame as that which increases preference of specialist beetles(Fig. 5) (Chrysomelidae: Galerucinae; Brzozowski et al. 2016;Ferguson et al. 1983; Hoffmann et al. 1996). Literatureon the preference of these beetles has been overwhelm-ingly associated with cucurbitacin content, where thesespecialists compulsively feed on and sequester cucurbitacins(Metcalf et al. 1980), and cucurbitacins increase larval perfor-mance (Tallamy and Gorski 1997; Halaweish et al. 1999).However, loss of fruit bitterness is a hallmark of all six do-mestications of the cultivated Cucurbita spp. (i.e. C. pepo,C. moschata, C. maxima, etc) (Decker 1988; Nee 1990;Sanjur et al. 2002), and we demonstrate here that leafcucurbitacin content is minimal in domesticated C. pepo.These results are inconsistent with cucurbitacins being theprincipal determinant of generalist or specialist herbivore be-havior, yet these diverse herbivores still find common groundin increased susceptibility of C. p. pepo.

Our results have interesting implications for how specialistand generalist herbivores respond to plant resistance traits inan agricultural context, and whether there is a dichotomy be-tween them. For instance, while many specialists haveevolved to overcome specific chemical defenses that are

Fig. 4 Mass of T. ni feeding onartificial diet for three days whenexposed to volatiles from leavesalone, leaves with T. ni feeding, orcontrols (Bempty^). Shown aremeans ±1 SE and asterisksindicates P < 0.05 for effect oftreatment (leaf alone, or with T. nifeeding) in varietal-specificANOVAs described in the text.# indicates the two cultivars thatdemonstrated the strongest in-duction response in previousassays

Fig. 5 Correlation of mass of T. ni after feeding for five days on control(non-induced) plants compared to percent defoliation by A. vittatum infield plots (Brzozowski et al. 2016) of the same cultivars (Table 1). Eachpoint represents the mean of an individual cultivar ±1 SE

322 J Chem Ecol (2019) 45:313–325

effective against generalist herbivores through sequestrationor avoidance (Ali and Agrawal 2012), such Bresistance^ traitsmay often be lost in the domestication process (Chen et al.2015a; Whitehead et al. 2017). In the current study, we founda similar outcome of the breeding process for well-adaptedspecialists and non-adapted generalists of C. pepo, indicatingsubstantial cross resistance.

Overall, this work highlights the benefits of studyingthe chemical ecology of diverse pools of cultivatedgermplasm with distinct breeding histories for discover-ing new mechanisms of resistance to insect herbivores.Dogma in plant breeding is that we must look to thegenetic diversity of wild species for traits to introgressinto elite germplasm to steel it against our most press-ing biotic and abiotic challenges (Dempewolf et al. 2017;Harlan 1976; McCouch 2004; Rick 1978; Tanksley andMcCouch 1997). With this approach, more than 2000 bioticstress resistance traits have been identified in crop wild rela-tives; however, the vast majority of traits identified are fordisease resistance, and less than one quarter of these targetinsect pests (Dempewolf et al. 2017). Thus, strategies forbreeding for resistance to insect pests must also be in-clusive of secondary centers of diversity, and contempo-rary breeding pools. The context in which these breed-ing pools were developed may also better reflect thecontext of agricultural plant-herbivore interactions thanwild systems, where major secondary metabolites (likecucurbitacins) may have different effects. In the diversepools of cultivated germplasm with distinct breedinghistories, plant breeders may discover alternative, per-haps quantitative, and likely smaller-impact resistancetraits. Screening material for the most promising, butless obvious traits will benefit by being informed bychemical ecology, and incorporating these traits intoagroecosystems will require tools from associated disciplines(Brzozowski and Mazourek 2018).

In conclusion, by using two independent domesticationevents to evaluate divergence in plant resistance traits inC. pepo, we identified differences in resistance that spanneddiverse leaf-chewing herbivores. We found that thesedifferences did not align to major known resistancetraits, like cucurbitacins, or nitrogen, but instead foundvarietal-specific induced volatile deterrents in both sub-species. This work contributes to the growing evidencethat plant defenses in the context of cultivated systemsmay be distinct from those in the wild (Chen et al.2017), and has implications for plant breeders. By ex-ploring defensive novelty through contrasting domestica-tions and breeding histories in C. pepo, we found sus-ceptibility in one lineage that persisted through the be-havior of diverse herbivores, and discovered a previous-ly unknown defense trait in C. pepo that may provide a newtarget for plant breeders.

Acknowledgements We thankWendy Kain and PingWang for providingT. ni, Georg Jander for providing S. exigua, William Holdsworth forassistance with cultivar selection, Amy Hastings for supporting laborato-ry and greenhouse work, Katalin Boroczky for development of HPLC-MS methods for cucurbitacin detection, Taylor Anderson for de-velopment of cucurbitacin solid phase extraction protocol, and theCornell University Agricultural Experiment Station greenhousestaff for providing excellent care of plant material. The manu-script was improved by thoughtful comments from Katja Povedaand two anonymous reviewers. LB was supported by a CornellUniversity Presidential Life Science Fellowship (2014-2015) and aSeed Matters Graduate Student Fellowship (2015-2019). Thiswork was supported by the United States Department of AgricultureNational Institute of Food and Agriculture Multi-State Hatch Project1008470, Harnessing Chemical Ecology to Address Agricultural Pestand Pollinator Priorities.

Funding LB was supported by a Cornell University Presidential LifeScience Fellowship (2014–2015), and a Seed Matters Graduate StudentFellowship (2015–2019). This work was supported by the USDANational Institute of Food and Agriculture, Multi-State Hatch Project1008470, Harnessing Chemical Ecology to Address Agricultural Pestand Pollinator Priorities.

Compliance with Ethical Standards

Conflict of Interest LB and AA declare that they have no conflict ofinterest. MM is the co-founder of, but has no financial stake in, Row 7, acompany that sells organic seed.

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