A Specialist Root Herbivore Reduces Plant Resistance and Uses an Induced Plant Volatile to Aggregate in a Density-Dependent Manner

Functional Ecology 2012, 26, 1429–1440 doi: 10.1111/j.1365-2435.2012.02030.x A specialist root herbivore reduces plant resistance and uses an induced plant volatile to aggregate in a density-dependent manner

Christelle A. M. Robert1,2, Matthias Erb2, Bruce E. Hibbard3, B. Wade French4, Claudia Zwahlen1 and Ted C. J. Turlings*,1

1Laboratory for Fundamental and Applied Research in Chemical Ecology (FARCE), University of Neuchâtel, Rue Emile- Argand 11, 2000, Neuchâtel, Switzerland; 2Root-Herbivore Interactions Group, Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Str. 8, 07745, Jena, Germany; 3United States Department of Agriculture, Agricultural Research Service, Plant Genetics Research Unit, University of Missouri, 205 Curtis Hall, Columbia, MO, 65211 USA; and 4United States Department of Agriculture, Agricultural Research Service, North Central Agricultural Research Laboratory, 2923 Medary Avenue, Brookings, SD, 57006 USA

Summary 1. Leaf-herbivore attack often triggers induced resistance in plants. However, certain specialist herbivores can also take advantage of the induced metabolic changes. In some cases, they even manipulate plant resistance, leading to a phenomenon called induced susceptibility. Compared to above-ground plant-insect interactions, little is known about the prevalence and consequences of induced responses below-ground. 2. A recent study suggested that feeding by the specialist root herbivore Diabrotica virgifera virgifera makes maize roots more susceptible to conspecifics. To better understand this phenomenon, we conducted a series of experiments to study the behavioural responses and elucidate the underlying biochemical mechanisms. 3. We found that D. virgifera benefitted from feeding on a root system in groups of intermediate size (3–9 larvae/plant in the laboratory), whereas its performance was reduced in large groups (12 larvae/plant). Interestingly, the herbivore was able to select host plants with a suitable density of conspecifics by using the induced plant volatile (E)-b-caryophyllene in a dose-dependent manner. Using a split root experiment, we show that the plant-induced susceptibility is systemic and, therefore, plant mediated. Chemical analyses on plant resource reallocation and defences upon herbivory showed that the systemic induced-susceptibility is likely to stem from a combination of (i) increased free amino acid concentrations and (ii) relaxation of defence inducibility. 4. These findings show that herbivores can use induced plant volatiles in a density-dependent manner to aggregate on a host plant and change its metabolism to their own benefit. Our study furthermore helps to explain the remarkable ecological success of D. virgifera in maize fields around the world.

Key-words: defence inducibility relaxation, Diabrotica virgifera virgifera, induced susceptibility, resource reallocation, root–herbivore interactions, Zea mays

mary compounds (Babst et al. 2005; Orians, Thorn & Introduction Gomez 2011). In many cases, the induced changes increase To withstand herbivory, plants reconfigure their metabo- the plant’s resistance against the attacking herbivore (Step- lism (Karban & Baldwin 1997; Walling 2000; Schwachtje puhn et al. 2004; Erb et al. 2009; Glauser et al. 2011). & Baldwin 2008). This reconfiguration includes the pro- However, in some cases, herbivore attack can also reduce duction of toxic secondary metabolites (Steppuhn et al. plant resistance. Most of the time, such plant susceptibility 2004; Glauser et al. 2011), as well as reallocation of pri- is induced by specialist herbivores, which have, over evolutionary time, adapted to specific host plants. Several *Correspondence author. E-mail: [email protected] mechanisms have been proposed to contribute to induced

© 2012 The Authors. Functional Ecology © 2012 British Ecological Society 1430 C. A. M. Robert et al. susceptibility (Karban & Agrawal 2002). First, herbivores 1987; Hibbard et al. 2003, 2004), possibly upon overex- may be able to suppress plant defences, by physically ploitation of their initial food source (Hibbard et al. 2004). severing defensive structures (Berryman et al. 1989; Raffa In the current study, we tested whether D. virgifera can 2001; Wallin & Raffa 2001; Kane & Kolb 2010) or inter- use the induced volatiles to select plants with an optimal ference with defensive signalling (Musser et al. 2002; Bede density of attacking conspecifics. We further investigated et al. 2006; Sarmento et al. 2011). Second, herbivores can whether the increase in growth on infested plants is attrib- evolve resistance to induced secondary metabolites utable to a stimulating effect of the induced volatiles, but (Ehrlich & Raven 1964; Rausher 1996; Berenbaum & Zan- also if possible changes in primary metabolism may be gerl 1998; Stout & Bostock 1999; Glauser et al. 2011; Rob- responsible for the increase in performance. For instance, ert et al. 2012b) and even use them to their own advantage D. virgifera has been shown to induce water-stress in maize (Hopkins, Ekbom & Henkow 1998; Agrawal & Sherriffs (Godfrey, Meinke & Wright 1993; Dunn & Frommelt 2001; Smallegange et al. 2007; Howe & Jander 2008; Rob- 1998; Erb et al. 2011b), a condition that can lead to an ert et al. 2012b). Third, herbivores can induce reallocation increase in shoot–root assimilate flow (Farooq et al. 2009). of primary metabolites to their feeding site (Way & To test this, we quantified the levels of free amino acids Cammell 1970; Larson & Whitham 1991; Giron et al. and sugars as well as the expression of marker genes asso- 2007; Kaiser et al. 2010; Compson et al. 2011). ciated with carbon transport and partitioning. Finally, we One important aspect of induced susceptibility is density investigated whether attack by high densities of D. virgif- dependence. Most herbivores can only benefit from induced era reduces the capacity of maize plants to mobilize changes in their host plant as long as resources are suffi- defences in response to subsequent herbivory by quantify- ciently abundant (Katano et al. 2007). High densities of ing the expression of maize defence marker genes. herbivores invariably lead to intraspecific competition and resource overexploitation, which reduces herbivore fitness (Ellner et al. 2001). Yet, it remains unclear to what extent Materials and methods specialist herbivores can select host plants on the basis of an optimal density of attacking conspecifics. Herbivore- PLANTS AND INSECTS induced plant volatiles that are emitted in a density-depen- Maize plants (Zea mays, variety Delprim) were sown in plastic dent manner (Shiojiri et al. 2010) can provide information pots (11 cm high, 4 cm diameter) by placing them on moist about the status of the host plant and may be used by her- washed sand (0–4 mm; Jumbo, Marin-Epagnier, Switzerland) and bivore to optimize host selection in this context. covering them with 2 cm of commercial potting soil (Aussaaterde, Ricoter, Aarberg, Switzerland). Seedlings were grown in a climate While it is generally accepted that induced resistance is chamber (23 ± 2 °C, 60% relative humidity, 16:8 h L/D and more common than induced susceptibility in above-ground 350 lmol mÀ2 sÀ1), and MioPlant Vegetable and Herbal Fertilizer plant–insect interactions (Karban & Agrawal 2002), even (Migros, Neuchaˆ tel, Switzerland) was added every 2 days after for specialist herbivores (Agrawal & Kurashige 2003), little plant emergence. Twelve-day-old plants with two fully developed is known about induced changes in resistance below- leaves were used for the experiments. Diabrotica virgifera eggs were obtained from the USDA-ARS-NCARL (Brookings, SD, ground, despite the fact that root herbivores are common in USA) and kept on freshly germinated maize until use. Second, many ecosystems and are among the most important agri- instar larvae were used in all laboratory experiments. cultural pests (Hunter 2001). In a recent study, feeding by larvae of the vine weevil, Otiorhynchus sulcatus, on rasp- DENSITY-DEPENDENT PERFORMANCE OF berry plants caused a 19% reduction in growth of subsequently attacking weevil larvae (Clark, Hartley & D. VIRGIFERA Johnson 2011). On the other hand, slightly damaged onion To assess the effect of egg density on D. virgifera under natural bulbs support higher survival of Delia antiqua larvae (Haus- conditions, a field study was conducted at the University of Mis- mann & Miller 1989), and D. radicum larvae grow better on souri Bradford Research and Extension Center, 9 km east of Columbia, MO, USA in 2005. Field design and soil conditions previously attacked turnip plants (Pierre et al. 2011). It has were described elsewhere (Hibbard et al. 2010). For this field been speculated that induced resistance below-ground may experiment, the line DKC 60–17 (RR) was used. Briefly, plots of be less frequent than above-ground, as alternative trading- 64 maize plants were planted using at 76·2 cm row spacing and off strategies like induced tolerance, such as induced 17 cm seed spacing. Densities of 25, 50, 100, 300, 600, 1200 and · resource sequestration and root regrowth after herbivory, 2400 viable D. virgifera eggs per 30 5 cm of maize row were applied into the soil and left to develop. Plots were covered with a may provide a greater benefit to the plant (Erb et al. 2012). screen tent (3·05 9 3·66 m; Coleman, Rye, NY, USA) prior adult We previously found that the specialist root herbivore emergence. Emerging beetles were collected two to three times per Diabrotica virgifera virgifera LeConte (Coleoptera: Chryso- week using either mouth aspirators (BioQuip, Rancho Domin- melidae) induces susceptibility in its host plant, Zea mays guez, CA, USA) or battery-operated aspirators (BioQuip). Adults L. (Poaceae), and that the herbivore uses induced volatiles were immediately transferred into 95% ethanol, and head capsule widths of the collected beetles were measured. Each egg density to find infested host plants (Robert et al. 2012a). Diabroti- was replicated four to six times. To evaluate the effect of larval ca virgifera larvae remain highly mobile during their devel- density on D. virgifera in the laboratory, maize seedlings were opment, and several studies show that they change host infested with one, three, six, nine or twelve larvae (n = 7), pre- plants and redistribute at later instars (Strnad & Bergman weighed using a XP2U micro-scale (Maximum capacity 6·1g,

© 2012 The Authors. Functional Ecology © 2012 British Ecological Society, Functional Ecology, 26, 1429–1440 Below-ground herbivory reduces root resistance 1431 readability: 0·1 lg; Mettler-Toledo International Inc., LLC, system. Synthetic (E)-b-caryophyllene (Sigma Aldrich Chemie Columbus, OH, USA). After 2 days, all larvae were collected by GmbH, Buchs SG, Switzerland) was continuously released into hand sorting and weighted again to determine their performance. the rhizosphere of one of the healthy plants using slow-release capillary dispensers as described by von Me´ rey et al.(2011). Capil-

laries of 0·5, 1, 2, 3, 6 and 25 lL were used (n0·5 and 2 lL = 11, DENSITY-DEPENDENT ATTRACTION OF D. VIRGIFERA n1, 3, 6 and 25 lL = 10). One microlitre capillary dispensers continuously release up to 40 ng hÀ1 (E)-b-caryophyllene, which corre- To evaluate the attraction of D. virgifera to infested plants at sponds to the emission of a D. virgifera infested root system different densities, healthy plants and plants infested with one, (Robert et al. 2012a). Thus, the amount of (E)-b-caryophyllene three, six, nine or twelve larvae were potted in two-arm below- released by the 0·5-, 1- and 2-lL dispensers are within the physio- ground olfactometers as described elsewhere (Robert et al. 2012a). logical range of infested maize seedlings, while the dose of (E)-b- All pots were filled using moist white sand (10% water; Migros) caryophyllene released by bigger capillaries would be aberrant in and covered with aluminium foil to avoid light stress and soil desic- nature. All pots were wrapped in aluminium foil to avoid light cation. After 2 days, the two pots were connected via an empty and desiccation of the system. After 48 h, the two pots were con- glass tube with a vertically connected access in the middle and one nected using an empty glass tube and Teflon connectors. After Teflon connector at both sides of the glass tube as previously 30 min, six D. virgifera larvae were inserted into the below-ground described. The Teflon connectors contained a fine metal screen olfactometers central part and their choice pattern was recorded. (2300 mesh; Small Parts Inc., Miami Lakes, FL, USA) that allowed volatile dispersion but avoided any visual or liquid cues for the herbivore. Furthermore, Teflon connectors prevented the larvae from D. VIRGIFERA PERFORMANCE ON SYSTEMIC ROOTS reaching the roots. A group of six larvae was released in the central OF INFESTED PLANTS glass connector of the olfactometer, and the first choice of the insects towards one or the other plant was recorded. In total, 346 To investigate whether the increased growth of D. virgifera on larvae were used for the choice experiments. Larvae that did not previously infested plants is attributable to plant-mediated effects choose after 15 min were noted as;no choice’. Leaf wilting was or direct, physical interaction with conspecifics, a split root system recorded for all plants and scored from zero (no symptoms) to four was designed. Maize root systems were gently washed with tap (complete loss of turgidity) as previously described (Erb et al. water and potted in two separate glass vials (15 cm long, 2 cm = 2011a). The experiment was repeated twice (nI1, I3, I6, I12 17, diameter) filled with moist white sand (10% water). Five D. virgifera = nI9 8). At the end of the first assay, CO2 emissions were evaluated larvae were added to one of the glass tubes (local roots). Control as described below and root systems were collected and gently plants were left uninfested. After 4 days, a second batch of five washed with tap water to determine their fresh biomass. In the sec- D. virgifera larvae was weighted as described above and placed on ond repetition, root systems were collected at the end of the choice the second (systemic) side of the root system of all plants. Six experiment, washed with tap water and immediately frozen in hours later, larvae were recovered and re-weighed. liquid nitrogen to determine induced volatiles as described below.

VOLATILE-MEDIATED FEEDING STIMULATION QUANTIFICATION OF VOLATILES To determine whether the increased growth of D. virgifera is To investigate the potential signals that D. virgifera uses to assess attributable to volatile-mediated feeding stimulation, we measured the infestation level of a plant, volatile emissions of seedlings that the growth of the herbivore upon exposure to induced volatiles. were used in the above two-arm olfactometers assays were deter- For this, two glass pots (5 cm diameter, 11 cm deep) were con- mined. CO2 emission was evaluated from the plants of the first nected as described above. The first pot contained the odour choice experiment (see above) by connecting the below-ground source: a healthy plant, a plant infested with five D. virgifera lar- glass pots to an additional glass vessel (28 cm long, 5 cm diame- vae or five larvae feeding on artificial diet (Pleau et al. 2002). ter) via the connector and a glass joint (n = 8). The glass vessel After 24 h, five D. virgifera larvae were weighted and allowed to was closed using parafilm and left to stabilize for 1 h. A CO2 gas feed on artificial diet in the second pot while exposed to the differ- meter (Voltcraft, CM-100; Conrad Electronics, Dietlikon, Switzer- ent odour sources at the same time (ndiet = 6, nhealthy plants = 9, land) was then introduced into the connected vessel for 3 min, ninfested plants = 12). One day later, the larvae were collected and and CO2 levels were recorded. Induced volatiles were determined re-weighed. from the second repetition of the choice experiment (see above) using SPME GC-MS following a previously described protocol (Erb et al. 2011a) (n = 8). The obtained peaks were analysed and INDUCED CHANGES IN PRIMARY METABOLISM identified by comparing volatile retention times and mass spectra with those of the NIST05 Mass Spectral Library (Agilent Techno- To evaluate whether changes in plant primary metabolism benefits logies Life Sciences and Chemical Analysis Group, Santa Clara, D. virgifera, the local and systemic response of maize roots follow- CA, USA) and those of pure compounds. ing infestation was investigated using the split-root system as described above. Five D. virgifera larvae were added to one of the glass tubes (local roots). Control plants were left uninfested DOSE-DEPENDENT RESPONSES OF D. VIRGIFERA TO (n = 7). The local and systemic parts of the root system were col- (E)-B -CARYOPHYLLENE lected separately 4 days after infestation, washed with tap water and immediately frozen in liquid nitrogen and stored at À80 °C. As (E)-b-caryophyllene can be detected in vivo in the headspace To ensure enough material for analyses, roots from three plants of D. virgifera-induced roots (Hiltpold et al. 2011), has superior were pooled together. Roots were ground into a fine powder diffusion properties in the soil (Hiltpold & Turlings 2008) and was under liquid nitrogen. Free amino acids were determined as previ- previously reported to be attractive for D. virgifera larvae (Robert ously described (Knill et al. 2008). Sucrose and hexose contents et al. 2012a), we focused on this compound and investigated were determined enzymatically using a Sucrose, D-fructose and whether D. virgifera can use it in a dose-dependent manner to D-glucose assay kit (Megazyme International Ireland Limited, detect infested plants. Two-arm olfactometers were used as Bray Business Park, Bray, Co. Wicklow, Ireland) following the described above. Healthy plants were potted in both arms of the manufacturer’s instructions (Beutler 1988; Kunst, Draeger &

Ziegernhorn 1988; Outlaw & Mitchell 1988). The concentrations ear regressions (P < 0.10) and the independency of the variables. of D-glucose, D-fructose and sucrose were calculated using the Diabrotica virgifera performance on healthy or infested plants in megazyme Mega-CalcTM software (Megazyme International Ire- the split root design was compared using Student’s t-tests. The land Limited, Bray Business Park, Bray Co. Wicklow, Ireland). effect of volatiles on D. virgifera growth on artificial diet was anal- The expression of marker genes involved in carbohydrate trans- ysed using a Kruskal-Wallis on ranks test (H-test), and the com- port and metabolism was assessed using previously established parison between the growth of larvae exposed to plant odours methods and primers listed in Table S1 (Supporting information; with the growth of larvae exposed to the volatile bouquet of con- Erb et al. 2010). specifics feeding on artificial diet was compared using a t-contrast on ranks test. The effect of D. virgifera feeding on amino acids and carbohydrate contents as well as marker gene expression in INDUCED CHANGES IN PLANT DEFENCES maize roots were investigated using t-tests when the data fulfilled the heteroscedasticity of error variance and normality conditions, To test whether D. virgifera suppresses plant defences in maize otherwise, Mann–Whitney rank sum tests (U-tests) were con- roots, we analysed the expression of marker genes involved in ducted. plant direct defences, hormonal signalling and volatile production using previously established methods and primers (Erb et al. 2009). For this experiment, the same cDNA as for the experiment Results above was used.

INDUCED SUSCEPTIBILITY BY D. VIRGIFERA IS ROOT DEFENCE RESPONSE TO SUBSEQUENT ATTACK DENSITY-DEPENDENT

To test whether the root infestation impacts the ability of healthy In the field, D. virgifera head capsule width, which can be roots from the same plant to respond to subsequent attack, maize used as a fitness indicator (French & Hammack 2010), was root systems were split by washing and transplanting them in two found to be density-dependent: D. virgifera adults tended glass tubes as described above. Five D. virgifera larvae were added in one of the tubes. Control plants remained uninfested on both to have smaller head capsules when feeding together in sides. As D. virgifera feeding would be influenced by the previous small densities than in medium densities. Furthermore, infestation treatment (see above), we used jasmonic acid (JA) as a adults that fed in medium densities had larger head capsules second inducer. JA is produced in the roots after D. virgifera than those feeding in large densities (n25 = 6, n50, 100 = 5, attack (Erb et al. 2009) and induces root volatiles in a similar n = 4; PROC mixed, P = 0·018; Fig. 1a). manner as the herbivore (Erb et al. 2011a), which is why we used 300, 600, 1200, 2400 this hormone as a herbivore-mimic for this particular experiment. Diabrotica virgifera larval performance was also found to Four days after infestation, 10 mL aqueous solution of 100 lM jasmonic acid or 10 mL of water only were added to the systemic (a) P = 0·07 side of the root system (n = 8). Twelve hours later, roots of the 1·1 two treatments (C?JA, D. virgifera ?JA) were collected, washed a a a a with tap water, frozen in liquid nitrogen and ground into a fine ab powder as described above. The expression of marker genes that ab are directly involved in the production of secondary metabolites b and defensive proteins and, consequently, are expected to contrib- 1 ute to induced resistance, was determined.

STATISTICAL ANALYSES Head capsule with (mm) 0·9 Analyses were performed on the software package R, version 2·8·1 25 50 100 300 600 1200 2400 and SAS Statistical Package (2004; SAS Institute Inc., Cary, NC, Egg density per 30·5 cm USA). All data were first analysed with a Levene’s and a Kol- (b) mogorov–Smirnov test to determine heteroscedasticity of error 70 variance and normality. The effect of D. virgifera density on head b 60 capsule width in the field and performance in laboratory were b b analysed using the PROC Mixed model of the SAS Statistical Pack- 50 ab age. Diabrotica virgifera choice was evaluated using a log linear 40 model (glm) using R. As the data did not fit to simple variance 30 assumptions implied in using a binomial distribution, quasi-likeli- hood functions were used to compensate for the over-dispersion 20 a of the larvae in the system. The two repetitions of the experiment 10 were included as a co-factor in the analysis. As the repetition of Relative weight gain (%) the experiment had no effect on the model, the factor ‘experiment’ 0 136912 was removed from the analysis. Root biomasses and volatile pro- Larval density duction of healthy and infested plants were compared using one- way ANOVAs followed by post hoc Tukey’s HSD tests. If the data Fig. 1. Diabrotica virgifera performance is density-dependent. did not pass the two tests, Kruskal-Wallis one-way ANOVAson (a) D. virgifera adult head capsule width after developing on ranks were performed, followed by pairwise Dunn’s tests. The plants infested with different egg densities. (b) D. virgifera larvae effect of the emitted volatiles on the larval choice was evaluated performance when feeding on plants with different larval densities by performing multiple one-way ANCOVAs with one volatile as a in laboratory. Mean ± SE are presented. Different letters indicate co-factor in each analysis, after testing the assumptions of hetero- significant differences (P < 0·05) within each larval density using scedasticity of error variance and normality, significance of the lin- the differences of least squares means.

© 2012 The Authors. Functional Ecology © 2012 British Ecological Society, Functional Ecology, 26, 1429–1440 Below-ground herbivory reduces root resistance 1433 be density-dependent in the laboratory, where larvae grew production of (E)-b-caryophyllene, a-humulene, a-copae- better when feeding in groups of three, six or nine larvae ne, tetradecane, heptadecane, 4-methyl nonane, 4-methyl than alone (n = 7; PROC mixed, P = 0·197; Fig. 1b). heptane and tetradecene (n = 8; Kruskal-Wallis one-way analysis of variance on ranks: P < 0·05). Among those compounds, only (E)-b-caryophyllene, a-humulene and D. VIRGIFERA IS ATTRACTED TO PLANTS WITH A a-copaene are actually released into the rhizosphere (data MEDIUM DENSITY OF CONSPECIFICS not shown). When included as co-variates into the choice Diabrotica virgifera larvae preferentially oriented towards pattern model, only (E)-b-caryophyllene and a-humulene healthy plants rather than plants that were infested with improved the model fit of the ANCOVA compared to a sim- low (1 larvae) or high (12 larvae) density of conspecifics, ple ANOVA (one-way ANOVA: P = 0·021; one-way ANCOVAs, but they were significantly attracted to plants that were (E)-b-caryophyllene: P = 0·008; a-humulene: P = 0·03; infested with a medium density of 6 larvae a-copaene: P = 0·603; Fig. 3), indicating that they are

[nI1, I3, I6, I12 = 17, nI9 = 8; glm, Control (C) vs. I1,I6 and most likely to explain the density-dependent D. virgifera

I12: P < 0·05; Fig. 2]. High density D. virgifera infestation attraction. Only the model for (E)-b-caryophyllene and led to clear wilting symptoms in the leaves (see Fig. S1a, a-humulene resulted in linear regressions, indicating that Supporting information). When we compared the choice the P-value for a-copaene should be interpreted of the larvae according to the severity of wilting symptoms cautiously. induced by conspecifics (using leaf-wilting as a grouping factor rather than infestation number), we found that the THE ATTRACTION OF D. VIRGIFERA TO (E)- B - extent of water-stress did not influence D. virgifera prefer- CARYOPHYLLENE IS DOSE-DEPENDENT ence, with the exception of plants that had completely lost their turgidity, which were avoided by the larvae (see Fig. Diabrotica virgifera was slightly attracted to 0·5 lL (E)-b- S1b, Supporting information). caryophyllene dispensers (n = 11; glm: P = 0·064; Fig. 4), and strongly preferred 1 lL (E)-b-caryophyllene dispensers over controls (n = 10; glm: P < 0·001; Fig. 4). The (E)- B -CARYOPHYLLENE AND A -HUMULENE, BUT NOT larvae were not attracted to dispensers with a bigger capil- CO2 EMISSIONS CORRELATE WITH LARVAL CHOICE lary volume (n l = 11, n l = 10; glm: P > 0·05; PATTERNS 2 L 3, 6 and 25 L Fig. 4). At high infestation density (9 and 12 larvae per plant), root biomass was decreased significantly, while low to D. VIRGIFERA INDUCES SYSTEMIC SUSCEPTIBILITY IN medium densities had no measurable impact (see Fig. S2, MAIZE ROOTS Supporting information).

High infestation also increased the CO2 production per Larvae feeding on the systemic side of infested roots grew gram of fresh roots (see Fig. S3a, Supporting informa- more than five times better than larvae feeding on systemic tion). However, the total amount of emitted CO2 was not roots of healthy plants (n = 7; t-test: P = 0·008; Fig. 5). influenced by infestation density (see Fig. S3b, Supporting The effect was still present when the sand moisture level information). Diabrotica virgifera attack induced the was increased to 20% to avoid the induction of water- stress (see Fig. S4, Supporting information). Although the general exposure to plant volatiles stimulated D. virgifera = = = Healthy plant Infested plant larvae to feed (ndiet 6, nhealthy 9, ninfested 12; Kruskal- Wallis on ranks: P = 0·039; ndiet = 6, nplants = 21; t con- 1 = · * on the infested plant trast on ranks: P 0 050; Fig. 6), no difference was found

Larval density between the performance of larvae exposed to the volatile 3 bouquet of healthy or infested plants. ** 6 ROOT RESOURCE ALLOCATION IS ALTERED BOTH 9 LOCALLY AND SYSTEMICALLY UPON ROOT HERBIVORY 12 *** Diabrotica virgifera attack led to an increase in free 6420246 amino acid concentrations, both locally and systemically. D. virgifera choice Locally, asparagine, aspartic acid, glutamine, histidine, (Number of larvae) phenylalanine and tryptophane significantly increased (n = 6, t-tests or Mann–Whitney rank sum tests: Fig. 2. Diabrotica virgifera selectively orients towards suitable host P < 0·05; Fig. 7a), and leucine, serine and tyrosine ± plants. Number of larvae (mean SE) that oriented towards a = · > > · healthy plant or a plant infested with different densities of D. vir- showed similar trends (n 6; t-tests: 0 05 P 0 10; gifera larvae. Stars indicate significant differences (*P 0·05, Fig. 7a). Systemically, the concentrations of histidine, **P 0·01; ***P 0·001). phenylalanine, tryptophan and tyrosine increased upon

b 20 0·8 b 0·6 b 10 0·4 ab

-humulene 0·2 -caryophyllene -caryophyllene α

β a (Peak area E06) 0 (Peak area E06) 0

(E)- 013612 013612

b 0·8 b 1 ab b 0·6 ab ab ab ab 0·4 0·5 a

-copaene 0·2 α

Tetradecane a (Peak area E06)

(Peak area E06) 0 0 013612 013612

1·5 1·5

1 1

0·5 0·5 Heptadecane (Peak area E06) 4-methyl nonane 4-methyl (Peak area E06) 0 0 013612 013612

b 30 ab 1 b ab b 20 a ab ab 0·5 10 a a Tetradecene

(Peak area E06) 0 0 4-methyl heptane 4-methyl 013612 (Peak area E06) 013612 Larval density Larval density

Fig. 3. Diabrotica virgifera induces plant volatiles. SPME GC-MS peak areas (xE06) (mean ± SE) of plant-induced volatiles upon D. virgifera attack. Different letters indicate significant differences within each larval density using a post hoc Dunn’s test.

Empty dispenser EβC dispenser 60 ** 50 0·5 μL P = 0·064 40 1 μL *** 30

2 μL on systemic roots 20

3 μL 10 Relative weight gain (%)

6 μL D.virgifera 0 Capillary dispenser

of Healthy D.virgifera 25 μL infested Local root status 5 4 3 2 1 0 1 2 3 4 5 D. virgifera choice Fig. 5. Diabrotica virgifera larvae benefit from plant-mediated (Number of larvae) interactions with spatially separated conspecifics. Diabrotica virgifera growth (mean ± SE) over a 6-h feeding period on healthy Fig. 4. Diabrotica virgifera attraction to (E)-b-caryophyllene is plants or on the systemic undamaged roots of a plant that had dose-dependent. Number of larvae (mean ± SE) that oriented been infested for 4 days with conspecifics. Stars indicate signifi- towards a healthy plant or a healthy plant whose rhizosphere was cant differences (*P 0·05, **P 0·01; ***P 0·001). complemented with different amounts of (E)-b-caryophyllene using slow-release capillary dispensers. Stars indicate significant differences (*P 0·05, **P 0·01; ***P 0·001). roots: glucose contents were reduced by 50% and more sucrose accumulated (n = 6; t-tests or Mann–Whitney infestation (n = 6; t-tests: P < 0·05; Fig. 7b). Asparagine, rank sum tests, sucrose and glucose: P < 0·05; fructose: aspartic acid and glutamic acid concentrations also P > 0·05; Fig. 7c). On the other hand, the partitioning of showed similar trends upon infestation (n = 6; t-tests: sucrose and hexoses was not affected in the undamaged 0·05 > P > 0·10; Fig. 7b). Carbohydrate partitioning in systemic roots of infested plant (n = 6; t-tests or Mann– D. virgifera attacked roots was affected only in the local Whitney rank sum tests: P > 0·05; Fig. 7d). In accordance

* INFESTATION ATTENUATES THE PLANT’ S 20 RESPONSIVENESS TO FUTURE ATTACKS b ab Following the infestation of one side of the root system by 15 D. virgifera larvae, the undamaged systemic root side responded less to jasmonic acid application than the sys- 10 temic roots of uninfested plants (n = 8; t-tests: cysII; cyst a and bx1: P < 0·05; Fig. 10), indicating a relaxation of 5 defensive inducibility. Relative weight gain (%) 0 Conspecifics Healthy Infested Discussion on diet plant plant Volatile source This study demonstrates that plant-mediated facilitation occurs between D. virgifera conspecifics, a phenomenon Fig. 6. Exposure to Diabrotica virgifera-induced plant volatile that is likely the result of a combination of volatile-medi- does not stimulate larval performance. Diabrotica virgifera larvae ated host location, plant resource reallocation and weak- ± relative weight gain (mean SE) when feeding for 24 h on artifi- ened plant defences. Diabrotica virgifera larvae were cial diet (Pleau et al. 2002) and exposed to volatiles from conspe- found to benefit from feeding in groups, as they per- cifics feeding on diet, healthy plant or D. virgifera infested plant volatiles. Stars indicate significant differences (*P 0·05, formed better on plants infested with other larvae in labo- **P 0·01; ***P 0·001) within each larval density using a ratory and field conditions. Yet the observed feeding post hoc Dunn’s test. facilitation was reversed when D. virgifera fed in large groups: in our assays, both larval performance and head capsule width of emerging adults decreased at high densities. Negative density-dependent effects are likely due to with the sugar measurements, both vacuolar (ivr) and cell competition for limiting plant-resources. For instance, in wall (incw) invertase genes were downregulated locally our assays, high densities of larvae considerably decreased upon herbivory (n = 7, t-tests: P < 0·05) with the exception the root biomass available for conspecifics, and the strong of ivr1, whose expression was enhanced (n = 7, t-tests, wilting of the leaves may have reduced overall plant qual- P < 0·05; Fig. 8a). ity. Similar effects were reported in previous field studies, The expression of carbohydrate transporter homologues where adult D. virgifera emergence decreases at high egg was induced locally upon infestation (n = 7; t-tests: density (Onstad et al. 2006; Hibbard et al. 2010). Interest- P < 0·05), except for c4, which showed lower expression, ingly, our laboratory assays show that the specialist and zifl2, which showed no significant change (n = 7; t- D. virgifera was able to select host plant with a suitable tests: P < 0·05 and 0·05 < P < 0·10 respectively; Fig. 8b). density of root herbivores: When given a choice between No change in invertases and carbohydrate transporters a healthy plant and a plant infested with different num- was detected in the systemic roots of D. virgifera infested bers of conspecifics, D. virgifera larvae preferentially ori- plants (n = 7; t-tests: P > 0·05; Fig. 8c,d). ented towards plants infested with an intermediate number (six larvae), but were not attracted to plants DOES NOT SUPPRESS DEFENCE D. VIRGIFERA infested with low or high densities. This behaviour can be MARKERS beneficial for the root herbivore, because it enables it to Roots responded strongly to the infestation by D. virgif- locate the best host plants. Together with our previous era larvae. Marker genes of volatiles [(E)-b-caryophyl- study showing that D. virgifera can distinguish host plants lene (tps23) and indole (igl), lipoxygenases (lox3, lox5 of different quality (Robert et al. 2012a), these results and lox8)], direct defences (proteinase inhibitors: cysII, demonstrate the remarkable ability of this root herbivore serpin, mpi and benzoxazinones: bx1) and pathogenesis- for host selection. related proteins (pr1 and pr5) were upregulated upon We propose here that D. virgifera uses plant volatiles to herbivory (n = 7; t-tests and Mann–Whitney rank tests: find plants with a suitable infestation density. Although P < 0·05; Fig. 9a). On the other hand, the expression of CO2 is known to be highly attractive to the herbivore marker genes of hormones like ethylene (acs6), auxin (Bernklau & Bjostad 1998), our results suggest that other (saur2), abscisic acid (nced) and jasmonic acid (opr7) plant volatiles also play a key role in host selection by remained unaffected upon infestation (n = 7; t-tests: D. virgifera, as in our experiments CO2 emissions remained P > 0·05; Fig. 9a). Systemically, only a few of the mark- constant upon infestation by different densities, but several ers responded: igl, acs6 and lox8 were upregulated induced volatiles were produced in a density-dependent (n = 7; t-tests and Mann–Whitney rank tests: P < 0·05), manner. (E)-b-caryophyllene, a-humulene, a-copaene, while all the other tested marker genes expression tetradecane and tetradecene in particular showed a para- remained unchanged (n = 7; t-tests and Mann–Whitney bolic pattern, with peak emission occurring at medium rank tests: P > 0·05; Fig. 9b). densities of infestation. It should be taken into account

15 (a) ** Local roots

FW) 10 –1

5 ** P = 0·09 * ** P = 0·08 * ** P = 0·06 0 ** Ile His Ala Tyr Gly Trp Thr Gln Arg Glu Ser Leu Asn Asp Phe Met-Val

15 (b) P = 0·08 Systemic roots

10 FW) AA (ng g –1

5 P = 0·07 P = 0·06 AA (ng g * * * * * 0 Ile His Ala Tyr Gly Trp Thr Arg Gln Glu Ser Leu Asn Asp Phe Met-Val

1·2 FW) 1·2 (c) Local roots (d) Systemic

–1 roots 0·8 0·8

0·4 * 0·4 * 0 0 Carbohydrate (mg g Sucrose Glucose Sucrose Glucose Fructose Fructose

Healthy Infested

Fig. 7. Root herbivory leads to reconfiguration of the primary metabolism. (a) Local amino acid contents in healthy or infested roots. (b) Systemic amino acid contents in roots of healthy or infested plants. (c) Local carbohydrate contents in healthy or infested roots. (d) Systemic carbohydrate contents in roots of healthy or infested plants. Mean ± SE are presented (ng gÀ1 of fresh weight). Stars indicate significant differences (*P 0·05, **P 0·01; ***P 0·001).

that as root biomass decreases upon infestation in higher pene synthase, tps23, and well known to be induced upon densities, our analyses likely over-estimated the total infestation by the root herbivore (Kollner et al. 2008). As amounts emitted from plants infested by nine or twelve (E)-b-caryophyllene is emitted in much higher amounts larvae. In vivo analyses of root volatile emission of D. vir- than a-humulene (Erb et al. 2011a) and was shown to be gifera infested plants show that among the compounds an attractant for D. virgifera larvae (Robert et al. 2012a), detected in the present study, only (E)-b-caryophyllene, we focused on that compound to investigate its dose- a-humulene and a-copaene are actually released into the dependent effect on the root herbivore. (E)-b-caryophyl- rhizosphere (data not shown), which nicely matches the lene was attractive to D. virgifera only when released at a results from a diffusion study by Hiltpold & Turlings rate of 40 ng hÀ1 (1 lL capillary dispensers), which corre- (2008). Analyses of covariance (ANCOVA) using these three sponds to the release rate of plants infested with six con- compounds as covariates showed that (E)-b-caryophyllene specifics (Robert et al. 2012a). Although the attractiveness and a-humulene, but not a-copaene, can improve the fit of of other chemical remains to be tested, it is highly proba- the model of larval choice, suggesting that these two com- ble that D. virgifera uses (E)-b-caryophyllene in a dose- pounds may be used by D. virgifera to distinguish plants dependent manner to locate good host plants. infested by different densities of conspecifics. (E)-b-caryo- The feeding facilitation of D. virgifera when feeding in phyllene and a-humulene are both products of a single ter- medium-sized group may be attributed to either

(a) (c) adjustment (Navari-Izzo, Quartacci & Izzo 1990; Marur, 2 Local roots Systemic roots * Sodek & Magalhes 1994), for example in response to the 1 water-stress imposed by the root herbivore (Dunn & 0 Frommelt 1998; Erb et al. 2011b), (ii) defence (D’Auria & 1 Gershenzon 2005; Tzin & Galili 2010; Vogt 2010) or (iii) 2 * P = 0·06 nitrogen transport away from the roots (Paine, Redak & 3

Ln fold changes Trumble 1993; Trumble, Kolodny-Hirsch & Ting 1993).

in gene expression 4 ** At the same time, amino acids are known to be the 5 *** growth-limiting factor of herbivorous insects (Behmer

Ivr1 Ivr2 2006) and their accumulation in the roots of infested ivr1 ivr2 Incw2 Incw3 Incw4

Incw2 Incw3 Incw4 plants may, therefore, explain the better performance of (b) (d) D. virgifera. Apart from nitrogen metabolism, D. virgifera Local roots Systemic roots 2·5 ** also aﬀected carbon distribution: Upon infestation, 2 attacked roots accumulated more sucrose, but less glucose 1·5 *** 1 ** than roots of healthy plants. Three putative carbohydrate 0·5 transporter genes were more strongly expressed in 0 attacked roots, while invertases were generally downregu- 0·5 lated. Ivr1 and Ivr2 showed divergent expression patterns, Ln fold changes in gene expression 1 supporting previous work showing that the depletion of 1·5 * carbohydrates upregulates the expression of Ivr1 but C4 C4 Zifl2

Stp1 downregulates the expression of Ivr2 (Xu et al. 1996). Zifl2 Stp1 Mss1 Mss1 Mtrans

Mtrans Invertases play a key role in regulating root sink strength (Weil & Rausch 1990; Miller & Chourey 1992; Kim et al. Fig. 8. Local carbohydrate metabolism changes upon root infestation. (a) Local ln fold changes in expression of vacuolar (ivr) and 2000; Roitsch et al. 2003; von Schweinichen & Buttner cell wall (incw) invertases. (b) Local ln fold changes in expression 2005). Upon water-stress, vacuolar invertases were of carbohydrate transporters. (c) Systemic ln fold changes in reported to be strongly induced in maize roots, resulting expression of vacuolar (ivr) and cell wall (incw) invertases. (d) Sys- in higher ratios between hexoses and sucrose that contrib- temic ln fold changes in expression of carbohydrate transporters. utes to the osmotic adjustment (Kim et al. 2000). The gen- Mean ± SE are presented. Stars indicate significant differences (*P 0·05, **P 0·01; ***P 0·001). eral downregulation of the invertase marker genes observed in our assays suggests that D. virgifera attack may reduce the plants’ capacity to react to the accompa- plant-mediated effects or the direct influence of conspecif- nying water-stress conditions, as has been observed before ics. The split root experiment shows that spatially sepa- (Erb et al. 2009). However, as the changes in carbohydrate rated larvae grew, over a period of only 6 h, five times concentrations were limited to the local tissue, they bigger on previously infested plants compared to healthy are vunlikely to explain the differential performance of plants, showing that plant-mediated effects are sufficient to D. virgifera on systemic roots. explain the positive density dependence observed in the Diabrotica virgifera attack induced a pronounced local field. As upon below-ground attack, (E)-b-caryophyllene defence response, as indicated by the increased expression is produced both locally and systemically (Hiltpold et al. of marker genes involved in hormonal signalling, such as 2011), we first tested the hypothesis that this sesquiterpene lipoxygenases (lox genes), direct defences, such as protein- may directly stimulate feeding. Many lepidopteran leaf- ase inhibitors (mpi, serpin, cysII), benzoxazinoids (bx1), herbivores for example are stimulated by green leaf vola- pathogenesis-related proteins (pr1 and pr5), and indirect tiles released from fresh wounds (Meldau, Wu & Baldwin defences such as volatile production (igl and tps23). These 2009) or by volatile breakdown products of induced sec- results show that D. virgifera does not strongly, if at all, ondary metabolites like glucosinolates (Agrawal & Sher- manipulate host defences (Zarate, Kempema & Walling riffs 2001; Nielsen et al. 2001). We found that larvae 2007; Sarmento et al. 2011). Although the undamaged part exposed to D. virgifera-induced volatiles grew similarly of the root system was barely induced, with the exception than larvae exposed to the volatile bouquet of healthy igl, acs6 and lox8 whose expression was slightly upregulat- plants, which shows that induced plant volatiles do not ed, a second inductiovn of the undamaged part of infested stimulate larvae to feed. Interestingly, exposure to plant root systems by jasmonic acid resulted in a lower induction volatiles in general increased larval weight gain, suggesting of direct defence marker genes compared to plants that that constitutive volatile compounds do have a stimulatory had not been previously infested. This indicates that effect on D. virgifera. D. virgifera larvae that attack an already infested plant Diabrotica virgifera attack led to changes in the primary may encounter a plant immune system that is less induc- metabolism of maize roots. Larval feeding induced the ible, and consequently, less resistant. It remains to be accumulation of free amino acids both locally and system- determined whether this relaxation of inducibility can ically. Free amino acids can be involved in (i) osmotic explain the higher performance of D. virgifera larvae on

Local roots (a) 6 *** *** *** ** 4 *** *** * ** *** 2 *** **

Ln fold changes * in gene expression

(b) 6 Systemic roots

4 **

2 * * 0

2 Ln fold changes in gene expression 4

6 igl pal pr1 pr5 bx1 mpi cyst lox3 lox5 lox8 opr7 cysII acs6 nced lox10 tps23 saur2 serpin

Volatiles Hormonal signaling Direct defenses

Fig. 9. Plant defences are strongly induced locally. (a) Local ln fold changes in expression of volatile, hormone signalling and direct defence marker genes. (b) Systemic ln fold changes in expression of volatile, hormone signalling and direct defence marker genes. Mean ± SE are presented. Stars indicate signiﬁcant diﬀerences (*P 0·05, **P 0·01; ***P 0·001).

0·5 Conclusions

0 Overall, our study shows that D. virgifera attack changes the root metabolism of maize plants, leading to systemi- 0·5 * cally induced susceptibility. Diabrotica virgifera was found *** P = 0·051 1 to use (E)-b-caryophyllene as a signal to locate plants 1·5 with a suitable density of conspecifics. The presented Ln fold changes in gene expression 2 experiments allow us to rule out volatile-mediated stimula- ** tion of feeding and direct effects of larval behaviour as 2·5 cysII cyst serpin mpi bx1 pal explanations for the increase in larval growth. The hypoth- eses that either the higher amino acid levels or the relaxa- Fig. 10. A first infestation by Diabrotica virgifera attenuates sub- tion of inducibility may be responsible for the enhanced sequent defence responses. Ln-transformed fold-induction values D. virgifera performance remain to be tested. Understand- (mean ± SE) in the expression of marker genes involved in plant direct defence in systemic roots of infested plants relative to ing the mechanisms behind induced susceptibility is likely healthy plants. Systemic roots of infested and healthy plants were to improve our understanding of the extraordinary success induced with 100 lM of jasmonic acid for 12 h. Stars indicate of D. virgifera as a maize pest. significant differences (*P 0·05, **P 0·01; ***P 0·001). attacked plants. Testing this hypothesis would need a more Acknowledgements detailed understanding of the mechanisms of induced We thank Matt Higdon, Rebecca Higdon, Sarah Zukoff, Julie Barry and resistance in maize roots. the whole student crew from the USDA-ARS Plant Genetics Research Unit

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Ferrieri, R.A., Turlings, T.C.J. & Erb, M. (2012b) A specialist root her- Fig. S3. Total CO2 emissions are similar at different densities of bivore exploits defensive metabolites to locate nutritious tissues. Ecology feeding herbivores. Letters, 15,55–64. Roitsch, T., Balibrea, M.E., Hofmann, M., Proels, R. & Sinha, A.K. (2003) Fig. S4. Diabrotica virgifera larval growth is not affected by the Extracellular invertase: key metabolic enzyme and PR protein. Journal host plant resistance to hydric stress. of Experimental Botany, 54, 513–524. Table S1. Primer list for q-RT-PCR used to assess the defence Sarmento, R.A., Lemos, F., Bleeker, P.M., Schuurink, R.C., Pallini, A., response and carbohydrate allocation patterns in this study. Oliveira, M.G.A., Lima, E.R., Kant, M., Sabelis, M.W. & Janssen, A. (2011) A herbivore that manipulates plant defence. Ecology Letters, 14, As a service to our authors and readers, this journal provides sup- – 229 236. porting information supplied by the authors. Such materials may Schwachtje, J. & Baldwin, I.T. (2008) Why does herbivore attack reconfigure primary metabolism? Plant Physiology, 146, 845–851. be re-organized for online delivery, but are not copy-edited or von Schweinichen, C. & Buttner, M. (2005) Expression of a plant cell wall typeset. Technical support issues arising from supporting informa- invertase in roots of Arabidopsis leads to early flowering and an increase tion (other than missing files) should be addressed to the authors. in whole plant biomass. Plant Biology, 7, 469–475. Shiojiri, K., Ozawa, R., Kugimiya, S., Uefune, M., van Wijk, M., Sabelis, M.W. & Takabayashi, J. (2010) Herbivore-specific, density-dependent