Herbivore-Induced Indirect Plant Defences of Lima Bean (Phaseolus Lunatus, Fabaceae)

Herbivore-induced indirect plant defences of Lima bean (Phaseolus lunatus, Fabaceae)

Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.)

vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller-Universität Jena

von Diplom-Biologe Christian Kost

geboren am 9. September 1975 in Bad Sobernheim

Gutachter

1. Prof. Dr. Martin Heil (Universität Duisburg-Essen, Deutschland)

2. Prof. Dr. Wolfgang W. Weisser (Friedrich-Schiller-Universität Jena, Deutschland)

3. Prof. Dr. Paulo S. M. C. de Oliveira (Universidade Estadual de Campinas, Brasilien)

Tag der Doktorprüfung: 30.01.2006

Tag der öffentlichen Verteidigung: 21.04.2006

Preface I

Preface This thesis is the end of a journey that lasted almost three years and that took me along the way twice to Mexico. When I look back and say that I learned a lot during this time, I mean not only scientific skills and knowledge that I acquired in the course of this interesting, inter-disciplinary project, but I also appreciate invaluable Just as much I owe thanks to Prof. experiences that I gained during my stays Wilhelm Boland (MPI for Chemical Ecol- abroad and my work in Jena. ogy) for teaching organic chemistry to an An enterprise like this Ph.D. project ecologist, for always having an open door, can certainly not be accomplished without his contagious enthusiasm, stimulating the help of numerous people that contrib- discussions and his unrestricted support. uted in numerous ways either directly or Beyond I am indebted to Prof. Wolf- indirectly to its success. To these people, gang W. Weisser (FSU Jena) for kindly that crossed my path during the past three accepting the official supervision as a rep- years and made this time unforgettable, I resentative of the FSU Jena and giving me would like to express my warm gratitude. the opportunity to present and discuss my First of all, I wish to thank my doctoral results in his group’s seminar. advisor Prof. Martin Heil (University Du- To Silvia Schmidt I am endlessly isburg-Essen) for the opportunity to work grateful for her love, company during all on this exciting project in Jena and Mex- the years, lots of encouraging words when ico, his invaluable support, and stimulating things were tough and giving me the feel- discussions throughout all stages of this ing of not being alone. thesis. His advice and encouragement I owe substantial debt to Sven were an added spur to my ambition. Adolph and Mesmin Mekem Sonwa who patiently taught me organic synthesis and

always kept an eye on me. Also Stefan Photograph: Cerotoma ruficornis OLIVIER on a leaf of Phaseolus lunatus. Source: Bartram, Christoph Beckmann, An- CHRISTIAN KOST.

II Preface

dreas Habel, Georg Pohnert, Doreen (University of Duisburg-Essen), Paulo S. Schachtschabel and Dieter Spiteller Oliveira (Universidade Estadual de always stood by me with moral and practi- Campinas, Brazil), Victor Rico-Gray cal support. (Instituto de Ecología, Veracruz, Mexico), I am especially grateful to Anja Bie- Flavio Roces (University of Würzburg), dermann for introducing me into various Georg Pohnert (Ecole polytechnique methodological techniques and assisting fédérale de Lausanne, Switzerland), Wolf- me with laboratory work. Furthermore, I gang W. Weisser (Friedrich-Schiller- am thankful to Angelika Berg for taking University Jena), Ian T. Baldwin, Wilhelm considerable care for the Mexican bean Boland, Jesse Colangelo-Lillis, Rüdiger beetles. A great deal of credit goes to Udo Dietrich, Maritta Kuhnert, Heiko Mai- Kornmesser, Andreas Weber, Tamara schak, Axel Mithöfer, Birgit Schulze, Krügel, and the rest of the greenhouse Silvia Schmidt, Thomas Wichard (all team for raising countless plants. Max Planck Institute of Chemical Ecology, For there company, help and a great Jena) as well as a number of anonymous time in Mexico I thank Kerstin Ploss, referees. Martin Heil and Ralf Krüger. I am espe- I especially enjoyed lively scientific cially grateful to Perla Monica Martinez and non-scientific discussions as well as Cruz (Universidad del Mar, Puerto Escon- tantalising games evenings with my cof- dido, Mexico) for a warm welcome in Mex- fee-round Lars Clement and Heiko Mai- ico as well as logistic support in the field. schak as well as with my group-spanning Janine Rattke (Elsie Widdowson Labora- interest and discussion group Markus tory, Cambridge) is thanked for helping Benderoth, Caroline C. von Dahl, Mar- with the GC-MS analysis of nectar sam- kus Hartl, Anja Paschold and Silvia ples and Manfred Verhaagh (Staatliches Schmidt. Museum für Naturkunde, Karlsruhe) for Finally I would like to thank my family the identification of collected ants. Further, for their love, support, encouragement and I want to express my thanks to Birgit the privilege to realize my dream of be- Schulze for introducing me into the se- coming a natural scientist. crets of JA-extraction and lots of inspiring Financial support by the German discussions. Research Foundation (DFG Grant He Several people benefited this thesis 3169/2-1, 2, 3, 4) and the Max Planck by discussions or reading drafts of manu- Society is gratefully acknowledged. scripts and commenting on them. Their This work would not have been possi- advice was time-consuming to them but ble without your help! immensely valuable to me: Martin Heil

Contents III

Contents

Preface………………………………………………………………………………………………....I Abbreviations, acronyms and symbols……………………………………………………………IV

1. General introduction……………………………………………………………………………….1 2. Thesis outline – List of manuscripts and author’s contribution………………………………13 3. Manuscript I Herbivore-induced, indirect plant defences………………………………………….…18

4. Manuscript II Protection of Lima beans by extrafloral nectar…………………………………...... 47

5. Manuscript III Airborne volatiles induce indirect plant defence……………………………………….65

6. Manuscript IV Two indirect defences benefit Lima bean in nature……………………………………84

7. Manuscript V Artefact formation during headspace sampling………………………………………102

8. General discussion ……………………………………………………………………………..111 9. Synthesis…………………………………………………………………………………………121 10. Summary 10.1. Summary…………………………………………………………………………….….123 10.2 Zusammenfassung…………..………………………………………………………....126 11. References………………………………………………………………………………..……129 12. Selbständigkeitserklärung……………………………………………………………….……157 13. Curriculum vitae…………...… ……………...……………………………………………..…158

IV Abbreviations, acronyms and symbols

Abbreviations, acronyms and symbols

ACC 1-aminocyclopropane-1-carboxylic acid

AIS peak area of the internal standard (1-bromodecane) AFLP amplified fragment length polymorphism AOC allene oxide cyclase AOS allene oxide cyclase ALA alamethicin

AVOC peak area of a volatile organic compound CAM calmodulin CDPK calcium-dependent protein kinase CLSM closed-loop-stripping method DDQ 4,5-Dichloro-3,6-dioxo-1,4-cyclohexadiene-1,2-dicarbonitrile

DMNT (3E)-4,8-dimethylnona-1,3,7-triene (C11-homoterpene) DW dry weight EAG electroantennogram EF extrafloral EFN extrafloral nectar EI-MS electron impact mass spectrometer EI-MS electron impact high resolution mass spectrometer ERF ethylene-responsive transcription factor ET ethylene eq equivalent FAC fatty acid–amino acid conjugate FAD ω-3 fatty acid desaturase Fig. figure FPP farnesyl diphosphate GC gas chromatography GC-MS gas chromatograph coupled to a mass spectrometer GPP geranyl diphosphate HI herbivore-induced HI-VOCs Herbivore-induced volatile organic compounds HIPV herbivore-induced plant volatile 13-HPOT 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octa-decanoid acid HPL 13(S)-hydroperoxy-hydroperoxid lyase HRMS high resolution mass spectrometer

Abbreviations, acronyms and symbols V

IGP indole-3-glycerol phosphate Ind individual JA jasmonic acid JAs jasmonates JMT jasmonic acid carboxyl methyltransferase LOX 13-lipoxygenase M+• molecular ion MAP mitogen activated protein MAPK mitogen-activated protein kinase 1-MCP 1-methylcyclopropene MeJA methyl jasmonate MeSA methyl salicylate MKP mitogen-activated protein kinase phosphatase MS mass spectrometer MSTFA N-methyl-N-trimethylsilyl-trifluoroacetamide NMR nuclear magnetic resonance OPC 8:0 3-oxo-2-(2'(Z)-pentenyl)-cyclo pentane-1-octanoic acid OPDA 12,13(S)-epoxy-octadecatrienoic acid OPR 12-oxo-phytodieonic acid reductase ORCA octadecanoid-derivative responsive Catharanthus AP2-domain P450 P450 monooxygenase PI proteinase inhibitor PLA phospholipase A PLD phospholipase D r.t. room temperature SA salicylic acid SAM S-adenosylmethionine SAR systemic acquired resistance SIPK salicylic acid-induced protein kinase SMT salicylic acid carboxyl methyltransferase Tab table

TMTT (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (C16-homoterpene)

tret retention time TPS terpene synthase VOC volatile organic compound WIPK wounding-induced protein kinase

VI Abbreviations, acronyms and symbols

Symbols and letters used in statistics

GLM general linear model LSD Fisher’s protected least significant differences test (i.e. post-hoc test) n sample size P probability r Pearson product moment correlation (i.e. measure of correlation; varies from -1 to 0) SEM standard error of the mean SD standard deviation * indicates a significant result (P < 0.05) ** indicates a significant result (P < 0.01) *** indicates a significant result (P < 0.001)

Abbreviations used for NMR assignment

d dublett q quartett δ chemical resonance shift s singulett t triplett

General introduction 1

1. General introduction led to coadaptation in which the evolving Ever since the greening of a strip of equa- parties continually responded and counter- torial coastline that fringed tropical oceans responded to the selection pressures im- approximately 425 million years ago, both posed by each other. Such positive feed- plants and animals have expanded their back loops caused rapid evolutionary ecological reach on land. Today their changes in the interacting partners and terrestrial representatives constitute the thus enhanced speciation (LUNAU, 2004). overwhelming bulk of macroscopic diver- Besides relationships which are mutually sity on the planet which consists of more beneficial, such as between plants and than 30 million species with many of them their insect pollinators, the most common being as yet unstudied and unnamed. Two interaction involves insects preying on groups, namely flowering plants (Angio- plants, and plant defences against her- sperms) and insects (Insecta), contribute bivorous insects. This predator-host rela- preponderance to the terrestrial macro- tionship is so common that virtually every scopic diversity we can observe to date. plant species is preyed on by at least one If the earliest forms of land plants and insect species, and, according to the co- insects are included, plants and insects evolutionary theory of EHRLICH and RA- have coexisted for as long as 350 million VEN (1964), insect feeding on plants has years and have, since then, developed been a determining factor in increasing myriads of relationships. The extraordinary species diversity in both herbivores and radiation of flowering plants for example hosts (HARBORNE, 1988). On the basis was clearly the impact of a coevolutionary of this long-standing relationship, it is not interaction with pollinating insects surprising that the strategies employed by (REGAL, 1977). In the Cretaceous, about plants that attempt to resist or evade their 100 million years ago, when the first insect herbivores are very diverse. widely foraging seed dispersers came into existence (initially birds), insect pollination Plant defence reactions allowed flowering plants to produce out- The spectrum of defensive mechanisms crossed offspring with conspecifics, even that plants have evolved against herbi- when they were patchy and widely dis- vores has been classified as direct (bot- persed. Plants offered nectar to insects, tom-up) or indirect defences (top-down). thus creating new niches for nectivores Direct defences prevent herbivores from with this diversification in turn creating new feeding via physical barriers, such as evolutionary possibilities for the plants. A spines, thorns, trichomes, and waxes or permanent reciprocal selection pressure chemically, via the production of toxins or between pairs of single or multiple species anti-digestive and anti-nutritive com-

2 General introduction

pounds. Additionally, plants may express their defence response to the particular traits that facilitate top-down control over herbivore as has been shown on the level the herbivore population by attracting of transcriptional responses (VOELCKEL natural enemies of the herbivore that indi- & BALDWIN, 2004), signal molecules (e.g. rectly defend them against their attackers jasmonic acid: HALITSCHKE et al., 2001; (i.e. indirect defences, see Box 1). Direct SCHITTKO et al., 2000; ethylene: KAHL et and indirect defences can be expressed al., 2000) and defence-related metabolites constitutively (i.e. permanently) or be such as e.g. nicotine (WINZ & BALDWIN, induced upon mechanical wounding or 2001), proteinase inhibitors (STOUT et al., herbivore damage. 1994; TAMAYO et al., 2000), as well as Defence responses channel re- volatile organic compounds (VOCs) (DE sources from vegetative and reproductive MORAES et al., 1998; TAKABAYASHI et growth into protective mechanisms. There- al., 1995). Elicitation and orchestration of fore, the plant must attain a balance to such complex biochemical and physio- ensure survival from immediate and sub- logical responses requires at first the re- sequent attacks without sacrificing plant cognition of the currently feeding herbivore vitality and reproduction. The finding that via physical and chemical signals which many direct and indirect defences are activate subsequent signal transduction inducible suggests that these defences cascades and finally up-regulate defence- may incur costs to the plant. Fitness costs related genes. can arise directly from ‘internal’ mechanisms such as allocation costs, genetic Herbivore recognition and signal costs and autotoxicity costs (i.e. the resis- transduction tance trait itself is toxic to the plant) or The study of how plants recognize an indirectly from ecological interactions with attacking insect herbivore has mainly mutualists or antagonistic species (HEIL & focused on the emission of induced vola- BALDWIN, 2002; STRAUSS et al., 2002). tiles. Here it could be shown that the sig- These costs have been discussed as a nals generated by the herbivore are first driving force for the evolution of induced the mechanical damage that is inflicted, resistance (HEIL, 2001; HEIL & BALD- and second the chemical compounds of WIN, 2002). the herbivore’s oral secretions which are In order to minimize costs associated introduced at the site of wounding. with induced resistance, plants must be Mechanical damage triggers the re- able to identify their attacker to mount the lease of several VOCs. Immediately after most efficacious defence strategy. Indeed wounding, the so-called green-leaf odours it has been shown that plants may tailor are released (i.e. saturated and un-

General introduction 3

saturated C6-carbon aldehydes, esters, autolytic oxidative breakdown of mem- and alcohols), which are produced by the brane lipids (PARÉ & TUMLINSON, 1999).

Box 1: Indirect defences

Indirect plant defences are traits that attract blend of volatile organic compounds (VOCs) carnivorous arthropods to the trait-expres- that may be used by carnivorous arthropods sing plant to increase the predation pressure as a host-location cue (KESSLER & BALD- on herbivores (KESSLER & BALDWIN, WIN, 2001; TURLINGS et al., 1990). In this 2001) and thus enhance plant fitness case, the herbivore itself serves as a reward (FRITSCHE-HOBALLAH & TURLINGS, for the attracted plant defender. Indirect 2001; VAN LOON et al., 2000). Attraction of defences can be expressed constitutively beneficial arthropods to the plant may be (i.e. permanently) or be induced after herbi- actively mediated by rewards such as attrac- vore damage. Induced indirect defences are tive food sources like extrafloral nectar mainly the secretion of EFN and the emis- (EFN; KOPTUR, 1992) and food bodies sion of VOCs. Other indirect defences, such (JANZEN, 1966; RICKSON, 1971) or the as the formation of food bodies or leaf do- provision of shelter in so-called leaf domatia matia, are often involved in obligate ant- (AGRAWAL & KARBAN, 1997). Further- plant interactions and thus expressed consti- more, herbivore feeding can induce de novo tutively (i.e. myrmecophytism; for review see production and emission of a highly specific HEIL & MCKEY, 2003). ffffffghg

Principle of induced indirect plant defences: An * herbivore induces plant volatiles (V) that attract para- * sitoids and elicits the secretion of extrafloral nectar (E) that attracts carnivores such as ants. * Extrafloral * nectary.

Sources: Ant (Crematogaster laeviscula var. clara MAYR) from SMITH (1947), caterpillar (Macrothyliacia rubi LINNAEUS) from HONOMICHL et al. (1996), and wasp (Austrotoxeuma kuscheli BOUCEK) from NOYES (2003).

4 General introduction

Some plant species (e.g. cotton, Gos- et al., 1997). However, Lima bean leaves sypium hirsutum) also maintain chemical do not respond to volicitin or other FACs reserves such as trichomes or specialized (SPITELLER et al., 2001), indicating the glands, in which compounds accumulate to high levels and are released upon cell- wall disrupture (GANG et al., 2001; PARÉ Mechanical wounding Herbivory & TUMLINSON, 1997). Usually the bouquets emitted after mechanical damage PLASMA are much weaker than what is emitted MEMBRANE upon herbivore damage (MATTIACCI et al., 1994; PARÉ & TUMLINSON, 1997; Phospholipase TURLINGS et al., 1990). One explanation Linolenic acid (18:3) for this observation could be that plants LOX are able to discriminate between a single 13-HPOT AOS HPL event of mechanical damage and continu- AOC

12-OPDA 12-OPDA C6 aldehydes ous feeding of a folivore. Indeed, adjusting OPR PLASTID the process of mechanical wounding to OPC 8:0 mimic the feeding mode of natural herbi- PEROXYSOME vores in terms of damage time and dam- OPC 8:0 aged leaf-area provoked a VOC emission 3 x β-oxidation which strongly resembled the bouquet Jasmonic acid induced by herbivores (MITHÖFER et al., Direct defences 2005). Defence gene Indirect defences Chemical elicitors (i.e. compounds activation Amplification NUCLEUS that trigger a certain plant response) of of further signal cascades VOC emission which have been isolated from the oral secretion or regurgitate of Figure 1. Schematic overview over the bio- phytophagous insects comprise certain synthesis of jasmonic acid (octadecanoid pathway) enzymes (FELTON & EICHENSEER, and green leaf volatiles (C6 aldehydes). Abbreviated 1999; FUNK, 2001; MATTIACCI et al., enzymes involved are LOX, 13-lipoxygenase; AOS, 1995) or fatty acid-amino acid conjugates allene oxide synthase; AOC, allene oxide cyclase; (FACs) such as e.g. volicitin (ALBORN et OPR, 12-oxo-phytodieonic acid reductase; HPL, 13(S)-hydroperoxy-hydroperoxid lyase. Substrates al., 1997). Volicitin which was originally are 13-HPOT, 13(S)-hydroperoxy-9(Z),11(E),15(Z)- isolated from corn (Zea mays) shows a octadecatrienoic acid; 12-OPDA, 9(S)/13(S)-12-oxo- VOC-inducing activity when added to me- phytodienoic acid; OPC 8:0, 3-oxo-2-(2'-pentenyl)- chanically wounded corn plants (ALBORN cyclopentane-1-octanoic acid.

General introduction 5

involvement of specific recognition defences. When planted into native habi- mechanisms (TRUITT et al., 2004). tats, lipoxygenase-deficient plants were Both physical and chemical signals more vulnerable to adapted herbivores are perceived at the outer membranes of and attracted novel herbivore species, the damaged cell layers, thus generating underlining the key role of the octa- secondary signals such as a depolariza- decanoid pathway (Fig. 1) for plant tion of the membrane potential (MAFFEI et defence and host selection of herbivores al., 2004), an intracellular calcium influx (KESSLER et al., 2004). (ZIMMERMANN et al., 1999), the genera- Interactions of the octadecanoid tion of reactive oxygen species (OROZ- pathway with additional signalling cas- CO-CÁRDENAS et al., 2001) or the acti- cades, which can be either synergistic or vation of protein kinase/phosphatase cas- antagonistic, help the plant to prioritise the cades (KODAMA et al., 2000; SEO et al., activation of a particular defence pathway 1995). Further cell signalling cascades over another one. For instance SA, which allow the amplification of these signals as is known to be involved in mediating the well as a regulation of the defence development of systemic acquired resis- response in local (i.e. site of attack) and tance (SAR) in response to pathogen at- systemic tissues (i.e. remote, undamaged tack, acts antagonistically to JA (STOUT tissues). The signalling molecules involved et al., 1999; THALER et al., 1999). Signal- in the downstream responses are mainly ling compounds may also differentially peptides (e.g. systemin) and phytohor- affect direct and indirect defences: ET for mones like jasmonic acid (JA), salicylic example can suppress the induction of acid (SA), ethylene (ET) as well as lipid- direct defence without negative effects on derived oxylipins. Among them, JA and its the induction of indirect defence (KAHL et derivatives, collectively called jasmonates al., 2000). This complex and fine-tuned (JAs), are of central importance for the interplay of different signal transduction regulation of both direct and indirect plant pathways which is still not perfectly under- defence responses against wounding and stood, represents an adaptive tailoring of a herbivory (HALITSCHKE & BALDWIN, plant's defence response against a par- 2004). This has been elegantly demon- ticular attacker. strated by BALDWIN and co-workers who silenced the lipoxygenase3 gene in the Ecology of indirect defences wild tobacco (Nicotiana attenuata), render- Even if mechanistic knowledge on en- ing the transformed plants impaired in dogenous processes associated with her- oxylipin signalling and thus unable to acti- bivore attack is accumulating, the ecology vate their direct and indirect anti-herbivore of indirect defences is still relatively poor

6 General introduction

understood. Herbivore-induced plant vola- BALDWIN, 2001) and not in simplified tiles, for example, may affect many organ- agro-ecosystems (DE MORAES et al., isms in the environment of the emitting 1998; DRUKKER et al., 1995; SHIMODA plant such as herbivores, carnivores or et al., 1997). This lack emphasizes the pollinators. So far, these interactions have need for field studies on the ecological been mainly studied under laboratory con- relevance of herbivore-induced volatile ditions, thereby focussing on the behavi- organic compounds (HI-VOCs) for the oural and electrophysiological responses plant as well as for the arthropods of herbivores and carnivores to the VOCs involved in the interaction as both herbi- emitted from various herbivore-infested vores and carnivores. plant species. These analyses revealed The situation is different for extrafloral that the released VOC bouquets can be nectar (EFN). The first ecological field ex- used by arthropod predators and parasi- periments on this indirect defence trait had toids to locate their particular hosts or prey been conducted already in 1889: VON (for review see TURLINGS & BENREY, WETTSTEIN excluded ants from Jurinea 1998; VAN POECKE & DICKE, 2004). In mollis, thereby demonstrating that nectary- some cases, the released VOCs also func- visiting ants reduce levels of plant dam- tioned as a direct defence by repelling age. Since then, the ant-plant protection herbivores (BERNASCONI et al., 1998; hypothesis has been repeatedly tested. DICKE, 1986; LANDOLT, 1993) or reduc- The majority of these studies has shown ing herbivore performance (TURLINGS & that ants benefit plants, yet with the mag- TUMLINSON, 1991). However, the few nitude of the benefit varying greatly, de- arthropod species which were involved as pending on factors such as the need for herbivores and carnivores in these investi- mutualistic ants or a varying abundance gations represent a restricted range of and identity of ant species (for review see feeding guilds. Moreover, in most of the BRONSTEIN, 1998; HEIL & MCKEY, cases, the arthropod species used did not 2003; OLIVEIRA & FREITAS, 2004). co-occur with the focal plant species in However, not only ants, but also other nature, or it remained unclear whether the arthropods such as wasps (BUGG et al., investigated plant-animal interaction was 1989; STAPEL et al., 1997), flies (HEIL et also of significance in the wild. Among the al., 2004a), or spiders (RUHREN & HAN- field studies, which are available to date DEL, 1999) are attracted to the secreted on the attraction of carnivores to VOCs EFN. Whether these non-ant visitors of emitted from herbivore-infested plants, extrafloral (EF) nectaries also protect the only one has been performed on naturally plant or act as parasites, because they growing plant populations (KESSLER & consume EFN without contributing to plant

General introduction 7

fitness, remains largely unclear. Among captured the imagination of many re- the first reports that considered multi- searchers. species interactions between plants and Initially harshly criticised for limited EF nectary-visiting arthropods is an ele- replication and the failure to consider al- gant field study of CUAUTLE and RICO- ternative hypotheses (FOWLER & LAW- GRAY (2003), which demonstrated that TON, 1985), major advances in recent both ants and wasps positively affect plant years have considerably supported the fitness. This study highlights the need to hypothesis that plants can adaptively re- take not only ants, but also other predators spond to chemical information emitted and parasitoids into account when the from their neighbour. Experimental evi- effectiveness of EFN as an indirect dence suggests that eavesdropping be- defence (PEMBERTON & LEE, 1996) is tween plants may occur: Exposing plants estimated. to increased levels of putative volatile signals indicated transcriptional changes of Inter-plant signalling defence-related genes (ARIMURA et al., HI-VOCs provide chemical information on 2005; ARIMURA et al., 2001; ARIMURA et the status of attack of the emitting plant al., 2000b; BATE & ROTHSTEIN, 1998; which might be used not only by higher PENG et al., 2005) as well as changes in trophic levels, but also by neighbouring the abundance of phytohormones (ARI- plants of the same or other species. Two MURA et al., 2002; ENGELBERTH et al., decades ago, researchers indeed reported 2004) and defence-related metabolites that wounding or herbivore attack resulted such as terpenoids (ENGELBERTH et al., in an altered herbivore resistance with 2004), proteinase inhibitors (FARMER & detrimental effects on populations of insect RYAN, 1990; TSCHARNTKE et al., 2001) herbivores not only of the attacked plants, or phenolic compounds (BALDWIN & but also of plants growing nearby (BALD- SCHULTZ, 1983). However, most of these WIN & SCHULTZ, 1983; RHOADES, studies were conducted under artificial 1983). In some of these experiments, an laboratory conditions, while rigorous verifi- aerial transfer of information was the most cation of the plant-plant communication- parsimonious explanation for the observed hypothesis under field conditions was phenomena. Since then, the idea of ‘talk- largely lacking (BRUIN & DICKE, 2001). ing trees’ that convey the information Among the studies addressing this issue, about their state of attack to their undam- DOLCH and TSCHARNTKE (2000) could aged neighbour that in turn ‘eavesdrops’ show that partial defoliation of alder trees on these cues and responds with the in- (Alnus glutinosa) to simulate herbivory duction of its own defence (Fig. 2), has resulted in an induced resistance in the

8 General introduction

Auto- Interspecific signalling signalling Intraspecific signalling

Attacked plant

Figure 2. Three modes of airborne signalling from or within herbivore-damaged plants are indicated: signalling to undamaged congeners, signalling to members of other species, and auto-signalling outside the plant body. Arrows represent herbivore-induced plant volatiles. Modified after FARMER (2001). Source: caterpillar (Macro- thylia rubi) from HONOMICHL et al. (1996). defoliated trees as well as in conspecific zyme (polyphenol oxidase) in downwind neighbours. More detailed analyses in the tobacco plants (KARBAN et al., 2000). laboratory identified changes in the leaf So far, the study of KARBAN and col- chemistry as possible responses causal leagues (2000) is the only to show the for the observed resistance (TSCHARN- VOC-mediated expression of defence- TKE et al., 2001). In another field study, related plant metabolites in undamaged experimental clipping of sagebrush downwind plants under field conditions. increased the herbivore resistance of Furthermore, most studies on plant-plant neighbouring tobacco plants, which communication focused on the induction of resulted in a reduced herbivory and an direct defences, ignoring the possibility increased life-time seed production com- that also indirect plant defences may be pared to control plants next to undamaged affected. sagebrush plants (KARBAN et al., 2000; KARBAN & MARON, 2002). Here, the The Lima bean (Phaseolus lunatus) - biologically active enantiomer (3R,7S) A model system in chemical ecology methyl jasmonate has been proposed as Like many other Fabaceae, Lima bean has the aerially transmitted signal that caused evolved cyanogenic glycosides as a direct an elevated activity of an anti-nutritive en- defence against herbivores (BALLHORN

General introduction 9

et al., 2005). Tissue break-down exposes such as mites (SABELIS & VAN DE the vacuole-located glucosides to β- BAAN, 1983), thrips (SHIMODA et al., glucosidases and hydroxy-nitrile lyases, 1997), staphylinid beetles (SHIMODA et with subsequent hydroylsis leading to the al., 2002) and braconid wasps (PETITT et release of cyanide, which is a universal al., 1992), but also on herbivorous beetles respriratory poison (BENNETT & WALLS- (HEIL, 2004b). Moreover, VOCs may deter GROVE, 1994). herbivores such as chrysomelid beetles The reason, however, why Lima bean (HEIL, 2004b) and thus act as a direct became an attractive model system for defence. chemical ecologists, is the observation In addition to the herbivore-induced that it emits significantly increased emission of VOCs, Lima bean bears extra- amounts of about 10 different VOCs after floral (EF) nectaries (Fig. 3). These spe- herbivore damage. These are likely to be cialized, nectar-secreting organs are synthesized de novo (BOLAND et al., located on its bracts, or arranged pairwise 1995; BOUWMEESTER et al., 1999; at the stipules of the trifoliate leaves as DONATH & BOLAND, 1995) and are well as at the petioles of the individual derived from the lipoxygenase pathway, the shikimic acid pathway, and the isoprenoid pathway (DICKE et al., 1999; for details on the pathways involved see manuscript I, Fig. 3). Like in many other plant species, herbivore damage in Lima bean is accompanied by a transient increase in endogenous JA-levels (KOCH, 2001). Exogenous application of JA Figure 3. Location of the extrafloral nectar- secreting stipules (i.e. extrafloral nectaries) within a (DICKE et al., 1999; HOPKE et al., 1994; trifoliate leaf of Phaseolus lunatus. Source: CHRIS- OZAWA et al., 2000a) or the early inter- TIAN KOST. mediates of jasmonate biosynthesis α-linolenic acid and OPDA (KOCH et al., leaflets (HEIL, 2004a). The secretion rate 1999) to plants also results in the emission of EFN is also inducible by JA, thus pro- of VOC blends which are similar, yet not viding the Lima bean with the two induc- identical to those released after herbivore ible, indirect defences: emission of HI- damage. VOCs released from herbivore- VOCs and secretion of EFN. Besides few infested or JA-treated Lima bean plants other plant species such as e.g. broad have been shown to exhibit an attractive bean (Vicia faba; COLAZZA et al., 2004; effect not only on predatory arthropods FIGIER, 1971) or cotton (PARÉ & TUM-

10 General introduction

LINSON, 1997; WÄCKERS & BONIFAY, have been described, this combination of 2004) for which both indirect defences traits makes Lima bean an ideal system

Box 2: The natural history of Lima bean

Lima bean (Phaseolus lunatus LINNAEUS, in many laboratory studies as well as the Fabaceae) is a food legume that originated wild type plants that have been used in all of in Central and South America and is now my experiments belong to the Meso- cultivated in all tropical regions. Studies american genotype (M. HEIL, unpublished based on morphological and biochemical data). The wild forms are self-compatible observations (DEBOUCK et al., 1989; with a mixed mating system, i.e. predomi- GUTIÉRREZ SALGADO et al., 1995; MA- nantly self-pollinating but with a fair amount QUET et al., 1990) as well as on molecular of outcrossing (HARDING & TUCKER, data (FOFANA et al., 2001; FOFANA et al., 1969; ZORRO BI et al., 2005). Plants in 1997) indicate the existence of two distinct natural populations grow as annuals or gene pools: the ‘Mesoamerican’ type being short-living perennials, although the above- widely distributed in neotropical lowlands, ground parts usually die during the dry sea- while the ‘Andean’ type appears to be son. Wild Lima bean forms soil seed banks restricted to the western Andes. Since both with the dormancy of seed being broken by gene pools comprise closely related wild drastic temperature changes and high pre- and cultivated forms, two independent cipitation rates (DEGREEF et al., 2002): In domestication events were suggested June or July the plants start to germinate or (GUTIÉRREZ SALGADO et al., 1995). Ac- bud and the first inflorescences usually cording to AFLP analyses (i.e. amplified appear in October or November. Depending fragment length polymorphism, a method to on water supply, the production of flowers examine variation in DNA sequences), the and fruits (see below) ends between Febru- variety ‘Jackson Wonder Bush’ that is used ary and April (HEIL, 2004a). kkkk A B C D

Lima bean (Phaseolus lunatus): A Inflorescence, B Pod, C Seeds, and D Seed- ling. Sources: B MARTIN HEIL; A,C,D CHRISTIAN KOST.

General introduction 11

Figure 4. Lima bean (Phaseolus lunatus) growing in its natural habitat. Wild plants entwine trees and shrubs along a roadside near Puerto Escondido, Mexico. Source: CHRISTIAN KOST.

to study the importance of these two 2004a), thus nearly no ecological informa- defensive traits in nature (see Box 2 for tion is available on this trait until now. information on the natural history of the These shortcomings highlight the need for Lima bean). evaluating the role of both indirect defensive traits under natural conditions. This Aim of this thesis should be realized in the present thesis. Most of the knowledge on the ecology of In a pioneering study, HEIL (2004a) HI-VOCs released from Lima bean has could experimentally demonstrate that wild been derived from laboratory-based growing Lima bean plants, which have approaches using Lima bean seedlings. A been repeatedly induced with exogenous look at the size and growth structure of application of JA, responded to this treat- Lima bean growing in nature (Fig. 4) ment with an increased seed set. Since leaves uncertainty as to which extend the the JA-treatment induced both the emis- results gathered under artificial laboratory sion of VOCs and the secretion of EFN, it conditions are also valid for the natural remained unclear how the two indirect situation in the field. Moreover, the exis- defences had contributed to the tence of EF nectaries in Lima bean has observed beneficial effect. Therefore, this just recently been discovered (HEIL,

12 General introduction

study provided an ideal starting point for site of Lima bean in Mexico? Do wild further field experiments. plants of P. lunatus benefit from either Based on the findings of HEIL VOCs or EFN alone under natural (2004a), the present work applies an inte- growing conditions? grative approach, in which the function of (2) Which animals are involved in the tri- the two indirect defences, emission of trophic interaction as plant defenders? VOCs and secretion of EFN, is analysed Which animals are attracted to VOCs on the level of i) the trait-expressing plant, and which to EFN? ii) the attracted arthropod community, and (3) Is there a VOC-mediated interaction iii) conspecific plant neighbours. In detail, between herbivore-damaged and un- the following three main sets of problems damaged Lima bean plants? Does the were addressed from a phytocentric per- emission of HI-VOCs induce the spective: secretion of EFN in undamaged, neighbouring tendrils? Which com- (1) How effective are the two inducible, pounds within the complex blend of indirect defences - VOC emission and HI-VOCs are responsible for the EFN secretion - at the natural growing induction of EFN secretion?

Thesis outline 13

2. Thesis outline – List of manuscripts and author’s contribution

Manuscript I

Herbivore-induced, indirect plant defences

Gen-ichiro Arimura, Christian Kost, and Wilhelm Boland

Biochimica et Biophysica Acta (2005) 1734, 91-111

This review summarizes the current highlights perspectives for future research. knowledge on herbivore-induced, indirect Thus this review provides the theoretical plant defences on the levels of signal per- background of the thesis. ception and transduction, gene expression The article was a joint effort by all and biosynthesis of defence-related me- three co-authors. I was responsible for tabolites as well as it examines indirect writing the ‘Abstract’, the ‘Introduction’ defences in the light of their ecology and (section 1), the sections ‘Ecology of in- evolution. Focussing on the two defensive duced indirect defences’ (section 4) and traits emission of herbivore-induced plant ‘Conclusions’ (section 5) as well as for the volatiles (HIPVs) and secretion of EFN, it preparation of table 1 and Fig. 5. The final provides a comprehensive overview over draft of the article was optimized in the latest developments in the field and agreement with all three co-authors.

14 Thesis outline

Manuscript II

Increased availability of extrafloral nectar reduces herbivory in Lima bean plants (Phaseolus lunatus, Fabaceae)

Christian Kost and Martin Heil

Basic and Applied Ecology (2005) 6, 237-248

This manuscript describes field experi- work, data evaluation, arthropod identifica- ments on the defensive effect of EFN in tion, and statistical analysis. MARTIN Phaseolus lunatus, identifies putative plant HEIL helped with the establishment of the defenders and examines insect responses experimental plots, counting of plant de- towards an artificial mixture of EFN. The fenders, and estimation of the herbivory results of this study suggest that Lima rate. Analysis of EFN composition was bean benefits of increased amounts of done by JANINE RATTKE and me and EFN and that ants and also flying defend- MANFRED VERHAAGH determined the ers like wasps or flies contribute to the collected ant species. I wrote the first draft observed beneficial effect. of the manuscript, which was refined in I was responsible for experimental collaboration with MARTIN HEIL.

Thesis outline 15

Manuscript III

Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants

Christian Kost and Martin Heil

Journal of Ecology, accepted

This manuscript analyses whether plant- realization of the nectar-induction experi- plant communication can occur in Lima ments as well as the chemical and statisti- bean under natural growing conditions and cal analysis were done by me. The whether this has defensive effects for the experimental design of the long-term receiver of the signal. In several inde- experiment was a joint effort by MARTIN pendent experiments an inductive effect of HEIL and me. MARTIN HEIL also helped entire volatile blends and of single blend with the establishment of the experimental constituents on the EFN secretion rate of plots, counting of plant defenders, and conspecific plants is verified. Furthermore, estimation of fitness-relevant plant para- the long-term consequences of a volatile- meters. Organic synthesis of VOCs was induced EFN secretion are assessed on done by me. SVEN ADOLPH and MES- the level of fitness-relevant plant parame- MIN MEKEM SONWA helped with the ters and number of plant defenders development and optimization of synthesis attracted, and compared to the defensive strategies. The manuscript was written by effect of EFN alone (manuscript II). me, optimized after suggestions of MAR- I had the idea of a potential induction TIN HEIL. of EFN by HI-VOCs. The planning and

16 Thesis outline

Manuscript IV

The defensive value of two indirect defences of Lima bean in nature

Christian Kost and Martin Heil

In preparation for Oecologia

This manuscript, which is based on field The experimental design was a joint experiments at the Lima bean’s natural effort by me and MARTIN HEIL who also growing site, contrasts the defensive effect helped with the establishment of the of EFN secretion and VOC emission. In experimental plots, counting of plant de- groups of neighbouring tendrils the fenders, and estimation of fitness-relevant amount of EFN and VOCs were artificially plant parameters. I was respon-sible for increased and compared to JA-treated preceding experiments on the emission of and control tendrils. The development of VOCs out of lanolin paste, the realisation fitness-relevant plant parameters was of the field experiments as well as the sta- monitored as well as the arthropod com- tistical data analysis. Organic synthesis of munity visiting the studied plants was VOCs was done by me with support of assessed by counting and sticky-trap cap- SVEN ADOLPH and MESMIN MEKEM tures. The interaction of the two indirect SONWA. The manuscript was written by defences is discussed in the light of the me and optimized after suggestions of functional groups of arthropods attracted. MARTIN HEIL.

Thesis outline 17

Manuscript V

Dehydrogenation of ocimene by active carbon: Artefact formation during headspace sampling from leaves of Phaseolus lunatus

Mesmin Mekem Sonwa, Christian Kost, Anja Biedermann, Robert Wegener, Stefan Schulz, and Wilhelm Boland

Submitted to Tetrahedron

This manuscript examines the observation I discovered the phenomenon and that during volatile-collection of induced performed the experiment in which differ- Lima bean plants, two compounds can ent VOC-sampling methods are compared only be detected when the headspace is (Tab. 1). The experiments on the mecha- sampled with charcoal traps, but not when nistic elucidation (radical versus hydride) other adsorption materials such as e.g. were performed by MESMIN MEKEM SPME or Tenax are used. It is shown that SONWA. Experiments on the functionali- ocimene, a major constituent of the plant’s sation of ocimene with different solvents volatile blend, reacts with the active car- and with DDQ (3.7.5) were done by ANJA bon of the trapping adsorbant leading to BIEDERMANN and me. Writing of the artefact formation. A mechanism for this manuscript was a joint effort of MESMIN chemical reaction is proposed as well as MEKEM SONWA, WILHELM BOLAND an exploitation of this new reaction for a and me. rapid functionalisation of ocimene is verified.

18 Herbivore-induced, indirect plant defences

Manuscript I

Herbivore-induced, indirect plant defences

Gen-ichiro Arimura, Christian Kost, Wilhelm Boland*

Biochimica et Biophysica Acta (2005) 1734, 91-111

Accepted: 1 March 2005

Department of Bioorganic Chemistry Max Planck Institute for Chemical Ecology Beutenberg Campus Hans-Knöll-Str. 8 D-07745 Jena, Germany

* Corresponding author: Wilhelm Boland Department of Bioorganic Chemistry Max Planck Institute for Chemical Ecology Beutenberg Campus, Hans-Knöll-Str. 8 D-07745 Jena, Germany Phone: + + 49 - 3641 - 57 12 00 Fax: + + 49 - 3641 - 57 12 02 E-mail: [email protected]

Manuscript I 19

Abstract Indirect responses are defensive strategies by which plants attract natural enemies of their herbivores that act as plant defending agents. Such defences can be either constitutively expressed or induced by the combined action of mechanical damage and low- or high-molecular weight elicitors from the attacking herbivore. Here, we focus on two induced indirect defences, namely the de novo production of volatiles and the secretion of extrafloral nectar, which both mediate interactions with organisms from higher trophic levels (i.e. parasitoids or carnivores). We give an overview on elicitors, early signals, and signal transduction resulting in a complex regulation of indirect defences and discuss effects of cross-talks between the signalling pathways (synergistic and antagonistic effects). In the light of recent findings, we review molecular and genetic aspects of the biosynthesis of herbivore-induced plant volatiles comprising terpenoids, aro- matic compounds, and metabolites of fatty acids which act as infochemicals for animals and some of which even induce defence genes in neighbouring plants. Finally, ecological aspects of these two indirect defences such as their variability, specificity, evolution as well as their ecological relevance in nature are discussed.

Key words: Extrafloral nectar; herbivore-induced plant volatile; indirect defence; oxylipin; signalling pathway

Abbreviations: ACC, 1-aminocyclopropane-1- methylcyclopropene; MeJA, methyl jasmonate; carboxylic acid; ALA, alamethicin; CAM, MKP, MAPK phosphatase; OPDA, 12- calmodulin; CDPK, calcium-dependent protein oxophytodienoic acid; ORCA, octadecanoid- kinase; DMNT, (E)-4,8-dimethyl-1,3,7- derivative responsive Catharanthus AP2- nonatriene; EAG, electroantennogram; EF, domain; P450, P450 monooxygenases; PI, extrafloral; EFN, extrafloral nectar; ERF, ethyl- proteinase inhibitor; PLA, phospholipase A; ene-responsive transcription factor; FAC, fatty PLD, phospholipase D; SA, salicylic acid; acid–amino acid conjugate; FAD, ω-3 fatty SAM, S-adenosylmethionine; SAR, systemic acid desaturase; FPP, farnesyl diphosphate; acquired resistance; SIPK, salicylic acid- GPP, geranyl diphosphate; HIPV, herbivore- induced protein kinase; SMT, SA carboxyl induced plant volatile; HPL, fatty acid hydro- methyltransferase; TMTT, (3E,7E)-4,8,12- peroxide lyase; IGP, indole-3-glycerol phos- trimethyl-trideca-1,3,7,11-tetraene; TPS, ter- phate; JA, jasmonic acid; JMT, JA carboxyl pene synthase; WIPK, wounding-induced pro- methyltransferase; LOX, lipoxygenase; MAPK, tein kinase. mitogen-activated protein kinase; 1-MCP, 1-

20 Herbivore-induced, indirect plant defences

1. Introduction including herbivore recognition, signal Plants have evolved a wide spectrum of elicitation, and early signalling steps, and strategies to defend themselves against followed by a description of the signalling herbivores. Such defensive strategies can cascades and biosynthesis pathways in- be classified as direct defences, which volved, we will end with an overview of the immediately exert a negative impact on ecology and evolution of induced, indirect herbivores, or indirect defences, which defensive plant traits. include higher trophic levels, thus fulfilling the defensive function (PRICE et al., 1980). Direct defences may prevent herbi- 2. Cell- and long-distance signalling in vores from feeding via physical barriers, plants in response to herbivory such as spines, thorns, trichomes, and Plants have evolved a large array of inter- waxes or chemical ones, with secondary connected cell-signalling cascades, result- plant metabolites (e.g. phenylpropanoids, ing in local resistance and long-distance terpenoids, alkaloids, and fatty acids); or signalling for systemic acquired resistance via specialized defence proteins (e.g. pro- (SAR). Such responses were initiated with teinase inhibitors, so-called PIs). On the the recognition of physical and chemical other hand, indirect defences work by at- signals of the feeding herbivores, activate tracting the herbivores’ enemies, such as subsequent signal transduction cascades, parasitoids or predators, which actively and finally lead to an activation of genes reduce the number of feeding herbivores. involved in defence responses that conse- Both strategies can be either constitutive, quently enhance feedback signalling and meaning that they are always expressed, metabolic pathways (Fig. 1). This section or inducible, meaning that they appear describes the aspects of cell signalling only when needed, namely, following her- involved in the induction of indirect herbi- bivory. vore responses. In recent years especially, induced, indirect defences have received increasing 2.1. Elicitors attention and have been studied on the In the 1990s and earlier, many chemical genetic, biochemical, physiological, and ecologists believed that herbivorous oral ecological levels. The aim of this review is secretions and regurgitants elicited insect- to summarize the current state of knowl- induced plant responses, since simple edge, merge results from different biologi- mechanical wound stimuli, in many cases, cal fields, and point to new possibilities for could not mimic plant responses following future research. Beginning with the first insect attack (BALDWIN, 1988; BOLAND contact between plant and herbivore, and et al., 1992; PARÉ & TUMLINSON, 1997;

Manuscript I 21

Figure 1. Schematic representation of the signalling pathways required for herbivore-induced responses in plants. This scheme merges the evidence obtained from several plant taxa. The overall scenario may differ in certain plants; in particular the existence and the extent of synergistic and antagonist interaction between pathways may vary significantly. Elements in blue represent enzymes. Broken arrows indicate possible steps not yet described. Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; ACS, ACC synthase; DAG; diacyl- glycerol; FAC, fatty acid-amino acid conjugate; FAD, ω-3 fatty acid desaturase; HIPV, herbivore-induced plant volatiles; JA, jasmonic acid; JMT, JA carboxyl methyl transferase; LOX, lipoxygenase; MAPK, mitogen-activated protein kinase; MeJA, methyl JA; MeSA, methyl SA; OPDA, 12-oxophytodienoic acid; PL, phospholipase; PA, phosphatidic acid; SA, salicylic acid; SAM, S-adenosyl-methionine; SAMT, SA carboxyl methyl transferase; TF, transcription factor.

TURLINGS et al., 1990). β-Glucosidase application of this β-glucosidase to me- derived from regurgitate of Pieris brassi- chanically wounded parts of cabbage cae larvae was identified as a potential leaves resulted in the emission of a vola- elicitor of herbivore-induced plant volatiles tile blend that attracted parasitic wasps (HIPVs) (MATTIACCI et al., 1995). The (Cotesia glomerata). On the other hand,

22 Herbivore-induced, indirect plant defences

the application of glucose oxidase, a sali- similar to that emitted when caterpillars fed vary gland enzyme, inhibited the synthesis on them. While volicitin was isolated from of nicotine in tobacco leaves and may thus S. exigua, other FACs with biological activ- be responsible for the observed resistance ity, such as N-acyl Gln/Glu, have been of feeding herbivores (MUSSER et al., isolated from the regurgitate of several 2002). The suppressive effect, which lepidopteran species (HALITSCHKE et al., maintains the plant palatable, may be due 2001; POHNERT et al., 1999; SPITEL- to the products of glucose oxidase, namely LER & BOLAND, 2003). For example, the hydrogen peroxide (H2O2) and gluconic FAC N-linolenoyl-Glu in the regurgitate of acid. the tobacco hornworm (Manduca sexta) N-(17-hydroxylinolenoyl)-L-glutamine was characterised as a potential elicitor of (volicitin), a fatty acid-amino acid conju- volatile emission in tobacco plants. FACs gate (FAC), was detected in the oral se- can induce the accumulation of 7-epi- cretion of beet armyworm larvae (Spodop- jasmonic acid (JA) an octadecanoid- tera exigua) (ALBORN et al., 1997) derived phytohormone that elicited tran- (Fig. 2). This FAC was identified as the scripts of herbivore-responsive genes in main elicitor inducing the emission of a the tobacco plants (HALITSCHKE et al., volatile blend in maize plants (Zea mays) 2001). 7-Epi-jasmonic acid is the originally

Figure. 2. N-acyl glutamines from oral secretions of lepidopteran larvae.

Manuscript I 23

produced and biologically active form of wounding of the leaves also caused Vm JA, which is isomerised in planta to the depolarization, yet without a concomitant less active trans-isomer (see Fig. 1). Re- influx of calcium. Hence, already at this cently, a plasma membrane protein from early step of signal recognition and trans- maize was suggested as a volicitin-binding duction, the pathways offer different protein (TRUITT et al., 2004). Plants that responses to wounding and herbivore have been either pre-treated with methyl attack. Thus, both wounding and the intro- jasmonate (MeJA) or S. exigua feeding duction of herbivore-specific elicitors showed increased binding activity of appear to be essential for the full induction labelled volicitin to the plasma membrane. of defence responses. These findings suggest that volicitin- On the other hand, recent studies induced JA enhanced the interactions of applying a continuous rather than a single the plasma membrane protein with volicitin instance of mechanical damage (pattern as a ligand or the induced formation of the wheel) to Lima bean leaves clearly re- proteins after the recognition of volicitin. sulted in the emission of volatile blends However, volicitin is not generally active. resembling those that occur after herbi- Leaves of Lima bean (Phaseolus lunatus), vore damage (MITHÖFER et al., 2005). As for example, do not respond to volicitin or mentioned above, Lima bean and cotton to other FACs with the induction of volatile (Gossypium hirsutum) do not respond to emission (SPITELLER et al., 2001). FACs with increased volatile emissions (SPITELLER et al., 2001). This observa- 2.2. Early and secondary signal tion may be consistent with a high internal transduction pathways threshold for mechanical damage which, in After mechanical damage, reactive oxygen some plant species, is reached only after species were generated near cell walls of continuous wounding and which triggers tomato vascular bundle cells and the re- the typical repertoire of defence responses sulting H2O2 was shown to act as a sec- caused by elicitors. cDNA was isolated ond messenger for the activation of defen- from a species of tobacco, encoding a sive genes in mesophyll cells which are mitogen-activated protein kinase (MAPK), expressed later (OROZCO-CÁRDENAS et whose transcript begins to accumulate in al., 2001). In Lima bean leaves infested the leaves 1 min after a single mechanical with Spodoptera littoralis larvae, a depo- wounding event (SEO et al., 1995). This larization of the membrane potential (Vm) protein kinase (WIPK) is considered and intracellular calcium influx were ob- essential for JA formation and JA-induced served only at the site of damage (MAF- responses in tobacco, since transgenic FEI et al., 2004). Simple mechanical plants in which WIPK has been silenced

24 Herbivore-induced, indirect plant defences

show impaired accumulation of JA and -SIPK/WIPK pathway. This discrepancy MeJA after wounding (SEO et al., 1995). may be not only due to the complicated In contrast, the loss of WIPK function in interactions of protein kinase/phosphatase this transgenic line resulted in an in- cascades, but also to cross-talk between creased accumulation of salicylic acid (SA) the JA-, SA-, and ethylene-signalling and its sugar conjugate salicylic acid pathways (see below). Since calcium- β-glucoside after wounding; however, dependent protein kinases (CDPKs) are such accumulation was not observed in regularly involved in signal transduction of wild-type plants. The signalling pathways a variety of biotic and abiotic stresses associated with JA and SA are generally (LUDWIG et al., 2004), their involvement thought to cross-communicate antagonis- as active protein cascades in herbi- tically (see below). Moreover, WIPK has vore/wound responses cannot be ex- also been reported to elicit the transcrip- cluded. CDPKs compose a large family of tion of a gene for a ω-3 fatty acid desatu- serine/threonine kinases in plants (for rase (FAD7), which catalyses the con- example, 34 members in Arabidopsis) version of linoleic acid to linolenic acid, a (CHENG et al., 2002; HARMON et al., precursor of JA (KODAMA et al., 2000). 2000). JA has been reported to affect Thus, WIPK may be an early activator of CDPK transcript and activity in potato the octadecanoid pathway. WIPK and the plants (ULLOA et al., 2002). SA-induced protein kinase (SIPK) were A transgenic Arabidopsis line in which found to share an upstream MAPKK, the expression level of phospholipase Dα NtMEK2 (LIU et al., 2003), and to interact (PLDα) is suppressed has been reported with the calmodulin (CaM)-binding MAPK to show a decreased wound-induced ac- phosphatase (NtMKP1) (YAMAKAWA et cumulation of phosphatidic acid and JA, as al., 2004). Both kinases are commonly well as JA-inducible transcript activation involved in wound-mediated defence re- (WANG et al., 2000). Curiously, this loss- sponses in tobacco (LIU et al., 2003; SEO of-function mutant also featured de- et al., 1999; ZHANG & KLESSIG, 1998). If creased expression of the gene coding the MAPK cascades in tobacco were only lipoxygenase2 (LOX2) but not of any other interlinked as described above, an active gene involved in the octadecanoid path- mutant of the upstream kinase of SIPK way. LOX2 is one of the key enzymes for (NtMEK2DD) should have increased the the wound-induced synthesis of JA in accumulation of JA and MeJA, but it did chloroplasts (BELL et al., 1995). There- not (KIM et al., 2003). Instead, ethylene fore, PLD may specifically regulate LOX2 was assumed to be involved in plant de- upstream of the octadecanoid pathway fence responses mediated by the NtMEK2 that leads to JA. In addition, the involve-

Manuscript I 25

ment of phospholipase A (PLA), which (KRUMM et al., 1995; STASWICK & mediates the release of linolenic acid from TIRYAKI, 2004), but the significance of cell membranes and is inducible by wound such conjugates still remains to be estab- stimuli, has been also proposed for tomato lished. Inhibiting the performance of JA or (Lycopersicon esculentum) and other spe- SA obviously renders plants more suscep- cies (NARVÁEZ-VÁSQUEZ et al., 1999). tible to herbivore damage and pathogen Altogether, early and secondary cell infection (MCCONN et al., 1997; ROYO et signalling for herbivore-induced plant al., 1999; RYALS et al., 1996; VIJAYAN et responses comprise: (1) the reception of al., 1998). JA and SA act antagonistically, an extracellular signal(s) such as high- or and are both required for the induced re- low-molecular weight factors from the her- sponse following herbivore feeding or bivore (e.g. FACs), (2) Vm depolarization pathogen attack (CIPOLLINI et al., 2004; and an intracellular calcium influx, (3) the ENGELBERTH et al., 2001; MORAN & activation of protein kinase/phosphatase THOMPSON, 2001; OZAWA et al., 2000a; cascades, and (4) the release of linolenic SPOEL et al., 2003). For instance, the acid from the cell membrane and subse- graduated increase of SA accumulation in quent activation of the octadecanoid path- Lima bean leaves treated with alamethicin way which leads finally to the synthesis of (ALA), a potent fungal elicitor of plant vola- JA and other oxylipins. However, multiple tile emission, interferes with steps in the signals, signal circulation by, for example, biosynthetic pathway downstream of protein phosphorylation/dephosphoryla- 12-oxophytodienoic acid (OPDA), thereby tion, and feedback regulation of signalling reducing the rapid accumulation of JA and metabolic pathways are likely to com- (~ 40 min) (ENGELBERTH et al., 2001) plicate the understanding of the accurate and, finally, the biosynthesis and emission mechanism. of all JA-linked volatiles. On the other hand, enhanced levels of OPDA selec-

2.3. Oxylipin, SA, and their antagonism tively induce the emission of the C16- JA and SA function as signalling mole- tetranorditerpene 4,8,12-trimethyltrideca- cules, which mediate induced plant re- 1,3,7,11-tetraene (TMTT) in Lima bean sponses toward herbivory and pathogen (KOCH et al., 1999). Consequently, the infection, resulting in the activation of dis- treatment of Lima bean leaves with vari- tinct sets of defence genes (MALECK & ous amounts of JA and SA caused the DIETRICH, 1999; REYMOND & FARMER, emission of characteristic blends of vola- 1998; TURNER et al., 2002). Besides free tiles including TMTT, which strongly JA, also a conjugate with the amino acid attracted the carnivorous natural enemies isoleucine may be involved in signalling of spider mites (DICKE et al., 1990;

26 Herbivore-induced, indirect plant defences

OZAWA et al., 2000a; SHIMODA et al., vory. For example, transgenic potato 2002). Hence, JA and SA pathways may plants over-expressing an allene oxide vie with each other to control and coordi- synthase gene, which is involved in the JA nate induced defences, since the emission formation, failed to express a PI gene of characteristic blends of herbivore- (Pin2) constitutively, despite the fact that induced volatiles from attacked plants transgenic plants exhibited six- to twelve- strongly depends on the delicate balance fold higher levels of endogenous JA than between JA and SA (ENGELBERTH et al., did non-transgenic plants (HARMS et al., 2001). Alternatively, both pathways may 1995). This observation indicates that JA play a role in discriminating insect biting is not always involved in the signalling from mechanical wounding and thus may pathways of wound/herbivore responses be a kind of filter that prevents unneces- and that other mediators may play a role in sary defence activation. the respective signalling cascades. As Whether or not the recently discov- described above, SA is an example of a ered phytoprostanes, which result from signalling molecule that acts antagonisti- non-enzymatic oxidative transformation of cally towards JA. In addition, other signal- linolenic acid into prostaglandine-type ling pathways may positively affect JA octadecanoids (THOMA et al., 2003), are action. Ethylene is such a candidate, as it involved in defence responses against induces the expression of defence genes, herbivores remains to be established. At and its synthesis is induced by wounding, least, gene expression analysis of Arabi- herbivore feeding, and JA treatment (ARI- dopsis cell cultures treated with the phyto- MURA et al., 2002; O'DONNELL et al.,

prostanes PPB1B -I or -II revealed that both 1996). Hints of a synergy between JA and compounds triggered a massive detoxifi- ethylene in the herbivory- or wound- cation and defence response covering the induced responses are: (1) plant defence expression of several glutathione S-trans- genes are synergistically induced by eth- ferases, glycosyl transferases and putative ylene and JA/MeJA in tobacco and in ATP-binding cassette transporters (LOEF- wounded tomato plants (O'DONNELL et FLER et al., 2005). al., 1996; XU et al., 1994), (2) treatment of maize plants with 1-methylcyclopropene 2.4. Synergy between JA and ethylene (1-MCP) reduced the production of ethyl- Undoubtedly, JA is among the most impor- ene and volatile emission following tant signalling molecules of herbivore- M. sexta feeding (SCHMELZ et al., 2003), induced responses. However, the activa- and (3) treatment of maize plants and tion of JA formation alone cannot explain Lima bean leaves with ethylene or its pre- all changes following wounding or herbi- cursor 1-aminocyclopropane-1-carboxylic

Manuscript I 27

acid (ACC) significantly promoted volatile Arabidopsis and tomato (LAUDERT & emission induced by JA (HORIUCHI et al., WEILER, 1998; O'DONNELL et al., 1996; 2001; SCHMELZ et al., 2003). In addition, SIVASANKAR et al., 2000). Also, the anta- simultaneous application of JA and ACC to gonistic interaction between ethylene and Lima bean leaves increased the attrac- JA on defence gene expression, resulting tiveness of the pre-treated Lima bean leaf in resistance of Arabidopsis plants to her- for predatory mites (e.g. Phytoseiulus per- bivory, has been proposed (ROJO et al., similis) as compared to leaves treated with 2003). These mechanisms are different JA alone (HORIUCHI et al., 2001). All among different plant species (e.g. tomato these results suggest that the cross-talk and Arabidopsis) and the other conditions between JA and ethylene is required for (ROJO et al., 2003; WANG et al., 2002). both direct and indirect defence responses Several other signalling molecules to herbivory. The application of ethylene may function synergistically with JA and and ethephon (an ethylene-releasing ethylene in plant defence responses. For compound) together with the application of example, in potato abscisic acid has been MeJA to tobacco plants suppressed the shown to increase JA levels following induction of nicotine accumulation yet mechanical damage to leaf tissues (DAM- hardly affected the emission of berga- MANN et al., 1997), whereas abscisic acid motene, a volatile sesquiterpene (KAHL et did not regulate any defence-related al., 2000). Moreover, the application of genes by itself (BIRKENMEIER & RYAN, 1-MCP to tobacco plants treated with oral 1998). Among other plant hormones, auxin secretions of M. sexta larvae increased is a likely candidate, as it also has a nega- the induced accumulation of nicotine but tive effect on the JA pathway (LEÓN et al., reduced fitness-relevant plant parameters 2001). Furthermore, spermine is consid- such as the rate of stalk elongation or life- ered as an integral constituent of time capsule production (VOELCKEL et wound/herbivory responses, because (1) al., 2001). In this case, the interplay spermine activates WIPK and SIPK in to- between ethylene and JA may reduce the bacco leaves and mitochondrial dysfunc- level of nicotine present in a plant, thus tion via a signalling pathway in which reac- reducing its autotoxicity as well as the tive oxygen species and a calcium influx physiological costs of this compound. are involved (TAKAHASHI et al., 2003), Moreover, ethylene has been reported to and (2) the expression of a S-adenosyl- positively regulate the induction of allene methionine (SAM) decarboxylase gene oxide synthase, an enzyme which cata- involved in the polyamine synthesis is in- lyzes the production of a precursor of duced in Lima bean leaves infested with OPDA in the octadecanoid pathway, in spider mites (ARIMURA et al., 2002).

28 Herbivore-induced, indirect plant defences

Considering that spider mite-infested Lima Arabidopsis, ERF1 and AtERF1, AtERF2, bean leaves do not accumulate endoge- and AtERF5 are known as active elements nous spermine, this polyamine may, how- of GCC-associated transcriptions, while ever, play a minor role in wound/herbivory AtERF3 and AtERF4 act as suppressive responses. Alternatively, polyamines such elements of these transcriptions (FUJI- as spermine may accumulate locally in MOTO et al., 2000). As they bind to the intercellular spaces, as has been shown same target sequence, their transcriptional for tobacco leaves infected with tobacco regulation is thought to depend on either mosaic virus (YAMAKAWA et al., 1998). the DNA binding affinity or on the relative More evidence is still required to fully un- abundance of active ERFs in the nucleus, derstand the role of polyamines in stress or on both factors. Moreover, such GCC- responses. associated transcripts have been shown to be coordinated not only by ethylene, but 2.5. Transcription also by JA (BROWN et al., 2003). Con- A subset of cis- and trans-transcription versely, JA-inducible transcription factors elements, which are involved in the JA- ORCAs (the octadecanoid-derivative re- ethylene- and the SA-signalling pathways, sponsive Catharanthus AP2-domain) have has been characterised. In several plant been characterised as a JA-responsive species, the GCC box (AGCCGCC), an APETALA2 (AP2)/ERF-domain transcrip- ethylene-inducible element, has been tion factor in Catharanthus roseus found in the promoter region of ethylene- (MENKE et al., 1999; VAN DER FITS & inducible defence genes (OHME-TAKAGI MEMELINK, 2000). The overexpression of et al., 2000). Four and six ethylene- Orca3 constitutively increased the expres- responsive transcription factors (ERF) that sion of genes involved in several meta- specifically interact with the GCC box were bolic pathways (tryptophan decarboxylase, characterised from tobacco and Arabidop- 1-deoxy-D-xylulose-5-phosphate synthase sis, respectively (FUJIMOTO et al., 2000; and desacetoxyvindoline 4-hydroxylase) OHME-TAKAGI & SHINSHI, 1995; SO- involved in terpenoids and indole alkaloid LANO et al., 1998). ERF genes can re- formations (VAN DER FITS & MEMELINK, spond to various extracellular stimuli, such 2000). Recently, a WRKY transcription as wounding, pathogen infection, and factor was shown to bind to a W-box, with drought stresses, by changing of their the latter consisting of two reversed TAGC transcriptional levels and have so far been repeats and located in the promoter region described for higher plants but not for of the cotton (+)-δ-cadinene (sesquiter- yeast and other fungi (FUJIMOTO et al., pene) synthase gene CAD1 (XU et al., 2000; OHME-TAKAGI et al., 2000). In 2004). The expression of this transcription

Manuscript I 29

factor was induced by JA and an elicitor species, such as tomato, for which an derived from Verticillium dahliae. Direct/ 18-amino-acid peptide called systemin indirect synergisms or antagonisms be- was discovered and claimed to represent tween multiple trans-factors are likely to an intercellular signalling molecule regulate genes via signalling mediators (PEARCE et al., 1991). Systemin is de- such as JA, ethylene, and SA. rived from a 200-aminoacid precursor pro- The recent development of high- systemin that has been originally discov- throughput microarray technology allows ered in tomato. Transgenic tomato plants the simultaneous and systematic monitor- that constitutively express antisense pro- ing of the expression pattern of an im- systemin RNA reduce wound-induced mense number of plant genes. Such transcript accumulation of PIs in systemic analyses have shown comprehensive leaves and are more susceptible to M. transcript profiles in plants responding to sexta attack than are wild-type plants insect feeding, mechanical wounding, JA, (OROZCO-CÁRDENAS et al., 1993). Fur-

H2O2, and plant volatiles (ARIMURA et al., thermore, a 160-kDa plasma membrane- 2000b; HALITSCHKE et al., 2003; QU et bound, leucine-rich repeat receptor kinase al., 2004; REYMOND et al., 2000, 2004). was recently characterised as a systemin For instance, the transcription of several receptor (SR160) from suspension cul- genes was induced in Arabidopsis follow- tures of tomato cells (SCHEER & RYAN, ing mechanical wounding and Pieris rapae 2002). Surprisingly, this receptor is identi- feeding (REYMOND et al., 2000). Curi- cal to tBRI1, a brassinosteroid receptor in ously, one gene encoding a hevein-like tomato, and homologous to BRI1 in Arabi- protein was induced by P. rapae, but not dopsis (MONTOYA et al., 2002; by mechanical wounding. As described SZEKERES, 2003; WANG & HE, 2004). above, induced plant responses to The dual function of SR160/tBRI1, which mechanical wounding and feeding herbi- remains to be unequivocally established, vores may differ due to varying degrees is very unique, as this receptor has two and types of damage and the presence or ligands (systemin and brassinosteroid) absence of salivary compounds. and apparently functions in development and defence responses (WANG & HE, 2.6. Systemic response 2004). Additional analyses demonstrated It is well known that plants respond to her- that the overexpression of SR160 in sus- bivory and mechanical wounding not only pension-cultured tobacco cells yielded at the site of damage, but also in remote, systemin-binding activity and a systemin- undamaged leaves. The best systems induced alkalinisation response in the studied in this context are solanaceous cells. These results indicate that post-

30 Herbivore-induced, indirect plant defences

receptional steps of the signal transduc- signalling. Moreover, it was also found that tion are also present in tobacco, another the rapid and transient activation of early member of the family Solanaceae. In to- genes in response to leaf excision or bacco, two 18-amino-acid hydroxyproline- wounding was not affected in spr1 plants, rich glycoproteins (TobHypSys I and II), indicating the existence of a Spr1- and which are potential PI-inducers in a similar systemin-independent pathway for wound manner to systemin, have been discov- signalling (LEE & HOWE, 2003). ered (PEARCE et al., 2001a, b). These Herbivore-induced systemic respon- two peptides are derived from a 165- ses can be observed not only in solana- amino-acid precursor that does not exhibit ceous species but also in many other plant any homology to prosystemin from tomato, families. Poplar trees (Populus trichocarpa but is analogous to peptide hormones x deltoides) are able to trigger the activa- from animals and yeast. Recently, this tion of gene expressions systemically in type of peptide was also discovered in undamaged leaves far from those that had tomato, raising the question of whether been damaged by the forest tent caterpil- these peptides represent a general type of lar (Malacosoma disstria) (ARIMURA et defence signal in the plant kingdom al., 2004a). This systemic response re- (PEARCE & RYAN, 2003). vealed acropetal (upper) but not basipetal Today, systemin is no longer consid- (lower) direction, but the transport mecha- ered as a long-distance signal. A sys- nism remains obscure. A putative apoplas- temin-insensitive tomato mutant (spr1, tic lipid transfer protein DIR1 has been suppressor of prosystemin-mediated re- suggested as a mobile signal, mediating sponses1), deficient in a signalling step an interaction between a leaf of Arabidop- that couples systemin perception to the sis infected with Pseudomonas syringae subsequent activation of the octadecanoid and distant, uninfected leaves (MALDO- pathway, showed that the peptide acts at NADO et al., 2002). It was speculated that or near the local site of wounding, increas- DIR1 could be a co-signal with other lipids, ing JA-synthesis above the threshold that such as oxylipins (JA), phosphatidic acid, is required for the systemic response in and N-acylethanolamines, which travel tomato (LEE & HOWE, 2003). According through the vascular system of the plant. to these results, systemin is now thought However, actual evidence for a possible to up-regulate the JA biosynthetic pathway involvement of DIR1 in an herbivore- to generate a long-distance signal at the induced defence is still lacking. In addition, local site (for example, JA or OPDA as the electrical signals have been noted and transmissible signals), whose recognition claimed to mediate long-distance interac- in distal leaves is linked to octadecanoid tions in wounded tomato seedlings (WIL-

Manuscript I 31

DON et al., 1992). Plants may have multi- some function as mono-TPSs and sesqui- ple systems that enable accurate long- TPSs (BOHLMANN et al., 2000; CHEN et distance signalling. Thus, JA itself can act al., 2004; CHEN et al., 2003a; FÄLDT et as a systemic signal in tobacco, which is al., 2003). Terpenoid formation is gener- formed in the wounded leaves and travels ally assumed to be regulated on the tran- to the undamaged distal leaves and roots script level of the TPS genes (ARIMURA where the expression of PI and the nico- et al., 2004a; DUDAREVA et al., 2003; tine biosynthesis are induced, respectively MCKAY et al., 2003; SHARON-ASA et al., (BALDWIN et al., 1994; LI et al., 2002). 2003). However, the regulation mechanism seems to be rather complex, because her- 3. Biosynthesis of herbivore-induced bivore-induced TPS transcripts and ter- plant volatiles (HIPVs) pene emissions are affected by several 3.1. Volatile terpenoids factors (for example, by diurnal rhythmicity Volatile terpenoids which can be induced and distance to herbivore-damaged tissue) by herbivore-feeding comprise monoter- (ARIMURA et al., 2004b; MILLER et al., penes (C10), sesquiterpenes (C15) and 2005). Figure 4 shows temporal patterns homoterpenes (C11 or C16). All terpenoids of volatile emissions in Lima bean leaves are synthesised through the condensation following herbivore attack by S. littoralis of isopentenyl diphosphate and its allylic over 4 days. The release of terpenoids isomer dimethylallyl diphosphate by cata- and the C6-volatile (3Z)-hex-3-enyl acetate lysis of farnesyl diphosphate (FPP) syn- follows diurnal cycles with increased emis- thase via the mevalonate pathway (cytsol/ sions during the light period and reduced endoplasmic reticulum) or geranyl diphos- emissions during darkness. This result is phate (GPP) and geranylgeranyl diphos- in line with findings in Lima beans treated phate via the methyld-erythritol-1-phos- with ALA and poplar leaves infested with phate pathway in plastids (LICHTEN- forest tent caterpillars, where the volatile THALER, 1999; RODRÍGUEZ-CONCEP- emissions or the TPS expressions follows CIÓN & BORONAT, 2002) (Fig. 3). A diurnal cycles (ARIMURA et al., 2004a; large, structurally diverse number of ter- KUNERT et al., 2002). In this context it penoids are yielded by a large family of would be interesting to study to what ex- terpene synthases (TPS) using GPP and tent volatile emissions are linked to the FPP as substrates. In Arabidopsis, 32 endogenous biological clock. genes including two gibberellin biosyn- On the other hand, a single event of thetic genes are putative members of the mechanical damage or the application of TPS family (AUBOURG et al., 2002), ALA to Lotus japonicus plants was not

32 Herbivore-induced, indirect plant defences

Figure 3. Biosynthetic pathways required for herbivore-induced plant volatiles. Elements in bold are enzymes. Abbreviations: ALDH, aldehyde dehydrogenase; DMAPP, dimethylallyl diphosphate; DMNT, (E)-4,8-dimethyl- 1,3,7-nonatriene; DXP, 1-deoxy-D-xylulose-5-phosphate; DXR, DXP reductoisomerase; DXS, DXP synthase; FPP, farnesyl diphosphate; FPS, FPP synthase; GGPP, geranylgeranyl diphosphate; GGPS, GGPP synthase; GPP; geranyl diphosphate; GPS, GPP synthase; HMG-CoA, 3-hydroxy-3-methyl-glutaryl CoA; HMGR, HMG-CoA reductase; HPL, fatty acid hydroperoxide lyase; IDI, IPP isomerase; IGL, indole-3-glycerol phosphate lyase; IPP, isopentenyl diphosphate; JA, jasmonic acid; JMT, JA carboxyl methyl transferase; LOX, lipoxygenase; MeJA, methyl jasmonate; MEP, 2-C-methyl-D-erythritol-4-phosphate; TMTT, 4,8,12-trimethyltrideca-1,3,7,11-tetraene; TPS, terpene synthase.

shown to result in an increased release of genic Arabidopsis plants overexpressing (E)-β-ocimene from the plants, despite strawberry linalool/nerolidol synthase increased transcript levels of a (E)-β- (NaNES1) produced not only volatile ocimene synthase (LjEβOS) gene (ARI- linalool but also its glycosylated and MURA et al., 2004b). Furthermore, trans- hydroxylated derivatives (AHARONI et al., genic Petunia hybrida plants that over- 2003). Several volatile terpenoids such as expressed Clarkia breweri S-linalool syn- linalool can be glucosylated or hydroxy- thase (lis) produced minor amounts of lated by the catalysis of glucosyl trans- volatile S-linalool, but conjugation to the ferase and cytochrome P450 monooxy- non-volatile S-linalyl-β-D-glucopyranoside genases (P450), respectively. The biosyn- which is absent in wild-type C. breweri thesis of two tetranorterpenoids (homo- (LÜCKER et al., 2001). Similarly, trans- terpenes), (E)-4,8-dimethyl-1,3,7-nonatri-

Manuscript I 33

ene (DMNT) and TMTT, has been pro- and enhancing the resistance against spi- posed to proceed via an oxidative degra- der mites in the leaves of neighbouring dation by P450 enzymes of the sesqui- conspecific plants (ARIMURA et al., terpene (E)-nerolidol and the diterpene 2000a, 2002). The signalling pathway in- geranyl linalool as precursors (DONATH & volved in the inter-plant communication is BOLAND, 1995). Herbivore-induced for- mediated by calcium influxes into cells and mation of DMNT is regulated at the level of protein-phosphorylation and dephosphory- both a (E)-nerolidol synthase and a P450- lation in the receiver plants. Similarly, the catalysed process in maize (DEGEN- non-volatile C20 diterpene (11E,13E)-lab- HARDT & GERSHENZON, 2000). Hence, da-11,13-diene-8α,15-diol (WAF-1) acti- multiple mechanisms including TPS tran- vates WIPK, SIPK, other MAPK and en- scripts and enzymatic modifications seem hanced transcript levels of wound- and to contribute to the control of terpenoids pathogen-inducible genes in tobacco synthesis. (SEO et al., 2003). The treatment of the Terpenoid compounds emitted from tobacco leaves with WAF-1 enhanced Lima bean plants infested with spider resistance to the tobacco mosaic virus mites act as airborne signals, activating infection. Thus, both volatile and non- JA/ethylene-induced defence responses volatile terpenoids can mediate between-

Figure 4. HIPV emission from Lima bean leaves during insect feeding. Volatile emissions released from five fully developed (2-week-old) plantlets infested with twelve Spodoptera littoralis larvae (third instar) were continuously monitored using an automated gas chromatographic system equipped with a preconcentration unit (zNose™) (KUNERT et al., 2002).

34 Herbivore-induced, indirect plant defences

cell and inter-plant signalling of induced well as the expression of defence genes in plant defences. several other plant species (ARIMURA et al., 2001; BATE & ROTHSTEIN, 1998;

3.2. C6-volatiles ENGELBERTH et al., 2004). It has also

Six-carbon (C6)-volatiles or ‘green leaf been reported that both C6-aldehydes and volatiles’, as they are also called, when -alcohols reduce tobacco aphid fecundity they are released from plant leaves imme- on tobacco leaves (HILDEBRAND et al., diately after wounding, include isomers of 1993). Moreover, antisense-mediated HPL hexenol, hexenal, and hexenyl acetate depletion significantly reduced the levels (HATANAKA, 1993). Amazingly, the for- of hexanal and hexenal and increased the mation of (3Z)-hex-3-enal, an initially aphid fecundity on potato plants (VAN- formed C6-volatile, can be observed within CANNEYT et al., 2001), suggesting that

20 s after the disruption of Arabidopsis leaf C6-volatiles play a role as either direct tissues (MATSUI et al., 2000). This forma- plant defence agents or signalling mole- tion is probably due to an array of enzy- cules which elicit defence reactions. matic activations (phospholipase, LOX, However, C6-volatiles may not always and fatty acid hydroperoxide lyase [HPL]) benefit plants. (2E)-Hex-2-enal inhibits the and/or the encounter of these enzymes germination and subsequent growth of with their substrates α-linolenic acid, soybeans as well as the germination of linoleic acid or 13-hydroperoxide (MATSUI apple pollen (GARDNER & AGRAWAL, et al., 2000). Pre-treatment of the Arabi- 2002; HAMILTON-KEMP et al., 1991). dopsis leaves with quinacrine, a lipase Furthermore, (2E)-hex-2-enal reduces the inhibitor, suppressed the formation of (3Z)- germination frequency of the MeJA- hex-3-enal but increased the accumulation insensitive Arabidopsis mutant jar1-1, of α-linolenic acid. These results appar- suggesting that (2E)-hex-2-enal and MeJA ently indicate that the cleavage of free are recognized in plants by different unsaturated fatty acids from membrane mechanisms (BATE & ROTHSTEIN, lipids is the rate-limiting step for the rapid 1998). A rapid formation of C6-volatiles formation of hexenal. The aldehyde iso- after wounding not only serves as protec- mers (3Z)-hex-3-enal and (2E)-hex-2-enal tion against herbivores or pathogens, but contain an α,β-unsaturated carbonyl may also be toxic for the plant itself. This group, known as reactive electrophile effect could be interpreted as some kind of (ALMÉRAS et al., 2003), which induces strategic retreat, as has been observed for the emission of volatile terpenoids in hypersensitive cell death in response to tomato plants (FARAG & PARE, 2002), pathogen infection. It remains to be inves- the accumulation of endogenous JA as tigated whether the transient burst of

Manuscript I 35

wound-induced C6-volatiles is detrimental plants exhibited constitutive transcript ac- for the growth and development of a plant cumulation of JA-responsible genes and in the long term. enhanced resistance against the virulent

In contrast to C6-aldehydes and -alco- fungus Botrytis cinere (SEO et al., 2001). hols, the emission of (3Z)-hex-3-enyl ace- These results indicate the importance of tate can frequently be observed a few MeJA as a cellular signal regulator and hours after the first impact of herbivore potential inter-cellular, and intra- and inter- feeding or mechanical damage (ARIMURA plant signal transducers. The other JA et al., 2000a; RÖSE & TUMLINSON, derivative, (Z)-jasmone, has been found to 2004; TURLINGS et al., 1995), as seen in repel the lettuce aphid (Nasonovia ribisni- the emission of herbivore-induced terpe- gri) and to induce (E)-β-ocimene, resulting noids (Fig. 2). Since the emission of both in an increased attractivity of the plants to (3Z)-hex-3-enyl acetate and β-ocimene the aphid parasitoid Aphidius ervi can be induced by JA and ethylene (BIRKETT et al., 2000). However, it has (HORIUCHI et al., 2001), the signalling been also reported that MeJA and pathway may be identical. However, noth- (Z)-jasmone are only moderately induced ing is known about whether or not the bio- in Nicotiana attenuata plants in response logical function of hexenyl acetate differs to feeding herbivores (9% of the JA accu- from the two early C6-volatiles. mulation) (VON DAHL & BALDWIN, 2004). Functionally, these JA derivatives 3.3. Volatile phytohormones and indole may be of minor importance for defence Like JA, MeJA is also considered one of and signalling. Otherwise, the importance the most important signalling molecules of may be limited in a few plant species such herbivore-induced plant defence. An early as A. tridentata. study showed that synthesis of PI proteins The Arabidopsis genome contains 24 in the leaves of solanaceous species is genes, which belong to a structurally re- induced by exogenous application of lated group of methyl transferases MeJA or natural emission of MeJA from (D’AURIA et al., 2003). These have been sagebrush plants (Artemisia tridentata) classified as one gene family termed (FARMER & RYAN, 1990). Transgenic AtSABATH, of which JMT is a member. Arabidopsis overexpressing JA carboxyl AtBSMT1, another member of this family, methyl transferase (JMT), an enzyme that has recently been characterised as SA methylates JA to form MeJA, showed a carboxyl methyl transferase, an enzyme three-fold increase of the endogenous that methylates SA to form MeSA (CHEN MeJA level without altering JA levels (SEO et al., 2003b). The transcript accumulation et al., 2001). Furthermore, transgenic of AtBSMT1 can be induced by any of the

36 Herbivore-induced, indirect plant defences

following: the fungal elicitor ALA; feeding constituents (DICKE, 1999b). Different Plutella xylostella; uprooting; mechanical plant species vary in the headspace com- wounding; and exogenous application of position (BARATA et al., 2000; TURLINGS MeJA to Arabidopsis leaves. In some of et al., 1993) yet also share compounds, for these cases, the emission of MeSA is also example, (E)-β-ocimene, (3Z)-hex-3-enyl induced. Besides its role as HIPV in at- acetate, and DMNT in Lima beans and tracting carnivores, such as the mites P. cucumber (TAKABAYASHI et al., 1991). persimilis and Amblyseius potentillae Even within a species, the volatile blends (DICKE et al., 1990c), MeSA can also in- emitted upon herbivore damage differ both duce defence responses in plants (SHU- quantitatively and qualitatively depending LAEV et al., 1997). on the genotype or cultivar (GOUIN- Occasionally indole has been ob- GUENE et al., 2001; KRIPS et al., 2001; served as a minor constituent of herbivore- SCUTAREANU et al., 2003), the leaf de- induced volatile blends. In maize plants, a velopmental stage (TAKABAYASHI et al., gene has been characterised as indole-3- 1994b), or the plant tissue attacked by an glycerol phosphate (IGP) lyase, which herbivore (TURLINGS et al., 1993), as catalyses the formation of free indole from well as on abiotic conditions (light inten- IGP (FREY et al., 2000). Both volicitin and sity, time of year, water stress) (TAKABA- MeJA were shown as selective inducers of YASHI et al., 1994a). Beyond that, the the gene expression and the indole emis- time of the day also influences the compo- sion from maize plants (FREY et al., 2000, sition of the emitted volatile blend (Fig. 4): 2004). for example, Nicotiana tabacum releases several HIPVs exclusively at night. These nocturnally emitted compounds were re- 4. Ecology of induced indirect defences pellent to female moths (Heliothis vires- 4.1. Variability of herbivore-induced plant cens), which search for oviposition sites volatiles during the night (DE MORAES et al., The emission of volatile organic com- 2001). Moreover, the blend composition pounds seems to be very common across strongly depends on the type of damage the plant kingdom and is not limited to (e.g. herbivore feeding versus oviposition certain life forms (see (DICKE & VET, (HILKER & MEINERS, 2002)) inflicted 1999) for a compilation of species). Odour upon a given plant individual as well as on blends emitted by herbivore-infested the time elapsed after leaf damage (Fig. plants are complex mixtures, often com- 4). To all the variability that already exists posed of more than 100 different com- on the plant level, several studies have pounds, many of which occur as minor added that the emitted blends also vary

Manuscript I 37

with the herbivore species (DICKE, attracting, repelling, or neutral effect on 1999a), and even different ontogenetic the behaviour of the focal arthropod. stages of the same herbivore (GOUIN- GUENE et al., 2003; TAKABAYASHI et 4.2. Specificity and behavioural responses al., 1995) may influence the headspace to HIPVs composition. However, differences be- Much has been learned from such behav- tween volatile blends seem to be largest ioural assays on the ability of arthropods among different plant species and smallest to discriminate different odour blends. As among plants of one species infested by the production of HIPVs has been origi- different herbivores (DICKE, 1999a; nally described in the context of host- TAKABAYASHI et al., 1991). location cues for parasitic wasps The high variability that characterises (TURLINGS et al., 1990), most research- the chemical composition of emitted ers have focussed on the role of plant- blends raises the question of the ecologi- derived volatiles in determining the ability cal relevance of these differences and of different parasitoid or carnivore species whether arthropods can discriminate to detect their herbivorous hosts or preys. among these complex mixtures. Electro- Several studies of an increasing number of antennogram (EAG) analyses allow indi- tritrophic plant-herbivore-carnivore sys- vidual blend components to be differenti- tems have indicated that the ability of the ated in order to determine which com- carnivores and parasitoids to discriminate pounds or combination of different com- different odour blends depends very much pounds can be perceived by an arthropod on their degree of dietary specialization (DU et al., 1998; WEISSBECKER et al., (VET & DICKE, 1992), and on their level of 2000). Further information on the physio- deprivation, as well as on their previous logical perception of different plant odours experience (DICKE, 1999a). The particular by arthropods have been obtained from in behavioural response - that is whether an vivo calcium-imaging measurements that herbivore or carnivore is attracted or re- provide additional information on how the pelled, or reacts neutrally to a specific olfactory information is processed in the blend of HIPVs - seems to depend arthropod brain (GALIZIA et al., 2000; strongly on the level of plant induction SKIRI et al., 2004). However, only the ad- (GOLS et al., 2003; HEIL, 2004b; HORIU- ditional inclusion of behavioural assays, in CHI et al., 2003b). which the arthropods are simultaneously However, plants do not only interact exposed to two or more odours among with other organisms above ground. Re- which they have to choose, provides in- cently, evidence has shown for the first formation on whether an odour has an time that mechanisms similar to the ones

38 Herbivore-induced, indirect plant defences

described above- may also work below- Caterpillars (S. exigua) placed on tomato ground: Entomopathogenic nematodes plants (L. esculentum var. Ace) that had (Heterohabditis megidis) are attracted as been grown in an agricultural system and yet unidentified chemicals that are induced previously with exogenously released from roots of a coniferous plant applied JA, suffered from higher para- (Thuja occidentalis) when weevil larvae sitism rates by an endoparasitic wasp (Otiorhynchus sulcatus) attack (VAN TOL (Hyposoter exiguae) than control plants et al., 2001). Moreover, systemically re- (THALER, 1999). A further field study of leased HIPVs have been shown to attract native tobacco (N. attenuata) demon- the specialist parasitoids of root-feeding strated that, indeed, the release of vola- larvae (NEVEU et al., 2002). Although tiles increased the predation rates of currently multitrophic below-ground inter- tobacco hornworm eggs by a generalist actions and the links between multitrophic predator (Geocoris pallens) (KESSLER & above- and below-ground interactions are BALDWIN, 2001). Recently, Lima bean poorly understood, future research will plants grown in their natural environment, undoubtedly address these questions followed by repeated treatments with JA (VAN DER PUTTEN et al., 2001). solutions for 5 weeks were shown to respond with an increased seed set (HEIL, 4.3. Ecological function of HIPVs in nature 2004a). Since both the emission of HIPV Most studies regarding HIPV emission as well as the secretion of extrafloral nec- have been conducted under artificial tar had been up-regulated by this treat- greenhouse or laboratory conditions. Fac- ment, it remained unclear which indirect ing the above-mentioned variability, the defence was responsible for the observed question arises whether plants benefit beneficial effect. More field trials are from the emission of HIPVs under natural clearly necessary to determine the eco- growing conditions. To date, few studies logical relevance of individual factors and exist that show predators are attracted to to verify results that have been gathered in herbivore-infested plants under field condi- laboratory or greenhouse experiments. tions (DRUKKER et al., 1995; DU et al., 1998; SCUTAREANU et al., 1997; SHI- 4.4. Extrafloral nectar MODA et al., 1997). Moreover, specialist In addition to releasing induced volatiles, parasitoid wasps were able to distinguish some plant species have developed other among maize, cotton, and tobacco plants ways to attract predatory arthropods that that were infested by their herbivorous will defend them. These include both the hosts from those which were under non- provision of shelter in so-called leaf doma- host attack (DE MORAES et al., 1998). tia (AGRAWAL & KARBAN, 1997), and

Manuscript I 39

the offer of attractive food sources such as metabolic energy (LÜTTGE, 1971), and food bodies or extrafloral nectar (EFN). EF nectaries strongly resemble secretory Extrafloral (EF) nectaries are specialized cells which contain large numbers of mito- nectar-secreting organs that may occur on chondria and show dense protoplasts and virtually all vegetative and reproductive large nuclei (BENTLEY, 1977a). Further- plant structures, yet do not involve pollina- more, the secretion of EFN can be in- tion (ELIAS, 1983). They have been de- duced by JA treatment. Originally discov- scribed for approximately a thousand plant ered in Macaranga tanarius (HEIL et al., species ranging over 93 plant families 2001), nectar secretion is suggested to be (KOPTUR, 1992) of flowering plants and inducible by leaf damage. This seems to ferns, but they are absent in gymnosperms be a widespread phenomenon (HEIL, (ELIAS, 1983). EFNs are aqueous solu- 2004a; NESS, 2003; WÄCKERS & tions that comprise mainly sucrose, glu- BEZEMER, 2003; WÄCKERS et al., cose, and fructose, but other sugars, 2001), which may be linked to the same or amino acids and other organic compounds similar signalling pathways as those dis- may be present in some species (BENT- cussed for the induction of volatile biosyn- LEY, 1977a). The secreted sugars are thesis. Inhibitors that suppress the release mainly derived from the phloem (FAHN, of linolenic acid or interfere with the pro- 2000; FREY-WYSSLING, 1955) or syn- duction of linolenic acid hydroperoxides thesised in the region of the nectary completely suppress the damage-induced (BARGONI, 1972). EF nectaries secrete flow of EFN and, hence, clearly demon- small amounts of nectar throughout the strate the involvement of oxylipin-based day. The secretion of nectar can follow signalling in EFN-induction. Interestingly, circadian rhythms (ELIAS, 1972; HEIL et also the attack of a below-ground herbi- al., 2000b; PASCAL & BELIN-DEPOUX, vore (Agriotes lineatus) on cotton plants 1991; RAINE et al., 2002) or be relatively (Gossypium herbaceum) induced above- constant throughout day and night (BENT- ground production of EFN (WÄCKERS & LEY, 1976, 1977b). BEZEMER, 2003). In Vicia faba, even the The mechanisms of nectar secretion number of EF nectaries increased follow- are poorly understood: Some researchers ing leaf damage (MONDOR & ADDICOTT, have described secretion as a passive 2003). process, whereas others discuss it as an Generally, the secretion of EFN is elimination of surplus sugars (BENTLEY, believed to function as an indirect anti- 1977a). However, there is evidence that herbivore defence by attracting and hence nectar secretion is an active secretory increasing the presence of putative plant process: It requires the expenditure of defenders on nectar-secreting plant parts.

40 Herbivore-induced, indirect plant defences

Most studies focussed on the protective reduce the number of herbivores. Both effect of ants on entire plants or individual ants and wasps exerted beneficial effects plant parts, demonstrating a beneficial on the EF nectary-bearing plant Turnera function of these insects (BENTLEY, ulmifolia when selectively excluded, yet no 1977a; KOPTUR, 1992). However, con- additive increase was observed when both tradictory observations that detected no insect groups had access (CUAUTLE & measurable preventive effect of EFN- RICO-GRAY, 2003). attracted ants also exist (BECERRA & The emission of floral odours by VENABLE, 1989; BOECKLEN, 1984; plants is very important for attracting polli- MODY & LINSENMAIR, 2004; O'DOWD & nators to their floral nectars. Some of CATCHPOLE, 1983; RASHBROOK et al., these floral nectars also scent by them- 1992). In these cases, the lack of protec- selves (RAGUSO, 2004), whereas nothing tion could be explained by, for example, is known about the headspace of EFNs. (1) differences in the aggressiveness of Beyond these odours, which communicate the attracted ant species (DEJEAN et al., the location, abundance, and quality of 2000; HORVITZ & SCHEMSKE, 1984; these nectars to higher trophic levels, MODY & LINSENMAIR, 2004), (2) differ- other mechanisms may help to guide EFN- ences in foraging behaviour of ant species feeding arthropods to remote nectar from different habitats (BARTON, 1986; sources. First, some EF nectaries are col- INOUYE & TAYLOR, 1979), and (3) a oured providing visual cues for foraging varying susceptibility of the herbivores to arthropods (KOPTUR, 1992). Additionally, ant predation (FOWLER & MACGARVIN, increased amounts of both HIPVs and 1985; HEADS & LAWTON, 1985). Besides EFN (Table 1) would allow foraging arthro- ants, the EF nectaries attract a diverse pods to use the emitted volatile organic spectrum of other arthropods including compounds as a cue to detect nectar Araneae, Diptera, Coleoptera, and Hy- sources from longer distances. Future menoptera (see (KOPTUR, 1992) for re- research should try to disentangle and view). Due to their predatory or parasitoid quantify the costs and benefits of one of ways of life, many of these non-ants, such these indirect defences for a plant and as ichneumonid, braconid and chalcid figure out to what extent each of them con- wasps (BUGG et al., 1989; KOST & HEIL, tributes to plant defence in nature. 2005a; STAPEL et al., 1997), jumping spiders (Salticidae) (RUHREN & HANDEL, 4.5. Evolution of HIPVs and EFN 1999), phytoseiid mites (VAN RIJN & The evolutionary origin of HIPV emission TANIGOSHI, 1999) or tachinid flies and EFN secretion remains unknown. As

(PEMBERTON & LEE, 1996), can as well described above, C6-volatiles or terpe-

Manuscript I 41

Table 1 Compilation of species which both emit HIPVs and feature extrafloral nectaries. Additional information on the inducibility is presented (for abbreviations see below).

Family Species Nectar inducible HIPVs inducible Reference

Euphorbiaceae Manihot esculenta n.i. n.i. + H 1 GARY & FOSTER, 2004; GNANVOSSOU et al., 2001

Leguminosae Phaseolus lunatus + JA + JA HEIL, 2004a

Vicia faba n.i. n.i. + H 2 COLAZZA et al., 2004; FIGIER, 1971

Vignia unguiculata n.i. n.i. + H3 KUO & PATE, 1985; VAN DEN BOOM et al., 2004

Malvaceae Gossypium herbaceum + M, H 4, 5 + H 5, 6 BEZEMER et al., 2004; WÄCKERS & BEZEMER, 2003; WÄCKERS et al., 2001

Gossypium hirsutum n.i. n.i. + H 6 , 7, 8 LANDOLT, 1993; LOUGHRIN et al., 1995; MCCALL et al., 1994; STAPEL et al., 1997

Rosaceae Prunus serotina n.i. n.i. + H 9 RUTHER et al., 2002; TILMAN, 1978

Salicaceae Populus deltoides n.i. n.i. + H 10 ARIMURA et al., 2004a; CURTIS & LERSTEN, 1974

Populus trichocarpa n.i. n.i. + H 10 ARIMURA et al., 2004a; TRELEASE, 1881

Solanacea Solanum nigrum n.i. n.i. + H 11, 12 KEELER, 1979; SCHMIDT et al., 2004

M mechanical damage, H 1 Spider mite (Mononychellus tanajoa BONDAR), H 2 Pentatomid bug (Nezara viridula LINNAEUS), H 3 Spider mite (Tetranychus urticae KOCH), H 4 Mediterranean Brocade (Spodoptera littoralis BOIS- DUVAL), H 5 Wireworms (Agriotes lineatus LINNAEUS), H 6 Beet armyworm (Spodoptera exigua HÜBNER), H 7 Tobacco budworm (Heliothis virescens FABRICIUS), H 8 Corn earworm (Helicoverpa zea BODDIE), H 9 Forest cockchafer (Melolontha hippocastani FABRICIUS), H 10 Forest tent caterpillar (Malacosoma disstria HÜBNER), H 11 Colorado potato beetle (Leptinotarsa decemlineata SAY), H 12 Death's Head Hawkmoth (Acherontia atropos LINNAEUS). noids may repel herbivores (AVÉ et al., (DICKE, 1999c). However, in this case, 1987; VANCANNEYT et al., 2001), be non-volatile defensive compounds rather toxic for phytophages (CRANKSHAW & than volatile substances should have LANGENHEIM, 1981; HOBALLAH et al., evolved to fulfil this purpose, as they 2004), or act anti-microbially (CROFT et would not diffuse so easily in the environ- al., 1993). HIPVs are therefore discussed ment (JANSSEN et al., 2002). HIPV emis- as having originally functioned as a direct sion is an active process that does not defence against pathogens or herbivores necessarily depend on cell damage at the

42 Herbivore-induced, indirect plant defences

site of emission (DICKE & VAN LOON, ferred by two pairs of recessive genes (i.e. 2000). Even if the first volatile release by ne-1 and ne-2) (MEYER & MEYER, 1961; an herbivore-damaged plant was inci- RHYNE, 1965). dental, natural enemies might have been In addition to the benefits discussed immediately able to use this cue to locate above, the concept of fitness costs pro- the responsible herbivores; hence, natural vides a powerful explanation for the evolu- selection should operate on the plant to tion of induced plants defences (CIPOL- optimize these signals (JANSSEN et al., LINI et al., 2003; HEIL, 2002): Costs cover 2002). any trade-off between resistance and The evolution of EF nectaries is con- other fitness-relevant processes (HEIL, sidered a very simple process (BENTLEY, 2002). Inducibility itself (i.e. the ability to 1977a) that may have occurred several change per se) can raise fitness costs by, times independently (BENTLEY, 1977a; for example, increasing the lag time until ELIAS, 1983). This development has re- the defence is up-regulated, during which sulted in an extreme variety of plant taxa the plants remains susceptible to herbi- that feature a wide structural diversity of vores (DEWITT et al., 1998; ZANGERL, EF nectaries (ZIMMERMANN, 1932); yet 2003). Further evolutionarily relevant costs these structures may be absent in one of comprise allocation costs (HERMS & two very closely related species (BENT- MATTSON, 1992), genetic costs (AGRA- LEY, 1977a). Recently, a 2-year study has WAL et al., 2002a; MITCHELL-OLDS et shown that traits of EF nectaries were al., 1996), autotoxicity costs (BALDWIN & heritable and that casual ant associates CALLAHAN, 1993), opportunity costs acted as agents of selection on these traits (BALDWIN & HAMILTON, 2000), and eco- (RUDGERS, 2004). Even if corresponding logical costs (Fig. 5) (AGRAWAL et al., studies on other EF nectary-bearing plants 2002b; PRICE et al., 1980). Inducible de- or plants that feature HIPVs are missing, fence strategies are generally assumed to these two indirect defences seem to be reduce the fitness of a plant when expres- evolutionarily plastic and to remain stable sed under enemy-free conditions (HAUKI- due to strong selection pressure. How- OJA & HAKALA, 1975) as compared to ever, the genetic basis of these two traits costs of a constitutive (i.e. permanent) has been investigated only for the devel- expression. The physiological expendi- opment of EF nectaries in cultivated cotton tures of EFN production that could cause (MEYER & MEYER, 1961; RHYNE, 1965) allocation trade-offs within the plant are and Acacia koa (DAEHLER et al., 1999): assumed to be very low, yet this has rarely For cotton crossing, studies showed that been tested: For the Balsa tree Ochroma the inheritance of “nectaryless” was trans- pyramidale, the physiological expenses of

Manuscript I 43

EFN secretion have been calculated to source availabilities for plants may also account for 1 % of the total energy invest- lead to trade-offs between direct and in- ment per leaf (O'DOWD, 1979). In con- direct defences (HEIL et al., 2002; RUDG- trast, a phylogenetical analysis of Ameri- ERS et al., 2004); these seem to be more can Acacia species revealed that constitu- common in obligate ant–plant mutualisms tive EFN secretion was found to be the (HEIL et al., 1999, 2000a; KOPTUR, 1985) derived state which developed as an than in facultative associations (RUDG- adaptation to obligate myrmecophytism. ERS et al., 2004). This may indicate that a constitutive flow Both EFN and HIPVs are openly pre- of EFN is costly and only profitable when sented cues that may non-specifically rewarded by permanent ant-protection attract members of higher trophic levels. (HEIL et al., 2004b). The actual costs Such loose forms of facultative mutualisms probably depend on the level of EFN ex- are especially prone to exploitation by un- cretion and physiological limitations such desired arthropods. For example, the at- as the availability of water or nutrients. traction of non-beneficial or even herbivo- EFF secretion even ceases in the absence rous species feeding on EFN results in of nectar feeders, yet this may be a pas- costs rather than benefits for the respec- sive process and not necessarily a plant- tive plant (Fig. 5) (DEVRIES & BAKER, regulated strategy to save costs (HEIL et 1989; HEIL et al., 2004a; KOPTUR & al., 2000b). The physiological expendi- LAWTON, 1988). HIPVs emitted from tures of HIPV are expected to be relatively strongly induced plants denote not only high (GERSHENZON, 1994a, b; GULMON parasitoids or predators, the presence of & MOONEY, 1986), yet predictions remain potential prey organisms but, additionally, controversial (DICKE & SABELIS, 1989; a hint at increased numbers of competitors GODFRAY, 1995; SABELIS & DE JONG, (JANSSEN et al., 1997, 1995a, b). Fur- 1988) and experimental evidence is rare. thermore, by calling on strongly induced In maize plants, the first hints of allocation plants, carnivores or parasitoids may well costs for the production of HIPVs have run the risk of falling prey themselves to a been seen (HOBALLAH et al., 2004). hyperparasite or a predator (HÖLLER et However, to what extent simultaneously al., 1994). The resulting deterrent effect of induced direct defences accounted for the HIPVs on beneficial arthropods represents measured costs remained unknown. Inter- an ecologically relevant fitness cost for the estingly, these allocation costs were de- herbivore-attacked plant (Fig. 5). tectable only in young plants, with com- Signals emitted from infested plants pensation for their metabolic investment can also provide information to other tro- occurring during maturation. Finite re- phic levels (Fig. 5): Conspecific herbi-

44 Herbivore-induced, indirect plant defences

vores, for example, can be attracted to fic herbivores may be deterred by the infested plants (BERNASCONI et al., emitted volatiles (BERNASCONI et al., 1998; BOLTER et al., 1997; HORIUCHI et 1998; DE MORAES et al., 2001), as al., 2003b), because this cue denotes the strongly induced plants may point to com- location of palatable host plants (INUI et petition with conspecific or heterospecific al., 2003) and suitable oviposition sites herbivores or to an enemy-dense patch (STANJEK et al., 1997) as well as the (HORIUCHI et al., 2003a). In this case, the presence of potential mating partners release of HIPVs would act as a direct (RUTHER et al., 2002). Such a ‘miscue’ defence (DE MORAES et al., 2001; from the plant’s perspective results as well DICKE & VAN LOON, 2000), thereby in ecological fitness costs for the plant. On benefiting the plant. Moreover, hyperpara- the other hand, conspecific or heterospeci- sitoides or carnivores of the fourth trophic

Figure 5. Potential interactions of plants with higher trophic levels via herbivore-induced plant volatiles (HIPVs) and extrafloral nectar (EFN). The stipules at the petioles of the leaves represent extrafloral nectaries. Effects mediated by HIPVs or EFN can be attraction (+) or deterrence (-) that consequently affect trophic interactions such as herbivory or predation (Arrows). Additionally, the ecological fitness costs (C) and benefits (B) of each interaction from the plant’s perspective are indicated.

Manuscript I 45

level could use plant-derived volatiles for mechanism or strategy that herbivores the search on larger spatial scales to find might evolve to cope with these highly their prey or host by locating potential host efficient plant defenders. plants (VÖLKL & SULLIVAN, 2000). Such attraction would again impose the respec- 5. Conclusions tive plant with ecological costs (Fig. 5). We described what is currently known Several plant species have the poten- about molecular mechanisms of cell sig- tial to defend themselves indirectly using nalling and signal travelling, as well as the HIPV emission and EFN secretion; both ecology and evolution of herbivore- indirect defences are inducible via the oc- induced plant responses. Despite the ef- tadecanoid pathway (Table 1). Comparing fort that has been brought to bear on this the widespread distribution of EF nectaries topic, the exact mechanisms as well as the across the plant kingdom (KOPTUR, ecological and evolutionary relevance still 1992) with the small number of nearly ex- remain elusive. One reason is the diversity clusively crop species for which HIPVs that exists on the genetic, biochemical, have been studied (DICKE, 1999c; VAN physiological, and phenotypical levels and POECKE & DICKE, 2004), an increasing that differs with the evolutionary back- number of plant species that features both ground of the studied plant species. In indirect defences can be expected. ecological studies, even the geographical Beyond that, a wide taxonomical distribu- location with its particular biotic and abiotic tion of these plant traits may indicate the conditions influences the outcome of a ecological success of these two induced, field experiment. We therefore suggest indirect defences. adopting model systems beyond the most In the ecological arms race between frequently used ones, namely tobacco, plants and herbivores, the latter are known Arabidopsis, maize, and Lima bean to dis- for rapidly adapting to novel host plants or tinguish in a more comparative way toxins (AGRAWAL, 2000; FRY, 1989; particular phenomena from general KARBAN, 1989) by evolving specific mechanisms. Such a biodiversity-oriented mechanisms to circumvent direct defences approach should include more non-crop with physiological tolerance (DUFFEY, species since in this case a stronger co- 1980; NISHIDA, 2002) or behavioural re- evolution with natural herbivores is to be sponses (DUSSOURD & DENNO, 1991; expected. DUSSOURD & EISNER, 1987). In view of Connectivity exists between multiple the diverse spectrum of carnivorous spe- defences, as they are often regulated via cies which is attracted to plants expressing the same signalling cascade (BALDWIN, indirect defences, it is hard to imagine any 2001; BALDWIN & PRESTON, 1999),

46 Herbivore-induced, indirect plant defences

leading to considerable methodological will contribute to separate the different problems when individual defensive plant defence responses. These new ap- traits are studied. For example, the clear proaches have to be complemented by distinction between pure pathogen and creative field experiments and compara- herbivore defence pathways was under- tive approaches to gain better insights into mined by the finding that the type of dam- emerging areas like e.g. the macroecology age inflicted by an herbivore (chewing or community ecology of induced, indirect caterpillars versus sucking aphids) deter- defences. Finally, the interactions of plants mines which defence pathway (SA versus with microorganisms inside the herbivores’ JA) is activated. Further identification of gut or with micro- or macrobiota below- specific elicitors and the incorporation of ground are promising fields of future re- response mutants and transgenic plants search.

Manuscript II 47

Manuscript II

Increased availability of extrafloral nectar reduces herbivory in Lima bean plants (Phaseolus lunatus, Fabaceae)

Christian Kost* and Martin Heil

Basic and Applied Ecology (2005) 6, 237-248

Accepted: 5 November 2004

Department of Bioorganic Chemistry Max Planck Institute for Chemical Ecology Beutenberg Campus, Hans-Knöll-Str. 8 D-07745 Jena, Germany

* Corresponding author: Christian Kost Dept. of Bioorganic Chemistry Max Planck Institute for Chemical Ecology Beutenberg Campus, Hans-Knöll-Str. 8 D-07745 Jena, Germany Phone: + + 49 - 3641 - 57 18 20 Fax: + + 49 - 3641 - 57 12 02 E-mail: [email protected]

48 Protection of Lima beans by extrafloral nectar

Abstract Lima bean (Phaseolus lunatus) features two inducible indirect defences to protect itself against herbivores. Besides the emission of plant volatiles, extrafloral nectar is secreted to attract carnivorous arthropods to herbivore-damaged plants. The activation of both putative defences efficiently protects Lima beans from leaf damage. In a field experiment in Mexico, we studied whether extrafloral nectar alone can benefit the Lima bean under natural conditions. An artificial blend mim- icking natural nectar both qualitatively and quantitatively was repeatedly applied to Lima bean tendrils. Ants, wasps and flies were significantly more abundant on treated tendrils than on untreated controls already after one week (i.e. after two treatment applications). Sticky traps were used to assess the functional groups of flying insects attracted to the Lima beans. After 24 h, 71 % of all trapped flies and 98 % of all wasps belonged to families comprising either parasitoid or predatory species. This observation suggests that also some of the flying visitors have played a role as putative defenders of Lima beans. Most of the trapped flies belonged to the families Dolichopodidae and Phoridae (each ca. one third of all individuals). Two thirds of the wasps belonged to Chalcidoidea (68 %). All ant species that had been collected manually belonged to generalist genera with Camponotus novogranadensis and Cephalotes minutus being most regularly encountered on study tendrils. An additional experiment, where both ‘nectar’ and ‘control’ tendrils were treated with artificial nectar, revealed that ants responded with an increased abundance on tendrils that had experienced the ‘nectar’ treatment before. After 25 days, the treated tendrils showed a significantly reduced herbivory as compared to controls. The mere presence of increased amounts of extrafloral nectar thus can benefit the Lima bean under natural conditions.

Zusammenfassung Die Limabohne (Phaseolus lunatus) verfügt über zwei induzierbare, indirekte Verteidigungsformen zur Abwehr von Herbivoren. Neben der Emission volatiler Verbindungen ist die Limabohne zusätzlich dazu in der Lage, extrafloralen Nek- tar zu sezernieren. Beides dient der Anlockung von Fraßfeinden zu den von Her- bivoren befallenen Pflanzen. In einem Freilandexperiment in Mexiko wurde untersucht, ob die Limabohne unter natürlichen Bedingungen von der Sekretion extrafloralen Nektars profitiert. Hierzu wurde ein künstliches Nektargemisch wie- derholt auf Limabohnenranken aufgetragen, welches natürlichen Nektar quantita-

Manuscript II 49

tiv und qualitativ imitierte. Bereits nach einer Woche (d.h. nach zwei Behandlun- gen) war die Abundanz von Ameisen, Fliegen und Wespen auf behandelten Ranken signifikant höher als auf unbehandelten Kontrollranken. Zur Erfassung der zur Limabohne angelockten fliegender Insekten sowie deren Zugehörigkeit zu funktionellen Gruppen wurden die Versuchsranken mit Klebefallen bestückt. Mehr als zwei Drittel der nach 24 h gefangenen Fliegen und 98 % aller Wespen gehörten parasitoidisch oder räuberisch lebenden Fliegen- bzw. Wespen- Familien an. Diese Beobachtung legt nahe, dass nicht nur Ameisen, sondern auch einige der gefangenen fliegenden Besucher eine Rolle als potentielle Ver- teidiger der Limabohne gespielt haben könnten. Von den gefangen Fliegen ge- hörten die meisten den Familien Dolichopodidae und Phoridae (je ca. ein Drittel aller gefangenen Individuen) an, wogegen die Chalcidoidea zwei Drittel (68 %) der gefangenen Wespen ausmachten. Unter den durch Handaufsammlung gefangenen Ameisen gehörten Camponotus novogranadensis und Cephalotes minutus zu den am häufigsten auf behandelten Ranken angetroffen Arten. Ein zusätzliches Experiment, in dem das künstliche Nektargemisch sowohl auf ‘Nek- tar’- als auch auf ‘Kontroll’-Ranken aufgebracht wurde, ergab, dass die Ameisen mit einer erhöhten Abundanz auf solchen Ranken reagierten, die bereits vorher die ‚Nektar’-Behandlung erfahren hatten. Nach 25 Tagen zeigten behandelte Ranken signifikant weniger Blattfraß im Vergleich zu unbehandelten Kontrollran- ken. Die bloße Erhöhung der Menge an extrafloralem Nektar reichte offensicht- lich dazu aus, unter natürlichen Bedingungen wachsenden Limabohnen einen Vorteil zu verschaffen.

Key words: ants, anti-herbivore defence, artificial nectar, induced defence, Leguminosae, plant-herbivore interaction, sugar, Mexico

50 Protection of Lima beans by extrafloral nectar

Introduction tion of entire plants or plant parts (BENT- Plants have developed a large number of LEY, 1977a; HEIL et al., 2001; SO- different strategies to defend themselves BRINHO et al., 2002), whereas other visi- against herbivores. Such strategies can be tors may act as commensals or even divided into direct or indirect defences. parasites (HEIL et al., 2004a; O'DOWD, Direct defences per definition directly exert 1979). negative impacts on herbivores. They Studies investigating the role of extra- comprise, e.g., spines, thorns, trichomes, floral nectar as an indirect defence ace waxes and a large diversity of secondary considerable methodological problems. In plant metabolites. On the other hand, indi- the majority of studies this issue was ad- rect defences are plant characteristics that dressed by excluding ants using sticky include higher trophic levels such as para- barriers such as Tangletrap® (CUAUTLE & sitoids or predators of the herbivores, RICO-GRAY, 2003; LABEYRIE et al., which then fulfil the defensive function 2001; STEPHENSON, 1982). The draw- (PRICE et al., 1980). back of this attempt is that crawling herbi- Extrafloral nectaries are nectar- vores are excluded as well (FREITAS et secreting organs that are not directly in- al., 2000; MACKAY & WHALEN, 1998) volved in pollination, yet play a vital role in and that this systematic error finally can maintaining other mutually beneficial rela- lead to an underestimation of the ‘true’ tionships among plants and animals effect. The second way to approach this (ELIAS, 1983). In general, extrafloral nec- issue is using plant-derived elicitors such tar (EFN) is believed to function as an indi- as jasmonic acid that induce extrafloral rect defensive mechanism. It is mainly nectar flow (HEIL et al., 2001) or to induce composed of mono- and disaccharides EFN secretion by natural herbivory and amino acids (GALETTO & BER- (NESS, 2003). Such induction phenomena NARDELLO, 1992; HEIL et al., 2000b; often act via the octadecanoid pathway, RUFFNER & CLARK, 1986). Besides which is well known to not only regulate ants, which constitute the main portion, the flow of extrafloral nectar, but also to other insects such as ichneumonid and mediate many other induced direct or indi- braconid wasps (BUGG et al., 1989; rect defences, such as, e.g., the change of GENTRY, 2003; STAPEL et al., 1997) or chemical constituents in attacked plant mosquitoes (FOSTER, 1995) utilize this tissues, or the production of volatile or- food source as well, resulting in varying ganic compounds (VOCs) to attract natu- degrees of anti-herbivore protection. The ral enemies of the herbivores (KARBAN & presence of ants, for example, has been BALDWIN, 1997). Consequently, the stud- repeatedly shown to enhance the protec- ies conducted so far suffered from differ-

Manuscript II 51

ent methodological problems: Exclusion A previous study demonstrated that regu- experiments run the risk of accidentally larly treating wild Lima bean with jasmonic barring crawling herbivores and thus un- acid and thereby inducing both the pro- derestimating the defensive effect, those duction of extrafloral nectar and VOCs inducing EFN secretion may overestimate benefits the plants at their natural growing it due to undesigned inductions of addi- site (HEIL, 2004a). However, it remained tional direct or indirect defences. unclear which insects were involved in this A possibility that circumvents all the interaction on both the side of herbivores abovementioned difficulties is the artificial and defenders. Moreover, it was not clear increase of extrafloral nectar by exoge- whether extrafloral nectar alone could also nous application of sugar solution. exert a defensive effect. Amongst others (BENTLEY, 1976; TEM- The aim of the present study was to PEL, 1983), this approach was followed follow the approach of JACOB and EV- up by Jacob & Evans (1998), who sprayed ANS (1998), yet applying artificial nectar alfalfa plants (Medicago sativa) with an directly onto or nearby extrafloral nectaries aqueous solution of sucrose. Two days in an amount and quality that mimicked later, significantly higher numbers of adult the natural occurrence of extrafloral nec- parasitoids as well as an increase in the tar. The following questions were addres- parasitation rate of herbivorous weevils sed: i) What kinds of insects are attracted were observed in plots sprayed with sugar to the artificial nectar applied to Lima solution as compared to control plots, beans? ii) Does extrafloral nectar alone which had been sprayed with water only. exhibit a beneficial effect for the Lima Phaseolus lunatus L. (Lima bean, bean at its natural growing site?, and iii) Fabaceae) is a common model plant used Does the presence of ants on Lima bean in genetic, biochemical or ecological stud- tendrils depend on their previous foraging ies. Besides the emission of VOCs that experience (i.e. do they appear in in- follows herbivore attack and attracts car- creased numbers where more nectar can nivorous animals (DICKE, 1994; DICKE et be expected)? al., 1990b; DICKE et al., 1993), the Lima bean features extrafloral nectaries (HEIL, 2004a). Although several aspects of VOC Material and Methods emission of Lima beans have been stud- Study site and species ied intensively in laboratory or greenhouse The study was conducted in the coastal experiments, almost nothing is known on area of the state of Oaxaca, Mexico, a the ecological function of its extrafloral location close to the centre of the genetic nectaries and of VOC-emission in nature. diversity of wild Lima beans (SALGADO et

52 Protection of Lima beans by extrafloral nectar

al., 1995). Two sites, some 15 km north- foliate leaves as well as at the petioles of west of Puerto Escondido, were selected. the individual leaflets (HEIL, 2004a). These two sites were about 3 km apart Voucher specimens are deposited at Her- from each other and 1 to 2 km away from bario MEXU (UNAM, Mexico City, Mexico) the Pacific Ocean. Both study sites repre- and in the personal collection of M. HEIL. sented waysides along dirt roads leading to extensively used pastures or planta- Experimental design tions, with site 1 featuring denser vegeta- In the beginning, 23 pairs of Lima bean tion and site 2 being much more exposed tendrils were selected. Eleven of these to direct sunlight. The two sites are identi- pairs were located at site 1 and twelve at cal with those of a previous study (HEIL, site 2. Due to the tangled growth of Lima 2004a). The climate in the study area is bean it was not possible to distinguish characterized by one main rainy season single plant individuals. Therefore, paired from June to October, which follows a bi- tendrils either belonged to one single plant modal distribution peaking in July and individual or to two adjacent plants with a September. Annual rainfall averages be- maximum distance of 2 m. Tendrils of tween 1,000 and 1,400 mm and the mean each pair were stretched along two strings annual temperature is 28 °C (STRÄßNER, for further growth. One tendril per pair was 1999). randomly assigned to treatment ‘nectar’, Wild Lima bean (Phaseolus lunatus the other one served as an untreated con- L., Fabacecae) grows as a twining vine or trol. ‘Nectar treatment’ means an aqueous herbaceous bush, entwining trees and solution of artificial nectar was applied shrubs along open sites such as forest directly on the extrafloral nectaries using edges or along roadsides. The Lima bean an Eppendorf pipette. ‘Nectar’ tendrils grows in part as a perennial plant, al- were treated every 3 to 4 d from though the aboveground parts usually die 27.10.2003 until 18.11.2003, resulting in during the dry season. In June or July the six application events. During this time, plants start to germinate or bud, and the the initial number of pairs was reduced to first inflorescences usually appear in Oc- a final sample size of 17 due to human tober or November. Depending on water impact. supply, the production of flowers and fruits ends between February and April (HEIL, Artificial nectar 2004a). As many other Fabaceae, the To mimic the extrafloral nectar production Lima bean bears extrafloral nectaries. of the Lima beans qualitatively and quanti- These are located on its bracts, or ar- tatively, both the secretion rate and the ranged pairwise at the stipules of the tri- chemical composition of extrafloral nectar

Manuscript II 53

had to be assessed. Lima bean plants Samples were analysed on a GC- growing at the aforementioned study sites Trace mass spectrometer (Thermo Finni- had been induced by spraying with 1 gan) using a DB 5 column (15 m x 0.25 mmol aqueous JA-solution (HEIL, 2004a). mm x 0.25 µm; Alltech, Unterhaching, After 24 h, the nectar production rate of Germany). Sugars were separated with these plants was quantified as amounts of the following program: 120 °C for 2 min secreted soluble solids, by quantifying the isothermal, then at 7 °C min-1 to 250 °C, nectar volume with micro capillaries and temperature of the injection port of 220 °C the nectar concentration with a refracto- and temperature of the GC-interface at meter (HEIL et al., 2000b; 2001). The nec- 280 °C. Simultaneous analysis of sugars tar secretion rate was then related to the and amino acids required a modified pro- secreting leaves and their respective dry gram: 40 °C for 2 min isothermal, then at weight. The estimated secretion rate for a 10 °C min-1 to 120 °C, after that at 7 °C strongly induced Lima bean leaf averaged min -1 to 250 °C and a GC operating with a about 1.5 mg soluble solids g-1 leaf dry split-ratio of 1:10. This analysis identified mass 24 h-1 as compared to 0.02 mg g-1 glucose, fructose and sucrose as the main 24 h-1 for an uninduced control leaf (HEIL, components of the extrafloral nectar 2004a). blend, with amino acids being below the The subsequent analysis of EFN fo- detection limit (i.e. < 1.3 pmol/µl). The cused on sugars and amino acids, as they blend of artificial nectar, which was ad- comprise the most important components justed to the cumulated secretion rate of of extrafloral nectar (BAKER et al., 1978; one leaf for three days, consisted of 4.01 g GALETTO & BERNARDELLO, 1992; sucrose l-1, and 24.24 g l-1 of each glucose RUFFNER & CLARK, 1986). Other poten- and fructose. Every three to four days, 40 tial nectar constituents such as proteins, µl of this blend were applied directly to the vitamins or lipids were not considered. To extrafloral nectaries of every leaf of the analyse sugars and amino acids as their ‘nectar’ tendrils. trimethylsilylated derivatives, 50 µl N-Methyl-N-(trimethylsilyl)-rifluoracetamide Insect counts (MSTFA) was added to a 2 µl nectar sam- The first census of the arthropods on the ple and heated for 1 h to 60 °C. The sam- 23 selected pairs of tendrils was per- ples were diluted with dichloromethane in formed immediately before the first treat- a rate of 1:100. Sugars from commercial ment (27.10.03 for site 2 and 29.10.03 for sources (Sigma) served as standards: 2 µl site 1). Both censuses were performed at of an aqueous solution (100 mg ml-1) was the same time of day. Numbers of ants, treated as described above. wasps or flies present on the plants were

54 Protection of Lima beans by extrafloral nectar

recorded, as they represented the most mated when the total leaf number per ten- abundant groups. dril was < 25, otherwise 25 leaves were Two additional censuses were per- randomly selected and the herbivory rate formed in the course of the experiment, to assessed. test the influence of the ‘nectar’ treatment Finally, average values were calcu- on the insect community. The first census lated for the two estimations and the dif- started d 7 of the experiment (7.11.03) at ference between these two was used to site 1. Ten pairs of tendrils were selected, measure the development of leaf damage. which had experienced two treatments To test the accuracy of this method, the until then. The first census was performed missing leaf area of 74 randomly chosen prior to the nectar application at 8:00 am. leaves was both estimated as mentioned Thereafter all ‘nectar’ tendrils were treated above and quantified as follows: First, and insects on all pairs were counted re- leaves were dried between sheets of pa- peatedly every 2 or 3 h until midnight. Two per for transport (leaves shrink less than additional censuses were performed at 5 % during this procedure). The leaves 9:00 and 10:00 am on the following two then were scanned together with a refer- days, resulting in a total of 14 monitorings. ence area of 1 cm2 (hp scanjet 8200 scan- The same was done with 10 pairs of ner). The resulting digital pictures were plants at site 2. This second series started analyzed with the graphics software pack- at d 18 after the onset of the experiment age Corel photo paint version 8.232. By (14.11.03) and consisted of a total of 13 outlining the area of both a given trifoliate censuses. leaf and the reference area, the corresponding leaf area could be determined, Defensive effect as indicated by the number of pixels. The The herbivory rate in percent leaf loss was missing leaf area was outlined accord- chosen as a fitness-relevant parameter. ingly. In cases of complete leaflets miss- This parameter was quantified by assigning, the corresponding area was estimated ing leaves to one of the following ranges since both the areas of the left and the of missing leaf area: 0 %, 1 - 5 %, 6 - 10 central leaflet (r = 0.93, p < 0.001, n = 58, %, 11 - 25 %, 26 - 50 % and 51 - 100 %. y = 0.8397 x + 20786) as well as the two At the beginning of the experiment lateral leaflets showed a strong correlation (24.10.03), the herbivory rate for all leaves (r = 0.95, p < 0.001, n = 56, y = 0.8361 x + present on the studied tendrils was as- 13361). Finally, the percentage of missing sessed as a reference. After 25 days, the leaf area was computed from missing and remaining tendrils were checked again: total area. The close linear relationship The herbivory rate of all leaves was esti- between the measured and estimated

Manuscript II 55

80 70 60 50 40 30 20 y = x 10 y = 0,8524x + 0,178

Measured missing leaf area (%) 0 0 10 20 30 40 50 60 70 80

Estimated missing leaf area (%)

Figure 1. Comparison between estimated and measured leaf area missing of 74 leaves of Phaseolus lunatus. Leaf area was estimated by assigning leaves to the closest of six different classes of missing leaf area (0 %, 1 - 5 %, 6 - 10 %, 11 - 25 %, 26 - 50 % and 51 - 100 %), of which the corresponding mid-points are displayed. For exact measurements digitized pictures of leaves were analysed by relating pixel numbers of outlined leaves to a reference area. A regression was calculated (solid line) to relate the estimated to the measured leaf area (r = 0.981, p < 0.001, n = 74). The axial bisector (dashed line) shows the theoretically perfect correlation. missing leaf areas (Fig. 1) allowed the consisted of 100 cm2 pieces of green plas- simplified determination of the missing leaf tic foil that had been coated with a thin area by estimation. layer of a trapping adhesive (Tangletrap®). Two such sticky traps were attached with Insect collection plastic strings to each of 14 ‘nectar’ and Ants, wasps and flies were the insect ‘control’ tendrils that were equally distrib- groups most regularly encountered on uted between the two study sites. After 24 experimental Lima bean tendrils. To study h of exposure, the traps were re-collected their affiliation to functional groups, speci- and the insects transferred to 75 % etha- mens of all three groups were collected. nol. Ants were determined to species level Ants were sampled at each of the two whenever possible by MANFRED VER- study sites from randomly chosen Lima HAAGH, Staatl. Museum für Naturkunde bean clusters that were directly neighbour- Karlsruhe. Flies and wasps were identified ing the study tendrils. All ants that showed to family level using keys and information up within approximately 45 min were col- provided by ARNETT (2000) and SCHAE- lected manually with tweezers. Flies and FER et al. (1994) for insect families and wasps were collected with sticky traps that NOYES (2003) for chalcid families. On the

56 Protection of Lima beans by extrafloral nectar

basis of the natural history information was verified by applying Mann-Whitney provided by HONOMICHL (1996) for flies U-tests to the census data of different in- and wasps, RETTENMEYER (1961) and sect groups which were assessed before DISNEY (1994) for phorid flies as well as the onset of the experiment (i.e. 27.10.03 RICO-GRAY (1993) and BROWN (2000) for site 2 and 29.10.03 for site 1). Data for ants, the collected insects were as- were pooled for tendrils that afterwards signed to the following guilds according to were assigned to the two treatments since nutritional or functional aspects: Detrivore no treatment had been administered at (D), Frugivore (F), Herbivore (H), Parasi- that time. Tests for the factor ‘time’ were toid (P), Predator (R) or utilization of other done by comparing insect numbers as- plant-derived resources including floral or sessed before the start of the experiment, extrafloral nectar, pollen and honeydew with one comparable census (i.e. same (S). time of day) seven days (site 1) or 18 days (site 2) after the start of the experiment. All Nectar experiment statistical evaluations were done using To assess whether increased ant numbers SPSS 10.0. on the nectar-treated tendrils resulted from the actual amount of artificial nectar alone, or whether the frequency of ant foragers Results had been adjusted to their previous forag- Effects of the ‘nectar’ treatment on insect ing experience, nine pairs of tendrils (three abundance at site 1 and six at site 2) were selected The application of artificial nectar resulted and both the ‘nectar’ and the ‘control’ ten- in a significant increase in the abundance dril treated with artificial nectar. The abun- of ants, wasps and flies on nectar tendrils dance of ants, wasps and flies was as- compared to control tendrils already after sessed in advance, and every 30 to 45 seven days (i.e. after two treatments, minutes after the treatment, resulting in 9 Fig. 2). The second assessment of insect to 11 countings per tendril pair. abundance starting from day 18 revealed an even more pronounced increase in the Statistical analysis number of ants. However, no statistically The approach of using adjacent pairs of significant difference between ‘nectar’ and ‘nectar’ and ‘control’ tendrils allowed the ‘control’ tendrils could be detected for use of the Wilcoxon test for matched pairs wasps and flies during the second census. whenever single or averaged values be- The three focal insect groups showed a tween the two treatments had to be com- strong diurnal activity pattern with a sub- pared. The influence of the factor ‘site’ sequent decline during the night for wasps

Manuscript II 57

Day 7 (day) 7 (night) 8 9 18 (day) 18 (night) 19 20 12 Control 10 * Nectar 8 * ** * * * 6 * * ** * * * 4 * *

Ants pertendril Ants 2 0 1.0

0.8 * 0.6 * 0.4

0.2 Wasps perWasps tendril 0.0 2.5

2.0 *

1.5 * 1.0 0.5

Flies per tendril 0.0 08.00 12.00 14.00 18.00 22.00 09.00 09.00 09.00 13.00 16.00 20.00 00.00 10.00 11.00

Time of day

Figure 2. Temporal pattern of the three most abundant insect groups on tendrils treated with artificial nectar and untreated controls, recorded at Site 1 (left) and Site 2 (right). The first census at each site shows insect numbers before the application of artificial nectar; the following censuses represent insect abundance thereafter. Insect numbers for 10 pairs of plants at a time were averaged for each census. Censuses were performed at three consecutive days starting at Site 1 on day seven after the beginning of the experiment and on Site 2 on day 18. Sig- nificant differences between corresponding pairs of tendrils are indicated as * p < 0.05, or ** = < 0.01; Wilcoxon pair test. and flies, but no strong relation to the time and the changes of insect abundance in of day for ant activity (Fig. 2). the course of the experiment (factor ‘time’) Insect censuses represented not only were assessed. Differences between sites two different dates (i.e. 7-9.11.03 and 14- were determined for the abundances of 16.11.03), but they were also performed at ants, wasps and flies present on study different sites. Therefore, a priori differ- tendrils at the onset of the experiment. As ences of insect abundance (factor ‘site’) no treatment had been administered at

58 Protection of Lima beans by extrafloral nectar

that time, insect abundances on the ten- ‘nectar’ tendrils). Artificial nectar was ap- drils which were later subjected to the plied to both the ‘nectar’ tendrils, which so treatments ‘nectar’ and ‘control’ were far had been treated six times every 2 to 3 pooled. The corresponding analysis in- days, and to the ‘control’ tendrils. The cluded 23 tendrils (n = 11 at site 1 versus subsequent monitoring of ant activity on n = 12 at site 2) and revealed that abun- both classes of tendrils revealed a very dances of wasps and flies were signifi- similar pattern of ant response: Ant activity cantly higher at site 1 as compared to site peaked 1.3 h after nectar application, 2 (Mann-Whitney-U-tests in both cases P thereafter declined (Fig. 3). However, the < 0.001), whereas no such difference be- intensity of ant response was significantly tween sites could be detected for ants lower on ‘control’ compared to ‘nectar’ (Mann-Whitney-U-test P = 0.515). tendrils. The putative influence of the factor ‘time’ was determined by comparing insect Visitors of the Lima bean abundances on ‘control’ tendrils before the The sticky trap experiment confirmed the onset of the experiment with those found observation that flies and wasps played an seven (site 1) or 18 days (site 2) later on important role as visitors of the Lima bean: the same untreated tendrils. No statisti- 44 % of all insects trapped were dipterans cally significant influence of the factor and 26 % hymenopterans (Table 1). Other ‘time’ could be detected for the abundance groups trapped were mainly Coleoptera of any of the focal insect groups (P > 0.05 (11 %), Auchenorrhyncha (9 %) or Ara- according to Wilcoxon pair tests with n = neida (6 %). Although there was no sig- 10 for both sites). Hence the abundance of nificant difference detectable between ants was neither influenced by the factors control and nectar tendrils for any of the ‘site’ nor ‘time’, whereas the abundance of trapped arthropod groups (Wilcoxon wasps and flies was heavily influenced by signed rank test P > 0.05), a more detailed a priori ‘site’-effects. analysis of the functional groups indicated As ants responded strongest to the a large proportion of putative beneficial application of artificial extrafloral nectar, insects visiting the Lima bean. Among the an additional experiment was conducted flies, Dolichopodidae (31 %) and Phoridae to clarify whether their increased presence (27 %) were the most abundant families on nectar tendrils depended on their pre- (Table 1). The Dolichopodidae are known vious foraging experience? In this case, to prey on smaller insects or insect larvae the presence of ants would be higher on that live in the vegetation along coastal tendrils on which increased amounts of areas. The family Phoridae covers many nectar had been available before (i.e. predatory or parasitoid species of mainly

Manuscript II 59

TableFormicidae, 1. Wasps but and also flies of trapped Orthoptera on experimental or Blat- clusters with sticky traps and ants collected manually from Limatodea. bean In clusters total, adjacent71 % toof theall experimental flies trapped clusters. belonged to fly families that comprise spe- Number Guild ciesOrder with Taxon (ind.) R P S F H D

Diptera

Dolichopodidae 47 l/a a Phoridae 40 l a a Tachinidae 11 l a Chloropidae 9 l l a l Culicidae 6 a l Drosophilidae 4 l/a

Platystomatidae 3 a l Agromyzidae 2 a l/a Canacidae 2 ? Lauxaniidae 2 a a Muscidae 2 l a l

Psychodidae 2 a l

Scenopinidae 2 l a l Syrphidae 2 l a l Others 14

Hymenoptera

Wasps Chalcidoidea 60 l/a a Braconidae 8 l/a a a Ichneumonidae 3 l/a a a Dryinidae 2 l/a a Sphecidae 2 l/a

Others 5

Ants Camponotus novogranadensis n. d. l a Camponotus (Myrmobrachys) sp. n. d. l a Cephalotes minutus n. d. l a Crematogaster sp. n. d. l a a Monomorium sp. n. d. l a a

Paratrechina longicornis n. d. l a Pseudomyrmex sp. 1 n. d. l a Pseudomyrmex sp. 2 n. d. l a

Numbers of wasps and flies refer to the total number of insects trapped and was not determined for ants (n. d.). The observed flies, wasps and ants were assigned to the fo llowing guilds according to nutritional or functional aspects: D, detrivore; F, frugivore; H, herbivore; P, parasitoid; R, predator/entomophaga; or S, utilization of other plant-derived resources including floral or extrafloral nectar, pollen and honeydew. The affiliation to a guild is further differentiated by the developmental stage of each taxon (l = larval, a = adult) that features the respective trait.

60 Protection of Lima beans by extrafloral nectar

25 All ants collected were generalists, which Control 20 Nectar apart from their preference for sugar

15 * sources such as extrafloral nectar, are 10 * * well known to prey on various arthropods * *

Ants pertendril 5 (Table 1).

0 5+ 0.0 0.3 0.8 1.3 1.8 2.3 2.8 3.3 3.8 4.3 4.8 Effect of the ‘nectar’ treatment on the her- Time after treatment (h) bivory rate The application of artificial nectar resulted Figure 3. Temporal pattern of the presence of ants in a significant reduction of the herbivory on nine pairs of tendrils (three from Site 1 and six from Site 2) after treating both ‘control’ and ‘nectar’ rate in treated versus control tendrils tendrils with artificial nectar. Ant numbers were (Fig. 4, P = 0.01; Wilcoxon pair test for averaged for each census. Significant differences pooled pairs). However, this protective between corresponding pairs of tendrils are indi- effect differed between the two sites cated as * p < 0.05; Wilcoxon pair test. (Fig. 4): It was very obvious at site 1 (Wil- coxon pair test: P = 0.047), whereas the predatory and parasitoid life habits. Among the Hymenoptera this trend became even more conspicuous: Except one 45 single individual of the Cephidae (Sym- phyta), all trapped Hymenoptera (98 %) 30 showed 15 either parasitoid or predatory life habits.

Here, members of the superfamily Chalci- 0 * doidea were the most abundant group, -15 which alone already contributed 68 % to all hymenopterans trapped. These parasi- -30 Control toid wasps belonged to 14 different fami- Difference in Herbivory (%) -45 lies with the Pteromalidae being the most Nectar abundant (27 %). -60 Site 1 Site 2 Among ants, Camponotus novogranadensis, Cephalotes minutus and Figure 4. Effect of the application of artificial nectar Crematogaster sp. were most regularly on herbivory of Phaseolus lunatus at the two study encountered on study tendrils (C. KOST, sites. Herbivory is presented as relative differences (day 0/day 34) in the estimated herbivory rate (%) personal observation). The remaining spe- for 17 pairs of Lima bean tendrils (n = 10 pairs for cies could only be observed occasionally Site 1 and n = 7 pairs for Site 2, * P < 0.05; and thus seem to be of minor importance. Wilcoxon pair test).

Manuscript II 61

same trend but no significant difference The following findings may also point into could be detected for site 2 (Wilcoxon pair this direction: A comparison of insect test: P = 0.091). abundances between the two sites before the start of the experiment revealed significantly higher frequencies of wasps and Discussion flies at site 1 than at site 2, whereas no The main purpose of the present study such difference between sites could be was to unravel whether the secretion of detected for ants. At the end of the extrafloral nectar benefits the Lima bean experiment, site 1 showed a strongly at its natural growing site. Many studies on reduced herbivory level, while at site 2 this topic have already been conducted only a trend towards a reduced herbivory before, but most were hampered by meth- level of treated tendrils could be observed odological problems such as i) unintended (Fig. 4). The protective effect was thus exclusion of crawling herbivores, ii) not strong at the site where, besides ants, taking into account putative influences of also wasps and flies showed increased flying plant defenders, and iii) simultane- activities, but it was weak at the site where ous induction of other defensive mecha- mainly ants were attendant to fulfil a de- nisms. In order to focus on defensive ef- fensive function. Obviously, flying defend- fects of EFN only, artificial nectar that ers added significantly to the overall de- mimicked the nectar secretion of induced fensive effect. Which of these two groups Lima bean plants in terms of amount and (flies and wasps) contributed most to the chemical composition was applied to Lima observed defensive effect? bean plants. It seems likely that the attracted The application of artificial nectar re- wasps contributed more to this effect than sulted in a significantly increased abun- the attracted flies. All flies belonging to the dance of ants, wasps and flies on treated family of Dolichopodidae were relatively compared to control tendrils after only small and thus may not have been able to seven days (Fig. 2). Most of the wasp and prey on larger herbivores. Furthermore, fly families encountered belonged to fami- the Phoridae are known not only to parasi- lies that exclusively or at least partially tize herbivorous species (RETTEN- comprise parasitoid or predatory species MEYER, 1961) but also ants (DISNEY, (Table 1). On this account, the observed 1994). An increased abundance of phorid defensive effect may not only be due to flies might therefore also have led to a the increased presence of ants, but could reduction of the ‘true’ defensive effect. also be exerted by flying defenders that Most of the studies regarding the de- are attracted to the extrafloral nectaries. fensive effect of EFN focused on ants,

62 Protection of Lima beans by extrafloral nectar

without taking into account influences of Even though the applied nectar was other putative plant defenders such as consumed very rapidly (C. KOST, per- spiders (RUHREN & HANDEL, 1999) or sonal observation), the abundance of ants, parasitoids (PEMBERTON & LEE, 1996). wasps and flies was sustainably increased Besides this study, only one study is avail- during the time of application: Even at the able to date where the defensive roles of third day following nectar application, all several groups of plant defenders have three insect groups still showed increased been studied simultaneously: CUAUTLE & abundances (Fig. 2). RICO-GRAY (2003) showed that either As ants were most dominant, an ex- ants or wasps exerted beneficial effects on periment was performed to verify, whether the extrafloral nectary bearing plant this sustainable increase in ant numbers Turnera ulmifolia when selectively ex- on ‘nectar’ tendrils was due to their ability cluded; when both insect groups had ac- to respond with a modification of their for- cess, however, their effects were not addi- aging behaviour based on previous ex- tive. periences. The ‘nectar experiment’, in Extrafloral nectaries secrete small which artificial nectar was applied to both amounts of nectar throughout the day. ‘nectar’ and ‘control’ tendrils at the end of Nectar can be secreted in diurnal patterns the experiment, revealed a similar pattern that are characterised by short peaks of ant response for both types of tendrils (HEIL et al., 2000b; RAINE et al., 2002) or (Fig. 3). However, the intensity of the re- at relatively constant rates throughout day sponse was significantly increased for and night (BENTLEY, 1976, 1977b). The tendrils that had experienced the nectar exact mode of nectar secretion is not treatment before. Obviously, the ants re- known for Phaseolus lunatus. An applica- sponded positively by preferentially forag- tion of artificial nectar at intervals of 3 to ing on tendrils that usually were character- 4 d, however, does most likely not match ised by an increased availability of extra- the natural situation. Yet, the attracted floral nectar. insects may have responded to the in- This effect could be explained by increased sugar amounts itself rather than creased time periods the ants spend on being visually attracted to the relatively plants that supply EFN more regularly, large droplets of sugar solution, since al- with increased visitation frequencies of ready 10 to 15 minutes after nectar appli- foraging ants, or with a more efficient re- cation the solvent water was completely cruitment to these tendrils. Experimental evaporated leaving behind only the sug- findings seem to support the second alter- ars. native: The time foraging ants spend at a food source depends on flow rates experi-

Manuscript II 63

enced previously and decreases with in- comparison illustrates that the total creasing nectar flow rates (SCHILMAN & amount of nectar, which was available on ROCES, 2003). Increased flow rates lead ‘nectar’ tendrils, did not exceed the at the same time to an increase of the vol- physiological limits of nectar production in ume collected. The ecological function of Lima beans. Moreover, since also ‘control’ such a foraging behaviour may be a de- tendrils produced certain amounts of EFN, fence-by-exploitation strategy. Resident the protective effect of EFN secretion that ants avoid competition against other nec- became obvious at the end of the experi- tar consumers by systematic and frequent ment was systematically underestimated visitation, thus leading to an exploitation of and thus provides a conservative measure the food source (DREISIG, 2000). Such of this defensive trait. an increased visitation frequency advan- In the present study Lima bean plants ces the probability of ant attendance, benefited from EFN secretion in nature. thereby facilitating the protective effect. Since not only ants but also flying insects This strategy seems to be characteristic were attracted to the artificial nectar, these not only for the species tested in the stud- may also have contributed to the observed ies mentioned before, but for all generalis- protective effect. Further studies are re- tic ants (F. ROCES, personal communica- quired to elucidate which of the observed tion). flying insects are beneficial to the Lima The ‘control’ tendrils were character- bean and which ones even cause eco- ized by a certain level of ‘background’ logical costs by consuming EFN without EFN secretion. The secretion rate of 18 exerting a beneficial effect. Lima bean plants that were untreated and adjacent to the experimental tendrils at site 1 ranged about ca. 0.36 mg soluble Acknowledgements solids g-1 24 h-1 (C. KOST, unpublished The authors thank JANINE RATTKE for data). Adding the constitutive secretion of analysing nectar probes and MANFRED EFN to the amount of applied artificial nec- VERHAAGH for support in ant determina- tar results in c. 1.06 mg soluble solids g-1 tion. We are grateful to WILHELM 24 h-1 of nectar being available on ‘nectar’ BOLAND for the possibility to use all the tendrils. Earlier measurements (2002) at facilities of the MPI and to PAULO S. the same site revealed a secretion rate of OLIVEIRA, JANINE RATTKE, VICTOR c. 0.02 mg soluble solids g-1 24 h-1 for un- RICO-GRAY and two anonymous referees treated and ca. 1.5 mg soluble solids g-1 for helpful comments on previous versions 24 h-1 for Lima bean plants induced with of the manuscript. The authors especially jasmonic acid (JA) (HEIL, 2004a). This appreciate valuable logistic support in the

64 Protection of Lima beans by extrafloral nectar

field received by PERLA MONICA MAR- man Research Foundation (DFG grant TINEZ CRUZ (Universidad del Mar Puerto He3169/2-1, 2) and the Max-Planck- Escondido). Financial support by the Ger- Society is gratefully acknowledged.

Manuscript III 65

Manuscript III

Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants

C. Kost1* and M. Heil1,2

Journal of Ecology, accepted

Accepted: 6 December 2005

1 Department of Bioorganic Chemistry Max Planck Institute for Chemical Ecology Hans-Knöll-Str. 8 Beutenberg Campus D-07745 Jena, Germany

2 Present address: Department of General Botany University Duisburg-Essen FB BioGeo - Botanik Universitätsstr. 5 D-45117 Essen, Germany

* Corresponding author: Christian Kost Department of Bioorganic Chemistry Max Planck Institute for Chemical Ecology Beutenberg Campus Hans-Knöll-Str. 8 D-07745 Jena, Germany Phone: + + 49 - 3641 - 57 18 20 Fax: + + 49 - 3641 - 57 12 02 E-mail: [email protected]

66 Airborne volatiles induce indirect plant defence

Summary 1 Many plant species respond to herbivory with an increased emission of volatile organic compounds (VOCs), which attract carnivorous arthropods and thereby function as an indirect defence. Whether neighbouring plants positioned downwind ‘eavesdrop’ on such airborne cues and tailor their defences accordingly, remains controversial. 2 We used native Lima bean plants (Phaseolus lunatus) to investigate whether herbivore-induced VOCs induce another indirect defence strategy, i.e. the secretion of extrafloral nectar (EFN) in conspecific plant neighbours, and whether this enhances the defence status of the receiving plant under natural conditions. 3 EFN secretion was induced by VOCs released from herbivore-damaged bean tendrils as well as by a synthetic VOC mixture resembling the natural one. One constituent of the herbivore-induced blend - namely the green leaf volatile (3Z)-hex-3-enyl acetate - was identified as sufficient to elicit the defence reaction. 4 A long-term experiment was conducted to compare the defensive effect of EFN alone with the VOC-mediated effect (EFN induction plus attraction of plant defenders). The results indicate that Lima bean benefits from both indirect defences. Repeated treatment of tendrils with either an artificial blend of VOCs or EFN correlated with a higher cumulative number of predacious and parasitoid insects attracted (i.e. ants and wasps) as well as with less herbivore damage and increased productions of inflorescences and leaves. 5 Our results demonstrate that one indirect defence mechanism can induce another one in conspecific plants, and that Lima bean plants can benefit from this VOC-induced EFN secretion under natural conditions. Regarding the wide taxonomic distribution of both extrafloral nectaries and the capability of plants to release VOCs upon herbivory, airborne signalling may represent a common mechanism of plants to regulate the secretion rate of EFN in plant parts which face an increased risk of herbivory.

Keywords: Airborne signalling, ants, indirect defence, extrafloral nectar, herbivore-induced plant volatiles, herbivory, (3Z)-hex-3-enyl acetate, Lima bean, Mex- ico, plant-plant communication.

Manuscript III 67

Introduction Plants that can activate and tailor their A large number of plants respond to leaf defences accordingly to information damage with the induction of a variety of derived from their neighbour may gain a defences. Many of these defences are selective advantage over plants that are regulated via the octadecanoid pathway, unable to make use of this information. with jasmonic acid acting as a central sig- Whether plants positioned downwind are nalling molecule (CREELMAN & MULLET indeed capable of such con- or intra- 1997; WASTERNACK & PARTHIER 1997). specific ‘eavesdropping’ on airborne cues In addition to direct defences such as the has been debated intensively (FOWLER & production of leaf toxins, plants may also LAWTON 1985; SHONLE & BERGELSON respond to herbivory with the emission of 1995), yet evidence supporting such a volatile chemicals that serve as host- plant-plant communication hypothesis is location cues for carnivores (for review see accumulating (e.g. BRUIN et al. 1992; PICHERSKY & GERSHENZON 2002; KARBAN et al. 2000; TSCHARNTKE et al. ARIMURA et al. 2005). Plant-mediated 2001). attraction of carnivorous arthropods to An increasing number of laboratory actively feeding herbivores is generally studies on different plant species suggest believed to function as an indirect defence that plants can perceive volatile signals, as that enhances the fitness of the volatile- evidenced by transcriptional changes of emitting plant by increasing the predation defence-related genes (BATE & ROTH- pressure on the herbivores (TAKABAYA- STEIN 1998; ARIMURA et al. 2000b, 2001; SHI & DICKE 1996; PARÉ & TUMLINSON GOMI et al. 2003; ARIMURA et al. 2005; 1999). KISHIMOTO et al. 2005; PASCHOLD et al. Moreover, such plant volatiles provide 2005; PENG et al. 2005). Furthermore, chemical information about the status of both laboratory and field studies have attack of the emitting plant, which might be revealed that exposure to herbivore- used not only by higher trophic levels induced (HI) volatile organic compounds (TURLINGS et al. 1995; KESSLER & (VOCs) results in changes in the abun- BALDWIN 2001; VAN POECKE & DICKE dance of phytohormones (ARIMURA et al. 2004; ARIMURA et al. 2005), but also by 2002; ENGELBERTH et al. 2004), as well neighbouring plants of the same or another as in an increasing production of defence- species (BALDWIN & SCHULTZ 1983; related metabolites such as terpenoids ARIMURA et al. 2000a; KARBAN et al. (ENGELBERTH et al. 2004; RUTHER & 2000; FARMER 2001; KARBAN & MARON KLEIER 2005), proteinase inhibitors 2002). (FARMER & RYAN 1990; TSCHARNTKE et al. 2001) or phenolic compounds

68 Airborne volatiles induce indirect plant defence

(BALDWIN & SCHULTZ 1983; TSCHARN- sion of HI-VOCs induce the secretion of TKE et al. 2001). extrafloral nectar (EFN) in undamaged, Despite the number of laboratory stud- neighbouring tendrils? (2) Which com- ies that revealed such an aerial transfer of pounds within the complex blend of HI- information between plants, relatively few VOCs are responsible for the induction of studies report these effects under field EFN secretion? (3) Does the Lima bean conditions (e.g. FOWLER & LAWTON benefit from a volatile-induced secretion of 1985; PRESTON et al. 2001). Especially EFN under natural growing conditions, and evidence for the expression of defence- (4) Are putative plant defenders (ants and related plant metabolites after exposure to wasps) attracted to VOCs and EFN? VOCs under field conditions is largely lacking (but see KARBAN et al. 2000). Here we attempt to bridge this gap by Materials and methods identifying a new mechanism of plant-plant Study site and species communication between herbivore- Field work was done in 2003 and 2004 damaged and undamaged plants growing during the transition from wet to dry season under natural conditions. To investigate (October – December). All field experi- this phenomenon, we chose Lima bean ments were performed on a native popula- (Phaseolus lunatus L., Fabaceae), a model tion of Lima bean growing in the coastal plant commonly employed in studies of area near Puerto Escondido in the state of induced defences. Besides the emission of Oaxaca, Mexico. The plants investigated HI-VOCs, Lima bean additionally features were growing under natural field conditions extrafloral nectaries that function as an along dirt roads leading to extensively used indirect defence under natural conditions pastures or plantations at sites, where pre- (KOST & HEIL 2005a), and whose nectar vious experiments had been performed secretion rates are also inducible (HEIL (HEIL 2004a; KOST & HEIL 2005a). Due to 2004a). These traits make Lima bean an the tangled growth of Lima bean, it was not ideal system to investigate whether VOCs always possible to ensure that the bean can induce another indirect defence trait tendrils used in the experiment belonged to (i.e. secretion of extrafloral nectar) in un- one single plant individual. damaged plants and whether this has fit- All laboratory experiments were per- ness consequences for the receiver of the formed with potted plants which were signal. grown from seeds derived from plants of In field experiments performed at the our study sites. According to preliminary plants’ natural growing site we addressed analyses with AFLP markers (M. HEIL, the following questions: (1) Does the emis- unpublished data), these plants belong to

Manuscript III 69

the ‘Mesoamerican genotype’ (sensu bagged in a PET foil (‘Bratenschlauch’, GUTIÉRREZ SALGADO et al. 1995). Toppits, Minden, Germany) that does not Plants were grown in plastic pots with a emit detectable amounts of volatiles by diameter of 14 cm. Growing conditions itself. The emitted VOCs were collected were 23 °C, 60 % humidity, and continuously over 24 h on charcoal traps 270 µE m-2 s-1 during a 14 h photoperiod. (1.5 mg charcoal, CLSA-Filters, Le Ruis- Experiments in the laboratory were per- saeu de Montbrun, France) using air circu- formed with 6 week old plants (i.e. 7 - 8 lation as described previously (DONATH & leaf stage). BOLAND 1995). After 24 h, leaves were dried for dry mass determination and vola- Emission of HI-VOCs tiles were eluted from the carbon trap with Experiment 1. We enclosed pairs of natu- dichloromethane (40 µl) containing 1-bro- rally growing, basifixed tendrils with five modecane (200 ng µl-1) as an internal leaves in gauze bags (mesh size 0.5 mm) standard. Samples were then transferred to and supplied one of the two bags with her- glass capillaries, sealed by melting the bivores that had been previously observed open end and stored at < 5 °C for transport feeding on Lima bean. Herbivores involved to Germany. Samples were analyzed on a were mainly Cerotoma ruficornis and GC-Trace mass spectrometer (Thermo Gynandrobrotica guerreroensis (both Chry- Finnigan: www.thermofinnigan.com) ac- somelidae), Mexican bean beetle Epi- cording to KOCH et al. (1999). Individual lachna varivestis MULSANT (Coccinelli- compounds (peak areas) were quantified dae), a curculionid species as well as one with respect to the peak area of the internal species of both Ensifera and Caelifera. standard and related to the dry weight of Abundance and species composition of the the measured tendril. caged herbivores mirrored the frequency of Experiment 2. In a laboratory experi- occurrence during insect sampling and ment we verified whether the detachment thus was a representative cross-section of Lima bean tendrils alters the qualitative through the local herbivore community and quantitative blend composition of the typically attacking Lima bean plants. Each emitted HI-VOCs. Therefore, the five gauze bag was supplied with 6 Ensifera or youngest leaves of two potted Lima bean Caelifera as well as with 10 beetles of dif- plants were exposed to 5 adult E. varivestis ferent species. for 2 d at room temperature, and two con- After 48 h, the naturally induced ten- trol plants remained undamaged. After 24 drils were detached, immediately supplied h, the herbivory rate of the two herbivore- with a water reservoir and the herbivores damaged tendrils was estimated carefully removed. Then, the tendrils were according to KOST and HEIL (2005a) to

70 Airborne volatiles induce indirect plant defence

assure that the mean leaf area consumed EFN depended strongly on the dry weight was similar between either tendrils (paired of the secreting leaves of both control (lin- t-test: P > 0.05, n = 9). One damaged and ear correlation r = 0.811, n = 16, P < 0.001) one undamaged tendril were cut and sup- and JA-treated tendrils (linear correlation plied with a water reservoir and the two after reciprocal transformation r = 0.57, other plants remained potted. Volatiles n = 14, P < 0.05), the measured amounts were collected from the five youngest of EFN produced was related to the dry leaves of all four plants for the next 24 h as weight of the secreting leaves. described for Experiment 1. This experiment was replicated nine times. EFN-induction experiments The amount of secreted EFN strongly EFN collection: general procedure depends on parameters such as light in- The production rate of EFN was deter- tensity (C. KOST, personal observation; mined as amounts of secreted soluble sol- HELDER 1958; MICHAUD 1990; PACINI ids (i.e. sugars, amino acids; see e.g. HEIL et al. 2003) or leaf age (C. KOST, unpub- et al. 2000b), by quantifying the nectar vol- lished data; HEIL et al. 2000b). To reduce ume with micro capillaries and the nectar the variability caused by environmental concentration with a portable, temperature- influences (e.g. different light intensities), compensated refractometer (HEIL et al. we used a paired experimental design with 2000b, 2001). the maximum distance between two ten- In a preceding laboratory experiment drils of each pair being < 1 m. The leaf-age we verified whether the amount of secreted as factor influencing the EFN secretion rate EFN depends on the dry weight of the se- was taken into account by matching ten- creting leaf. Therefore, the accumulated drils pairs of similar leaf age. Due to this EFN of 30 potted Lima bean plants at the experimental approach, not the absolute 7 - 8 leaf stage was washed off with pure amount of EFN secreted, but the relative water. Fourteen of these plants were difference between treated and control sprayed with an aqueous solution of tendrils is the appropriate measure to 1 mmol jasmonic acid (JA) until the sur- assess any given effect. faces of all leaves were covered, while the Tendrils used for all EFN-induction remaining 16 control plants remained un- experiments were undamaged basifixed treated. After 24 h, the amount of newly tendrils with five leaves which were placed secreted EFN of the five youngest leaves in mesh bags (mesh size 0.5 mm). A ring was quantified as described above and the of sticky resin (Tangletrap®, Tanglefoot leaves were dried for dry weight (DW) de- Company, Grand Rapids, Michigan, USA) termination. Since the amount of secreted was applied at their base to protect the

Manuscript III 71

tendrils from flying and crawling nectar Experiment 4. A blend of synthetic consumers. VOCs was prepared and dissolved in lano- Experiment 3. Wild growing tendrils lin paste (Sigma-Aldrich: www.sigma- were exposed to herbivores naturally feed- aldrich.com) as a matrix, from which the ing on Lima bean as described above (Ex- volatiles could evaporate (KESSLER & periment 1). After 48 h, the naturally in- BALDWIN 2001). According to preceding duced tendrils (‘emitter tendrils’) were ab- measurements, this mixture was adjusted scised, immediately supplied with a water to mimic the herbivore-induced bouquet reservoir and the herbivores carefully re- both quantitatively and qualitatively as to moved. The detached emitter tendrils were emission within 24 h. The artificial blend then packed together with a basifixed bean consisted of 0.12 µg (R)-(-)-linalool, tendril (‘receiver tendril’) into a perforated 0.13 µg β-caryophyllene, 0.19 µg methyl PET-plastic bag (‘Braten-schlauch’, Top- salicylate, 0.26 µg (Z)-jasmone (all pur- pits, Minden, Germany). Receiver tendrils chased of Sigma-Aldrich), 0.02 µg (3Z)- packed together with detached, undam- hex-3-enyl acetate (Avocado Research aged emitter tendrils served as controls Chemicals Ltd., Leysham, Lancaster, UK), accordingly. The two tendrils (emitter and 0.85 µg (E,Z)-β-ocimene (mixture of receiver tendril) were arranged in a way (E)-isomer (70 %) and (Z)-isomer (30 %) that the emitter tendril entered the plastic kindly provided by ROGER SNOWDEN, bag from above and the receiver tendril Firmenich, Geneva, Switzerland), 0.63 µg from below. Both ends were tied up with a (3E)-4,8-dimethylnona-1,3,7-triene DMNT) cord with the water reservoir projecting out and 0.9 µg (3E,7E)-4,8,12-trimethyl- of the lower end of the assembly. EFN pro- trideca-1,3,7,11-tetraene (TMTT) (synthe- duction rates of receiver tendrils were sized by standard methods (PATTENDEN quantified after 24 h. The plastic bags used & WEEDON 1968)) per µl lanolin. Purity of for this experiment were perforated with a all compounds was > 98 %. conventional office hole-punch (hole area To verify whether the VOCs released 0.78 cm2) in regular intervals, resulting in a from the lanolin paste mimicked the herbi- perforation ratio of 11 %. This ratio was a vore-induced bouquet also under field con- result of previous trials, where the number ditions, seven pairs of detached Lima bean of punched holes was adjusted in a way leaves were supplied with green plastic that even after direct exposure to sunlight, strips on which 40 µl of either the artificial the packed tendril did neither show any VOC mixture or lanolin only were spotted. wilting symptoms nor any wetting at the The headspace of these leaves was sam- inside of the foil within 24 h. pled for 24 h as described for Experi-

72 Airborne volatiles induce indirect plant defence

ment 1. Use of the green plastic strips EFN alone to the effect mediated by air- should prevent any diffusion of compounds borne volatiles (i.e. EFN induction and into the plant. attraction of plant defenders). For this pur- Experiment 5. Lanolin paste containing pose, we used a paste containing volatiles the artificial volatile blend (40 µl) was ap- which was similar to the one used in plied to each of five leaves of a bean tendril Experiment 4 and prepared an artificial and compared to a neighbouring control EFN. Both mixtures were adjusted to mimic tendril to which lanolin paste only was the natural production rates within three applied. Both control paste and paste con- days both quantitatively and qualitatively as taining volatiles were spotted on green determined in previous experiments plastic strips attached to tendrils. The pro- (C. KOST, unpublished data; KOST & HEIL duction rate of EFN was quantified after 2005a). The artificial EFN consisted of an 24 h. This experiment has been conducted aqueous solution of 4.01 g sucrose l-1, in 2003 (n = 10) as well as in 2004 24.24 g l-1 of each fructose and glucose, (n = 12). which are the main constituents of Lima Experiment 6. Pairwise comparisons bean EFN (KOST & HEIL 2005a). between tendrils which have been treated Seventeen groups of four neighbouring with lanolin alone and untreated tendrils tendrils each growing < 3 m apart from allowed to test for an effect of lanolin on each other were selected as experimental the EFN production rate. Sample size for units. All tendrils selected were trained this experiment was twelve pairs of ten- along supporting ropes, and the tendrils of drils. each group were randomly assigned to one Experiment 7. With the same experi- of four treatments. Tendrils were either left mental design as has been used in untreated (control group) or treated every Experiment 5, we performed single-com- 3 d with lanolin paste only (treatment con- pound comparisons with all eight constitu- trol group), artificial volatile blend (volatile ents of the complete blend. For this pur- group) or artificial nectar mixture (nectar pose we mixed blends that contained 1 µg group). The lanolin paste was applied as of every compound per µl lanolin and 40 µl described above, whereas the EFN mimic of this mixture were applied per leaf as (40 µl per trifoliate leaf) was applied di- described above. Sample sizes for this rectly to the extrafloral nectaries of every experiment are given in table 1. leaf of each tendril of the nectar group. To determine the effect of the four treatments Long-term experiment on the fitness of the treated tendrils, we Experiment 8. The aim of this experiment quantified the fitness-relevant plant pa- was to compare the defensive effect of rameters, herbivory rate (as percent leaf

Manuscript III 73

loss) and the numbers of newly developed tests for matched pairs. Despite deploying leaves and inflorescences. The number of multiple comparisons to one data set, we leaves and inflorescences were quantified did not use a multiple-comparison correc- by counting and the herbivory rate as tion. By adjusting the type I error (i.e. error described in KOST and HEIL (2005a). All of incorrectly declaring a difference) down- three parameters were quantified at the wards, such a procedure would have beginning of the experiment and after 25 d. increased the chance of making a type II The differences between these two values error (i.e. no difference is declared, while in were calculated to determine the develop- fact there is a difference). Experiments 1, 2 ment of the respective parameter. and 4 aimed at comparing our treatments Experiment 9. The influence of our to the natural situation. Thus, accepting treatments on the presence of putative type I rather than type II errors was the plant defenders was investigated by more conservative approach. assessing the number of ants and wasps Data of the long-term experiment (Ex- that visited the study tendrils. Fourteen periments 8 and 9) were analysed by censuses were performed within 3 d at two applying a mixed-effect model with ‘treat- sites (at site 1 on day 7 and at site 2 on ment’ as a fixed and ‘tendril group’ as a day 18 after the beginning of the experi- random factor. The number of leaves were ment) and insect numbers were pooled for ln-transformed and the number of inflores- each tendril. cences log-transformed to meet the assumption of homogeneity of variances. Statistical analyses Post-hoc comparisons (LSD) were per- The high variability of the EFN secretion formed to test for statistically significant rates measured in Experiments 3, 5, 6 and differences between treatments. Data were 7, which is caused by environmental fac- analysed using SPSS 13.0 (SPSS for Win- tors (see above), allowed comparisons dows, SPSS Inc., Chicago, USA). between two groups of paired tendrils, yet no multiple statistical testing between different tendril pairs. These experiments Results were therefore considered independent Emission of HI-VOCs and analysed by using paired t-tests with- In response to herbivore attack, Lima bean out a multiple-comparison correction (such plants released a blend of VOCs com- as e.g. Bonferroni). Differences in the prised of eight main constituents (Experi- emission rate of VOCs (Experiments 1, 2 ments 1 and 2; Figs. 1 and 2). Detachment and 4) were estimated using exact Mann- of bean tendrils neither changed the total Whitney U tests and Wilcoxon signed rank amount of volatiles emitted (Wilcoxon

74 Airborne volatiles induce indirect plant defence

(a) IS 5 C H 3 IS 4 9 7 1 7 1 8 10 3 4 10 2 6

45 TC IS V IS 8 9 2 10 2 7 3 Relative abundance 2 1 1 7 9 10 6 3 6

4 6 8 10 12 14 4 6 8 10 12 14

3 (b) C (n = 7) 2 TC (n = 7) 1 )

-1 0 g

-1 7 H (n = 6) 6 V (n = 6) 24 h 24 -1

IS 5

A 4 VOC emission emission VOC VOC

(A 3 2 1 0 * 12345678910

Compound

Figure 1. Volatile blends resulting from different treatments (Experiments 1 and 4). (a) Representative gas chromatographic profiles of an undamaged bean tendril (C), an herbivore-induced bean tendril (H), a single leaf treated with lanolin paste (TC) or a single leaf treated with artificial volatile blend dissolved in lanolin paste (V). (b) Pairwise comparisons between mean (+ SEM) VOC emission of undamaged tendrils (C,) and tendrils treated with lanolin paste (TC) as well as between herbivore-induced bean tendrils (H) and tendrils treated with the artificial volatile blend dissolved in lanolin paste (V). The amount of emitted VOCs is given as peak area relative to the peak area of an internal standard per 24 h and per g dry weight. Asterisks denote significant differences between C and CT as well as between H and V for every analysed VOC (exact Mann-Whitney U test, *, P < 0.05). Identified compounds are: 1, (3Z)-hex-3-enyl acetate ; 2, (E,Z)-β-ocimene; 3, (R)-(-)-linalool; 4, DMNT;

5, C10H14; 6, methyl salicylate; 7, C10H16O; 8, (Z)-jasmone; 9, β-caryophyllene; 10, TMTT; IS, internal standard (1-bromodecane).

Manuscript III 75

6 Control basifixed 4 abscised

2 *

) ** -1

g 0 -1 10 Mexican bean beetle 24 h

-1 8 IS A VOC emission emission VOC

VOC 6 (A 4

0 12345678910

Compound

Figure 2. Comparison of the mean (+ SEM) VOC emission between basifixed and abscised tendrils from undamaged plants (Control) and plants damaged by five adult individuals of Epilachna varivestis (Mexican bean beetle) (Experiment 2). The amount of emitted VOCs is given as peak area relative to the peak area of an internal standard per 24 h and per g dry weight. The ten most dominant compounds are shown: 1, (3Z)-hex-3-enyl acetate; 2, (E,Z)-β-ocimene; 3, (R)-(-)-linalool; 4, DMNT; 5, C10H14; 6, methyl salicylate; 7, C10H16O; 8, (Z)- jasmone; 9, β-caryophyllene; 10, TMTT. Asterisks denote significant differences between basifixed and abscised tendrils for every analysed VOC (Wilcoxon signed rank test, *, P < 0.05; **, P < 0.01). Sample size was nine plants per treatment. signed rank test: P > 0.05, n = 9), nor the cant decrease in the emission rate of emission rate of the eight main constituents β-caryophyllene and the homoterpene of the blend in comparison to potted plants TMTT as compared to potted plants (Fig. 2, (Experiment 2; Fig. 2, Wilcoxon signed Wilcoxon signed rank tests: P < 0.01 and rank test for all eight compounds: P > 0.05, P < 0.05, n = 9). n = 9). This held true for both undamaged control plants and herbivore-damaged EFN-induction experiments plants. Solely in undamaged plants, de- To test whether HI-VOCs could induce the tachment of bean tendrils caused a signifi- secretion of EFN in neighbouring plants,

76 Airborne volatiles induce indirect plant defence

we exposed basifixed receiver tendrils of ficial volatile blend which we dissolved in wild growing plants to VOCs emitted from lanolin paste. Volatile collection under field detached, herbivore-damaged emitter ten- conditions confirmed that the artificial blend drils. Quantification of EFN after 24 h indi- indeed mimicked the blend emitted from cated that receiver tendrils exposed to naturally induced tendrils both qualitatively naturally induced VOCs showed a two-fold and - to a large degree - quantitatively (Ex- increase in EFN secretion rate as com- periment 4; Fig. 1). However, the artificial pared to controls (Experiment 3; Fig. 3, VOC-mixture released significantly lower paired t-test: P < 0.001, n = 9). amounts of (3Z)-hex-3-enyl acetate than If airborne VOCs were responsible for herbivore-damaged tendrils (Fig. 1, exact the observed effect, then also synthetic Mann-Whitney U test: P < 0.05). compounds should be capable of inducing Application of this artificial volatile EFN secretion in undamaged plants. We blend to bean tendrils, and as a conse- tested this hypothesis by preparing an arti- quence thereof, an increased amount of

7 *** ) -1 6 * 24 h *

-1 5

4 ** 3

1 EFN secretion (mg g (mg secretion EFN 0 TC V TC V TC HAC C TC C‘ H (n = 10) (n = 12) (n = 10) (n = 9) (n = 12) 2003 2004

Figure 3. Extrafloral nectar (EFN) secretion in response to different treatments. Mean (+ SEM) EFN secretion rates are given in mg soluble solids per g leaf dry mass per 24 h. Pairwise comparisons between tendrils treated with lanolin paste (TC) and the artificial volatile blend (V) (Experiment 5) or (3Z)-hex-3-enyl acetate (HAC) dissolved in lanolin (Experiment 7) are depicted. Furthermore, the EFN secretion rate of untreated tendrils (C) and tendrils treated with lanolin paste (TC) (Experiment 6) as well as between tendrils exposed to detached, undamaged tendrils (C’) and tendrils exposed to detached, herbivore-damaged tendrils (H) (Experiment 3) are displayed. Asterisks denote significant differences between pairs of tendrils (paired t-test, *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Manuscript III 77

Table 1. Extrafloral nectar (EFN) secretion from natural growing Lima bean tendrils after treatment with single

constituents of the herbivore-induced volatile blend.

-1 -1 EFN secretion rate (mg g 24 h )

Compound (No.) n Control Compound P

(3Z)-Hex-3-enyl acetate (1) 10 2.6 (± 0.6) 4.6 (± 0.9) 0.040

(E,Z)-β-Ocimene (2) 7 2.2 (± 0.8 2.5 (± 0.6 0.542

(R)-(-)-Linalool (3) 10 3.5 (± 0.9) 4.9 (± 0.9) 0.300

DMNT (4) 8 2.8 (± 0.9) 4.3 (± 0.8) 0.246

Methyl salicylate (6) 7 4.4 (± 0.7) 3.5 (± 0.4) 0.104

(Z)-Jasmone (8) 8 4.8 (± 0.7) 4.5 (± 0.7) 0.746

β-Caryophyllene (9) 6 3.9 (± 0.6) 3.1 (± 0.8) 0.296

TMTT (10) 8 4.7 (± 1.0) 6.3 (± 1.5) 0.405

Pairwise comparisons between wild growing, neighboring tendrils of five leaves, each treated with either 40 µl of lanolin paste (Control) or lanolin paste containing 1 µg µl-1 of a single compound (Compound), are displayed. Number of compounds accord to Figures 2 and 3. Mean (± SEM) EFN secretion rates in mg soluble solids per g leaf dry mass per 24 h and P values of paired t-tests between both treatment groups are given.

VOCs in the headspace of Lima bean ten- EFN secretion? To answer this question, drils, significantly increased the EFN secre- the EFN secretion rate of control tendrils tion rate of treated tendrils as compared to (lanolin only) were pairwise compared with controls that had been treated with lanolin tendrils that had been treated with a lanolin only (Experiment 5; Fig. 3, paired t-test: mixture containing only one of the eight P < 0.01, n = 10). Originally performed in main constituents of the complete herbi- November 2003, a replication of this vore-induced blend (Experiment 7; Table experiment in October 2004 validated the 1). Among these compounds, only (3Z)- initial result (Fig. 3, paired t-test: P < 0.05, hex-3-enyl acetate significantly increased n = 12). A comparison of the EFN produc- EFN secretion rates (Fig. 3, Table 1). Other tion rate between untreated tendrils and constituents tested, for example (R)-(-)- tendrils to which lanolin only had been linalool, DMNT and TMTT, showed a trend applied revealed no statistically significant towards an inductive effect, yet caused no difference between either groups (Experi- statistically significant increase in EFN ment 6; Fig. 3, paired t-test: P > 0.05, secretion rate (Table 1). The strength of n = 12). the EFN-induction effect of (3Z)-hex-3-enyl Which compounds of the applied vola- acetate resembled the one of the complete tile blend elicited the observed increase in mixture of synthetic VOCs (Fig. 3).

78 Airborne volatiles induce indirect plant defence

Long-term experiment 80 Number of leaves This experiment was conducted to verify 70 60 whether VOC-induced EFN secretion in- 50 deed enhances the defence status of wild 40 growing Lima bean plants (Experiments 8 30 and 9). Therefore we compared the defen- 20 10 sive effect of EFN alone to the volatile- a a b b 0 mediated effect, i.e. attraction of plant 30 defenders to VOCs and to VOC-induced Number of 25 inflorescences EFN. After 25 d of repeatedly applying a 20 synthetic mixture of EFN and VOCs (i.e. 15 every 3 d), fitness-relevant plant parameters such as the number of inflorescences 10 5 (Fig. 4, univariate ANOVA: P < 0.01, a a b b n = 17) and leaves (Fig. 4, univariate 0 Mean absolute difference ANOVA: P < 0.001, n = 17) were signifi- 15 Herbivory rate (%) cantly increased in the two treatment 10 groups (nectar and volatiles) as compared 5 a a to both control groups (control and treat- 0 b b ment control) (Experiment 8). Moreover, -5 tendrils treated with the artificial blends of -10 VOCs and EFN had received significantly -15 less damage by leaf-chewing herbivores C TC V N (Fig. 4, univariate ANOVA: P < 0.01, n = 17) than did control tendrils. Remark- Treatment ably, the intensity of the defensive effects Figure 4. Effects of volatile and nectar treatment on observed in the nectar and the volatile fitness-relevant plant parameters (Experiment 8). group were quantitatively indistinguishable Differences between measurements at t = 0 d and t (Fig. 4). = 25 d are displayed (mean + SEM). Groups of The numbers of ants and wasps that tendrils were left untreated (control, C), or were treated at regular intervals (3 d) with lanolin paste visited the studied tendrils at the end of the (treatment control, TC), artificial volatile blend dis- experiment were significantly increased on solved in lanolin paste (volatiles, V) or an artificial ants and wasps: P < 0.001, n = 17) as extrafloral nectar (nectar, N). Different letters indicate significant differences among treatments (uni- compared to controls (Experiment 9; Fig. variate ANOVA, P < 0.05 according to LSD post- 5). Again, no statistically significant differ- hoc test). Sample size was seventeen groups of ence could be detected between nectar tendrils.

Manuscript III 79

and volatile group. However, wasps were Discussion generally 25-times less frequently ob- Our results demonstrate that HI-VOCs served than ants (Fig. 5). were recognized by undamaged, neighbouring Lima bean plants as a signal for an impending herbivore attack, which 120 Ants subsequently prompted them to activate 100 their own defence, in our case the secre- 80 tion of EFN. Both naturally emitted VOCs 60 (Experiment 3) and a mixture of synthetic 40 VOCs (Experiment 5) were capable of inducing EFN secretion in receiver tendrils. 20 a a b b (3Z)-hex-3-enyl acetate was identified as a 0 biologically active substance (Experiment 5 Wasps 7) and the application of the mixture of synthetic VOCs benefited Lima bean under 4 natural growing conditions (Experiments 8 3 Mean cumulative number and 9). 2 Contrasting the VOC profiles emitted from Lima bean plants, which had been 1 attacked by either a mixture of natural a a b b 0 folivores (Experiment 1; Fig. 1) or Mexican CTCV N bean beetles E. varivestis (Experiment 2; Fig. 2) revealed quantitative rather than Treatment qualitative differences between the induced Figure 5. Effects of volatile and nectar treatment on blends. The VOCs released from P. luna- insect numbers (Experiment 9). Insects, residing on tus upon herbivore damage largely bean tendrils that were left untreated (control, C), or matched those of previous reports (DICKE were treated at regular intervals (3 d) with lanolin paste (treatment control, TC), artificial volatile blend et al. 1999; ARIMURA et al. 2000a; dissolved in lanolin paste (volatiles, V) or an artificial OZAWA et al. 2000; HEIL 2004a; MIT- mixture of extrafloral nectar (nectar, N), were HÖFER et al. 2005). However, it appears counted. Fourteen censuses were performed within that the particular type of damage inflicted 3 d at two sites (site 1 on day 7 and site 2 on day 18 after the beginning of the experiment). Cumulated (chewing vs. cell-content feeding) has a insect numbers (mean + SEM) are presented. Dif- major influence on the qualitative composi- ferent letters indicate significant differences among tion of the emitted VOC blend. Chewing treatments (univariate ANOVA, P < 0.05 according to LSD post-hoc test). Sample size was seventeen herbivores, no matter if caterpillar, beetle groups of tendrils. or even snail provoke a similar blend of

80 Airborne volatiles induce indirect plant defence

VOCs (this study; MITHÖFER et al. 2005), To reduce disturbing wind current and whereas cell-content feeders such as mites additional signalling from other VOC frequently induce compounds which are sources, we enclosed the receiver and the absent in the VOC profile induced by chew- naturally induced emitter tendrils in bags of ing herbivores (DICKE et al. 1999; ARI- PET foil (Experiment 3). Previous studies MURA et al. 2000a). As a first step in (e.g. BATE & ROTHSTEIN 1998; ARI- investigating whether VOCs can induce MURA et al. 2000a; TSCHARNTKE et al. EFN secretion, we focused our analysis on 2001) were often criticised for using airtight the eight most dominant compounds which chambers which may alter the physiologi- are induced independently of the feeding cal status of the enclosed plants by a short- mode of the attacking herbivore. age of CO2 (BALDWIN et al. 2002; DICKE Although it is known that ablation of et al. 2003a). Therefore, we perforated the leaves or branches may alter the amount PET foil (perforation ratio 11 %) with rela- and blend composition of VOCs emitted tively large holes of 0.78 cm2. The en- from herbivore-infested plants (ARIMURA closed tendrils did thus not show any et al. 2001; SCHMELZ et al. 2001), we symptoms of wilting or any wetting at the could experimentally demonstrate that the inside of the foil, even after exposure to HI-VOC profiles emitted from abscised direct sunlight. Our experimental conditions tendrils and basifixed plants were highly may have corresponded to the situation on similar (Experiment 2; Fig. 2). This finding a windless day. The observation that the allowed us to use detached tendrils as artificial VOC mixture, which closely mi- VOC-emitters for our EFN induction micked the naturally released blend (Ex- experiment (Experiment 3). periment 5), was capable of inducing EFN Studying airborne communication be- secretion also in tendrils which were not tween plants under field conditions is a enclosed at all, suggests a minor influence daunting task: Plants in their natural envi- of the enclosure. However, future investi- ronment are exposed to a multitude of gations in which Lima bean plants are ex- additional signals, and other factors such posed to herbivore-damaged neighbouring as e.g. disturbing wind current may hamper plants without any enclosure are necessary the detectability of a given effect. This to clarify, whether this phenomenon occurs study intended to verify whether airborne also under unconstrained environmental VOCs can induce EFN secretion in Lima conditions. bean plants at all. Our approach, thus, was The possibility of an information designed to increase the probability of exchange between emitter- and receiver- detecting a respective effect under field tendrils via a systemic signal within the conditions. same plant individual (DICKE & DIJKMAN

Manuscript III 81

2001; WÄCKERS et al. 2001) or via the damage by many plant species (ARIMURA rhizosphere between two different plant et al. 2000a; KALBERER et al. 2001; individuals (CHAMBERLAIN et al. 2001; KESSLER & BALDWIN 2001; RÖSE & PICKETT et al. 2003) seemed to be of TUMLINSON 2004). minor importance in our experiments. (3Z)-hex-3-enyl acetate has been previ- Since we used detached emitter tendrils to ously described to induce defence genes in expose basifixed receiver tendrils to the uninfested leaves of Lima bean (ARIMURA headspace of either undamaged or herbi- et al. 2001) and Arabidopsis (BATE & vore-damaged tendrils (Experiment 3), ROTHSTEIN 1998) and to prime corn systemic plant responses as well as plants against subsequent herbivore dam- rhizosphere signalling could be excluded age (ENGELBERTH et al. 2004). An due to the missing physical connection increased emission rate of (3Z)-hex-3-enyl between emitter- and receiver-tendrils. acetate directly after mechanical wounding However, in the EFN-induction experi- (i.e. within the first hour) of Lima bean ments with synthetic VOCs (Experiments 5 plants (ARIMURA et al. 2000a) may facili- and 7), within-plant- or rhizosphere- tate a fast induction of the EFN secretion in signalling may have occurred. In this case, neighbouring tendrils and thus allow to both mechanisms should have resulted in rapidly respond to a current threat. an induction of the control tendrils and thus Since (3Z)-hex-3-enyl acetate has to an underestimation of the observed been identified as biologically active sub- effect. This line of reasoning suggests that stance, the decreased amounts of this GLV the signal which elicited an increased EFN emitted from the mixture of synthetic VOCs secretion has been transmitted aerially, (Fig. 1) should have led to an under- rather than through the plant or the soil. estimation of the inductive effect and could The use of synthetic VOCs allowed explain the weaker effect of VOCs emitted dissecting the full blend of HI-VOCs into its from the synthetic VOC blend compared to active constituents and thereby identifica- herbivore-damaged bean tendrils (Fig. 3). tion of (3Z)-hex-3-enyl acetate as eliciting The fact that the synthetic VOC mixture the observed defence induction (Experi- was capable of inducing the EFN secretion ment 7; Table 1). (3Z)-hex-3-enyl acetate rate despite these decreased amounts of belongs to the group of so-called green leaf (3Z)-hex-3-enyl acetate (Fig. 1) indicates volatiles (GLVs), which are known to be that also the other constituents of the syn- emitted rapidly after leaf-damage thetic VOC mixture may have contributed (LOUGHRIN et al. 1994; TURLINGS et al. to EFN induction. Compounds that have 1995; ARIMURA et al. 2000a), but are also been identified in other studies as eliciting produced de novo in response to herbivore VOC-induced plant responses, but which

82 Airborne volatiles induce indirect plant defence

did not show an effect on the secretion rate an increased seed set and thus enhance of EFN in this study, involve (Z)-jasmone the Lima bean’s reproductive success. (BIRKETT et al. 2000; BRUCE et al. 2003), Since VOCs induced EFN secretion, (E,Z)-β-ocimene, DMNT, TMTT (ARIMURA the tendrils of the volatile group experi- et al. 2000a), and methyl salicylate (SHU- enced the combined defensive effect of LAEV et al. 1997). Our experimental both EFN and VOCs. Interestingly, the per- results neither exclude an inductive effect formance of all three fitness-relevant plant of a single, nor the combined action of parameters measured in the volatile and several of these other blend constituents or the nectar group developed similarly (Fig. compounds, which have not been detected 4), i.e. no statistically significant difference with the applied analytical methodology for could be detected. EFN obviously played a headspace sampling and VOC analysis. more important role as indirect defence in Few studies conducted under field our study system than did volatile chemi- conditions clearly demonstrated fitness cals. VOCs may thus have rather func- consequences for the receiver of the vola- tioned as EFN-inducing signals among tile signals (DOLCH & TSCHARNTKE bean tendrils located within a patch of in- 2000; KARBAN et al. 2000; KARBAN & creased herbivore pressure than as long- MARON 2002). To verify whether VOC- distant cues for flying plant defenders. induced EFN secretion enhances the However, our experimental approach does defence status of Lima bean plants and not exclude a protective effect exerted by thus plant fitness, we treated tendrils re- volatile-attracted defenders. This issue peatedly (every 3 d) with the artificial VOC should be addressed in future studies. mixture (Experiment 8). Already after 25 d, The growth architecture of the Lima these tendrils had lost less leaf area to bean at its natural growing site is charac- herbivores and had produced more leaves terized by (i) short distances among indi- and inflorescences than controls (Fig. 4). vidual bean tendrils and (ii) a tangled These parameters positively affect plant growth that creates microenvironments fitness. In a previous study conducted at with reduced wind current. Therefore, a the same sites, Lima bean plants that had systemic defence-inducing signal which is been repeatedly induced with the chemical transported within the plant would be very elicitor jasmonic acid suffered from less ineffective (JONES et al. 1993). In contrast, herbivore damage and showed a signifi- an airborne signal would create a gradient cant increase in the number of leaves, in- of the infochemical around the site of florescences, and fruits (HEIL 2004a). The attack, to which parts of the emitting plant increased number of inflorescences ob- or neighbouring plants can respond. More- served in this study did likely translate into over, the intricate growth structure may

Manuscript III 83

minimize dilution of airborne signals Acknowledgements (THISTLE et al. 2004) and thus favour We thank S. ADOLPH and M. MEKEM plant-plant communication. SONWA for help with organic synthesis. Regarding the widespread taxonomic We also thank I. T. BALDWIN, W. distribution of both extrafloral nectaries BOLAND, J. COLANGELO-LILLIS, R. (KOPTUR 1992) as well as the capability of DIETRICH, G. POHNERT, H. MAISCHAK, plants to respond to herbivore damage with A. MITHÖFER, B. SCHULZE, T. WICH- the emission of VOCs (VAN POECKE & ARD, W. W. WEISSER and four anony- DICKE 2004), such a plant-plant communi- mous referees for critically reading earlier cation response may represent a common versions of the manuscript. W. BOLAND is mechanisms which adds a new facet to our gratefully acknowledged for the possibility understanding of the complex interactions to use all the facilities of the MPI. This among different trophic levels. Thus, the study was financially supported by the elucidation of this mechanism opens up German Research Foundation (DFG grants new avenues for further studies that range HE 3169/2-1,2,3,4). from the underlying signalling cascades to the ecological relevance of this mechanism in our as well as in other study systems.

84 Two indirect defences benefit Lima bean in nature

Manuscript IV

The defensive value of two indirect defences of Lima bean in nature

Christian Kost1* and Martin Heil1,2

In preparation for Oecologia

1 Department of Bioorganic Chemistry Max Planck Institute for Chemical Ecology Beutenberg Campus, Hans-Knöll-Str. 8 D-07745 Jena, Germany

2 Present address: Department of General Botany University Duisburg-Essen FB BioGeo - Botanik Universitätsstr. 5 D-45117 Essen, Germany

Manuscript IV 85

Abstract The Lima bean (Phaseolus lunatus) features two indirect anti-herbivore defences - emission of volatile organic compounds (VOCs) and secretion of extrafloral nectar (EFN) - which are both inducible upon herbivore damage. In a previous study, Lima bean benefited of a simultaneous induction of both defences, yet it remained unclear whether both defences had contributed to plant protection. Our experimental approach aimed at separating the defensive function of both indirect defences. Tendrils sprayed with jasmonic acid (JA) to induce both indirect defences were compared to tendrils treated with an artificial blend of either EFN or VOCs as well as to control tendrils. Repeated application of EFN or VOCs benefited the treated tendrils as indicated by increased numbers of leaves, inflorescences and living shoot tips as well as a decreased herbivory rate and reduced numbers of dead shoot tips as compared to controls. Tendrils treated with JA showed a similar trend, yet some fitness parameters responded weaker to this treatment. This suggests that a putative JA-dependent direct defence was of minor importance, since otherwise JA-treated tendrils should have performed better than VOC- and EFN-treated tendrils. Comparing the amount of newly produced EFN between tendrils treated with JA and the artificial VOC mixture revealed that VOCs had induced EFN secretion in our experimental tendrils quantitatively similar to the JA treatment. Tendrils treated with synthetic EFN only, benefited equally or more of this treatment than did tendrils treated with VOCs or JA. Furthermore, field observation revealed that mainly ants and to a lower degree also wasps were significantly more attracted to tendrils treated with JA, VOCs and EFN than to control tendrils and were thus most likely responsible for the observed beneficial effect. Obviously, under our experimental conditions, EFN played a more important role as an indirect defence for Lima bean than a VOC-mediated arthropod attraction.

Key words: Ants, indirect defence, extrafloral nectar, plant-plant communication, volatile organic compounds.

86 Two indirect defences benefit Lima bean in nature

Introduction herbivore (for review see ARIMURA et al., Plants respond to herbivore attack with a 2005), thus providing highly reliable sig- bewildering array of changes in plant nals to the members of the third trophic chemistry, morphology, and physiology level. that frequently result in an increased resis- In many cases, plants do not only rely tance of plants to further attack (KARBAN on one single defence strategy, but use a & BALDWIN, 1997). Induced resistance complex array of different defensive may be due to direct effects on the herbi- mechanisms. For example, several plant vore with the plant producing toxic species have been identified that feature metabolites or anti-digestive and anti- both VOC emission and EFN secretion nutritive compounds. Furthermore, plants (ARIMURA et al., 2005). The presence of may utilize indirect defences that facilitate two indirect defences within one plant ”top-down” control of herbivore popula- individual gives rise to the questions tions by the herbivore's predators and whether or not both defences contribute to parasitoids. plant fitness and how they interact in One way for a plant to attract benefi- attracting beneficial arthropods (PRICE et cial arthropods is by providing suitable al., 1980). Studies on the benefit of in- food sources such as extrafloral nectar direct defence traits in nature are gener- (EFN; for review see KOPTUR, 1992). ally rare (HEIL, 2004a; KESSLER & EFN is an aqueous solution with sugars BALDWIN, 2001; THALER, 1999) and no and amino acids being the most abundant study to date has tried to disentangle the solutes (GALETTO & BERNARDELLO, defensive value of two defences within 1992; HEIL et al., 2000b; RUFFNER & one plant species. CLARK, 1986), which is secreted from The Lima bean (Phaseolus lunatus) is specialized organs, the so-called extra- a common model system in studies on floral nectaries. Additionally, plants may induced indirect defences. Recently, Heil increase the emission rate of volatile (2004a) could demonstrate that wild grow- organic compounds (VOCs) in response to ing Lima bean plants which have been herbivore attack that serve as cue guiding induced by exogenous application of the foraging parasitoids and predators to the phytohormone jasmonic acid (JA) feeding herbivore (PARÉ & TUMLINSON, responded with an increase of both the 1997; TURLINGS et al., 1990). The com- emission of VOCs and secretion of EFN. position of the herbivore-induced volatile Repeated application of JA (i.e. every 3 d) blend depends not only on the plant spe- resulted in a benefit for the treated plants cies or cultivar, but also varies with the as reflected e.g. by a decreased herbivory species and even the larval stage of the rate and an increased seed set. Further-

Manuscript IV 87

more, KOST and HEIL (2005a) demon- ing point to unravel whether both induced strated that artificially increasing the defence traits contribute to plant defence amount of available EFN benefits Lima or whether solely EFN secretion is bean in nature by attracting predacious responsible for Lima bean protection. and parasitoid arthropods. Thus, these Therefore we chose an experimental two pilot studies represent an ideal start- approach similar to the one used in HEIL (2004a): Groups of five tendrils were used as experimental units (Fig. 1, Table 1). C TC JA N V Within each group, two tendrils were either treated with JA or left untreated. The inclusion of two further bean tendrils in which the amount of either EFN or VOCs was artificially increased, enabled us to experimentally separate the protective effect of the two indirect defences. A fifth group served as a treatment control. Repeated application of these five Figure 1. Experimental design. Five groups of Lima bean tendrils (Phaseolus lunatus) served as ex- treatments allowed to answer the following perimental unit with C, no treatment; TC, application questions: (i) What is the relative contribu- of lanoline paste; JA, spraying with jasmonic acid; tion of the two indirect defences - extraflo- V, application of an artificial volatile blend dissolved in lanoline paste, and N, application of a artificial ral nectar secretion and volatile emission - mixture of EFN. See Table 1 for details. to the overall herbivore defence of the

Table 1. The five treatments and their effect on the treated tendrils.

Abbreviation Treatment Effect

C (Control) no treatment untreated control

TC (Treatment control) application of lanolin paste effect of lanolin paste

JA (JA-treatment) spraying with jasmonic acid induction of VOCs, EFN and putative JA-dependent direct defences

V (Volatile treatment) induction of EFN, attraction of application of a volatile mix- arthropods to VOCs and EFN ture dissolved in lanolin paste

N (Nectar treatment) application of an artificial mix- attraction of arthropods to

ture of EFN EFN

88 Two indirect defences benefit Lima bean in nature

Lima bean?, (ii) Is there an additional in- apart from each other were selected as fluence of a putative JA-dependent direct experimental unit (Fig.1). Eleven groups defence?, and (iii) What kinds of plant were located at site 1 and 12 at site 2. defenders are attracted to the treated ten- Due to the tangled growth of Lima bean it drils? was not always possible to ensure that all five tendrils of every group belonged to one single plant individual. All selected Material and Methods tendrils were trained along supporting Study site and species ropes, and the tendrils of each group were This study was conducted in the coastal randomly assigned to one of five treat- area near Puerto Escondido in the state of ments: Tendrils were either left untreated Oaxaca, Mexico. The climate in the study (control group) or treated every three to area is characterized by one main rainy four days with lanolin paste only (treat- season from June to October, which fol- ment control group), sprayed with a aque- lows a bimodal distribution peaking in July ous solution of JA (JA group), an artificial and September. Annual rainfall averages VOC blend (VOC group) or an artificial between 1 and 1.4 m and the mean an- mixture of EFN (EFN group) (Table 1). nual temperature is 28 °C (STRÄßNER, Starting 27th October 2003, the experiment 1999). Two sites were selected, which lasted 25 d resulting in a total of six appli- have already been used in previous stud- cation events. During this time, the initial ies (HEIL, 2004a; KOST & HEIL, 2005a). number of tendril groups was reduced to a These two sites were located 15 km final sample size of 17 due to cattle and northwest of Puerto Escondido and about human impact. 3 km apart from each other. Here, Lima bean grows naturally along dirt roads lead- Treatment of tendril groups ing to extensively used pastures or planta- The tendrils of the JA group were sprayed tions. All experiments were performed on with an aqueous solution of 1 mmol JA this native population of Lima bean plants. until the surfaces of all leaves were cov- Field work was done in 2003 and 2004 ered. The artificial volatile blend and the during the transition from wet to dry sea- synthetic mixture of EFN were adjusted to son (October - December). mimic its natural model within three days post-induction. Therefore, previous ex- Experimental design periments had been performed to deter- To reduce the variability that is caused by mine the qualitative and quantitative com- effects of site and genotype, 23 groups of position of the secreted EFN after induc- five Lima bean tendrils growing < 3 m tion with JA (KOST & HEIL, 2005a) and of

Manuscript IV 89

the volatile blend after herbivore feeding (KOST & HEIL, 2005b). Therefore, the aim (KOST & HEIL, 2005b). The applied artifi- of this experiment was to quantitatively cial EFN thus consisted of an aqueous compare the amount of EFN secreted solution of 4.01 g sucrose l-1, 24.24 g l-1 of from tendrils after exposure to airborne each fructose and glucose. 40 µl of this VOCs with the secretion rate of JA- blend were applied with an Eppendorf induced tendrils. Eleven groups of three pipette directly to the extrafloral nectaries tendrils spread across the two sites were of every trifoliate leaf of the EFN group. selected, which were located < 1 m apart. The artificial volatile blend consisted Each selected tendril was basifixed and of 0.12 µg (R)-(-)-linalool, 0.13 µg had 5 leaves. The first tendril within each β-caryophyllene, 0.19 µg methyl salicylate, group was treated with lanolin paste, the 0.26 µg (Z)-jasmone (all purchased of second with lanolin paste containing vola- Sigma-Aldrich), 0.02 µg (Z)-3-hex-3-enyl tiles and the third was sprayed with JA. All acetate (Avocado Research Chemicals treatments were similar to the ones Ltd., Leysham, Lancaster, UK), 0.85 µg described above. The three tendrils were (E,Z)-β-ocimene (mixture of E/Z-isomers then placed in gauze bags (mesh size ca. 70:30; kindly provided by Roger 0.5 mm) and a ring of sticky resin (Tangle- Snowden, Firmenich, Geneva, Switzer- trap®, Tanglefoot Company, Grand Rap- land), 0.63 µg (3E)-4,8-dimethyl-nona- ids, Michigan, USA) was applied at their 1,3,7-triene (DMNT) and 0.9 µg (3E,7E)- base as a protection against flying and 4,8,12-trimethyltrideca-1,3,7,11-tetraene crawling nectar consumers. After 24 h the (TMTT) (synthesized by standard methods amount of newly produced EFN was (PATTENDEN & WEEDON, 1968)) per µl measured as the amount of secreted lanolin. Purity of all compounds was soluble solids (i.e. sugars, amino acids; > 98 %. Pure lanolin and lanolin paste see HEIL et al., 2000b) by quantifying the containing volatiles were spotted on green nectar volume with micro capillaries and plastic strips attached to tendrils to pre- the nectar concentration with a portable, vent any diffusion of compounds into the temperature-compensated refractometer treated plants. 120 µl of both pastes were (HEIL et al., 2000b; HEIL et al., 2001). applied per five leaves of either volatile- or treatment control-group. Fitness-relevant plant parameters To assess the effect of our treatments on EFN-induction experiment the fitness of the study tendrils, the follow- Our previous work suggested that herbi- ing fitness-relevant plant parameters were vore-induced VOCs can induce the secre- considered: Number of leaves, inflores- tion of EFN in undamaged bean tendrils cences, living and dead shoot tips as well

90 Two indirect defences benefit Lima bean in nature

as herbivory rate. The herbivory rate was for an effect of the treatment on the num- estimated as percent leaf loss as de- ber of insects observed. scribed in KOST and HEIL (2005a), and To assess the functional groups of the remaining parameters were quantified insects attracted to the experimental ten- by counting. All five parameters were as- drils, two sticky traps were attached with sessed at the beginning of the experiment plastic strings to each tendril of 14 groups, and after 25 d. The differences between which were equally distributed between these two values were calculated to de- the two study sites. The sticky traps con- termine the development of the respective sisted of 100 cm2 pieces of green plastic parameter in the course of the study foil that had been coated with a thin layer period. of a trapping adhesive (Tangletrap®). After 24 h of exposure, the traps were re- Insect counts and sticky traps collected and the insects transferred to The insect community visiting the treated 75 % ethanol. Insects were identified to bean tendrils was assessed by counting order or family level using keys and infor- and with sticky traps. Two series of insect mation provided by ARNETT (2000), countings were performed in which twenty SCHAEFER et al. (1994) and NOYES groups of tendrils at both study sites were (2003). On the basis of the natural history repeatedly visited. The first series of information provided by HONOMICHL et countings started at day 7 and the second al. (1996), DALY et al. (1998), KELSEY on day 18 after the onset of the experi- (1969; 1981) and MATILE (1997), the col- ment. Ten groups of tendrils were selected lected arthropods were assigned to the at each study site and the number of ants, following guilds according to nutritional or wasps or flies present on the plants were functional aspects: Predator/entomo- recorded, as they represented the most phaga (R), parasitoid (P), utilization of abundant groups. The first census was plant-derived resources including floral or performed prior tendril treatment at 8:00 extrafloral nectar, pollen and honeydew am. Thereafter, all tendrils were treated (S), frugivore (F), herbivore and flower and insects on all pairs were counted feeder (H), detrivore including phyto- repeatedly every 2 or 3 h until midnight. saprophage and zoosaprophage (D), Two additional censuses were performed blood-sucking and ectoparasitic (B), and at 9:00 and 10:00 am on the following 2 fungivore (M). days, resulting in a total of 14 monitorings. The number of all insects counted per Statistical analysis experimental tendril was cumulated to test Our randomized complete block design allowed analysing the data of the EFN-

Manuscript IV 91

) 1 induction experiment, the fitness-relevant - 16 Box-plot: 14 Maximum plant parameters and the cumulative 1st Quartile (25 %) 24 h

1 b - 12 Median insect numbers with a mixed-effect model 3rd Quartile (25 %) 10 Minimum b (univariate GLM procedure) with ‘treat- 8 a ment’ as fixed and ‘tendril group’ as ran- 6 dom factor. The following variables have 4 2 been transformed to meet the assumption

EFN secretion (mg g EFN secretion 0 of homogeneity of variances (transforma- TC JA V tion in brackets): number of living shot tips Treatment and number of wasps (square root), number of inflorescences and dead shoot tips Figure 2. Effect of different treatments on the secretion rate of extrafloral nectar (EFN) given in mg (log), number of leaves, cumulative num- soluble solids per g leaf dry mass per 24 h. The ber of ants and flies (ln). Post-hoc com- comparisons between tendrils treated with lanolin parisons (LSD) were performed to test for paste (TC), the artificial volatile blend dissolved in statistically significant differences between lanolin (V), and jasmonic acid (JA) are displayed. Different letters indicate significant differences treatments. All statistical analysis was among treatments (univariate ANOVA, P < 0.05 done using SPSS 13.0 (SPSS for Win- according to LSD post-hoc test). Sample size was dows, SPSS Inc., Chicago, USA). eleven groups of tendrils. See insert for an explanation of box-whisker plots.

Results Effect of the treatments on fitness-relevant Nectar-induction experiment plant parameters Treatment of bean tendrils with either the Already after 25 d, our three treatments artificial volatile mixture dissolved in lano- JA, volatiles, and EFN had significant ef- lin or with JA had a significant effect on fects on the measured vegetative and re- the EFN secretion rate within 24 h as productive plant traits of the treated ten- compared to controls treated with lanolin drils: Tendril groups treated with volatiles only (univariate ANOVA: P < 0.01, n = 11). and EFN showed a significant increase in Both treatments doubled the EFN secre- the numbers of newly produced leaves, tion rate compared to the treatment control shoot tips and inflorescences, bore less (Fig. 2). However, no statistically signifi- dead shoot tips, and had suffered of less cant difference could be detected between herbivore damage than the two controls the volatile and the JA-treated group. (Fig. 3). JA-treatment also significantly Thus, in the long-term experiment, tendrils decreased the number of dead shoot tips of the JA and the volatile group had pro- and the herbivory rate suffered as com- duced comparable amounts of EFN. pared to the two control groups, yet no

92 Two indirect defences benefit Lima bean in nature

such difference could be detected for the abundant groups visiting the study ten- number of living shoot tips, leaves, and drils. Among them, ants were the most inflorescences. According to a LSD post- dominant group: Comparing the number of hoc test for these three parameters, the ants with those of wasps and flies ob- tendrils of the JA group took an intermedi- served among the different treatments ate position between EFN and volatile reveals that the number of ants counted group on one hand and the two controls on the experimental tendrils exceeded the on the other side (Fig. 3). number of flies 5 - 15-fold and the number of wasps even 20 - 30-fold (Fig. 4). Effect of the treatments on insect The five treatments significantly af- abundance fected the insect visitation rate of the ex- Ants, wasps and flies were the most perimental tendrils. The mean cumulative

30 6 Number of living shoot tips Number of dead shoot tips Figure 3. Effect of the five treatments ** *** on fitness-relevant plant parameters. b 20 ab 4 b a b Each tendril within groups of five a b Lima bean tendrils received one of 10 a ab 2 b five treatments: C, no treatment; TC, treatment with lanoline paste; JA, 0 0 spraying with jasmonic acid; V,

Mean absolute difference application of an artificial volatile -10 -2 C TC JA V N C TC JA V N blend dissolved in lanoline paste and 140 70 b Number of b Number of b N, application of an artificial mixture 120 60 leaves inflorescences of EFN. See Tab.1 for details. Mean 100 *** ab 50 * a absolute differences between day 0 80 a 40 b and day 25 of the experiment are 60 30 ab ab a displayed. All values above zero 40 20 (dashed line) indicate an increase, all 20 10 values below a decrease in 0 0 Mean absolute difference comparison to the starting situation. -20 -10 C TC JA V N C TC JA V N Asterisks indicate significant 60

Treatment treatment effects (univariate ANOVA, 50 Herbivory rate 40 *** *, P < 0.05; **, P < 0.01; ***, P < 30 a a 0.001) and different letters indicate 20 b significant differences among 10 b b 0 treatments (LSD post-hoc test, P < -10 0.05). Sample size was twenty -20 groups of tendrils. See insert in Fig. 2 -30 Mean absolute difference -40 for an explanation of box-whisker

C TC JA V N plots. Treatment

Manuscript IV 93

ant number was significantly increased on 2 (volatile group) and 2.5 (EFN group), tendrils treated with JA, volatiles and EFN while these insects were rarely encoun- as compared to the two control tendrils tered on control tendrils (Fig. 4). This dif- (Fig. 4, LSD post-hoc test after univariate ference between treatments was also sig- ANOVA, P < 0.05). Volatile treatment nificant (LSD post-hoc test after univariate doubled the median cumulative number of ANOVA, P < 0.05). Flies responded ants on experimental tendrils, while the JA weaker to the treatment of the experimen- and nectar treatment even lead to a three- tal tendrils. Despite a significantly in- fold increase compared to the untreated creased visitation rate of flies to JA-treated state. Correspondingly, median wasp tendrils as compared to controls (Fig. 4, number ranged between 1.5 (JA group), LSD post-hoc test after univariate ANOVA,

300 Ants 250 *** b

200 b 150 b

100 a

50 a Cumulative number Cumulative

0 C TC JA V N 15 Wasps *** b 12 b Figure 4. Effect of the five treatments on insect numbers 9 b visiting the Lima bean. Insects residing on Lima bean ten- 6 drils that received one of five treatments were counted. a Treatments were: C, no treatment; TC, treatment with lano- 3 a

Cumulative number Cumulative line paste; JA, spraying with jasmonic acid; V, application 0 of an artificial volatile blend dissolved in lanoline paste and C TC JA V N N, application of an artificial mixture of EFN. See Tab. 1 for 30 Flies details. Fourteen censuses were performed within 3 d at 25 *** two sites (site 1 on day 7 and site 2 on day 18 after the ab b 20 beginning of the experiment). Insect numbers were pooled ab per counted tendril. Asterisks indicate significant treatment 15 a effects (univariate ANOVA, *, P < 0.05; **, P < 0.01; ***, P a 10 < 0.001) and different letters indicate significant differences 5

Cumulative number Cumulative among treatments (LSD post-hoc test, P < 0.05). Sample

0 size was twenty tendril groups. See insert in Fig. 2 for an C TC JA V N explanation of box-whisker plots. Treatment

94 Two indirect defences benefit Lima bean in nature

P < 0.05), no such effect could be ob- Coleoptera (6 %), Araneida (5 %) and served for VOC- and EFN- treated tendrils Thysanoptera (3 %). Interestingly, 73 % of (Fig. 4). The two latter groups were statis- all trapped arthropods were characterized tically indistinguishable from the JA- by parasitoid or predacious life habits and treated tendrils and both control groups. may thus be classified as potentially beneficial to the Lima bean (Fig. 5). On the Community composition of arthropods other hand, only 16 % of the trapped ar- visiting Lima bean thropods were herbivores or flower feed- A total number of 899 arthropods were ers and therefore potentially detrimental to caught on the sticky traps and > 94 % the plant. Other plant-derived food could be identified to the order or family sources like EFN, pollen or honeydew are level. Among them, Diptera (55 %) and known to be used by 67 % of all trapped Hymenoptera (26 %) were the most abun- arthropods and 19 % of all trapped taxa dant insect groups captured (Table 1, Fig. rely on other food sources such as e.g. 5). Other groups trapped were mainly fungi, fruits or detritus. The two latter

100 potentially beneficial 500 80 potentially detrimental neutral 60 400 40

300 20 Guild affiliation (%) affiliation Guild 0 200 RPHSFDBM

100 Guild

Absolute numer trappedAbsolute 0

ra ra ra ra ra era te te tera tera e te e t p p p rina pt o op a o Dip leo rop h c eno Araneidaan e t Ac o m Co Homopt Or hys Het Ps Hy T

Taxon

Figure 5. Arthropods taxa trapped on the sticky traps. Insert: Affiliation of trapped taxa to different guilds: R, predator/entomophaga; P, parasitoid; S, utilization of plant-derived resources including floral or extrafloral nectar, pollen and honeydew; F, frugivore; H, herbivore and flower feeder; D, detrivore including phytosaprophage and zoosaprophage; B, blood-sucking and ectoparasitic; M, fungivore. Arthropod groups were assigned to guilds according to nutritional or functional aspects whenever larval or adult stage feature the respective trait. Addition- ally, putative effects on the Lima bean are encoded in different colours of bars. Multiple affiliations per taxon were allowed. Sample size was two sticky traps per 14 tendril groups.

Manuscript IV 95

Table 2. Arthropod taxa trapped on the experimental tendrils with sticky traps.

Number Guild

Order Taxon (ind.) R P S F H D B M

Diptera Dolichopodidae 150 l/a a

Phoridae 103 l a a

Chloropidae 31 l a a l

Tachinidae 24 l a a

Scatopsidae 22 a l/a

Sciaridae 12 l l/a l/a

Cecidiomyidae 12 l a a l l/a

Psychodidae 11 a l

Drosophilidae 10 l/a l/a

Culicidae 9 l a l l a

Canacidae 7 ?

Platystomatidae 5 a l l

Asilidae 4 l/a

Asteiidae 3 l/a

Micropezidae 3 a l/a

Tipulidae 3 a l

Agromyzidae 2 a l/a

Scenopinidae 2 l a l

Odiniidae 2 l

Others 22

Hymenoptera Chalcidoidea 136 l/a a a

Formicidae 25 l/a a

Braconidae 12 l/a a a

Sphecidae 7 l/a a a

Bethylidae 7 l/a a a

Pompilidae 6 l a a

Vespoidea 4 l/a a a a

Ichneumonidae 3 l/a a a

Dryinidae 3 l/a a a

Cephidae 2 a l/a

continued Æ

96 Two indirect defences benefit Lima bean in nature

Table 2. continued

Guild Number Order Taxon (ind.) R P S F H D B M Tiphiidae 2 l/a a a

Others 3

Coleoptera Lathridiidae 10 l/a

Coccinellidae 10 l/a l/a l/a

Chrysomelidae 9 l/a

Staphylinidae 4 l/a a a l l/a

Curculionidae 4 l/a

Cucujidae 3 l/a l/a l

Carabidae 2 l/a a l/a

Tenebrionidae 2 l/a a l/a l/a l/a l/a

Cleroidea 2 l/a

Others 101

Araneida 41 l/a

Thysanoptera 26 l/a l/a a

Heteroptera Tingidae 4 l/a

Hebridae 2 l/a

Others 2

Orthoptera Gryllidae 2 l/a l/a

Others 4

Acarina 3 l/a l/a l/a

Psocoptera 2 l/a l/a l/a

Numbers of wasps and flies refer to the total number of insects trapped. ‘Others’ comprise individuals that either could only be determined to the order level or taxa o f which just one in dividu al wa s trapped. The ob served arthropod groups were assigned to the following guilds according to nutrit ional o r func tional aspects: R, predator/entomophaga; P, parasitoid; S, utilization of plant-deri ved resou rces includ ing floral or extrafloral nectar, pollen and honeydew; F, frugivore; H, herbivore and flower feeder; D, detrivore including phytosaprophage and zoosaprophage; B, blood-sucking and ectoparasitic; an d M, fungivore. Th e affili ation to a guild is further differentiated by the developmental stage of each taxon (l = larval, a = ad ult) th at featu res th e respe ctive trait.

Manuscript IV 97

may be classified as ‘tourists’ of the Lima cially increased. Interestingly, it turned out bean and are likely to have a neutral effect in the course of the experiment that a on the plant (Table 2, Fig. 5). clear separation of the se two defences A closer look at the arthropod taxa was not possi ble: VOC s ind uced the trapped revealed that Dolichopodidae secretion of EFN (Fig. 2). Hence, the ten- (34 % of all trapped Di ptera), Phoridae drils of the vo latile grou p ha ve experi- (24 %) and Chloropidae (7 %) were the enced the combined defensive effect of most abundantly trapped Dipterans both VOCs and EFN to an extent, compa- (Table 2). All three groups share parasi- rable to the tendrils of the JA group. Obvi- toid and predacious life habits and are ously, these two defences are not only known to occasionally feed on EFN (Table connected by the shared signalling mole- 2). Members of the Chalcidoidea contrib- cule JA (HEIL, 2004a), but also airborne uted preponderance to the captured Hy- VOCs are implicated in the induction of menoptera (64 %). The individuals trapped EFN secretion within one o r b etween two of this superfamily of parasitoid wasps conspecific plant individuals . belonged to 16 different fam ilies with Eu- A quan titati ve co mpa rison of th e five lopidae (13 % of all tra pped Hymenop- experimental groups with regard to the tera), Encyrtidae (12 %) and Pteromalidae development of fitness-relevant plant (8 %) being most frequently trapped. parameters revealed that the tend rils of Among the hymen opterans, Formicidae the JA, the vola tile an d the EFN group had (12 %) and Braconidae (6 %) were the benefited of the respe ctive trea tmen t. most often captured non-chalcid families. These tendrils had suffere d of less herb i- vory by leaf-chewing herbivores and bore fewer dead shoot tips than the two c ontrol Discussion groups. The picture, however, changes Treatment effects on the plant when also the other measured fitness- The aim of the present study was to ex- relevant plant parameters are included. perimentally separate the protective effect For the number of living shoot tips, leaves, of the two indirect defences EFN secretion and inflorescences onl y th e tendrils and VOC emission in Lima bean under treated with VOCs and EFN differed sig- field conditions. The performance of nificantly from the controls (Fig. 3). A pos- plants, which have been induced by spray- sible explanation for the weaker defensive ing with JA and thus had increased effect experienced by the JA-treated ten- amounts of VOCs and EFN was moni- drils could be that induction with JA tored, and compared to plants where the incurred allocation costs to the treated amount of either EFN or VOCs was artifi- tendrils, which were greater then costs

98 Two indirect defences benefit Lima bean in nature

incurred by the VOC-induced EFN secre- not find evidence for an induced direct tion (HEIL & BALDWIN, 2002; STRAUSS resistance to spider mites. However, a et al., 2002). Beyond stress-related reac- protective effect of a direct defence, which tions such as insect and disease resis- in our long-term experiment could have tance, JA is well known to be also involved been induced after JA-application, cannot in various physiological or morphological be excluded. In this case, the direct changes which are not necessarily related defence did not significantly contribute to to resistance (CREELMAN & MULLET, plant protection, because tendrils with in- 1997). Given that these are costly in terms creased amounts of EFN only (EFN of metabolic resources, the induction of group) or EFN and volatiles combined such processes may have affected the (volatile group) performed better than the measured fitness parameters and thus tendrils with EFN, volatiles and the puta- could explain the weaker defensive status tive direct defence (JA group) (Fig. 3). of JA-treated tendrils. EFN and volatile treatment were ad- The development of the fitness- justed to resemble strongly induced Lima relevant plant parameters measured in the bean tendrils. The qualitative and quantita- JA-treated group and the two controls tive composition of the emitted VOC blend strongly resembled those of a preceding and the secreted EFN within 24 h, was study (HEIL, 2004a). In both studies, JA- determined in preceding experiments treatment led to an increase in the number (HEIL, 2004a; KOST & HEIL, 2005a, b). of newly produced leaves and a decrease Due to the sample size required to of both the number of dead shoot tips as achieve sufficient statistical power, the well as the herbivory rate. The induction of minimal interval between treatment rounds these two indirect defences has benefited was 3 to 4 days. Thus, the amount of both Lima bean plants in two independent stud- artificial VOC and EFN blend applied was ies performed in two consecutive years, scaled up to cover the mean amounts underlining the importance of EFN secre- naturally produced by a plant within this tion and VOC emission for Lima bean time period. Therefore, our mode of appli- defence in nature. cation did likely not meet the natural situa- JA-application could have additionally tion. Nevertheless, the amount of VOCs induced putative direct defences (HALIT- and EFN applied artificially ranged largely SCHKE & BALDWIN, 2004). However, no below the physiological expression limit of such alternative defence strategy has these two indirect defences (HEIL, 2004a; been described for Lima bean until now. In KOST & HEIL, 2005a, b). its close relative Phaseolus vulgaris, ENGLISH LOEB and KARBAN (1991) did

Manuscript IV 99

Treatment effects on the insect community creased presence of herbivores and Ants were by far the most dominant insect hence potential prey. However, prelimi- group observed on experimental tendrils nary experiments with Camponotus novo- and were significantly more attracted to granadensis, one of the three most domi- the tendrils of the JA, volatile and EFN nant ant species visiting Lima bean at the group (Fig. 4). Attraction of ants to EFN is two study sites (KOST & HEIL, 2005a), a well known defensive mechanism indicated that given the choice in a (BENTLEY, 1977a; BUCKLEY, 1982) that Y-olfactometer (HEIL, 2004b) between has been reported to translate into an en- lanolin paste and lanolin paste containing hanced plant protection in some studies volatiles, these ants did not preferentially (BENTLEY, 1977b; HEIL et al., 2001; choose the olfactometer arm with volatiles, LABEYRIE et al., 2001; OLIVEIRA et al., although they significantly chose an arm 1999), yet not in others (BECERRA & with mashed banana in previous trials VENABLE, 1989; BOECKLEN, 1984; (C. KOST, unpublished data). More ex- O'DOWD & CATCHPOLE, 1983; RASH- periments are needed to study the role of BROOK et al., 1992). Although chemical plant volatiles for ant foraging behaviour. cues are important for ants, since they are Also wasps were significantly more frequently involved in home range-marking attracted to the tendrils of the JA, volatile or mediating social interactions (KEELING and EFN group, yet in much smaller num- et al., 2004; VANDER MEER et al., 1998), bers than ants. The majority of the wasps relatively little is known on whether also trapped on the experimental tendrils were plant-derived volatiles can influence ant characterized by predacious or parasitoid behaviour. Some reports are available on life habits (Table 2), supporting a previous the role of yet unidentified plant chemicals study on sticky-trap captures of untreated for the orientation of ants to their host and EFN treated Lima bean tendrils plant (AGRAWAL & DUBIN-THALER, (KOST & HEIL, 2005a). These observa- 1999; DJIETO-LORDON & DEJEAN, tions suggest that not only ants, but also 1999a, b; FIALA & MASCHWITZ, 1990) or wasps have contributed to the protection the patrolling behaviour within a host plant of the treated tendrils. Unfortunately it re- (BROUAT et al., 2000). In Lima bean, mains unclear which indirect defence - herbivory induces both VOC emission and volatiles or EFN - was mainly responsible EFN secretion (HEIL, 2004a). Ants could for wasp attraction. Wasps feeding on use the herbivore-induced volatiles as EFN has been reported for several differ- long-distance cues to detect patches of ent plant species (see KOPTUR, 1992 for increased availability of EFN which at the review), yet so far only one study demon- same time are characterized by an in- strated that this mechanism may translate

100 Two indirect defences benefit Lima bean in nature

into a fitness-benefit for the EFN-secreting cates a clear functional assignment of the plant (CUAUTLE & RICO-GRAY, 2003). trapped flies. In most of the cases it ap- Comprehensive evidence for volatile- pears likely that the trapped flies simply mediated wasp attraction is available from exploited the offered EFN as an additional laboratory-based studies (e.g. DU et al., food source rather than contributing to a 1996; TAKABAYASHI et al., 1995; TUR- larger extent to plant protection. In this LINGS et al., 1990), yet relatively few ad- case, EFN consumption without providing dressed this issue in the field. Among the plant with any mutual benefit such as them, JAMES (2005) identified methyl plant protection could cause important salicylate as attractant for parasitic fami- ecological costs, because the EFN- lies such as Encyrtidae and Mymaridae. producing plant would be less protected Methyl salicylate was also constituent of against herbivores (HEIL, 2002; HEIL et the artificial volatile blend used in this al., 2004a). However, this is speculation study and these families were also and needs to be confirmed in future ex- trapped on our experimental tendrils. The periments. same holds true for Braconidae. These parasitic wasps were attracted to Synopsis (Z)-3-hex-3-enyl acetate and (Z)-jasmone Ecological studies on the benefit of indi- (JAMES, 2005), two further constituents of rect defences generally focussed on the our artificial VOC blend. A more detailed protective effect of either one single de- analysis of the attractive effects of EFN fensive trait or the combined effect of sev- and VOCs on wasps is required, in which eral defences such as after simultaneous especially single blend constituents should induction with elicitors like JA. While this be field-tested. simplification is easy to understand from Flies responded differently to tendril the viewpoint of experimental feasibility, treatment than ants and wasps, since they such univariate approaches may be inap- were only significantly attracted to the ten- propriate since they do not appreciate the drils of the nectar group (Fig. 4). This complex interplay of several plant de- observation suggests that flies were more fences within one plant species (DUFFEY attracted to the artificial nectar than to air- & STOUT, 1996). This study takes a first borne volatiles. The community of trapped step into this direction. Diptera covered a very diverse spectrum The mere application of artificial EFN of feeding habits ranging from predacious (nectar group) resulted in a fitness benefit or parasitoid taxa over herbivorous taxa to always stronger or quantitatively similar to taxa feeding on detritus or fungi (Table 2). the one experienced by the tendrils of the This heterogeneous composition compli- JA- and the VOC-treatment (Fig. 3).

Manuscript IV 101

Moreover, ants observed on the experi- ses. Furthermore, laboratory- and field- mental tendrils showed an overwhelming based experimentation is needed to study numerical superiority over all other arthro- whether inductive situations exist in which pod groups (Fig. 4). These two observa- either the volatile emission or the EFN tions suggest that under our experimental secretion is differentially up- or down- conditions the secretion of EFN was more regulated or if both defences always important for plant defence than the VOC- respond similarly to herbivore attack. mediated arthropod attraction. However, our experimental design does not allow excluding an attractive ef- Acknowledgements fect of airborne VOCs on flying or crawling We thank W. BOLAND for the possibility arthropods. This issue needs to be ad- to use all the facilities of the Department of dressed in future studies, which should Bioorganic Chemistry and S. ADOLPH especially focus on the role of volatiles and M. MEKEM SONWA for technical ad- and EFN for the short- and long-distance vice on organic synthesis. Financial sup- attraction of herbivores and plant defend- port by the German Research Foundation ers. Several of the arthropod taxa which (DFG Grant He 3169/2-1, 2) and the Max- have been identified in this study could Planck-Society is gratefully acknowledged. serve as possible targets for such analy-

102 Artefact formation during headspace sampling

Manuscript V

Dehydrogenation of ocimene by active carbon: Artefact formation during headspace sampling from leaves of Phaseolus lunatus

Mesmin Mekem Sonwa1, Christian Kost1, Anja Biedermann1, Robert Wegener2, Stefan Schulz2 and Wilhelm Boland1*

Submitted to Tetrahedron

1 Department of Bioorganic Chemistry Max Planck Institute for Chemical Ecology Beutenberg Campus Hans-Knöll-Str. 8 D-07745 Jena, Germany

2 Department of Organic Chemistry Technical University of Braunschweig Hagenring 30 D-38106 Braunschweig, Germany

Manuscript V 103

Graphical Abstract

Dehydrogenation of ocimene by active carbon: Artefact formation during headspace sampling from leaves of Phaseolus lunatus Mesmin Mekem Sonwa1, Christian Kost1, Anja Biedermann1, Robert Wegener2, Stefan Schulz2, and Wilhelm Boland1* 1 Max-Planck-Institut für Chemische Ökologie, Hans-Knöll-Str. 8, D-07745 Jena, Germany 2 Technical University of Braunschweig, Department of Organic Chemistry, Hagenring 30, D-38106 Braunschweig, Germany

ROH, charcoal, active carbon humid air

or H2O/acetone HO charcoal RO ocimene

Abstract The blend of volatiles emitted from jasmonate-treated Lima bean (Phaseolus lunatus) leaves comprised two ocimene-derived artefacts that were only present, if the compounds were collected on active carbon traps. Identified were (3E,5E)-2,6- dimethyl-3,5,7-octatrien-2-ol (6) and (3E,5E)-2,6-dimethyl-1,3,5,7-octatetraene (5) resulting from oxidation of ocimene (2) by active carbon in presence of humid air. The catalytical capacity of the active carbon could be exploited for a rapid and efficient functionalization of ocimene to give the (3E,5E)-2-alkoxy-3,5,7-octatrienes (14), (15), and (16) in methanol, ethanol, or isopropanol as solvents.

Keywords: Phaseolus lunatus, charcoal, induced volatiles, ocimene, closed-loop- stripping analysis (CLSA), SPME

104 Artefact formation during headspace sampling

1. Introduction after treatment with jasmonic acid (JA), we The Lima bean (Phaseolus lunatus) exhib- observed two rare monoterpenes that its selective response patterns to different were only present in the volatile blend col- herbivores (OZAWA et al., 2000) or lected with the closed-loop-stripping chemical elicitors like jasmonic acid method (CLSA) (DONATH & BOLAND, (KOCH et al., 1999). The different blends 1995) (Tab. 1), yet were absent if volatiles of volatiles which are emitted upon herbi- were collected on polydimethyl-siloxane- vore feeding are generally believed to coated fibres with solid-phase-micro- attract natural enemies of the herbivores extraction (SPME) (ARTHUR & PAWL- and thereby function as an indirect plant ISZYN, 1990). Trapping ocimene with defence (DE MORAES et al., 1998; GOU- commercial charcoal traps and subse- INGUENE et al., 2003). Thus, precise quent analysis of the desorbed com- knowledge on the qualitative and quantita- pounds with GC-MS again revealed the tive composition of the volatile blends is presence of 5 and 6, whereas simultan- essential for understanding multitrophic eous trapping of the same headspace with interactions. Analysing the volatile organic SPME did not result in the detection of compounds emitted by the Lima bean these compounds.

4 6

O 11 O 2 3 9 Standard

5 O 8

1 7 10

5 10 tret [min]

Figure 1. Gas chromatographic separation of volatiles collected from the gas phase around Lima bean leaves treated with jasmonic acid. Compounds were absorbed on charcoal traps (DONATH & BOLAND, 1995) and analyzed by GLC-MS after desorbtion with CH2Cl2.

Manuscript V 105

The two substances 5 and 6 have been redox reactions catalysed by carbon, previously identified in the headspace of halogenation and even polymerization. hyacinth flowers where they could only be Active carbon is also known to catalyse detected in headspace samples yet not in the oxidation of fluorene as well as halo- extracts or distillates of the same plant gen exchange reactions (YANG & JOHN- material (KAISER & LAMPARSKY, 1977). SON, 1977). MAKINO et al. (1992) dem- Furthermore, the substances could be onstrated that activated active carbon clearly identified as artefacts derived from catalyses the conversion of physalin B into (E)-β-ocimene (2), which is formed in the 25-hydroxyphysalin B under very mild presence of active charcoal (BRUNKE et conditions. Although the mode of action of al., 1993). Here we report on this carbon- active carbon has not yet been estab- mediated reaction that rapidly and stereo- lished, carbon-oxygen surface complexes selectively leads to a functionalisation of have been postulated. Other reactions 1,3-dienes. may be due to the acidic or alkaline nature of the carbon surface (YANG & JOHN-

Table 1. Amount of 2, 5 and 6 released by three SON, 1977). TOMITA et al. (1971) postu- Lima bean plants 24 h after spraying with jas- lated free radicals as carbon surface ac- monic acid. Area has been referenced to plant dry tive sites catalysing the reaction. weight (DW). To clarify whether radicals play a role Mean area ± SD 8 -1 in the active carbon catalysed hydroxyla- (co un t seconds 10 *g DW ) tion of ocimene, active carbon was either Sampling method 2 5 6 treated with the radical starter AIBN or

Char coal trap 24 ± 15 2.9 ± 1 6.5 ± 5 with the radical scavenger BHT prior to the addition of ocimene. In both cases, no SPME 2.0 ± 1 - - change was found in the catalytic activity a Charcoal trap 34.2 0.4 4.7 of active carbon, suggesting that radicals a eluate re-trapped with SPME, - not detected are not involved in the oxidation process. Alternatively, oxygen-functionalized 1,4- quinonoid centres on the surface of active 2. Results and discussion carbon have been recently discussed for 2.1. Active carbon catalysed oxidation of the dehydrogenation of ethylbenzene to ocimene. styrene (ZACHARIA, 2004; ZHU et al., Active carbon is a well known support for 2002). Accordingly, the initial step of the noble metals, such as palladium and plati- dehydrogenation of ocimene is the trans- num (RODRIGUEZ-REINOSO, 1998). fer of a hydride to a surface carbonyl COUGHLIN et al. (1969) have reported (Scheme 1).

106 Artefact formation during headspace sampling

CH3 (30 %), the presence of the alcohol 6

HH 2 CH3 CH3 (35 %) along with the tetraene 5 (30 %), CH3 H CH3 OO OH O demonstrating that the presence of active carbon is sufficient to achieve the reaction.

carbon surface In a more polar medium (acetone/water, access of air), the oxidation proceeded CH3 faster, and after 48 h most of 2 (60 %) was 5 CH3 CH2 OH HO converted to the alcohol 6 and the hydrocarbon 5. Control experiments without active carbon did not result in oxidation of ocimene. Since the cationic intermediate Scheme 1. Postulated mechanism of the dehydrogenation of ocimene by 1,4-quinonoid centres on of Scheme 1 principally allows a reaction the surface of active carbon; modified after with nucleophiles, we tested several alco- ZACHARIA (2004). The proposed mechanism re- hols as solvents and reactants (Scheme sembles dehydrogenation reactions mediated by 2). DDQ (BRAUDE et al., 1960).

Subsequent loss of a proton from the

active carbon, resonance-stabilized cationic intermediate ROH RO mostly (3E,5E)

6 R = H to the resulting phenolate anion completes 14 R = CH3 15 R = C2H5 16 R = CH(CH3)2 the sequence and yields the tetraene 5. If E:Z = 85:15 2 a nucleophile is present, this could com- acidic charcoal acetone (3E,5E) 85% pete with the elimination and lead to func- 5 (3E,5Z) 15% tionalized octatrienes as shown in Scheme Scheme 2. Oxidative transformations of ocimene 2. The observation that the same dehydro- with active carbon. genation could be also achieved by DDQ (BRAUDE et al., 1960) by simply stirring of In fact, simple stirring of ocimene with 1 eq. of ocimene in tetrachloromethane active carbon in methanol generated with 3 eq. of DDQ at r. t. suggests that the 2-methoxy-2,6-dimethyl-octa-3,5,7-triene transformation may indeed follow the (14), known as a minor constituent of the mechanistic model of Scheme 1. To verify essential oil of Narcissus geranium (VAN this reaction course, moistened active car- DORT et al., 1993). Previous syntheses of bon was stirred in hexane and oxygen was 14 required lengthy multi-step procedures removed by passing argon through the (VAN DORT et al., 1993). The starting suspension (1 h) followed by addition of mixture of (3E,Z)-ocimene showed a faster ocimene (2). After 72 h, GLC-MS analysis conversion of the (3E)-isomer resulting in revealed, besides unreacted ocimene oxidation products with higher configu-

Manuscript V 107

rational purity than the educts. According with an Alltech DB5 (0.25 mm x 30 m) to NMR, the new double bond was exclu- column. Helium at 30 cm min-1 served as sively trans. A similar reaction was per- carrier gas. Compounds were eluted un- formed in ethanol and yielded (3Z,5E)-7- der programmed conditions starting from ethoxy-3,7-dimethylocta-1,3,5-triene (15) 40 °C (4 min) and then at 10 °C min-1 to with a non-optimized yield of 13 % after 200 °C followed 55 °C min-1 to 280 °C 48 h and 18 % after 72 h. In isopropanol (3 min). Activated carbon was purchased (3Z,5E)-2-isopropxy-2,6-dimethyl-octa-3,5- from Aldrich (Cat.: 16,155-1, 82024 diene (16) was obtained albeit in low yield Taufkirchen, Germany). (5 % after 48 h and 6 % after 72 h). In t-butanol only trace amounts of the corres- 3.2. Plant material ponding t-butylether were detected (ca. Experiments using jasmonic acid- 1 %), demonstrating that only primary and treatments were performed with Lima secondary alcohols can be used. bean plants (Phaseolus lunatus var. Jack- If the acidic character of the active son Wonder Bush). Seeds were obtained carbon was increased by pre-treatment from Kelloggs Seed Inc., USA. Individual with nitric acid prior to use (acetone as plants were grown from seeds in plastic solvent), ocimene was directly transform- pots (Ø = 5.5 cm) with sterilized potting ed to the hydrocarbon 5. Scope and limita- soil at 21 - 23 °C and 50 - 60 % humidity tions of this novel and operationally sim- using daylight fluorescent tubes at apple, active carbon-mediated, functional- proximately 470 µE m-2s-1 with a photo- isation of 1,3-dienes are currently investi- phase of 14 hours. gated. 3.3. Induced biosynthesis of volatiles and collection of emitted compounds 3. Experimental 10- to 14-d-old plantlets of P. lunatus with 3.1. General information two fully developed primary leaves were 1H and 13C NMR: Bruker Avance 400 cut with razor blades and immediately spectrometer (Bruker, D-76287 Rhein- placed into glass vials containing an stetten, Karlsruhe, Germany). Chemical aqueous solution of jasmonic acid (4 ml of shifts of 1H and 13C NMR are given in ppm a 1 mmol aqueous solution of jasmonic

(δ) based on solvent peaks: DCCl3 7.26 acid). The vials with the plants were trans- ppm (1H NMR) and 77.70 ppm (13C NMR); ferred to 5 l desiccators and the emitted 1 CD3OD 3.31 ppm ( H NMR) and 49.00 volatiles collected over a period of 48 h ppm (13C NMR). GC-MS spectra were with the closed loop stripping method recorded on a Finnigan GCQ, equipped (CLSA; DONATH & BOLAND, 1995)

108 Artefact formation during headspace sampling

using active carbon traps (1 mg of char- mixture was stirred at room temperature. coal, CLSA-Filter, Le Ruisseau de Mont- After 48 hours, GC-MS analysis showed brun, F-09550 Daumazan sur Arize, that 60 % of ocimene (largely the (3E)-iso- France). At the end of the collection pe- mer) was transformed. After 72 hours, the riod, the trapped volatiles were eluted from (3E)-isomer and ca. 50 % of the (3Z)- the active carbon by washing three times isomer were hydroxylated. After 96 h both with 10 µl of dichloromethane. 1-Bromo- isomers were completely transformed. decane was used as an internal standard Following addition of water (20 ml) the (400 µg ml-1). The collected samples were product was extracted with pentane (3 x analysed by GLC-MS and indicated, be- 20 ml), concentrated in vacuo, and purified sides other compounds shown in Figure 1, by chromatography on silica gel. Yield: 14 the presence of 5 and 6. Alternatively, mg (41 %). The spectroscopic data were volatiles were collected with SPME fibres in agreement with literature data coated with carboxen™-polydimethyl-silox- (BRAUDE et al., 1960). ane (Supelco/Aldrich, Cat.: 57318, D-82024 Taufkirchen, Germany) (AR- 3.5. Functionalisation of ocimene, THUR & PAWLISZYN, 1990). Subsequent general procedure thermal desorbtion from the fibre and Activated carbon (2.5 g) was suspended GLC-MS analysis confirmed the presence with stirring in either methanol (120 ml), of all compounds shown in Figure 1 ex- ethanol or isopropanol (415 ml) followed cept of the ocimene-derived artefacts 5 by addition of ocimene (198 mg, mixture of and 6. The observation that re-trapping an 3E/Z isomers, ca. 85:15). After stirring at eluate of an active carbon trap measure- r.t. for 48 h or 72 h, the reactions were ment with SPME and analysing it with stopped and the activated carbon was GLC-MS, lead to the compounds 5 and 6 removed by filtration. GC-MS analysis de- suggested that both compounds originated monstrated the formation of 2-methoxy- from the first collection process using 2,6-dimethylocta-3,5,7-triene (14) from active carbon traps. methanol, 2-ethoxy-2,6-dimethylocta-3,5,7 -triene (15) from ethanol, and 2-iso- 3.4. Active carbon catalyzed hydroxylation propoxy-2,6-dimethylocta-3,5,7-triene (16) of ocimene to 2,6-dimethylocta-3,5,7- from isopropanol as solvent and nucleo- triene-2-ol (6) phile. For isolation of the ethers the sol- 30 mg (0.22 mmol) ocimene (mixture of vents were removed under reduced pres- 3E,Z-isomers, ca. 85:15) was added to a sure and the products purified by column suspension of active carbon (0.5 g) in chromatography. Owing to the high volatil- 31 ml acetone/water (30:1, v:v) and the ity of the ethers, removal of the rather

Manuscript V 109

large amounts of solvents resulted in low (23), 103 (11), 93 (14), 92 (20), 91 (91), 79 yields of isolated products. (24), 78 (11), 77 (33), 65 (15), 55 (11), 53 (9), 51 (9), 41 (15), 39 (15). 3.6. Dehydrogenation of ocimene with acidic active carbon 3.7.2. (3E,5E)-2-Methoxy-2,6-dimethyl- 100 ml of 66 % nitric acid was added to 3,5,7-octatriene (14) 20 g of activated carbon and the mixture Yield: 117 mg (60 %). (3E,5E)-14 (85 %) was stirred for 18 hours. Nitric acid was and (3E,5Z)-14 (15 %) according to NMR. 1 removed and the active carbon was (3E,5E)-14: H NMR (CDCl3, 400 MHz) washed with distilled water until the pH of δ ppm 6.41 (dd, 1H, J4,5 = 11.18 Hz, J4,3 = the suspension of active carbon in water 15.59 Hz, H-4), 6.33 (dd, 1H, J7,8a = 17.24 stabilized at 5.5. Water was removed and Hz, J7,8b = 11.18 Hz, H-7), 6.01 (d, 1H, the active carbon was dried at 80 °C for 16 H-5), 5.66 (d, 1H, H-3), 5.15 (d, 1H, H-8a), hours. 20 mg of the oxidized active carbon 4.98 (d, 1H, H-8b), 3.10 (s, 3H, O-CH3), was used for the transformation of oci- 1.81 (s, 3H, C6-CH3), 1.24 (s, 6H, 13 mene, following the same protocol as for C2-(CH3)2); C NMR (CDCl3, 100 MHz) δ untreated active carbon described in 3.7. (ppm) 141.54 (C-3), 140.32 (7), 135.70 After 24 hours, GC-MS analysis revealed (C6), 131,18 (C5), 125.97 (C-4), 113.07 complete conversion of ocimene to the (C-8), 75.51 (C-2), 50.85 (O-CH3), 26.31 hydrocarbon 5. (C2-(CH3)2), 12.45 (C6-CH3) ; EIMS (m/z) 166 (68), 165 (11), 152 (7), 151 (82), 150 3.7. Spectroscopic data (15), 137 (13), 136 (12), 135 (35), 133 3.7.1. (3E,5E)-2,6-Dimethyl-1,3,5,7-octa- (26), 123 (18), 119 (100), 118 (14), 117 tetraene (5): (3E,5E)-(5) (24), 109 (10), 107 (35), 105 (33), 93 (46), 1 H NMR (CDCl3, 400 MHz) δ 6.55 (dd, 1H, 92 (22), 91 (79), 90 (21), 86 (25), 85 (11),

J4,5 = 15.4 Hz, J4,5 = 11.5 Hz, H-4), 6.45 79 (28), 76 (54), 73 (17), 67 (6), 65 (11),

(dd, 1H, J7,8a = 10.6 Hz, J7,8b = 17.4 Hz, 59 (64), 58 (25), 55 (13), 43 (46). H-7), 6.57 (d, 1H, H-5), 6.14 (d, 1H, H-5), 5.41 (d, 1H, H-8b), 5.04 (d, 1H, H-8a), 3.7.3. (3Z/5E)-2-Ethoxy-2,6-dimethylocta-

5.00 (s, 4H, H-1), 1.90 (d, 5H, CH3), 1.89 3,5,7-triene (15) 13 1 (s, 5H, CH3); C NMR (CDCl3, 100 MHz) H NMR (CDCl3, 400 MHz) δ 6.49 (dd, 1H,

δ 142.9 (C-4), 141.5 (C-7), 136.5 (C-3), J7,8a = 17.1 Hz, J7,8b = 11.0 Hz, H-7), 6.40

136.3 (C-6), 131.9 (C-5), 125.7 (C-4), (ddd, 1H, J3,4 = 17,3 Hz, H-7), 6.10 (d, 1H,

117.1 (C-1), 112.7 (C-8), 18.6 (C6-CH3), J4,5 = 10.4 Hz, H-5), 5.78 (d, 1H, J3,4 =

12.2 (C2-CH3); EIMS (m/z) 134 (63), 120 15.4 Hz, H-3), 5.24 (d, 1H, J7,8a = 17.1 Hz,

(10), 119 (100), 117 (21), 106 (9), 105 H-8a), 5.07 (d, 1H, J7,8a = 11 Hz, H-8b),

110 Artefact formation during headspace sampling

3.35 (q, 2H, OCH2CH3), 1.90 (s, 3H, CH3), (2 x C-2’), 12.1 (C-6 CH3); EI-MS (m/z) +• 1.34 (d, 6H, CH3), 1.18 (t, 3H, -CH2CH3); 194 (M , 16), 179 (6), 152 (13), 151 (15), 13 C NMR (CDCl3, 100 MHz) δ 141.8 (C-3), 137 (25) 135 (24), 119 (22) 109 (100), 107 140.8 (C-7), 135.1 (C-6), 130.9 (C-5), (31), 93 (56), 91 (46), 81 (27), 77 (30), 67

125.6 (C-4), 112.5 (C-8), 74.9 (C-2), 58.0 (14), 55 (12). EI-HRMS calcd. for C13H22O

(C-1’), 26.5 (2 x C-2 CH3), 16.1 (C-6 CH3), 194.16707, found 194.16813. 12.0 (C-2’); EIMS (m/z) 180 (M+•, 54), 165 (66), 151 (19), 136 (51) 135 (52), 121 (31), 3.7.5. Dehydrogenation of ocimene with 119 (72), 109 (61), 107 (68), 105 (40), 93 DDQ (100), 91 (95), 79 (41), 77 (60), 59 (44), 55 Ocimene (40 mg, 0.3 mmol, mixture of

(34). EI-HRMS calcd. for C12H20O 3E,3Z isomers) was dissolved in carbon 180.15142, found 180.15157. tetrachloride (15 ml) and DDQ (0.20 g, 0.9 mmol) was added with stirring. Stirring 3.7.4. (3Z/5E)-2-Isopropxy-2,6-dimethyl- was continued for 48 h under argon at octa-3,5-diene (16) room temperature. After 48 h GC-MS 1 H NMR (CDCl3, 400 MHz) δ 6.38 (dd, 1H, revealed the exclusive formation of 5.

J7,8a = 17.1 Hz, J7,8b = 11.0 Hz, H-7), 6.34 Owing to the absence of water no con-

(dd, 1H, J3,4 = 17.3 Hz, H-4), 6.01 (d, 1H, comitant formation of the alcohol 6 was

J4,5 = 11.0 Hz, H-5), 5.72 (d, 1H, J3,4 = observed.

15.4 Hz, H-3), 5.20 (d, 1H, J7,8a = 17.1 Hz,

H-8a), 4.98 (d, 1H, J7,8a = 11 Hz, H-8b),

3.60 (sept., 1H, OCH(CH3)2), 1.71 (s, 3H, Acknowledgements

CH3), 1.22 (d, 6H, CH3), 1.18 (d, 6H, Rearing of plant and insect cultures by 13 -CH(CH3)2). C NMR (CDCl3, 100 MHz) ANGELIKA BERG is gratefully acknowl- δ 141.2 (C-3), 140.3 (C-7), 135.1 (C-6), edged. We thank the Fonds der 131.0 (C-5), 124.8 (C-4), 112.6 (C-8), 75.4 Chemischen Industrie, Frankfurt a. M., for

(C-2), 64.8 (C-1’), 27.1 (2 x C-2 CH3), 25.0 financial support.

General discussion 111

8. General discussion tween insect herbivores and natural enemies” generated a wealth of interesting The present thesis provides substantial studies demonstrating that members of evidence for the functioning of indirect the third trophic level can use plant- defences in nature and their role for medi- derived VOCs to locate their host or prey. ating intra- and interspecific interactions of Although the idea that plants and the natu- the Lima bean (Phaseolus lunatus). To ral enemies of their herbivores cooperate cope with its herbivorous enemies, Lima with the latter acting as bodyguards is in- bean has developed two indirect defence tuitively appealing (STRONG & LARS- strategies which are both inducible upon SON, 1994), this hypothesis has to bear herbivore damage (Manuscript I). These scrutiny of whether or not it fulfils the fol- are the secretion of extrafloral nectar lowing assumptions (DICKE & VET, 1999; (EFN) and the emission of volatile organic GODFRAY, 1995; JANSSEN et al., 2002; compounds (VOCs). Both of which VAN DER MEIJDEN & KLINKHAMER, attracted predacious and parasitoid arthro- 2000): i) the volatile signal should be suffi- pods that defended the plant against at- ciently specific to allow natural enemies of tacking herbivores and thus exerted a the herbivore an effective localisation of beneficial effect on the Lima bean (Manu- their hosts/prey, ii) attraction of predators/ script II, III and IV). Besides their role as parasitoids should result in a reduced her- host-location cue for members of the third bivore load that translates into an in- trophic level, VOCs are also implicated in creased plant fitness, and iii) the benefit of signalling processes between herbivore- volatile-attracted plant defenders should damaged and undamaged plants. VOCs outweigh potential costs for the plant such induced by feeding herbivores had an in- as e.g. attraction of additional herbivores. ductive effect on the EFN secretion rate of Evidence to support the first assump- neighbouring, conspecific plants or plant tion in the Phaseolus lunatus system is parts (Manuscript III and IV). Hence, they mainly derived from laboratory studies. In may serve as a signal for an impending Lima bean, the total amount of VOCs pro- herbivore attack which cause all bean ten- duced depends strongly on the quantity of drils positioned within a patch of increased herbivory inflicted (DE BOER et al., 2004; herbivore pressure to precautionary acti- HORIUCHI et al., 2003a). The resulting vate an indirect defence. bouquets were only attractive to predatory mites when being emitted from plants in- The role of VOCs for plant defence fested with median or high herbivore loads Price et al.’s (1980) seminal review on the (HORIUCHI et al., 2003a). Hence, forag- “influence of plants on interactions being carnivores may assess food patch

112 General discussion

quality via the quantity of VOCs emitted experienced by the VOC-treated tendrils. from attacked plants. Furthermore, testing A comparison of VOC-treated tendrils with the olfactory response of parasitoids and tendrils where an artificial mixture of EFN predators revealed their ability to discrimi- was applied revealed similar effects of nate between prey and non-prey herbi- both treatments (Manuscript III and IV), vores on Lima bean (DE BOER et al., indicating that the attractive effect of EFN 2004; DICKE & GROENEVELD, 1986; seemed to be more important than the one HORIUCHI et al., 2003a; SABELIS & VAN of VOCs. In this case, VOCs would pri- DE BAAN, 1983), between Lima bean and marily function as an intra-individual and other plant species attacked by the same intra-specific signal rather than as an inter- herbivore species (DICKE et al., 1990a; specific signal directed towards the mem- PETITT et al., 1992) as well as between bers of the third trophic level. This hy- different plant cultivars of the Lima bean’s pothesis needs to be addressed in further close relative Phaseolus vulgaris attacked studies. by the same herbivore species (DICKE et For other plant species such as Nico- al., 1990b). Although the ecological sig- tiana attenuata in which a volatile-induced nificance of these findings remains to be EFN secretion can be excluded due to the demonstrated under field conditions absence of extrafloral nectaries, VOCs (HUNTER, 2002), they impressively illus- attracted generalist predators which effec- trate the information content that is en- tively decreased the plant’s herbivore load coded in the plant-derived VOC plumes. (KESSLER & BALDWIN, 2001). Moreover, Until now it is unclear whether also plants that were fed upon by parasitized the second assumption of a VOC- lepidopteran larvae produced more seeds mediated top-down control is met in the than plants attacked by unparasitized lar- Phaseolus lunatus system. Application of vae (FRITSCHE HOBALLAH & TUR- an artificial VOC blend to wild growing LINGS, 2001; VAN LOON et al., 2000). Lima bean plants attracted a large number Thus it appears reasonable to assume of parasitoid and predacious insect spe- that also the parasitoid and predatory species which presumably caused the re- cies attracted to the Lima bean had similar duced herbivory rate and the increased effects on the plant. number of inflorescences as compared to The fitness benefit of the VOC- controls without VOCs (Manuscript III and treatment to tendrils outweighed costs IV). However, VOCs also induced EFN. associated with the emission of volatile Therefore, the attractive effects of both signals (Manuscript III and IV), and thus VOCs and the VOC-induced EFN may the third assumption was met. Potential have accounted for the beneficial effect costs of VOC emission that could have

General discussion 113

Table 1. Plant species which share constituents of their herbivore-induced volatile blend with the Lima bean

(Phaseolus lunatus). -ocimene β )-(-)-linalool )-3-hex-3-enyl acetate -caryophyllene ( Z Plant species ( E )- ( R DMNT methyl salicylate (Z)-jasmone β TMTT Reference

Glycine max1 X VAN DEN BOOM et al., 2004

Laburnum anagyroides1 X X X VAN DEN BOOM et al., 2004

Lotus japonicus1 X X X X OZAWA et al., 2000b

Medicago truncatula1 X X X X X LEITNER et al., 2005

Phaseolus vulgaris1 X X X X X COLAZZA et al., 2004

Robinia pseudo-acacia1 X X X VAN DEN BOOM et al., 2004

Vicia faba1 X X X X COLAZZA et al., 2004

Vigna unguiculata1 X X X VAN DEN BOOM et al., 2004

Capsicum annuum2 X X X X X VAN DEN BOOM et al., 2004

Datura stramonium2 X X X VAN DEN BOOM et al., 2004

Nicotiana attenuata2 X X X X KESSLER & BALDWIN, 2001

Nicotiana tabacum2 X X X X X X VAN DEN BOOM et al., 2004

Solanum nigrum2 X X SCHMIDT et al., 2004

Solanum melalonga2 X X X VAN DEN BOOM et al., 2004

Solanum tuberosum2 ni. ni. ni. ni. ni. ni. X ni. WEISSBECKER et al., 2000

Zea mays3 X X X X X X TAKABAYASHI et al., 1995

Cucumis sativus4 X X X X X TAKABAYASHI et al., 1994b

Arabidopis thaliana5 X X X VAN POECKE et al., 2001

Brassica olearacea5 X MATTIACCI et al., 1995

Gossypium hirsutum6 X X X X X X X RÖSE & TUMLINSON, 2004

Pinus sylvestris7 X MUMM et al., 2003

Humulus lupulus8 X X X VAN DEN BOOM et al., 2004

Ginkgo biloba9 X X VAN DEN BOOM et al., 2004

Vitis vinifera10 X X X X X VAN DEN BOOM et al., 2004

Adenostyles alliariae11 X X KALBERER et al., 2001

Petasites paradoxus11 KALBERER et al., 2001 Plant families: 1 Fabaceae, 2 Solanaceae, 3 Poaceae, 4 Cucurbitaceae, 5 Brassicaceae, 6 Malvaceae, 7 Pinaceae, 8 Moraceae, 9 Ginkgoaceae, 10 Vitaceae, 11 Asteraceae; X emission compound significantly increased upon herbivore damage; ni not investigated.

114 General discussion

occurred under our experimental condi- of future herbivory in order to tailor their tions are the attraction of herbivorous ar- responses accordingly. thropods to the applied VOCs (DICKE & Theory predicts that plants will only VET, 1999; HILKER & MEINERS, 2002). respond to information released by dam- Indeed, curculionid beetles, which are aged neighbours if the information is suffi- amongst the most important leaf-chewing ciently reliable (ADLER & KARBAN, 1994; herbivores of Lima bean in the study area, GETTY, 1996; KARBAN et al., 1999). As are attracted to VOCs of induced plants discussed previously, the amount of VOCs (HEIL, 2004b). Furthermore, VOCs could emitted by a plant reflects the intensity of have attracted members of the fourth tro- herbivore attack as well as the quantitative phic level such as e.g. hyperparasites that and qualitative composition of the VOC would have had detrimental effects on the plume encodes the species of both the third trophic level (Manuscript I). However, emitting plant and the attacking herbivore the net effect for the plant was positive (Manuscript I). Interestingly, the com- indicating that these costs were out- pound which caused the strongest in- weighed by the benefits of the applied crease in the EFN secretion rate in down- VOCs. wind tendrils is known to be produced by many different plant species upon herbi- The role of VOCs for plant-plant vore attack or mechanical damage: (Z)-3- communication hex-3-enyl acetate (Manuscript III, Tab. 1). The presented results provide several in- Consequently, this compound is not only novations in the field of plant-plant com- emitted from conspecifics, but also from munication (Manuscript III): First, they other plant species that provide the Lima demonstrate plant-plant communication bean with information on the risk of future for Lima bean. Second, they identify (Z)-3- attack. hex-3-enyl acetate (possibly amongst oth- Generally, inducible defences suffer ers) as an airborne signal responsible for an unavoidable drawback, namely the lag the observed defence reaction. Third, they time, which is needed to induce the de- are the first demonstration that an indirect fence. During this time the plant remains defence mechanism is induced in the re- susceptible to herbivores and is likely to ceiver plant, and fourth they provide field suffer greater tissue loss than a plant that evidence that the observed mechanism has its defences already activated may benefit Phaseolus lunatus under (JÄREMO et al., 1999). VOC-mediated natural growing conditions. Lima bean plant-plant communication may be an plants obviously can use volatile signals adaption to cope with this drawback, be- from their environment to assess the risk cause yet undamaged individuals activate

General discussion 115

their defences before they actually en- 1988). The same should apply when counter an herbivore. (Z)-3-hex-3-enyl sender and receiver belong to different acetate in particular is known to be emit- species and share the same phytophages ted rapidly after mechanical damage (BRUIN & DICKE, 2001). However, the (ARIMURA et al., 2000a) and thus allow situation reverses, if sender and receiver the Lima bean to quickly responds to a (Lima bean) belong to different species, current threat. yet the sender is attacked by a specialist Evolutionary considerations immedi- that would not attack the receiver of the ately pose the following question: What is signal. In this case, the receiver would pay the benefit for the sender of this signal? At the costs of activating a defence without its natural growing site, Lima bean forms an actual need. Our results have shown gregarious patches where it occurs closely that increased amounts of EFN, which entwined with conspecific individuals, were either applied experimentally or in- other vines and the supporting vegetation. duced by airborne VOCs, benefited Lima Many contact points with the surrounding bean under natural growing conditions vegetation facilitate the movement of (Manuscript II, III and IV). crawling herbivores between different If the Lima bean would have benefited plant individuals. Herbivore-induced VOC of increased amounts of EFN, why did it emission may not only attract plant de- not activate this defence by itself? The fenders to the sender, but also to closely answer to this interesting question cannot neighboured Lima bean plants which re- be answered based on the data available. ceive the information and subsequently A conceivable explanation could be that secrete increased amounts of EFN. KO- the ecological strategy of the liana Lima BAYASHI and YAMAMURA (2003) sug- bean is to grow rather than to defend and gested the term ‘‘cooperative signal’’ to that this strategy is more successful on describe such an interaction between un- longer time-scales (i.e. when more than damaged and herbivore infested plant one generation is regarded). The level of individuals that signal together as if they VOCs which have been applied experi- were collectively calling for bodyguards. A mentally to tendrils (Manuscript III and IV) potential conflict of interests between may have exceeded the amount of VOCs, sender and receiver is solved when the to which a Lima been tendril is naturally benefit of the emitted VOCs (i.e. effective exposed. If corresponding amounts of reduction of the herbivore pressure within VOCs reach a Lima bean tendril, it would a patch) is counterbalanced by the physio- allocate more resources to defence. This logical and ecological costs for the VOC- could be regulated via a certain threshold emitting plant (SABELIS & DE JONG, which is needed to activate EFN secretion.

116 General discussion

Chemical composition and specificity of scribed artefacts are also formed under the VOC blend natural conditions and if so, which factors Precise knowledge on the qualitative and favour their formation. quantitative composition of the emitted Besides this reaction for which it is not VOC blends is a prerequisite for under- yet clear whether it also plays a role under standing their role for mediating complex natural conditions, several other factors interactions between different trophic lev- hamper a reliable interpretation of the els. The example in Manuscript V has emitted VOCs. For example, the VOC pro- shown that two monoterpenes, which pre- file emitted from Lima bean plants upon viously have been considered as constitu- herbivore damage is subject to a consid- ents of the induced VOC blend of Lima erable variability both on the quantitative bean (ENGELBERTH et al., 2001; HEIL, and qualitative level (Manuscript I). Causal 2004a), could now be identified as degra- for this variation is a complex interplay of dation products resulting from a chemical numerous factors such as time of day, leaf reaction with the adsorption materials growth stage, species and life stage of the used for volatile-trapping. This finding attacking herbivore as well as abiotic con- highlights the need to always take artefact ditions (e.g. light and water availability; formation into account when using stan- TAKABAYASHI et al., 1994a). In its natu- dard methods for headspace sampling ral environment, Lima bean is surrounded (BRUNKE et al., 1993). by many other plant species which also Beyond, it remains elusive whether emit highly variable blends of VOCs, not the two observed compounds are pure only to attract carnivores, but also pollina- artefacts of the sampling methods or tors. For example, the eight most abun- whether they are also occasionally formed dant VOCs emitted from the Lima bean by the Lima bean under natural conditions. are very common constituents of the her- Such a reaction could proceed either ac- bivore-induced VOC plumes of many dif- tively by involving enzymatic activities or ferent plant species (Tab. 1). Thus, the passively under the influence of certain receiver of the emitted volatile signals, no abiotic factors or the catalytic activity of matter if insect or neighbouring plant, has the plant surface. In the latter case, the to cope with these difficulties. question arises how the transmission of In the case of arthropods, evidence informative airborne signals can be main- that mixing of odour blends interferes with tained despite interfering processes like host plant localization is controversial. the one described in manuscript V. The Among the few studies that addressed this very first step to answer this question issue, some showed masking for herbi- would be to analyse whether the two de- vores (VISSER, 1986; VISSER & AVÉ,

General discussion 117

1978) and carnivores (MONTEITH, 1960; despite this inevitable systematic error SHIOJIRI et al., 2000; VOS et al., 2001), suggests that the absence or presence of whereas others did not find evidence of certain compounds (such as (Z)-3-hex-3- such antagonistic effects (DICKE et al., enyl acetate) is responsible for the effect 2003b; RÖTTGER, 1979). The detectabil- rather than the relative composition of the ity of the emitted cue depends most likely whole blend. Preliminary trials for the on the amount of compounds shared with EFN-induction experiments (Manuscript the interfering blend, the tuning of the in- III) indicated that a certain threshold of sect receptors to specific compounds as VOCs is required to reliably induce EFN well as the wind speed within a given habi- secretion (results not shown), thereby pro- tat (VISSER, 1986). Associative learning viding first hints to a dose-response rela- is one possible strategy arthropods have tionship between airborne VOCs and the developed to cope with these difficulties amount of secreted EFN. More experi- (PAPAJ & LEWIS, 1993; VET et al., ments are definitely needed in which com- 1995). binations of different compounds in vary- The situation may be different for ing concentrations are tested for their plant-plant communication. Insects often EFN-inducing effect in Lima bean and need to be attracted from larger distances. other plant species. In contrast, for ‘eavesdropping’ plants it is more important to sense the immediate The role of EFN for plant defence vicinity for cues indicating an increased The hypothesis of an adaptive function of risk of herbivory. Plants should respond to extrafloral nectaries providing safeguards single or few compounds that are emitted from herbivores is much better supported rapidly after tissue damage in a sharp by field studies (for review see BEATTIE, dosage-dependent manner, rather than to 1985; BENTLEY, 1977a) than the indirect highly variable mixtures of different VOCs. defence hypothesis of VOCs. The specific- Indeed our results are consistent with ity that is needed for a volatile signal to these predictions. Despite our artificial attract plant defenders from longer dis- VOC mixture mimicked the quantitative tances does presumably not apply to and qualitative composition of a naturally EFNs. Due to the reward-based nature of induced blend as to emission within 24 h, this signal, the open presentation of a car- our mode of application may not have per- bohydrate-rich food source itself is suffi- fectly matched the natural situation at a cient to attract foraging ants or other plant given time point within this period (Manu- visitors from short distances. In the Lima script III). The fact that we observed sig- bean, the mere application of an artificial nificantly increased EFN secretion rates EFN mixture that mimicked the natural

118 General discussion

blend both quantitatively and qualitatively (DELPINO cited in VON WETTSTEIN, attracted natural enemies of the herbi- 1889). Since then, a growing body of re- vores (Manuscript II, III and IV). In this search has revealed ant-mediated plant context it would be interesting to see protection (for review see BENTLEY, whether the qualitative composition of 1977a; BUCKLEY, 1982; HEIL & MCKEY, Lima bean EFN changes in response to 2003; KOPTUR, 1992). Also the pre- different herbivores and whether these sented field experiments were consistent changes can alter the composition of the with these findings: ants were by far the attracted arthropod community (BLÜTH- most dominant arthropod groups attracted GEN & FIEDLER, 2004). to Lima bean tendrils thereby being most An increase in the availability of EFN likely responsible for the observed en- attracted more putative plant defenders to hancement of plant protection (Manuscript Lima bean plants (Manuscript II). In the II, III and IV). The ecological dominance of long run (i.e. after 25 d), experimentally ants may be caused by their territoriality increasing the amounts of EFN had a that facilitates monopolisation of food positive effect on plant fitness as com- sources (HÖLLDOBLER & LUMSDEN, pared to controls (Manuscript III and IV). A 1980). Furthermore, their capability of great diversity of arthropods was attracted learning (HARRISON & BREED, 1987) in to both EFN-treated and control tendrils combination with a foraging strategy that (Manuscript II). Furthermore, the positive involves frequent patrolling, efficient re- effects of the nectar treatment counterbal- cruitment and trail laying allows a highly anced any negative effects associated optimised exploitation of rewarding food with this defence, such as an attraction of sources (HÖLLDOBLER & WILSON, additional herbivores or hyper-parasitoids. 1990). Indeed, behavioural observations Thus in Lima bean, EFN can be consid- suggested a defence-by-exploitation strat- ered a functioning indirect defence mecha- egy (DREISIG, 2000): resident generalist nism. ant species avoided competition with other EFN-consumers by increasing the visita- Ants as plant defenders tion frequency of the extrafloral nectaries. The secretion of EFN represents a loose The increased presence of these ants on form of facultative mutualism that attracts plants with higher amounts of EFN facili- a broad spectrum of different arthropods. tated the defensive effect (Manuscript II). Among them, ants are most dominant. The attraction of ants through extrafloral nectaries acting as defensive agents has been suggested as early as 1874

General discussion 119

Wasps and flies as putative plant bean plants (Manuscript II and IV). These defenders findings suggest that P. lunatus does not Not all studies on the facultative mutua- exclusively rely on a highly specialised lism between ants and EFN-secreting interaction with a reduced number of de- plants detected measurable beneficial fending species or taxa, but is rather effects on the plant partner (BECERRA & loosely associated with a broad range of VENABLE, 1989; BOECKLEN, 1984; different organisms. For many of which it O'DOWD & CATCHPOLE, 1983; RASH- is not yet clear whether they have a posi- BROOK et al., 1992). In the majority of tive, negative or neutral effect on the plant. these studies, this issue was addressed A high diversity of arthropods visiting the by excluding ants using sticky barriers Lima bean may further hint to the absence such as Tangletrap® (BARTON, 1986; of regulatory mechanisms of EFN quality, MODY & LINSENMAIR, 2004; O'DON- as they are known from obligate ant-plant- NELL et al., 1996). The drawback of this mutualisms, which act as a filter to ex- attempt is that putative flying plant de- clude less desirable partners in multi- fenders remain unaffected by this ap- species associations (HEIL et al., 2005). proach. Given that - under certain conditions - these are more important for plant Predators versus parasitoids defence than ants, no protective effect Members of the third trophic level which would be detected with such an experi- are attracted by indirect defences such as mental design, since the flying plant de- HI-VOCs or EFN can be either predators fenders would have had access to both or parasitoids. Predators kill their herbivo- ant-exclusion and control plants. By ma- rous prey immediately, thus directly pre- nipulating the amount of available EFN, venting further damage to the plant. The we could circumvent these difficulties and benefit for the plant is less obvious in the demonstrate a beneficial effect of EFN on case of parasitoids. These lay their eggs the Lima bean (Manuscript II and IV). The on or in the herbivore, thus killing their increased abundance of predaceous and host after hatching of their larvae (STEID- parasitoid insects such as flies and wasps LE & VAN LOON, 2002). Depending on suggests that besides ants, also these the parasitoid species, egg laying can re- flying defenders may have contributed to duce or increase the feeding and growth increasing the plant’s fitness (Manuscript of their herbivorous host (HARVEY, 2000; II). VAN ALPHEN & JERVIS, 1996). Conse- Captures with sticky traps indicated quently, the benefit for the plant depends an enormous diversity of dipteran and hy- strongly on which plant defenders are at- menopteran families visiting the Lima tracted to VOCs or EFN. The defence

120 General discussion

strategy of the Lima bean relied predomi- & SCHEMSKE, 1984; INOUYE & TAY- nantly on ants, with other putative plant LOR, 1979). Hence, in facultative ant-plant defenders like non-ant predators or parasi- associations, the benefit for the EFN- toid species playing a minor role (Manu- secreting plant appears to be directly script II, III and IV). A proximate reason for linked to the availability of potential plant this observation could be that the liana-like defenders. Therefore, the performance of growth structure, which is characterized by Lima bean within different habitat types is a close contact to the surrounding vegeta- likely constrained by the local abundance tion, may favour ants as plant-defending of beneficial insects. agents, since they can be easily attracted Plants located within such a mosaic of in larger numbers from the vicinity. In con- environments with a varying presence of trast, the foraging movement of flying de- plant defenders adapt their defensive fenders could be hindered by the intricate phenotype according to the different con- growth architecture of the plant (PRICE et ditions. If nectar feeders are absent, EFN al., 1980). Another explanation could be secretion has been reported to decline the time-scales at which ant- and parasi- (HEIL et al., 2000b). The production rates toid-mediated plant defence operates at could be partially restored again when optimal effectiveness: Due to the relatively insects gained access to the EFN- short vegetation periods of the Lima bean secreting plant. Similar responses are at the experimental sites, ants may be known for the production of floral nectars more effective, because a beneficial effect (GILL, 1988; KOPTUR, 1983; PYKE, resulting from parasitoid defence requires 1991). In some plant species, nectars are a longer time. even reabsorbed in flowers where insect visitation has been prevented (BÚRQUEZ Benefit of indirect defences depends on & CORBET, 1991; FAHN, 1988). So far, the ecological context the regulatory mechanisms underlying Interestingly, the numbers of attracted these processes are unknown. However, arthropods varied considerably between such economy in the regulation of food two sites, resulting in a spatial variation of rewards may save a plant metabolic en- the protective effect (Manuscript II). Simi- ergy which can be invested in alternative lar observations have been reported for plant traits. Direct defence strategies for other EFN-secreting plant species (BENT- example may be increasingly activated LEY, 1976; COGNI et al., 2003; HORVITZ when insect plant defenders are absent.

Synthesis 121

9. Synthesis sive laboratory experiments, to the field, The chapters compiled in this thesis en- and to verify its applicability to wild grow- compass analyses on induced, indirect ing plant populations of the model plant plant defences against herbivory and their Lima bean. Field experiments at the role for mediating multispecies interac- plant’s natural growing site yielded results tions. The impetus for these studies was that could answer some of the initial ques- to transfer the knowledge on this issue, tions, yet several surprising observation which mainly has been gathered in exten- stimulated also several new hypotheses.

VOCs VOCs

Attraction

Airborne signalling

Putative airborne signalling

Extrafloral nectary

Figure 1. The role of induced, indirect defences for mediating intra- and interspecific interactions in the Phaseo- lus lunatus system. In the Lima bean (left plant), herbivory induces the secretion of EFN and the emission of VOCs. Both cues attract wasps and flies. Ants are mainly attracted to EFN. Beyond, VOCs are also implicated in intra-individual and intra-specific plant-plant communication. Parts of the same (left) or neighbouring plants (mid- dle) induce the secretion of EFN in response to airborne VOCs emitted from herbivore-damaged plants or plant- parts (left). Also VOC blends emitted from heterospecific plants (right) which overlap in their qualitative composition with the one emitted from Lima bean may induce defence responses in eavesdropping Lima bean plants. Sources: Ant (Crematogaster laeviscula var. clara MAYR (SMITH, 1947); caterpillar (Macrothylia rubi LIN- NAEUS; HONOMICHL et al., 1996); wasp (Austrotoxeuma kuscheli; NOYES, 2003), beetle (Cerotoma trifurcata FORSTER; http://ipm.ncsu.edu/AG295/html/bean_leaf_beetle.htm), fly (Ludovicius eucerus LOEW; SÉGUY, 1951).

122 Synthesis

In the following, the gathered results are of being attacked. Lima bean tendrils merged to develop a theoretical model receiving this cue cautionary increase which may serve as a basis for future ex- their secretion rate of EFN, thereby aminations (see also Fig. 1): benefiting from receiving this signal. (1) In the Lima bean (Phaseolus lunatus) (4) A secondary function of VOCs is the herbivory induces both the emission attraction of members of the third tro- of VOCs as well as the secretion of phic level which can associate the EFN. complex information encoded in the (2) Among these two indirect defences, emitted VOC plumes with the emitting EFN secretion is the more important plant species as well as with the defence mechanism for the Lima abundance and species of the attack- bean. The reward-based nature of this ing herbivore. Also the attraction of signal attracts – depending on their predacious or parasitoid plant defend- availability in different environments – ers benefits the VOC-emitting plant. a broad spectrum of flying and crawl- (5) (Z)-3-hex-3-enyl acetate is one signal ing arthropods with varying defensive (possibly among others) that conveys capabilities. Ants are likely to be most the information on an impending her- important for plant defence, but also bivore attack to yet undamaged Lima carnivorous wasps, flies and also bean tendrils. In face of the wide other taxonomic groups may contrib- taxonomic distribution of the ability of ute to prevent herbivory. Increasing plants to emit this compound, it ap- the amount of secreted EFN can pears reasonable to assume that the benefit the Lima bean in terms of an sender may not only be downwind- enhanced reproductive success. located tendrils of the same or other (3) The primary function of VOCs, which Lima bean individuals but also hetero- are increasingly emitted upon herbi- specific plants attacked by generalist vore damage, is the intra-individual herbivores come into question as pu- and intra-specific conveyance of in- tative senders. formation on a currently increased risk

Summary 123

10. Summary plant, several studies are available to date 10.1. Summary on mechanistic and functional aspects of Due to their sessile nature of growth, herbivore-induced VOC emission applying which does not allow them to escape un- ecological, analytical and molecular ap- favourable situations, plants have evolved proaches. However, the secretion of EFN a plethora of mechanisms to cope with has largely been neglected as an alterna- various abiotic and biotic stress factors. tive defensive strategy of Phaseolus luna- The defensive mechanisms against herbi- tus. Research on indirect defences of the vory, for example, cover a diverse spec- Lima bean has been mainly conducted trum of mechanical barriers such as thorns under laboratory conditions, thus evidence or hairs (trichomes), and chemical de- for the ecological role of these two indirect fences that are toxic to the attacking or- defences in nature is largely lacking. ganism or reduce the nutritional value of The aim of the present thesis was to the plant material. Such defence strate- study the function of the two indirect de- gies, which directly wield a negative im- fences - emission of HIPVs and secretion pact on the attacking herbivore, have been of VOCs - at the plant’s natural growing termed direct defences. In contrast, indi- site, and to unravel their role for mediating rect defences recruit members of the third intra- and interspecific interactions. A re- trophic level, which reduce the number of view on the mechanisms of herbivore rec- currently feeding herbivores. Attraction of ognition, signal transduction, the biosyn- beneficial arthropods to plants can be me- thesis of HIPVs as well as ecological and diated by herbivore-induced plant volatiles evolutionary aspects of induced, indirect (HIPVs), the provision of shelter in so- defences summarized already existing called domatia, or by plant-provided food knowledge. Field experiments on plants supplements such as extrafloral nectar growing in a native population near Puerto (EFN). Direct and indirect defences can Escondido (Oaxaca, Mexico) were con- either be expressed constitutively (i.e. ducted to investigate whether EFN secre- permanently) or be induced in response to tion and VOC emission benefit Lima bean herbivore attack. at its natural growing site, to assess the The Lima bean (Phaseolus lunatus) arthropod community which is attracted to features the emission of volatile organic VOCs and EFN, to scrutinize whether compounds (VOCs) and the secretion of HIPVs are involved in plant-plant commu- EFN. Both indirect defences are inducible nication, and to contrast the defensive role upon herbivore damage as well as after of EFN to that of VOCs. Laboratory ex- treatment with the phytohormone jasmonic periments were performed on the forma- acid (JA). Using Lima bean as a model tion of artefacts which have been ob-

124 Summary

served during headspace sampling of in- treated tendrils should have performed duced Lima bean plants. better than VOC- and EFN-treated tendrils. VOC- and JA-treated tendrils had HIPVs induced the secretion of EFN in produced similar amounts of EFN. How- neighbouring Lima bean tendrils. Vola- ever, tendrils treated with synthetic EFN tiles released from herbivore-damaged only benefited equally or more of this treat- bean tendrils as well as an artificial volatile ment than did tendrils treated with VOCs blend resembling the naturally released or JA. This finding indicates that - under blend elicited the indirect defence in our experimental conditions - EFN had treated tendrils. A single-compound com- played a more important role as an indirect parison of the eight most important VOCs defence for Lima bean than did a VOC- within the herbivore-induced blend identi- mediated arthropod attraction. fied the green leaf volatile (Z)-3-hex-3-enyl acetate as being responsible for the ob- Ants were the most important plant served defence reaction. These results defenders. Treatment of Lima bean ten- suggest that plant-plant communication drils with JA or an artificial blend of either between different tendrils of the same or VOCs or EFN significantly increased the of several plant individuals occurs in Lima number of ants and wasps as compared to bean. controls. The numbers of flies visiting the experimental tendrils were significantly Increased amounts of EFN and VOCs increased only on tendrils treated with benefited Lima bean plants in nature. In artificial EFN. However, ants were ca. 25- a long-term experiment designed to disen- fold more abundant than wasps and ca. tangle the defensive effects of VOCs and 10-fold more frequently observed than EFN, tendrils sprayed with jasmonic acid flies. Sticky traps attached to the experi- (JA) to induce both indirect defences were mental tendrils trapped predominantly Dip- compared to tendrils treated with an artifi- tera and Hymenoptera (55 % and 26 % of cial blend of either EFN or VOCs as well all insects trapped). The observation that as to control tendrils. Repeated application 98 % of all hymenopterans and 77 % of all of EFN and VOCs benefited the experi- dipterans belonged to families with parasi- mental tendrils as indicated by increased toid or predacious life habits indicates that plant fitness correlates. Because tendrils besides ants and wasps also flies may treated with JA showed a similar yet have contributed to the observed benefi- weaker response, it is concluded that a cial effect. putative JA-dependent direct defence was of minor importance, since otherwise JA-

Summary 125

Active carbon, which is frequently used derived from (E)-β-ocimene. Based on as adsorption material during head- further experiments, the involvement of space sampling, dehydrogenates oci- radicals could be excluded. Instead, a mene and thus leads to artefact forma- mechanism is proposed in which the oci- tion. During headspace sampling of JA- mene is dehydrogenated by interactions induced Lima bean plants we observed with the surface of active carbon in a the two monoterpenes (3E,5E)-2,6- DDQ-like dehydrogenation reaction. The dimethyl-3,5,7-octatrien-2-ol and (3E,5E)- catalytic capacity of the active carbon 2,6-dimethyl-1,3,5,7-octatetraene only if could be further exploited for a rapid and volatiles were trapped using activated efficient functionalisation of ocimene to charcoal, but not with alternative adsorp- yield (3E,5E)-2-alkoxy-3,5,7-octatrienes tion materials such as SPME. These two using methanol, ethanol, or isopropanol as substances could be identified as artefacts solvents.

126 Zusammenfassung

10.2 Zusammenfassung können entweder konstitutiv (d.h. perma- Aufgrund ihrer sessilen Lebensweise sind nent) ausgebildet sein, oder erst durch Pflanzen nicht dazu in der Lage, ungünsti- Herbivorenbefall induziert werden. gen Bedingungen aktiv auszuweichen. Im Die Limabohne (Phaseolus lunatus) Laufe der Evolution haben sie daher eine emittiert Duftstoffe und sezerniert extraflo- Vielzahl von Abwehrreaktionen entwickelt, ralen Nektar. Beide indirekten Verteidi- um auf die verschiedensten biotischen gungen sind sowohl durch Herbivorie als und abiotischen Umweltfaktoren reagieren auch durch Behandlung mit dem Pflanzen- zu können. Verteidigungsstrategien gegen hormon Jasmonsäure (JA) induzierbar. In Herbivore zum Beispiel umfassen ein brei- mehreren ökologischen, analytischen oder tes Spektrum, welches von mechanischen molekularen Studien konnten bereits ver- Barrieren wie Dornen oder Haaren (Tri- schiedene mechanistische und funktionel- chome) bis hin zu chemischen Abwehr- le Aspekte der im Modellsystem Limaboh- mechanismen reicht. Letztere wirken ent- ne durch Herbivorbefall induzierten Duft- weder giftig auf den angreifenden Orga- stoffemission aufgeklärt werden. Im Ge- nismus oder reduzieren die Verwertbarkeit gensatz dazu wurde die Sekretion des pflanzlichen Materials. Solche Ab- extrafloralen Nektars als alternative Ver- wehrstrategien, die einen direkt negativen teidigungsstrategie von Phaseolus lunatus Einfluss auf den angreifenden Herbivoren bisher kaum beachtet. Die bisher durchge- ausüben, werden unter der Bezeichnung führten Untersuchungen zur indirekten direkte Abwehrmechanismen zusammen- Verteidigung der Limabohne wurden fast gefasst. Im Gegensatz dazu locken indi- ausschließlich unter Laborbedingungen rekte Pflanzenverteidigungen Vertreter der durchgeführt, weswegen Hinweise auf die dritten trophischen Stufe an, die dann die ökologische Rolle der beiden indirekten Anzahl der angreifenden Herbivoren redu- Verteidigungen unter Freilandbedingun- zieren. Eine solche Anlockung von für die gen bisher fehlen. Pflanze nützlichen Arthropoden kann z.B. Die Zielsetzung der vorliegenden Ar- durch leicht flüchtige organische Verbin- beit war es daher, die Funktion der beiden dungen (Duftstoffe) erreicht werden, wel- indirekten Verteidigungen, Duftstoffemis- che nach Herbivorenbefall vermehrt pro- sion und Nektarsekretion, am natürlichen duziert werden. Alternative Möglichkeiten Standort der Pflanze zu untersuchen, so- sind die Bereitstellung von Unterschlupf- wie deren Rolle für die Vermittlung intra- möglichkeiten in so genannten Domatien, und interspezifischer Interaktionen zu ana- oder das Anbieten attraktiver Nahrungs- lysieren. Hierzu wurde zunächst der der- quellen wie z.B. extrafloraler Nektar. zeitige Kenntnisstand zu den Mechanis- Direkte und indirekte Verteidigungsformen men der Herbivorenerkennung, Signalwei-

Zusammenfassung 127

terleitung, Duftstoffbiosynthese sowie öko- den. Diese Befunde weisen auf eine logischen und evolutionären Aspekten der „Kommunikation“ zwischen verschiedenen beiden indirekten Verteidigungen in einem Ranken desselben oder mehrerer unter- Übersichtsartikel zusammengefasst. Dar- schiedlicher Pflanzenindividuen der Lima- über hinaus wurden Feldexperimente an bohne hin. einer natürlich wachsenden, westlich von Puerto Escondido (Oaxaca, Mexiko) loka- Die Limabohne profitierte unter Frei- lisierten Population von Limabohnen landbedingungen von einer erhöhten durchgeführt. Hierbei sollte der jeweils aus Menge an extrafloralem Nektar und Duftstoffemission und Sekretion extraflora- Duftstoffen. In einem Langzeitexperiment len Nektars für die Limabohne resultieren- sollte die Verteidigungswirkung von Duft- de Vorteil quantifiziert, und einander ver- stoffen und extrafloralem Nektar experi- gleichend gegenübergestellt werden. Zu- mentell getrennt werden. Hierzu wurden sätzlich wurde die von extrafloralem Nek- Bohnenranken mit Jasmonsäure besprüht tar oder Duftstoffen angelockte Arthropo- um beide indirekte Verteidigungen zu akti- dengemeinschaft erfasst, sowie die Rolle vieren. Diese wurden dann sowohl mit der Duftstoffe für zwischenpflanzliche Kontrollranken als auch mit Ranken ver- Kommunikation untersucht. In Laborexpe- glichen, die mit einer künstlichen Mi- rimenten wurde die bei der Duftstoffsamm- schung aus entweder Duftstoffen oder lung induzierter Limabohnen beobachtete extrafloralem Nektar behandelt worden Artefaktbildung analysiert. waren. Wiederholte Behandlung von Ran- ken mit Duftstoffen oder extrafloralem Duftstoffe induzierten die Sekretion Nektar verbesserte die Entwicklung fit- extrafloralen Nektars in benachbarten ness-relevanter Pflanzenparameter, was Ranken der Limabohne. Die indirekte auf einen aus beiden indirekten Verteidi- Verteidigung konnte sowohl von Duftstof- gungen resultierenden Vorteil für die fen ausgelöst werden, die von durch Her- Pflanze deutet. Mit Jasmonsäure behan- bivoren beschädigten Ranken emittiert delte Ranken zeigten einen ähnlichen, wurden, als auch von einer künstlichen jedoch schwächeren Effekt. Dies deutet Mischung, deren Zusammensetzung das darauf hin dass eine eventuell durch Jas- natürlich emittierte Duftstoffspektrum monsäure induzierte direkte Verteidigung nachahmte. Bei einem Einzelvergleich der eine untergeordnete Rolle zu spielen acht wichtigsten Bestandteile des Lima- scheint, da sich mit Jasmonsäure behan- bohnenduftspektrums konnte (Z)-3-Hex-3- delte Ranken sonst hätten besser entwi- enyl acetat als Auslöser für die beobachte- ckeln müssen als die mit Duftstoffen oder te Verteidigungsreaktion identifiziert wer- extrafloralem Nektar behandelten Ranken.

128 Zusammenfassung

Jasmonsäure- und Duftstoffbehandlung toidisch oder räuberisch lebenden Famili- führte zu einer erhöhten Sekretionsrate en angehörten, deutet darauf hin, dass extrafloralen Nektars, wobei keine quanti- neben Wespen und Ameisen auch die tativen Unterschiede zwischen beiden angelockten Fliegen einen Beitrag zur Behandlungsgruppen festgestellt werden Verteidigung der Limabohne geleistet ha- konnten. Ranken, auf die künstlicher ben könnten. extrafloraler Nektar aufgebracht worden war, profitierten genauso stark oder stär- Aktivkohle, ein häufig bei der Duftstoff- ker von ihrer Behandlung als mit Duftstof- sammlung verwendetes Adsorptions- fen oder Jasmonsäure behandelte Ran- material dehydriert Ocimen und führt ken. Dieser Befund deutet darauf hin, so zur Artefaktbildung. Bei der Duftstoff- dass unter unseren experimentellen Be- sammlung von mit Jasmonsäure induzier- dingungen extrafloraler Nektar eine wich- ter Limabohnen konnten wir die beiden tigere Rolle als indirekte Pflanzenverteidi- Monoterpene (3E,5E)-2,6-Dimethyl-3,5,7- gung spielte als eine Duftstoff-vermittelte oktatrien-2-ol und (3E,5E)-2,6-Dimethyl- Anlockung von Arthropoden 1,3,5,7-oktatetraen nur beobachten, wenn Aktivkohle, nicht aber wenn alternative Ameisen waren die wichtigsten Pflan- Adsorptionsmaterialien wie z.B. SPME, zenverteidiger. Die Behandlung von Li- verwendet wurden. Die beiden Verbindun- mabohnenranken mit Jasmonsäure oder gen konnten als von (E)-β-Ocimen abge- der künstlichen Duftstoff- oder Nektarmi- leitete Artefakte identifiziert werden. Wei- schung führte im Vergleich zu Kontrollran- tere Experimente deuteten darauf hin, ken zu einer signifikant erhöhten Anzahl dass die bei der Artefaktbildung beteiligte angelockter Ameisen und Wespen. Die chemische Reaktion nicht über eine radi- Zahl der angelockten Fliegen war nur bei kalische Zwischenstufe verläuft. Stattdes- den mit künstlichem extrafloralem Nektar sen wird ein Mechanismus vorgeschlagen, behandelten Ranken signifikant erhöht. bei dem Ocimen in Wechselwirkung mit Generell wurden Ameisen etwa 25x häufi- der Aktivkohlenoberfläche in einem DDQ- ger als Wespen und ca. 10x häufiger als ähnlichen Reaktionsmechanismus dehyd- Fliegen beobachtet. In einer Erfassung der riert wird. Die entsprechenden Reaktions- zur Limabohne angelockten Insekten mit eigenschaften der Aktivkohle konnten dar- Klebefallen wurden hauptsächlich Diptera über hinaus zu einer schnellen und effi- und Hymenoptera (55 und 26 % aller ge- zienten Funktionalisierung von Ocimen zu fangener Insekten) gefangen. Die Tatsa- (3E,5E)-2-Alkoxy-3,5,7-octatrienen mit che dass 98 % aller gefangenen Hyme- Methanol, Ethanol oder Isopropanol als nopteren und 77 % aller Dipteren parasi- Lösungsmittel genutzt werden.dsdsdsdsd

References 129

11. References ALMÉRAS E, STOLZ S, VOLLENWEIDER S, REYMOND P, MENE-SAFFRANE L & ADLER FR & KARBAN R (1994) Defended FARMER EE (2003) Reactive electrophile fortresses or moving targets? Another species activate defense gene expression model of inducible defenses inspired by in Arabidopsis. Plant Journal, 34, 205- military metaphors. The American Natu- 216. ralist, 144, 813-832. ARIMURA GI, HUBER DPW & BOHLMANN J AGRAWAL AA (2000) Host-range evolution: (2004a) Forest tent caterpillars (Mala- Adaptation and trade-offs in fitness of cosoma disstria) induce local and sys- mites on alternative hosts. Ecology, 81, temic diurnal emissions of terpenoid vola- 500-508. tiles in hybrid poplar (Populus trichocarpa AGRAWAL AA & KARBAN R (1997) Domatia x deltoides): cDNA cloning, functional mediate plant-arthropod mutualism. characterization, and patterns of gene ex- Nature, 387, 562-563. pression of (-)-germacrene D synthase, AGRAWAL AA & DUBIN-THALER BJ (1999) PtdTPS1. Plant Journal, 37, 603-616. Induced responses to herbivory in the ARIMURA GI, KOST C & BOLAND W (2005) Neotropical ant-plant association between Herbivore-induced, indirect plant de- Azteca ants and Cecropia trees: Re- fences. Biochimica et Biophysica Acta - sponse of ants to potential inducing cues. Lipids and Lipid Metabolism, 1734, 91- Behavioral Ecology and Sociobiology, 45, 111. 47-54. ARIMURA GI, OZAWA R, HORIUCHI J, AGRAWAL AA, CONNER JK, JOHNSON MTJ NISHIOKA T & TAKABAYASHI J (2001) & WALLSGROVE R (2002a) Ecological Plant-plant interactions mediated by vola- genetics of an induced plant defense tiles emitted from plants infested by spider against herbivores: Additive genetic vari- mites. Biochemical Systematics and Ecol- ance and costs of phenotypic plasticity. ogy, 29, 1049-1061. Evolution, 56, 2206-2213. ARIMURA GI, OZAWA R, KUGIMIYA S, AGRAWAL AA, JANSSEN A, BRUIN J, TAKABAYASHI J & BOHLMANN J POSTHUMUS MA & SABELIS MW (2004b) Herbivore-induced defense re- (2002b) An ecological cost of plant de- sponse in a model legume. Two-spotted fence: Attractiveness of bitter cucumber spider mites induce emission of (E)-β- plants to natural enemies of herbivores. ocimene and transcript accumulation of Ecology Letters, 5, 377-385. (E)-β-ocimene synthase in Lotus japoni- AHARONI A, GIRI AP, DEUERLEIN S, GRIE- cus. Plant Physiology, 135, 1976-1983. PINK F, DE KOGEL WJ, VERSTAPPEN ARIMURA GI, OZAWA R, SHIMODA T, FWA, VERHOEVEN HA, JONGSMA MA, NISHIOKA T, BOLAND W & TAKABAYA- SCHWAB W & BOUWMEESTER HJ SHI J (2000a) Herbivory-induced volatiles (2003) Terpenoid metabolism in wild-type elicit defence genes in Lima bean leaves. and transgenic Arabidopsis plants. Plant Nature, 406, 512-515. Cell, 15, 2866-2884. ARIMURA GI, TASHIRO K, KUHARA S, ALBORN T, TURLINGS TCJ, JONES TH, NISHIOKA T, OZAWA R & TAKABAYA- STENHAGEN G, LOUGHRIN JH & TUM- SHI J (2000b) Gene responses in bean LINSON JH (1997) An elicitor of plant leaves induced by herbivory and by herbi- volatiles from beet armyworm oral secre- vore-induced volatiles. Biochemical and tion. Science, 276, 945-949.

130 References

Biophysical Research Communications, BALDWIN IT & PRESTON CA (1999) The eco- 277, 305-310. physiological complexity of plant re- ARIMURA GI, OZAWA R, NISHIOKA T, sponses to insect herbivores. Planta, 208, BOLAND W, KOCH T, KUHNEMANN F & 137-145. TAKABAYASHI J (2002) Herbivore- BALDWIN IT & HAMILTON W (2000) Jas- induced volatiles induce the emission of monate-induced responses of Nicotiana ethylene in neighboring Lima bean plants. sylvestris results in fitness costs due to Plant Journal, 29, 87-98. impaired competitive ability for nitrogen. ARNETT RH (2000) American insects - A Journal of Chemical Ecology, 26, 915- handbook of the insects of America north 952. of Mexico. CRC Press, Boca Raton. BALDWIN IT, SCHMELZ EA & OHNMEISS TE ARTHUR CL & PAWLISZYN J (1990) Solid- (1994) Wound-induced changes in root phase microextraction with thermal- and shoot jasmonic acid pools correlate desorption using fused-silica optical with induced nicotine synthesis in Nico- fibers. Analytical Chemistry, 62, 2145- tiana sylvestris Spegazzini and Comes. 2148. Journal of Chemical Ecology, 20, 2139- AUBOURG S, LECHARNY A & BOHLMANN J 2157. (2002) Genomic analysis of the terpenoid BALDWIN IT, KESSLER A & HALITSCHKE R synthase (AtTPS) gene family of Arabi- (2002) Volatile signaling in plant-plant- dopsis thaliana. Molecular Genetics and herbivore interactions: What is real? Genomics, 267, 730-745. Current Opinion in Plant Biology, 5, 351- AVÉ DA, GREGORY P & TINGEY WM (1987) 354. Aphid repellent sesquiterpenes in glandu- BALLHORN DJ, LIEBEREI R & GANZHORN lar trichomes of Solanum berthaultii and JU (2005) Plant cyanogenesis of Phaseo- Solanum tuberosum. Entomologia lus lunatus and its relevance for herbi- Experimentalis et Applicata, 44, 131-138. vore-plant interaction: The importance of BAKER HG, OPLER PA & BAKER I (1978) A quantitative data. Journal of Chemical comparison of the amino-acid comple- Ecology, 31, 1445-1473. ments of floral and extrafloral nectars. BARATA EN, PICKETT JA, WADHAMS LJ, Botanical Gazette, 139, 322-332. WOODCOCK CM & MUSTAPARTA H BALDWIN IT (1988) The alkaloidal responses (2000) Identification of host and nonhost of wild tobacco to real and simulated her- semiochemicals of eucalyptus woodborer bivory. Oecologia, 77, 378-381. Phoracantha semipunctata by gas chro- BALDWIN IT (2001) An ecologically motivated matography-electroantennography. Jour- analysis of plant-herbivore interactions in nal of Chemical Ecology, 26, 1877-1895. native tobacco. Plant Physiology, 127, BARGONI N (1972) Synthesis of sucrose in 1449-1458. the nectary of Convolvulus sepium. Bollet- BALDWIN IT & SCHULTZ JC (1983) Rapid tino Societa Italiana Biologia Sperimen- changes in tree leaf chemistry induced by tale, 48, 1159-1160. damage: Evidence for communication BARTON AM (1986) Spatial variation in the between plants. Science, 221, 277-279. effect of ants on an extrafloral nectary BALDWIN IT & CALLAHAN P (1993) Autotox- plant. Ecology, 67, 495-504.

icity and chemical defense - Nicotine BATE NJ & ROTHSTEIN SJ (1998) C6- accumulation and carbon gain in solana- volatiles derived from the lipoxygenase ceous plants. Oecologia, 94, 534-541.

References 131

pathway induce a subset of defense- leaf injury. Journal of Chemical Ecology, related genes. Plant Journal, 16, 561-569. 30, 53-67. BEATTIE AJ (1985) The evolutionary ecology BIRKENMEIER GF & RYAN CA (1998) Wound of ant-plant mutualisms. Cambridge Uni- signaling in tomato plants - Evidence that versity Press, Cambridge. ABA is not a primary signal for defense BECERRA JXI & VENABLE DL (1989) Extra- gene activation. Plant Physiology, 117, floral nectaries: A defense against ant- 687-693. Homoptera mutualisms? Oikos, 55, 276- BIRKETT MA, CAMPBELL CAM, CHAMBER- 280. LAIN K, GUERRIERI E, HICK AJ, MAR- BELL E, CREELMAN RA & MULLET JE TIN JL, MATTHES M, NAPIER JA, PET- (1995) A chloroplast lipoxygenase is re- TERSSON J, PICKETT JA, POPPY GM, quired for wound-induced jasmonic acid POW EM, PYE BJ, SMART LE, WAD- accumulation in Arabidopsis. Proceedings HAMS GH, WADHAMS LJ & WOOD- of the National Academy of Sciences of COCK CM (2000) New roles for cis- the United States of America, 92, 8675- jasmone as an insect semiochemical and 8679. in plant defense. Proceedings of the Na- BENNETT RN & WALLSGROVE RM (1994) tional Academy of Sciences of the United Tansley Review No. 72: Secondary me- States of America, 97, 9329-9334. tabolites in plant defence mechanisms. BLÜTHGEN N & FIEDLER K (2004) Prefer- New Phytologist, 127, 617-633. ences for sugars and amino acids and BENTLEY BL (1976) Plants bearing extrafloral their conditionality in a diverse nectar- nectaries and the associated ant commu- feeding ant community. Journal of Animal nity: Interhabitat differences in the reduc- Ecology, 73, 155-166. tion of herbivore damage. Ecology, 57, BOECKLEN WJ (1984) The role of extrafloral 815-820. nectaries in the herbivore defence of Cas- BENTLEY BL (1977a) Extrafloral nectaries and sia fasciculata. Ecological Entomology, 9, protection by pugnacious bodyguards. 243-249. Annual Review of Ecology and System- BOHLMANN J, MARTIN D, OLDHAM NJ & atics, 8, 407-427. GERSHENZON J (2000) Terpenoid sec- BENTLEY BL (1977b) The protective function ondary metabolism in Arabidopsis of ants visiting the extrafloral nectaries of thaliana: cDNA cloning, characterization, Bixa orellana (Bixaceae). Journal of Ecol- and functional expression of a myr- ogy, 65, 27-38. cene/(E)-β-ocimene synthase. Archives of BERNASCONI ML, TURLINGS TCJ, AM- Biochemistry and Biophysics, 375, 261- BROSETTI L, BASSETTI P & DORN S 269. (1998) Herbivore-induced emissions of BOLAND W, FENG Z, DONATH J & GAE-

maize volatiles repel the corn leaf aphid, BLER A (1992) Are acyclic C11 and C16 Rhopalosiphum maidis. Entomologia homoterpenes plant volatiles indicating Experimentalis et Applicata, 87, 133-142. herbivory? Naturwissenschaften, 79, 368- BEZEMER TM, WAGENAAR R, VAN DAM 371. NM, VAN DER PUTTEN WH & WÄCK- BOLAND W, HOPKE J, DONATH J, NUSKE J ERS FL (2004) Above- and below-ground & BUBLITZ F (1995) Jasmonic acid and terpenoid aldehyde induction in cotton, coronatin induce odor production in Gossypium herbaceum, following root and plants. Angewandte Chemie International Edition, 34, 1600-1602.

132 References

BOLTER CJ, DICKE M, VANLOON JJA, VIS- BRUCE TJA, MARTIN JL, PICKETT JA, PYE SER JH & POSTHUMUS MA (1997) BJ, SMART LE & WADHAMS LJ (2003) Attraction of Colorado potato beetle to Cis-Jasmone treatment induces resis- herbivore-damaged plants during herbi- tance in wheat plants against the grain vory and after its termination. Journal of aphid, Sitobion avenae (Fabricius) Chemical Ecology, 23, 1003-1023. (Homoptera: Aphididae). Pest Manage- BOUWMEESTER HJ, VERSTAPPEN FWA, ment Science, 59, 1031-1036. POSTHUMUS MA & DICKE M (1999) BRUIN J & DICKE M (2001) Chemical informa- Spider mite-induced (3S)-(E)-nerolidol tion transfer between wounded and un- synthase activity in cucumber and Lima wounded plants: Backing up the future. bean. The first dedicated step in acyclic Biochemical Systematics and Ecology,

C11-homoterpene biosynthesis. Plant 29, 1103-1113. Physiology, 131, 173-180. BRUIN J, DICKE M & SABELIS MW (1992) BRAUDE EA, JACKMAN LM, LINSTEAD RP & Plants are better protected against spider- LOWE G (1960) Hydrogen transfer. Part mites after exposure to volatiles from in- 13. The kinetics of dehydrogenation of fested conspecifics. Experientia, 48, 525- 1,1-disubstituted 1,2-dihydronaphthalenes 529. and tetralins by tetrachloro-1,2-benzo- BRUNKE EJ, HAMMERSCHMIDT FJ & quinone. Journal of the Chemical Society, SCHMAUS G (1993) Flower scent of 3133-3138. some traditional medicinal plants. ACS BRONSTEIN JL (1998) The contribution of Symposium Series, 525, 282-296. ant-plant protection studies to our under- BUCKLEY RC (1982) Ant-plant interactions: a standing of mutualism. Biotropica, 30, world review. Pages 111-162 in BUCK- 150-161. LEY RC (ed.) Ant-plant interactions in BROUAT C, MCKEY D, BESSIERE JM, PAS- Australia. Junk, The Hague. CAL L & HOSSAERT-MCKEY M (2000) BUGG RL, ELLIS RT & CARLSON RW (1989) Leaf volatile compounds and the distribu- Ichneumonidae (Hymenoptera) using tion of ant patrolling in an ant-plant protec- extrafloral nectar of Faba bean (Vicia faba tion mutualism: Preliminary results on L, Fabaceae) in Massachusetts. Biological Leonardoxa (Fabaceae: Caesalpinioi- Agriculture & Horticulture, 6, 107-114. deae) and Petalomyrmex (Formicidae: BÚRQUEZ A & CORBET SA (1991) Do flow- Formicinae). Acta Oecologica, 21, 349- ers reabsorb nectar? Functional Ecology, 357. 5, 369-379. BROWN RL, KAZAN K, MCGRATH KC, MAC- CHAMBERLAIN K, GUERRIERI E, PENNAC- LEAN DJ & MANNERS JM (2003) A role CHIO F, PETTERSSON J, PICKETT JA, for the GCC-box in jasmonate-mediated POPPY GM, POWELL W, WADHAMS LJ activation of the PDF1.2 gene of Arabi- & WOODCOCK CM (2001) Can aphid- dopsis. Plant Physiology, 132, 1020-1032. induced plant signals be transmitted aeri- BROWN WL (2000) Diversity of ants. Pages ally and through the rhizosphere? Bio- 45-79 in AGOSTI D, MAJER JD, chemical Systematics and Ecology, 29, ALONSO LE & SCHULTZ T (eds.) Ants - 1063-1074. Standard methods for measuring and CHEN F, THOLL D, D'AURIA JC, FAROOQ A, monitoring biodiversity. Smithonian Insti- PICHERSKY E & GERSHENZON J tution Press, Washington. (2003a) Biosynthesis and emission of ter-

References 133

penoid volatiles from Arabidopsis flowers. COUGHLIN RW (1969) Carbon as adsorbent Plant Cell, 15, 481-494. and catalyst. Industrial & Engineering CHEN F, D'AURIA JC, THOLL D, ROSS JR, Chemistry Product Research and Devel- GERSHENZON J, NOEL JP & PICHER- opment, 8, 12-23. SKY E (2003b) An Arabidopsis thaliana CRANKSHAW DR & LANGENHEIM JH (1981) gene for methylsalicylate biosynthesis, Variation in terpenes and phenolics identified by a biochemical genomics through leaf development in Hymenaea approach, has a role in defense. Plant and its possible significance to herbivory. Journal, 36, 577-588. Biochemical Systematics and Ecology, 9, CHEN F, RO DK, PETRI J, GERSHENZON J, 115-124. BOHLMANN J, PICHERSKY E & THOLL CREELMAN RA & MULLET JE (1997) Biosyn- D (2004) Characterization of a root- thesis and action of jasmonates in plants. specific Arabidopsis terpene synthase re- Annual Review of Plant Physiology and sponsible for the formation of the volatile Plant Molecular Biology, 48, 355-381. monoterpene 1,8-cineole. Plant Physiol- CROFT KPC, JUTTNER F & SLUSARENKO ogy, 135, 1956-1966. AJ (1993) Volatile products of the lipoxy- CHENG SH, WILLMANN MR, CHEN HC & genase pathway evolved from Phaseolus SHEEN J (2002) Calcium signaling vulgaris (L) leaves inoculated with Pseu- through protein kinases. The Arabidopsis domonas syringae pv Phaseolicola. Plant calcium-dependent protein kinase gene Physiology, 101, 13-24. family. Plant Physiology, 129, 469-485. CUAUTLE M & RICO-GRAY V (2003) The CIPOLLINI D, PURRINGTON CB & BERGEL- effect of wasps and ants on the reproduc- SON J (2003) Costs of induced responses tive success of the extrafloral nectaried in plants. Basic and Applied Ecology, 4, plant Turnera ulmifolia (Turneraceae). 79-85. Functional Ecology, 17, 417-423. CIPOLLINI D, ENRIGHT S, TRAW MB & BER- CURTIS JD & LERSTEN NR (1974) Morphol- GELSON J (2004) Salicylic acid inhibits ogy, seasonal variation, and function of jasmonic acid-induced resistance of Ara- resin glands on buds and leaves of Popu- bidopsis thaliana to Spodoptera exigua. lus deltoides (Salicaceae). American Jour- Molecular Ecology, 13, 1643-1653. nal of Botany, 61, 835-845. COGNI R, FREITAS AVL & OLIVEIRA PS D’AURIA JC, CHEN F & PICHERSKY E (2003) Interhabitat differences in ant activ- (2003) The SABATH family of MTs in ity on plant foliage: Ants at extrafloral nec- Arabidopsis thaliana and other plant spe- taries of Hibiscus pernambucensis in cies. Pages 253-283 in ROMEO JT (ed.) sandy and mangrove forests. Entomologia Recent advances in phytochemistry. El- Experimentalis et Applicata, 107, 125- sevier Science Ltd, Oxford. 131. DAEHLER CC, YORKSTON M, SUN WG & COLAZZA S, MCELFRESH JS & MILLAR JG DUDLEY N (1999) Genetic variation in (2004) Identification of volatile syno- morphology and growth characters of mones, induced by Nezara viridula feed- Acacia koa in the Hawaiian Islands. Inter- ing and oviposition on bean spp, that national Journal of Plant Sciences, 160, attract the egg parasitoid Trissolcus basa- 767-773. lis. Journal of Chemical Ecology, 30, 945- DALY HV, DOYEN JT & PURCELL AH (1998) 964. Introduction to insect biology and diversity. Oxford University Press, Oxford.

134 References

DAMMANN C, ROJO E & SANCHEZ- New York Entomological Society, 97, 332- SERRANO JJ (1997) Abscisic acid and 340. jasmonic acid activate wound-inducible DEWITT TJ, SIH A & WILSON DS (1998) genes in potato through separate, organ- Costs and limits of phenotypic plasticity. specific signal transduction pathways. Trends in Ecology and Evolution, 13, 77- Plant Journal, 11, 773-782. 81. DE BOER JG, POSTHUMUS MA & DICKE M DICKE M (1986) Volatile spider-mite phero- (2004) Identification of volatiles that are mone and host-plant kairomone, involved used in discrimination between plants in spaced-out gregariousness in the infested with prey or nonprey herbivores spider mite Tetranychus urticae. Physio- by a predatory mite. Journal of Chemical logical Entomology, 11, 251-262. Ecology, 30, 2215-2230. DICKE M (1994) Local and systemic produc- DE MORAES CM, MESCHER MC & TUM- tion of volatile herbivore-induced terpe- LINSON JH (2001) Caterpillar-induced noids: Their role in plant-carnivore mutual- nocturnal plant volatiles repel nonspecific ism. Journal of Plant Physiology, 143, females. Nature, 410, 577-580. 465-472. DE MORAES CM, LEWIS JM, PARÉ PW, DICKE M (1999a) Are herbivore-induced plant ALBORN HT & TUMLINSON JH (1998) volatiles reliable indicators of herbivore Herbivory-infested plants selectively identity to foraging carnivorous arthro- attract parasitoids. Nature, 393, 570-573. pods? Entomologia Experimentalis et DEBOUCK DG, MAQUET A & POESSO CE Applicata, 91, 131-142. (1989) Biochemical evidence for two dif- DICKE M (1999b) Specifity of herbivore- ferent gene pools in Lima bean, Phaseo- induced plant defences. Pages 43-54 in lus lunatus L. Annual Report of the Bean CHADWICK DJ & GOODE JA (eds.) Improvement Cooperation, 32, 58-59. Insect-plant interactions and induced plant DEGENHARDT J & GERSHENZON J (2000) defence. John Wiley & Sons Ltd, Chiches- Demonstration and characterization of ter. (E)-nerolidol synthase from maize: A her- DICKE M (1999c) Evolution of induced indirect bivore-inducible terpene synthase partici- defences of plants. Pages 62-88 in TOLL- pating in (3E)-4,8-dimethyl-1,3,7-nona- RIAN R & HARVELL CD (eds.) The ecol- triene biosynthesis. Planta, 210, 815-822. ogy and evolution of inducible DEGREEF J, ROCHA OJ, VANDERBORGHT defenses. Princeton University Press, T & BAUDOIN JP (2002) Soil seed bank Princeton. and seed dormancy in wild populations of DICKE M & GROENEVELD A (1986) Hierar- Lima bean (Fabaceae): Considerations for chical structure in kairomone preference in situ and ex situ conservation. American on the predatory mite Amblyseius potentil- Journal of Botany, 89, 1644-1650. lae: Dietary component indispensable for DEJEAN A, MCKEY D, GIBERNAU M & diapause induction affects prey location BELIN M (2000) The arboreal ant mosaic behaviour. Ecological Entomology, 11, in a Cameroonian rainforest (Hymenop- 131-138. tera: Formicidae). Sociobiology, 35, 403- DICKE M & SABELIS MW (1989) Does it pay 423. plants to advertize for bodyguards? DEVRIES PJ & BAKER I (1989) Butterfly Towards a cost-benefit analysis of in- exploitation of an ant-plant mutualism duced synomone production. Pages 341- adding insult to herbivory. Journal of the 358 in LAMBERS H, CAMBRIDGE ML,

References 135

KONINGS H & PONS TL (eds.) Variation and foraging success of carnivorous in growth rate and productivity of higher arthropods. Oikos, 101, 38-48. plants. SPB Academic Publishing, The DICKE M, SABELIS MW, TAKABAYASHI J, Hague. BRUIN J & POSTHUMUS MA (1990b) DICKE M & VET LEM (1999) Plant-carnivore Plant strategies of manipulating predator- interactions: Evolutionary and ecological prey interactions through allelochemicals: consequences for plant, herbivore and Prospects for application in pest control. carnivore. Pages 483-520 in OLFF H, Journal of Chemical Ecology, 16, 3091- BROWN VK & DRENT RH (eds.) Herbi- 3118. vores: Between plants and predators. DICKE M, VANBEEK TA, POSTHUMUS MA, Blackwell Science, Oxford. BENDOM N, VANBOKHOVEN H & DE- DICKE M & VAN LOON JJA (2000) Multi- GROOT AE (1990c) Isolation and identifi- trophic effects of herbivore-induced plant cation of volatile kairomone that affects volatiles in an evolutionary context. Ento- acarine predator-prey interactions - mologia Experimentalis et Applicata, 97, Involvement of host plant in its production. 237-249. Journal of Chemical Ecology, 16, 381- DICKE M & DIJKMAN H (2001) Within-plant 396. circulation of systemic elicitor of induced DISNEY RHL (1994) Scuttle flies: The Phori- defence and release from roots of elicitor dae. Chapman & Hall, London. that affects neighbouring plants. Bio- DJIETO-LORDON C & DEJEAN A (1999a) chemical Systematics and Ecology, 29, Tropical arboreal ant mosaics: Innate at- 1075-1087. traction and imprinting determine nest site DICKE M, AGRAWAL AA & BRUIN J (2003a) selection in dominant ants. Behavioral Plants talk, but are they deaf? Trends in Ecology and Sociobiology, 45, 219-225. Plant Science, 8, 403-405. DJIETO-LORDON C & DEJEAN A (1999b) DICKE M, VAN DER MAAS J, TAKABAYASHI Innate attraction supplants experience J & VET LEM (1990a) Learning affects during host selection in an obligate plant- response to volatile allelochemicals by ant. Behavioural Processes, 46, 181-187. predatory mites. Proceedings of Experi- DOLCH R & TSCHARNTKE T (2000) Defolia- mental and Applied Entomology, 1, 31-36. tion of alders (Alnus glutinosa) affects DICKE M, VANBAARLEN P, WESSELS R & herbivory by leaf beetles on undamaged DIJKMAN H (1993) Herbivory induces neighbours. Oecologia, 125, 504-511. systemic production of plant volatiles that DONATH J & BOLAND W (1995) Biosynthesis attract predators of the herbivore: Extrac- of acyclic homoterpenes: Enzyme selection of endogenous elicitor. Journal of tivity and absolute configuration of the Chemical Ecology, 19, 581-599. nerolidol precursor. Phytochemistry, 39, DICKE M, GOLS R, LUDEKING D & POST- 785-790. HUMUS MA (1999) Jasmonic acid and DREISIG H (2000) Defense by exploitation in herbivory differentially induce carnivore- the Florida carpenter ant, Camponotus attracting plant volatiles in Lima bean floridanus, at an extrafloral nectar re- plants. Journal of Chemical Ecology, 25, source. Behavioral Ecology and Sociobi- 1907-1922. ology, 47, 274-279. DICKE M, DE BOER JG, HOFTE M & RO- DRUKKER B, SCUTAREANU P & SABELIS CHA-GRANADOS MC (2003b) Mixed MW (1995) Do anthocorid predators re- blends of herbivore-induced plant volatiles spond to synomones from Psylla-infested

136 References

pear trees under field conditions. Ento- radenium (Leguminosae). Botanical Ga- mologia Experimentalis et Applicata, 77, zette, 133, 38-42. 193-203. ELIAS TS (1983) Extrafloral nectaries: Their DU YJ, POPPY GM & POWELL W (1996) structure and distribution. Pages 174-203 Relative importance of semiochemicals in BENTLEY B & ELIAS TS (eds.) The bi- from first and second trophic levels in host ology of nectaries. Columbia University foraging behavior of Aphidius ervi. Journal Press, New York. of Chemical Ecology, 22, 1591-1605. ENGELBERTH J, ALBORN HT, SCHMELZ EA DU YJ, POPPY GM, POWELL W, PICKETT & TUMLINSON JH (2004) Airborne sig- JA, WADHAMS LJ & WOODCOCK CM nals prime plants against insect herbivore (1998) Identification of semiochemicals attack. Proceedings of the National Acad- released during aphid feeding that attract emy of Sciences of the United States of parasitoid Aphidius ervi. Journal of America, 101, 1781-1785. Chemical Ecology, 24, 1355-1368. ENGELBERTH J, KOCH T, SCHÜLER G, DUDAREVA N, MARTIN D, KISH CM, KO- BACHMANN N, RECHTENBACH J & LOSOVA N, GORENSTEIN N, FÄLDT J, BOLAND W (2001) Ion channel-forming MILLER B & BOHLMANN J (2003) (E)-β- alamethicin is a potent elicitor of volatile Ocimene and myrcene synthase genes of biosynthesis and tendril coiling. Cross talk floral scent biosynthesis in snapdragon: between jasmonate and salicylate signal- Function and expression of three terpene ing in Lima bean. Plant Physiology, 125, synthase genes of a new terpene syn- 369-377. thase subfamily. Plant Cell, 15, 1227- ENGLISH LOEB GM & KARBAN R (1991) 1241. Consequences of mite feeding injury to DUFFEY SS (1980) Sequestration of plant beans on the fecundity and survivorship of natural products by insects. Annual Re- the two-spotted spider mite (Acari: view of Entomology, 25, 447-477. Tetranychidae). Experimental & Applied DUFFEY SS & STOUT MJ (1996) Antinutritive Acarology, 11, 125-136. and toxic components of plant defence FAHN A (1988) Secretory tissues in vascular against insects. Archives of Insect Bio- plants. New Phytologist, 108, 229-257. chemistry and Physiology, 32, 3-37. FAHN A (2000) Structure and function of sec- DUSSOURD DE & EISNER T (1987) Vein- retory cells. Advances in Botanical cutting behavior insect counterploy to the Research, 31, 37-75. latex defense of plants. Science, 237, FÄLDT J, ARIMURA G, GERSHENZON J, 898-901. TAKABAYASHI J & BOHLMANN J (2003) DUSSOURD DE & DENNO RF (1991) Deacti- Functional identification of AtTPS03 as vation of plant defense - Correspondence (E)-β-ocimene synthase: A monoterpene between insect behavior and secretory synthase catalyzing jasmonate- and canal architecture. Ecology, 72, 1383- wound-induced volatile formation in 1396. Arabidopsis thaliana. Planta, 216, 745- EHRLICH PR & RAVEN PH (1964) Butterflies 751.

and plants: A study in coevolution. Evolu- FARAG MA & PARÉ PW (2002) C6-Green leaf tion, 18, 586-608. volatiles trigger local and systemic VOC ELIAS TS (1972) Morphology and anatomy of emissions in tomato. Phytochemistry, 61, foliar nectaries of Pithecellobium mac- 545-554.

References 137

FARMER EE (2001) Surface-to-air signals. devil's advocate position. American Natu- Nature, 411, 854-856. ralist, 126, 181-195. FARMER EE & RYAN CA (1990) Interplant FREITAS L, GALETTO L, BERNARDELLO G communication - Airborne methyl jas- & PAOLI AAS (2000) Ant exclusion and monate induces synthesis of proteinase- reproduction of Croton sarcopetalus (Eu- inhibitors in plant-leaves. Proceedings of phorbiaceae). Flora, 195, 398-402. the National Academy of Sciences of the FREY M, SPITELLER D, BOLAND W & GIERL United States of America, 87, 7713-7716. A (2004) Transcriptional activation of Igl, FELTON GW & EICHENSEER H (1999) Her- the gene for indole formation in Zea mays: bivore saliva and its effects on plant A structure-activity study with elicitor- defense against herbivores and patho- active N-acyl glutamines from insects. gens. Pages 19-36 in AGRAWAL AA, Phytochemistry, 65, 1047-1055. TUZUN S & BENT E (eds.) Induced plant FREY M, STETTNER C, PARÉ PW, defenses against pathogens and herbi- SCHMELZ EA, TUMLINSON JH & GIERL vores: Ecology and agriculture. American A (2000) An herbivore elicitor activates Phytopathology Society Press, St. Paul. the gene for indole emission in maize. FIALA B & MASCHWITZ U (1990) Studies on Proceedings of the National Academy of the south east asian ant-plant association Sciences of the United States of America, Crematogaster borneensis/Macaranga: 97, 14801-14806. Adaptations of the ant partner. Insectes FREY-WYSSLING A (1955) The phloem sup- Sociaux, 37, 212-231. ply to nectaries. Acta Botanica Neer- FIGIER J (1971) Fine structure in extrafloral landica, 4, 358-369. nectary of Vicia faba L. Planta, 98, 31-49. FRITSCHE HOBALLAH ME & TURLINGS TCJ FOFANA B, DU JARDIN P & BAUDOIN JP (2001) Experimental evidence that plants (2001) Genetic diversity in the Lima bean under caterpillar attack may benefit from (Phaseolus lunatus L) as revealed by attracting parasitoids. Evolutionary Eco- chloroplast DNA (cpDNA) variations. logy Research, 3, 553-565. Genetic Resources and Crop Evolution, FRY JD (1989) Evolutionary adaptation to host 48, 437-445. plants in a laboratory population of the FOFANA B, VEKEMANS X, DU JARDIN P & phytophagous mite Tetranychus urticae BAUDOIN JP (1997) Genetic diversity in Koch. Oecologia, 81, 559-565. Lima bean (Phaseolus lunatus L) as FUJIMOTO SY, OHTA M, USUI A, SHINSHI H revealed by RAPD markers. Euphytica, & OHME-TAKAGI M (2000) Arabidopsis 95, 157–165. ethylene-responsive element binding fac- FOSTER WA (1995) Mosquito sugar feeding tors act as transcriptional activators or and reproductive energetics. Annual repressors of GCC box-mediated gene Review of Entomology, 40, 443-474. expression. Plant Cell, 12, 393-404. FOWLER SV & MACGARVIN M (1985) The FUNK CJ (2001) Alkaline phosphatase activity impact of hairy wood ants Formica lugu- in whitefly salivary glands and saliva. bris on the guild structure of herbivorous Archives of Insect Biochemistry and insects on birch, Betula pubescens. Jour- Physiology, 46, 165-174. nal of Animal Ecology, 54, 847-856. GALETTO L & BERNARDELLO LM (1992) FOWLER SV & LAWTON JH (1985) Rapidly Extrafloral nectaries that attract ants in induced defenses and talking trees: The Bromeliaceae: Structure and nectar com-

138 References

position. Canadian Journal of Botany, 70, GNANVOSSOU D, HANNA R, DICKE M & 1101-1106. YANINEK SJ (2001) Attraction of the GALIZIA CG, SACHSE S & MUSTAPARTA H predatory mites Typhlodromalus manihoti (2000) Calcium responses to pheromones and Typhlodromalus aripo to cassava and plant odours in the antennal lobe of plants infested by cassava green mite. the male and female moth Heliothis vires- Entomologia Experimentalis et Applicata, cens. Journal of Comparative Physiology 101, 291-298. A – Sensory, Neural, and Behavioral GODFRAY HCJ (1995) Communication Physiology, 186, 1049-1063. between the first and 3rd trophic levels - GANG DR, WANG JH, DUDAREVA N, NAM An analysis using biological signalling KH, SIMON JE, LEWINSOHN E & PICH- theory. Oikos, 72, 367-374. ERSKY E (2001) An investigation of the GOLS R, ROOSJEN M, DIJKMAN H & DICKE storage and biosynthesis of phenylpro- M (2003) Induction of direct and indirect penes in sweet basil. Plant Physiology, plant responses by jasmonic acid, low 125, 539-555. spider mite densities, or a combination of GARDNER SN & AGRAWAL AA (2002) jasmonic acid treatment and spider mite Induced plant defence and the evolution infestation. Journal of Chemical Ecology, of counter-defences in herbivores. Evolu- 29, 2651-2666. tionary Ecology Research, 4, 1131-1151. GOMI K, YAMASAKI Y, YAMAMOTO H & GARY RE & FOSTER WA (2004) Anopheles AKIMITSU K (2003) Characterization of a gambiae feeding and survival on honey- hydroperoxide lyase gene and effect of

dew and extrafloral nectar of peridomestic C6-volatiles on expression of genes of the plants. Medical and Veterinary Entomol- oxylipin metabolism in Citrus. Journal of ogy, 18, 102-107. Plant Physiology, 160, 1219-1131. GENTRY G (2003) Multiple parasitoid visitors GOUINGUENE S, DEGEN T & TURLINGS to the extrafloral nectaries of Solanum TCJ (2001) Variability in herbivore- adherens. Is S. adherens an insectary induced odour emissions among maize plant? Basic and Applied Ecology, 4, 405- cultivars and their wild ancestors 411. (teosinte). Chemoecology, 11, 9-16. GERSHENZON J (1994a) Metabolic costs of GOUINGUENE S, ALBORN H & TURLINGS terpenoid accumulation in higher plants. TCJ (2003) Induction of volatile emissions Journal of Chemical Ecology, 20, 1281- in maize by different larval instars of Spo- 1328. doptera littoralis. Journal of Chemical GERSHENZON J (1994b) The cost of plant Ecology, 29, 145-162. chemical defense against herbivores: A GULMON SL & MOONEY HA (1986) Costs of biochemical perspective. Pages 105-173 defense and their effects on plant produc- in BERNAYS EA (ed.) Insect-plant inter- tivity. Pages 151-197 in GIVNISH TJ (ed.) actions. CRC Press, Boca Raton. On the economy of plant form and func- GETTY T (1996) The maintenance of pheno- tion. Cambridge University Press, Cam- typic plasticity as a signal detection prob- bridge. lem. American Naturalist, 148, 378-385. GUTIÉRREZ-SALGADO A, GEPTS P & DE- GILL FB (1988) Effects of nectar removal on BOUCK DG (1995) Evidence for two gene nectar accumulation in flowers of Helico- pools of the Lima bean, Phaseolus luna- nia imbricata (Heliconiaceae). Biotropica, tus L, in the Americas. Genetic 20, 169-171. Resources and Crop Evolution, 42, 15-28.

References 139

HALITSCHKE R & BALDWIN IT (2004) Jas- PENA-CORTÉS H (1995) Expression of a monates and related compounds in plant- flax allene oxide synthase cDNA leads to insect interactions. Journal of Plant increased endogenous jasmonic acid (JA) Growth Regulation, 23, 238-245. levels in transgenic potato plants but not HALITSCHKE R, SCHITTKO U, POHNERT G, to a corresponding activation of JA- BOLAND W & BALDWIN IT (2001) Mo- responding genes. Plant Cell, 7, 1645- lecular interactions between the specialist 1654. herbivore Manduca sexta (Lepidoptera, HARRISON JM & BREED MD (1987) Tempo- Sphingidae) and its natural host Nicotiana ral learning in the giant tropical ant attenuata. III. Fatty acid-amino acid con- Paraponera clavata. Physiological Ento- jugates in herbivore oral secretions are mology, 12, 317-320. necessary and sufficient for herbivore- HARVEY JA (2000) Dynamic effects of parasit- specific plant responses. Plant Physiol- ism by an endoparasitoid wasp on the ogy, 125, 711-717. development of two host species: Implica- HALITSCHKE R, GASE K, HUI DQ, SCHMIDT tions for host quality and parasitoid fit- DD & BALDWIN IT (2003) Molecular ness. Ecological Entomology, 25, 267- interactions between the specialist herbi- 278. vore Manduca sexta (Lepidoptera, HATANAKA A (1993) The biogeneration of Sphingidae) and its natural host Nicotiana green odor by green leaves. Phytochem- attenuata. VI. Microarray analysis reveals istry, 34, 1201-1218. that most herbivore-specific transcriptional HAUKIOJA E & HAKALA T (1975) Herbivore changes are mediated by fatty acid-amino cycles and periodic outbreaks: Formula- acid conjugates. Plant Physiology, 131, tion of a general hypothesis. Reports from 1894-1902. the Kevo Subarctic Research Station, 12, HAMILTON-KEMP TR, LOUGHRIN JH, 1-9. ARCHBOLD DD, ANDERSEN RA & HEADS PA & LAWTON JH (1985) Bracken HILDEBRAND DF (1991) Inhibition of pol- ants and extrafloral nectaries 3. How len germination by volatile compounds insect herbivores avoid ant predation. including 2-hexenal and 3-hexenal. Jour- Ecological Entomology, 10, 29-42. nal of Agricultural and Food Chemistry, HEIL M (2001) The ecological concept of costs 39, 952-956. of induced systemic resistance (ISR). HARBORNE JB (1988) Introduction to ecologi- European Journal of Plant Pathology, cal biochemistry. Academic Press, Lon- 107, 137-146. don. HEIL M (2002) Ecological costs of induced HARDING J & TUCKER CL (1969) Quantita- resistance. Current Opinion in Plant Biol- tive studies on mating systems: III. Meth- ogy, 5, 345-350. ods for the estimation of male gameto- HEIL M (2004a) Induction of two indirect de- phytic selective values and differential fences benefits Lima bean (Phaseolus outcrossing rates. Evolution, 23, 85-95. lunatus, Fabaceae) in nature. Journal of HARMON AC, GRIBSKOV M & HARPER JF Ecology, 92, 527-536. (2000) CDPKs - A kinase for every Ca2+ HEIL M (2004b) Direct defense or ecological signal? Trends in Plant Science, 5, 154- costs: Responses of herbivorous beetles 159. to volatiles released by wild Lima bean. HARMS K, ATZORN R, BRASH A, KUHN H, Journal of Chemical Ecology, 30, 1289- WASTERNACK C, WILLMITZER L & 1295.

140 References

HEIL M & BALDWIN IT (2002) Fitness costs of plants? A critical re-evaluation of a widely induced resistance: Emerging experimen- accepted hypothesis. Oikos, 99, 457-468. tal support for a slippery concept. Trends HEIL M, GREINER S, MEIMBERG H, in Plant Science, 7, 61-67. KRÜGER R, NOYER JL, HEUBL G, LIN- HEIL M & MCKEY D (2003) Protective ant- SENMAIR KE & BOLAND W (2004b) plant interactions as model systems in Evolutionary change from induced to con- ecological and evolutionary research. stitutive expression of an indirect plant re- Annual Review of Ecology, Evolution, and sistance. Nature, 430, 205-208. Systematics, 34, 425-453. HELDER RJ (1958) The excretion of carbohy- HEIL M, STAEHELIN C & MCKEY D (2000a) drates (nectaries). Pages 978-990 in Low chitinase activity in Acacia myrmeco- RUHLAND W (ed.) Handbuch der Pflan- phytes: A potential trade-off between bi- zenphysiologie. Springer-Verlag, Berlin. otic and chemical defences? Naturwis- HERMS DA & MATTSON WJ (1992) The senschaften, 87, 555-558. dilemma of plants: To grow or to defend. HEIL M, RATTKE J & BOLAND W (2005) The Quarterly Review of Biology, 67, 283- Postsecretory hydrolysis of nectar su- 335. crose and specialization in ant/plant HILDEBRAND DF, BROWN GC, JACKSON mutualism. Science, 308, 560-563. DM & HAMILTON-KEMP TR (1993) HEIL M, FIALA B, BOLLER T & LINSENMAIR Effects of some leaf-emitted volatile com- KE (1999) Reduced chitinase activities in pounds on aphid population increase. ant plants of the genus Macaranga. Journal of Chemical Ecology, 19, 1875- Naturwissenschaften, 86, 146-149. 1887. HEIL M, FIALA B, BAUMANN B & LINSEN- HILKER M & MEINERS T (2002) Induction of MAIR KE (2000b) Temporal, spatial and plant responses to oviposition and feeding biotic variations in extrafloral nectar secre- by herbivorous arthropods: A comparison. tion by Macaranga tanarius. Functional Entomologia Experimentalis et Applicata, Ecology, 14, 749-757. 104, 181-192. HEIL M, HILPERT A, KRÜGER R & LINSEN- HOBALLAH ME, KÖLLNER TG, DEGEN- MAIR KE (2004a) Competition among HARDT J & TURLINGS TCJ (2004) Costs visitors to extrafloral nectaries as a source of induced volatile production in maize. of ecological costs of an indirect defence. Oikos, 105, 168-180. Journal of Tropical Ecology, 20, 201-208. HÖLLDOBLER B & LUMSDEN CJ (1980) HEIL M, KOCH T, HILPERT A, FIALA B, Territorial strategies in ants. Science, 210, BOLAND W & LINSENMAIR KE (2001) 732-739. Extrafloral nectar production of the ant- HÖLLDOBLER B & WILSON EO (1990) The associated plant, Macaranga tanarius, is ants. Springer-Verlag, Berlin. an induced, indirect, defensive response HÖLLER C, MICHA SG, SCHULZ S, elicited by jasmonic acid. Proceedings of FRANCKE W & PICKETT JA (1994) the National Academy of Sciences of the Enemy-induced dispersal in a parasitic United States of America, 98, 1083-1088. wasp. Experientia, 50, 182-185. HEIL M, DELSINNE T, HILPERT A, HONOMICHL K, BELLMANN H, JACOBS W & SCHURKENS S, ANDARY C, LINSEN- RENNER M (1996) Biologie und Ökologie MAIR KE, SOUSA MS & MCKEY D der Insekten. Gustav Fischer Verlag, (2002) Reduced chemical defence in ant- Stuttgart.

References 141

HOPKE J, DONATH J, BLECHERT S & INUI Y, MIYAMOTO Y & OHGUSHI T (2003) BOLAND W (1994) Herbivore-induced Comparison of volatile leaf compounds volatiles - The emission of acyclic homo- and herbivorous insect communities on terpenes from leaves of Phaseolus luna- three willow species. Population Ecology, tus and Zea mays can be triggered by a 45, 41-46. β-glucosidase and jasmonic acid. FEBS JACOB HS & EVANS EW (1998) Effects of Letters, 352, 146-150. sugar spray and aphid honeydew on field HORIUCHI J, ARIMURA G, OZAWA R, SHI- populations of the parasitoid Bathyplectes MODA T, TAKABAYASHI J & NISHIOKA curculionis (Hymenoptera: Ichneumoni- T (2001) Exogenous ACC enhances vola- dae). Environmental Entomology, 27, tiles production mediated by jasmonic 1563-1568. acid in Lima bean leaves. FEBS Letters, JAMES DG (2005) Further field evaluation of 509, 332-336. synthetic herbivore-induced plant volatiles HORIUCHI J, ARIMURA G, OZAWA R, SHI- as attractants for beneficial insects. Jour- MODA T, TAKABAYASHI J & NISHIOKA nal of Chemical Ecology, 31, 481-495. T (2003a) A comparison of the responses JANSSEN A, SABELIS MW & BRUIN J (2002) of Tetranychus urticae (Acari: Tetranychi- Evolution of herbivore-induced plant vola- dae) and Phytoseiulus persimilis (Acari: tiles. Oikos, 97, 134-138. Phytoseiidae) to volatiles emitted from JANSSEN A, VANALPHEN JJM, SABELIS Lima bean leaves with different levels of MW & BAKKER K (1995a) Odor-mediated damage made by T. urticae or Spodop- avoidance of competition in Drosophila tera exigua (Lepidoptera: Noctuidae). Ap- parasitoids: The ghost of competition. plied Entomology and Zoology, 38, 109- Oikos, 73, 356-366. 116. JANSSEN A, VANALPHEN JJM, SABELIS HORIUCHI JI, ARIMURA GI, OZAWA R, SHI- MW & BAKKER K (1995b) Specificity of MODA T, DICKE M, TAKABAYASHI J & odor mediated avoidance of competition NISHIOKA T (2003b) Lima bean leaves in Drosophila parasitoids. Behavioral exposed to herbivore-induced conspecific Ecology and Sociobiology, 36, 229-235. plant volatiles attract herbivores in addi- JANSSEN A, BRUIN J, JACOBS G, tion to carnivores. Applied Entomology SCHRAAG R & SABELIS MW (1997) and Zoology, 38, 365-368. Predators use volatiles to avoid prey HORVITZ CC & SCHEMSKE DW (1984) patches with conspecifics. Journal of Ani- Effects of ants and an ant-tended herbi- mal Ecology, 66, 223-232. vore on seed production of a neotropical JANZEN DH (1966) Coevolution of mutualism herb. Ecology, 65, 1369-1378. between ants and acacias in Central HUNTER MD (2002) A breath of fresh air: America. Evolution, 20, 249-275. Beyond laboratory studies of plant vola- JÄREMO J, TUOMI J & NILSSON P (1999) tile-natural enemy interactions. Agricul- Adaptive status of localized and systemic tural and Forest Entomology, 4, 81-86. defense responses in plants. Pages 31-44 INOUYE DW & TAYLOR ORJ (1979) A temin TOLLIAN R & HARVELL CD (eds.) The perate region plant ant seed predator sys- ecology and evolution of inducible tem: Consequences of extra floral nectar defenses. Princeton University Press, secretion by Helianthella quinquenervis. Princeton. Ecology, 60, 1-7. JONES CG, HOPPER RF, COLEMAN JS & KRISCHIK VA (1993) Control of systemi-

142 References

cally induced herbivore resistance by Topics in Current Chemistry, 239, 133- plant vascular architecture. Oecologia, 93, 177. 452-456. KELSEY LP (1969) A revision of the Sceno- KAHL J, SIEMENS DH, AERTS RJ, GABLER pinidae (Diptera) of the world. Bulletin of R, KÜHNEMANN F, PRESTON CA & the United States National Museum, 277, BALDWIN IT (2000) Herbivore-induced 1-336. ethylene suppresses a direct defense but KELSEY LP (1981) New Scenopinidae (Dip- not a putative indirect defense against an tera) from the Palearctic. Folia Ento- adapted herbivore. Planta, 210, 336-342. mologica Hungarica, 2, 85-93. KAISER R & LAMPARSKY D (1977) Nou- KESSLER A & BALDWIN I (2001) Defensive veaux constituants de l'absolue de jacin- function of herbivore-induced plant volatile the et leur comportment olfactif. Parfums, emissions in nature. Science, 291, 2141- Cosmétiques, Arômes, 17, 71-79. 2144. KALBERER NM, TURLINGS TCJ & RAHIER KESSLER A, HALITSCHKE R & BALDWIN IT M (2001) Attraction of a leaf beetle (Ore- (2004) Silencing the jasmonate cascade: ina cacaliae) to damaged host plants. Induced plant defenses and insect popu- Journal of Chemical Ecology, 27, 647- lations. Science, 305, 665-668. 661. KIM CY, LIU YD, THORNE ET, YANG HP, KARBAN R (1989) Fine-scale adaptation of FUKUSHIGE H, GASSMANN W, herbivorous thrips to individual host HILDEBRAND D, SHARP RE & ZHANG plants. Nature, 340, 60-61. SQ (2003) Activation of a stress- KARBAN R & BALDWIN IT (1997) Induced responsive mitogen-activated protein responses to herbivory. University of Chi- kinase cascade induces the biosynthesis cago Press, Chicago. of ethylene in plants. Plant Cell, 15, 2707- KARBAN R & MARON J (2002) The fitness 2718. consequences of interspecific eavesdrop- KISHIMOTO K, MATSUI K, OZAWA R &

ping between plants. Ecology, 83, 1209- TAKABAYASHI J (2005) Volatile C6- 1213. aldehydes and allo-ocimene activate KARBAN R, AGRAWAL AA, THALER JS & defense genes and induce resistance ADLER LS (1999) Induced plant against Botrytis cinerea in Arabidopsis responses and information content about thaliana. Plant and Cell Physiology, 46, risk of herbivory. Trends in Ecology and 1093-1102. Evolution, 14, 443-447. KOBAYASHI Y & YAMAMURA N (2003) Evo- KARBAN R, BALDWIN I, BAXTER K, LAUE G lution of signal emission by non-infested & FELTON G (2000) Communication plants growing near infested plants to between plants: Induced resistance in wild avoid future risk. Journal of Theoretical tobacco plants following clipping of Biology, 223, 489-503. neighboring sagebrush. Oecologia, 125, KOCH T (2001). Analytik pflanzlicher Sig- 66-71. nalstoffe: Phytohormone und Abwehr. KEELER KH (1979) Species with extrafloral PhD thesis, Bioorganic Chemistry, Max nectaries in a temperate flora (Nebraska). Planck Institute for Chemical Ecology, Prairie Naturalist, 11, 33-38. Jena. KEELING CI, PLETTNER E & SLESSOR KN KOCH T, KRUMM T, JUNG V, ENGELBERTH (2004) Hymenopteran semiochemicals. J & BOLAND W (1999) Differential induction of plant volatile biosynthesis in the

References 143

Lima bean by early and late intermediates leucine- and isoleucine conjugates of of the octadecanoid-signaling pathway. 1-oxo- and 1-hydroxyindan-4-carboxylic Plant Physiology, 121, 153-162. acid: Evidence for amino acid conjugates KODAMA H, NISHIUCHI T, SEO S, OHASHI Y of jasmonic acid as intermediates in the & IBA K (2000) Possible involvement of octadecanoid signalling pathway. FEBS protein phosphorylation in the wound- Letters, 377, 523-529. responsive expression of Arabidopsis KUNERT M, BIEDERMANN A, KOCH T & plastid ω-3 fatty acid desaturase gene. BOLAND W (2002) Ultrafast sampling and Plant Science, 155, 153-160. analysis of plant volatiles by a hand-held KOPTUR S (1983) Flowering phenology and miniaturised GC with pre-concentration floral biology of Inga (Fabaceae: Mimo- unit: Kinetic and quantitative aspects of soideae). Systematic Botany, 8, 354-368. plant volatile production. Journal of Sepa- KOPTUR S (1985) Alternative defenses ration Science, 25, 677-684. against herbivores in Inga (Fabaceae, KUO J & PATE JS (1985) The extrafloral nec- Mimosoideae) over an elevational gradi- taries of cowpea (Vigna unguiculata (L) ent. Ecology, 66, 1639-1650. Walp): I. Morphology, anatomy and fine KOPTUR S (1992) Extrafloral nectary- structure. Planta, 166, 15-27. mediated interactions between insects LABEYRIE E, PASCAL L, DELABIE J, and plants. Pages 81-129 in BERNAYS E ORIVEL J, DEJEAN A & HOSSAERT- (ed.) Insect-plant interactions. CRC Press, MCKEY M (2001) Protection of Passiflora Boca Raton. glandulosa (Passifloraceae) against her- KOPTUR S & LAWTON JH (1988) Interactions bivory: Impact of ants exploiting extrafloral among vetches bearing extrafloral nectar- nectaries. Sociobiology, 38, 317-321. ies their biotic protective agents and her- LANDOLT PJ (1993) Effects of host plant leaf bivores. Ecology, 69, 278-283. damage on cabbage looper moth attrac- KOST C & HEIL M (2005a) Increased avail- tion and oviposition. Entomologia Experi- ability of extrafloral nectar reduces herbi- mentalis et Applicata, 67, 79-85. vory in Lima beans (Phaseolus lunatus, LAUDERT D & WEILER EW (1998) Allene Fabaceae). Basic and Applied Ecology, 6, oxide synthase: A major control point in 237-248. Arabidopsis thaliana octadecanoid signal- KOST C & HEIL M (2005b) Herbivore-induced ling. Plant Journal, 15, 675-684. plant volatiles induce an indirect defence LEE GI & HOWE GA (2003) The tomato in neighbouring plants. Journal of Ecol- mutant spr1 is defective in systemin per- ogy, accepted. ception and the production of a systemic KRIPS OE, WILLEMS PEL, GOLS R, POST- wound signal for defense gene expres- HUMUS MA, GORT G & DICKE M (2001) sion. Plant Journal, 33, 567-576. Comparison of cultivars of ornamental LEITNER M, BOLAND W & MITHÖFER A crop Gerbera jamesonii on production of (2005) Direct and indirect defences spider mite-induced volatiles, and their induced by piercing-sucking and chewing attractiveness to the predator Phytoseiu- herbivores in Medicago truncatula. New lus persimilis. Journal of Chemical Ecol- Phytologist, 167, 597-606. ogy, 27, 1355-1372. LEÓN J, ROJO E & SÁNCHEZ-SERRANO JJ KRUMM T, BANDEMER K & BOLAND W (2001) Wound signalling in plants. Journal (1995) Induction of volatile biosynthesis in of Experimental Botany, 52, 1-9. the Lima bean (Phaseolus lunatus) by

144 References

LI L, LI CY, LEE GI & HOWE GA (2002) Dis- ways: Specificity and cross-talk. Journal tinct roles for jasmonate synthesis and ac- of Experimental Botany, 55, 181-188. tion in the systemic wound response of LUNAU K (2004) Adaptive radiation and co- tomato. Proceedings of the National evolution - Pollination biology case stud- Academy of Sciences of the United States ies. Organisms Diversity & Evolution, 4, of America, 99, 6416-6421. 207-224. LICHTENTHALER HK (1999) The 1-deoxy-D- LÜTTGE U (1971) Structure and function of xylulose-5-phosphate pathway of isopre- plant glands. Annual Review of Plant noid biosynthesis in plants. Annual Physiology, 22, 23-44. Review of Plant Physiology and Plant Mo- MACKAY DA & WHALEN MA (1998) Associa- lecular Biology, 50, 47-65. tions between ants (Hymenoptera: Formi- LIU YD, JIN HL, YANG KY, KIM CY, BAKER B cidae) and Adriana Gaudich. (Euphor- & ZHANG SQ (2003) Interaction between biaceae) in East Gippsland. Australian two mitogen-activated protein kinases Journal of Entomology, 37, 335-339. during tobacco defense signaling. Plant MAFFEI M, BOSSI S, SPITELLER D, Journal, 34, 149-160. MITHÖFER A & BOLAND W (2004) LOEFFLER C, BERGER S, GUY A, DURAND Effects of feeding Spodoptera littoralis on T, BRINGMANN G, DREYER M, VON Lima bean leaves: I. Membrane poten- RAD U, DURNER J & MUELLER MJ tials, intracellular calcium variations, oral

(2005) B1-phytoprostanes trigger plant secretions, and regurgitate components. defense and detoxification responses. Plant Physiology, 134, 1752-1762. Plant Physiology, 137, 328-340. MAKINO B, KAWAI M, YAMAMOTO T, YA- LOUGHRIN JH, MANUKIAN A, HEATH RR & MAMURA H, BUTSUGAN Y, HAYASHI M TUMLINSON JH (1995) Volatiles emitted & OGAWA K (1992) Activated charcoal- by different cotton varieties damaged by mediated hydroxylation under very mild feeding beet armyworm larvae. Journal of conditions - Conversion of physalin-B into Chemical Ecology, 21, 1217-1227. 25-hydroxyphysalin-B. Journal of the LOUGHRIN JH, MANUKIAN A, HEATH RR, Chemical Society-Chemical Communica- TURLINGS TCJ & TUMLINSON JH tions, 1430-1431. (1994) Diurnal cycle of emission of MALDONADO AM, DOERNER P, DIXON RA, induced volatile terpenoids by herbivore- LAMB CJ & CAMERON RK (2002) A injured cotton plants. Proceedings of the putative lipid transfer protein involved in National Academy of Sciences of the systemic resistance signalling in Arabi- United States of America, 91, 11836- dopsis. Nature, 419, 399-403. 11840. MALECK K & DIETRICH RA (1999) Defense LÜCKER J, BOUWMEESTER HJ, SCHWAB on multiple fronts: How do plants cope W, BLAAS J, VAN DER PLAS LHW & with diverse enemies? Trends in Plant VERHOEVEN HA (2001) Expression of Science, 4, 215-219. Clarkia S-linalool synthase in transgenic MAQUET A, GUTIERREZ A & DEBOUCK DG petunia plants results in the accumulation (1990) Further biochemical evidence for of S-linalyl-β-D-glucopyranoside. Plant the existence of two gene pools in Lima Journal, 27, 315-324. beans. Annual Reports of the Bean LUDWIG AA, ROMEIS T & JONES JDG Improvement Cooperation, 33, 128-129. (2004) CDPK-mediated signalling path- MATILE L (1997) Phylogeny and evolution of the larval diet in the Sciaroidea (Diptera,

References 145

Bibionomorpha) since the Mesozoic. and elicitor-inducible AP2-domain tran- Memoires du Museum National d'Histoire scription factor, ORCA2. EMBO Journal, Naturelle, 173, 273-303. 18, 4455-4463. MATSUI K, KURISHITA S, HISAMITSU A & MEYER CR & MEYER VG (1961) Origin and KAJIWARA T (2000) A lipid-hydrolysing inheritance in nectariless cotton. Crop activity involved in hexenal formation. Bio- science, 1, 167-170. chemical Society Transactions, 28, 857- MICHAUD JP (1990) Observations on nectar 860. secretion in fireweed, Epilobium angusti- MATTIACCI L, DICKE M & POSTHUMUS MA folium L (Onagraceae). Journal of Apicul- (1994) Induction of parasitoid attracting tural Research, 29, 132-137. synomone in Brussel sprouts plants by MILLER B, MADILAO LL, RALPH S & feeding Pieris brassicae larvae: Role of BOHLMANN J (2005) Insect-induced mechanical damage and herbivore elici- conifer defense. White pine weevil and tor. Journal of Chemical Ecology, 20, methyl jasmonate induce traumatic resi- 2229-2247. nosis, de novo formed volatile emissions, MATTIACCI L, DICKE M & POSTHUMUS MA and accumulation of terpenoid synthase (1995) β-Glucosidase: An elicitor of herbi- and putative octadecanoid pathway tran- vore-induced plant odor that attracts host- scripts in Sitka spruce. Plant Physiology, searching parasitic wasps. Proceedings of 137, 369-382. the National Academy of Sciences of the MITCHELL-OLDS T, SIEMENS D & PEDER- United States of America, 92, 2036-2040. SEN D (1996) Physiology and costs of re- MCCALL PJ, TURLINGS TCJ, LOUGHRIN J, sistance to herbivory and disease in Bras- PROVEAUX AT & TUMLINSON JH sica. Entomologia Experimentalis et Ap- (1994) Herbivore-induced volatile emis- plicata, 80, 231-237. sions from cotton (Gossypium hirsutum L) MITHÖFER A, WANNER G & BOLAND W seedlings. Journal of Chemical Ecology, (2005) Effects of feeding Spodoptera 20, 3039-3050. littoralis on Lima bean leaves. II. Continu- MCCONN M, CREELMAN RA, BELL E, MUL- ous mechanical wounding resembling LET JE & BROWSE J (1997) Jasmonate insect feeding is sufficient to elicit herbi- is essential for insect defense Arabidop- vory-related volatile emission. Plant sis. Proceedings of the National Academy Physiology, 137, 1160-1168. of Sciences of the United States of Amer- MODY K & LINSENMAIR KE (2004) Plant- ica, 94, 5473-5477. attracted ants affect arthropod community MCKAY SAB, HUNTER WL, GODARD KA, structure but not necessarily herbivory. WANG SX, MARTIN DM, BOHLMANN J Ecological Entomology, 29, 217-225. & PLANT AL (2003) Insect attack and MONDOR EB & ADDICOTT JF (2003) Con- wounding induce traumatic resin duct spicuous extra-floral nectaries are induc- development and gene expression of ible in Vicia faba. Ecology Letters, 6, 495- (-)-pinene synthase in Sitka spruce. Plant 497. Physiology, 133, 368-378. MONTEITH LG (1960) Influence of plants MENKE FLH, CHAMPION A, KIJNE JW & other than the food plants of their host on MEMELINK J (1999) A novel jasmonate- host-finding by tachinid parasites. Cana- and elicitor-responsive element in the dian Entomologist, 92, 641-652. periwinkle secondary metabolite biosyn- MONTOYA T, NOMURA T, FARRAR K, KA- thetic gene Str interacts with a jasmonate- NETA T, YOKOTA T & BISHOP GJ

146 References

(2002) Cloning the tomato curl3 gene lication. ww.nhm.ac.uk/entomology/chalci- highlights the putative dual role of the leu- doids/index.html [accessed 05-Sep-2003]. cine-rich repeat receptor kinase tBRI1/ O'DONNELL PJ, CALVERT C, ATZORN R, SR160 in plant steroid hormone and pep- WASTERNACK C, LEYSER HMO & tide hormone signaling. Plant Cell, 14, BOWLES DJ (1996) Ethylene as a signal 3163-3176. mediating the wound response of tomato MORAN PJ & THOMPSON GA (2001) Mo- plants. Science, 274, 1914-1917. lecular responses to aphid feeding in O'DOWD DJ (1979) Foliar nectar production Arabidopsis in relation to plant defense and ant activity on a neotropical tree, pathways. Plant Physiology, 125, 1074- Ochroma pyramidale. Oecologia, 43, 233- 1085. 248. MUMM R, SCHRANK K, WEGENER R, O'DOWD DJ & CATCHPOLE EA (1983) Ants SCHULZ S & HILKER M (2003) Chemical and extrafloral nectaries: No evidence for analysis of volatiles emitted from Pinus plant protection in Helichrysum spp-ant in- sylvestris after induction by insect oviposi- teractions. Oecologia, 59, 191-200. tion. Journal of Chemical Ecology, 29, OHME-TAKAGI M & SHINSHI H (1995) Ethyl- 1235-1252. ene-inducible DNA-binding proteins that MUSSER RO, HUM-MUSSER SM, EICHEN- interact with an ethylene-responsive ele- SEER H, PEIFFER M, ERVIN G, MUR- ment. Plant Cell, 7, 173-182. PHY JB & FELTON GW (2002) Herbivory: OHME-TAKAGI M, SUZUKI K & SHINSHI H Caterpillar saliva beats plant defences - A (2000) Regulation of ethylene-induced new weapon emerges in the evolutionary transcription of defense genes. Plant and arms race between plants and herbivores. Cell Physiology, 41, 1187-1192. Nature, 416, 599-600. OLIVEIRA PS & FREITAS AVL (2004) Ant- NARVÁEZ-VÁSQUEZ J, FLORIN-CHRISTEN- plant-herbivore interactions in the SEN J & RYAN CA (1999) Positional neotropical cerrado savanna. Naturwis- specificity of a phospholipase A activity senschaften, 91, 557-570. induced by wounding, systemin, and oli- OLIVEIRA PS, RICO-GRAY V, DIAZ- gosaccharide elicitors in tomato leaves. CASTELAZO C & CASTILLO-GUEVARA Plant Cell, 11, 2249-2260. C (1999) Interaction between ants, extra- NESS JH (2003) Catalpa bignonioides alters floral nectaries and insect herbivores in extrafloral nectar production after herbi- Neotropical coastal sand dunes: Herbi- vory and attracts ant bodyguards. vore deterrence by visiting ants increases Oecologia, 134, 210-218. fruit set in Opuntia stricta (Cactaceae). NEVEU N, GRANDGIRARD J, NENON JP & Functional Ecology, 13, 623-631. CORTESERO AM (2002) Systemic re- OROZCO-CÁRDENAS ML, MCGURL B & lease of herbivore-induced plant volatiles RYAN CA (1993) Expression of an by turnips infested by concealed root- antisense prosystemin gene in tomato feeding larvae Delia radicum L. Journal of plants reduces resistance toward Man- Chemical Ecology, 28, 1717-1732. duca sexta larvae. Proceedings of the Na- NISHIDA R (2002) Sequestration of defensive tional Academy of Sciences of the United substances from plants by Lepidoptera. States of America, 90, 8273-8276. Annual Review of Entomology, 47, 57-92. OROZCO-CÁRDENAS ML, NARVÁEZ- NOYES JS (2003) Universal chalcidoidea VÁSQUEZ J & RYAN CA (2001) Hydro- database. world wide web electronic pub- gen peroxide acts as a second messen-

References 147

ger for the induction of defense genes in the Chemical Society C-Organic, 16, tomato plants in response to wounding, 1984-1997. systemin, and methyl jasmonate. Plant PEARCE G & RYAN CA (2003) Systemic sig- Cell, 13, 179-191. naling in tomato plants for defense OZAWA R, ARIMURA G, TAKABAYASHI J, against herbivores - Isolation and charac- SHIMODA T & NISHIOKA T (2000a) terization of three novel defense-signaling Involvement of jasmonate- and salicylate- glycopeptide hormones coded in a single related signaling pathways for the produc- precursor gene. Journal of Biological tion of specific herbivore-induced volatiles Chemistry, 278, 30044-30050. in plants. Plant and Cell Physiology, 41, PEARCE G, STRYDOM D, JOHNSON S & 391-398. RYAN CA (1991) A polypeptide from OZAWA R, SHIMODA T, KAWAGUCHI M, tomato leaves induces wound-inducible ARIMURA G, HORIUCHI J, NISHIOKA T proteinase-inhibitor proteins. Science, & TAKABAYASHI J (2000b) Lotus japoni- 253, 895-898. cus infested with herbivorous mites emits PEARCE G, MOURA DS, STRATMANN J & volatile compounds that attract predatory RYAN CA (2001a) Production of multiple mites. Journal of Plant Research, 113, plant hormones from a single polyprotein 427-433. precursor. Nature, 411, 817-820. PACINI E, NEPI M & VESPRINI JL (2003) PEARCE G, MOURA DS, STRATMANN J & Nectar biodiversity: A short review. Plant RYAN CA (2001b) RALF, a 5-kDa ubiqui- Systematics and Evolution, 238, 7-21. tous polypeptide in plants, arrests root PAPAJ DR & LEWIS AC (1993) Insect learn- growth and development. Proceedings of ing: ecological and evolutionary perspec- the National Academy of Sciences of the tives. Chapman & Hall, New York. United States of America, 98, 12843- PARÉ PW & TUMLINSON JH (1997) De novo 12847. biosynthesis of volatiles induced by insect PEMBERTON RW & LEE JH (1996) The influ- herbivory in cotton plants. Plant Physiol- ence of extrafloral nectaries on parasitism ogy, 114, 1161-1167. of an insect herbivore. American Journal PARÉ PW & TUMLINSON JH (1999) Plant of Botany, 83, 1187-1194. volatiles as a defense against insect her- PENG JY, LI ZH, XIANG H, HUANG JH, JIA bivores. Plant Physiology, 121, 325-331. SH, MIAO XX & HUANG YP (2005) Pre- PASCAL L & BELIN-DEPOUX M (1991) On liminary studies on differential defense the biological rhythm correlation of plant- responses induced during plant communi- ant association - The case of extrafloral cation. Cell Research, 15, 187-192. nectaries of American Malpighiaceae. PETITT FL, TURLINGS TCJ & WOLF SP Comptes rendus de l'Académie des sci- (1992) Adult experience modifies attrac- ences. Serie III, Sciences de la vie, 312, tion of the leafminer parasitoid Opius dis- 49-54. situs (Hymenoptera: Braconidae) to vola- PASCHOLD A, HALITSCHKE R & BALDWIN tile semiochemicals. Journal of Insect IT (2005) Using "mute" plants to translate Behavior, 5, 623-634. volatile signals. Plant Journal, in press. PICHERSKY E & GERSHENZON J (2002) PATTENDEN G & WEEDON BLL (1968) Caro- The formation and function of plant vola- tenoids and related compounds. part tiles: Perfumes for pollinator attraction and XVIII. Synthesis of cis-and di-cis-polyenes defense. Current Opinion in Plant Biology, by reactions of the Wittig type. Journal of 5, 237-243.

148 References

PICKETT JA, RASMUSSEN HB, WOOD- REGAL PJ (1977) Ecology and evolution of COCK CM, MATTHES W & NAPIER JA flowering plant dominance. Science, 196, (2003) Plant stress signalling: Under- 622-629. standing and exploiting plant-plant inter- RETTENMEYER CW (1961) Observations on actions. Biochemical Society Transac- the biology and taxonomy of flies found tions, 31, 123-127. over swarm raids of army ants (Diptera: POHNERT G, JUNG V, HAUKIOJA E, LEMPA Tachinidae, Conopidae). University of K & BOLAND W (1999) New fatty acid Kansas Science Bulletin, 42, 993-1066. amides from regurgitant of lepidopteran REYMOND P & FARMER EE (1998) Jas- (Noctuidae, Geometridae) caterpillars. monate and salicylate as global signals Tetrahedron, 55, 11275-11280. for defense gene expression. Current PRESTON CA, LAUE G & BALDWIN IT (2001) Opinion in Plant Biology, 1, 404-411. Methyl jasmonate is blowing in the wind, REYMOND P, WEBER H, DAMOND M & but can it act as a plant-plant airborne FARMER EE (2000) Differential gene signal? Biochemical Systematics and expression in response to mechanical Ecology, 29, 1007-1023. wounding and insect feeding in Arabidop- PRICE PW, BOUTON CE, GROSS P, MC- sis. Plant Cell, 12, 707-719. PHERON BA, THOMPSON JN & WEIS REYMOND P, BODENHAUSEN N, VAN AE (1980) Interactions among 3 trophic POECKE RMP, KRISHNAMURTHY V, levels - Influence of plants on interactions DICKE M & FARMER EE (2004) A con- between insect herbivores and natural served transcript pattern in response to a enemies. Annual Review of Ecology and specialist and a generalist herbivore. Systematics, 11, 41-65. Plant Cell, 16, 3132-3147. PYKE GH (1991) What does it cost a plant to RHOADES DF (1983) Responses of alder and produce floral nectar? Nature, 350, 58-59. willow to attack by tent caterpillars and QU N, SCHITTKO U & BALDWIN IT (2004) webworms - Evidence for pheromonal Consistency of Nicotiana attenuata's her- sensitivity of willows. Pages 55-68 in bivore- and jasmonate-induced transcrip- HEDIN PA (ed.) Plant resistance to tional responses in the allotetraploid spe- insects. American Chemical Society, cies Nicotiana quadrivalvis and Nicotiana Washington DC. clevelandii. Plant Physiology, 135, 539- RHYNE CL (1965) Inheritance of extrafloral 548. nectaries in cotton. Advancing Frontiers in RAGUSO RA (2004) Why are some floral nec- Plant Science, 13, 121-135. tars scented? Ecology, 85, 1486-1494. RICKSON FR (1971) Glycogen plastids in RAINE NE, WILLMER P & STONE GN (2002) Müllerian body cells of Cecropia peltata - Spatial structuring and floral avoidance a higher green plant. Science, 173, 344- behavior prevent ant-pollinator conflict in 347. a Mexican ant-acacia. Ecology, 83, 3086- RICO-GRAY V (1993) Use of plant-derived 3096. food resources by ants in the dry tropical RASHBROOK VK, COMPTON SG & LAW- lowlands of coastal Veracruz, Mexico. TON JH (1992) Ant-herbivore interactions: Biotropica, 25, 301-315. Reasons for the absence of benefits to a RODRÍGUEZ-CONCEPCIÓN M & BORONAT fern with foliar nectaries. Ecology, 73, A (2002) Elucidation of the methylerythri- 2167-2174. tol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A

References 149

metabolic milestone achieved through ge- to ants. American Journal of Botany, 73, nomics. Plant Physiology, 130, 1079- 185-189. 1089. RUHREN S & HANDEL SN (1999) Jumping RODRIGUEZ-REINOSO F (1998) The role of spiders (Salticidae) enhance the seed carbon materials in heterogeneous ca- production of a plant with extrafloral nec- talysis. Carbon, 36, 159-175. taries. Oecologia, 119, 227-230. ROJO E, SOLANO R & SÁNCHEZ-SERRANO RUTHER J & KLEIER S (2005) Plant-plant JJ (2003) Interactions between signaling signaling: Ethylene synergizes volatile compounds involved in plant defense. emission in Zea mays induced by expo- Journal of Plant Growth Regulation, 22, sure to (Z)-3-hexen-1-ol. Journal of 82-98. Chemical Ecology, 31, 2217-2222. RÖSE USR & TUMLINSON JH (2004) Vola- RUTHER J, REINECKE A & HILKER M (2002) tiles released from cotton plants in Plant volatiles in the sexual communica- response to Helicoverpa zea feeding tion of Melolontha hippocastani: Re- damage on cotton flower buds. Planta, sponse towards time-dependent bouquets 218, 824-832. and novel function of (Z)-3-hexen-1-ol as RÖTTGER U (1979) Untersuchungen zur a sexual kairomone. Ecological Entomol- Wirtswahl der Rübenfliege Pegomya ogy, 27, 76-83. betae (Diptera: Anthomyidae). I. Olfak- RYALS JA, NEUENSCHWANDER UH, WIL- torische Orientierung zur Wirtspflanze. LITS MG, MOLINA A, STEINER HY & Zeitschrift für Angewandte Entomologie, HUNT MD (1996) Systemic acquired 87, 337-348. resistance. Plant Cell, 8, 1809-1819. ROYO J, LEÓN J, VANCANNEYT G, ALBAR SABELIS MW & VAN DE BAAN HE (1983) JP, ROSAHL S, ORTEGO F, CASTA- Location of distant spider mite colonies by NERA P & SÁNCHEZ-SERRANO JJ phytoseiid predators: Demonstration of (1999) Antisense-mediated depletion of a specific kairomones emitted by Tetrany- potato lipoxygenase reduces wound in- chus urticae and Panonychus ulmi. Ento- duction of proteinase inhibitors and mologia Experimentalis et Applicata, 33, increases weight gain of insect pests. Pro- 303-314. ceedings of the National Academy of Sci- SABELIS MW & DE JONG MCM (1988) ences of the United States of America, 96, Should all plants recruit bodyguards? 1146-1151. Conditions for a polymorphic ESS of RUDGERS JA (2004) Enemies of herbivores synomone production in plants. Oikos, 53, can shape plant traits: Selection in a fac- 247-252. ultative ant-plant mutualism. Ecology, 85, SALGADO AG, P. G & DEBOUCK DG (1995) 192-205. Evidence for two gene pools of the Lima RUDGERS JA, STRAUSS SY & WENDEL JE bean, Phaseolus lunatus L, in the Ameri- (2004) Trade-offs among anti-herbivore cas. Genetic Resources and crop Evolu- resistance traits: Insights from Gos- tion, 42, 15-28. sypieae (Malvaceae). American Journal of SCHAEFER M, BOHLKEN H, REICHSTEIN H, Botany, 91, 871-880. SATTLER K, SCHMIDT E, SICK F & TIS- RUFFNER GA & CLARK WD (1986) Extraflo- CHLER W (1994) Brohmer - Fauna von ral nectar of Ferocactus acanthodes (Cac- Deutschland. Quelle und Meyer Bestim- taceae): Composition and its importance mungsbücher, Heidelberg.

150 References

SCHEER JM & RYAN CA (2002) The systemin SÉGUY E (1951) Atlas des Diptères de receptor SR160 from Lycopersicon peru- France, Belgique, Suisse. Boubée, Paris. vianum is a member of the LRR receptor SEO HS, SONG JT, CHEONG JJ, LEE YH, kinase family. Proceedings of the National LEE YW, HWANG I, LEE JS & CHOI YD Academy of Sciences of the United States (2001) Jasmonic acid carboxyl methyl- of America, 99, 9585-9590. transferase: A key enzyme for jasmonate- SCHILMAN PE & ROCES F (2003) Assess- regulated plant responses. Proceedings of ment of nectar flow rate and memory for the National Academy of Sciences of the patch quality in the ant Camponotus United States of America, 98, 4788-4793. rufipes. Animal Behaviour, 66, 687-693. SEO S, SANO H & OHASHI Y (1999) Jas- SCHITTKO U, PRESTON CA & BALDWIN IT monate-based wound signal transduction (2000) Eating the evidence? Manduca requires activation of WIPK, a tobacco sexta larvae can not disrupt specific jas- mitogen-activated protein kinase. Plant monate induction in Nicotiana attenuata Cell, 11, 289-298. by rapid consumption. Planta, 210, 343- SEO S, SETO H, KOSHINO H, YOSHIDA S & 346. OHASHI Y (2003) A diterpene as an en- SCHMELZ EA, ALBORN HT & TUMLINSON dogenous signal for the activation of JH (2001) The influence of intact-plant defense responses to infection with To- and excised-leaf bioassay designs on bacco mosaic virus and wounding in to- volicitin- and jasmonic acid-induced ses- bacco. Plant Cell, 15, 863-873. quiterpene volatile release in Zea mays. SEO S, OKAMOTO M, SETO H, ISHIZUKA K, Planta, 214, 171-179. SANO H & OHASHI Y (1995) Tobacco SCHMELZ EA, ALBORN HT, BANCHIO E & MAP kinase: A possible mediator in TUMLINSON JH (2003) Quantitative rela- wound signal transduction pathways. tionships between induced jasmonic acid Science, 270, 1988-1992. levels and volatile emission in Zea mays SHARON-ASA L, SHALIT M, FRYDMAN A, during Spodoptera exigua herbivory. BAR E, HOLLAND D, OR E, LAVI U, Planta, 216, 665-673. LEWINSOHN E & EYAL Y (2003) Citrus SCHMIDT DD, KESSLER A, KESSLER D, fruit flavor and aroma biosynthesis: Isola- SCHMIDT S, LIM M, GASE K & BALD- tion, functional characterization, and de- WIN IT (2004) Solanum nigrum: A model velopmental regulation of Cstps1, a key ecological expression system and its gene in the production of the sesquiter- tools. Molecular Ecology, 13, 981-995. pene aroma compound valencene. Plant SCUTAREANU P, BRUIN J, POSTHUMUS Journal, 36, 664-674. MA & DRUKKER B (2003) Constitutive SHIMODA T, TAKABAYASHI J, ASHIHARA W and herbivore-induced volatiles in pear, & TAKAFUJI A (1997) Response of alder and hawthorn trees. Chemoecology, predatory insect Scolothrips takahashii 13, 63-74. toward herbivore-induced plant volatiles SCUTAREANU P, DRUKKER B, BRUIN J, under laboratory and field conditions. POSTHUMUS MA & SABELIS MW Journal of Chemical Ecology, 23, 2033- (1997) Volatiles from Psylla-infested pear 2048. trees and their possible involvement in at- SHIMODA T, OZAWA R, ARIMURA G, TAKA- traction of anthocorid predators. Journal BAYASHI J & NISHIOKA T (2002) Olfac- of Chemical Ecology, 23, 2241-2260. tory responses of two specialist insect predators of spider mites toward plant

References 151

volatiles from Lima bean leaves induced and ethylene-response-factor-1. Genes & by jasmonic acid and/or methyl salicylate. Development, 12, 3703-3714. Applied Entomology and Zoology, 37, SPITELLER D & BOLAND W (2003) N-(17- 535-541. acyloxy-acyl)-glutamines: Novel surfac- SHIOJIRI K, TAKABAYASHI J, YANO S & tants from oral secretions of lepidopteran TAKAFUJI A (2000) Flight response of larvae. Journal of Organic Chemistry, 68, parasitoids toward plant-herbivore com- 8743-8749. plexes: A comparative study of two para- SPITELLER D, POHNERT G & BOLAND W sitoid-herbivore systems on cabbage (2001) Absolute configuration of volicitin, plants. Applied Entomology and Zoology, an elicitor of plant volatile biosynthesis 35, 87-92. from lepidopteran larvae. Tetrahedron SHONLE I & BERGELSON J (1995) Interplant Letters, 42, 1483-1485. communication revisited. Ecology, 76, SPOEL SH, KOORNNEEF A, CLAESSENS 2660-2663. SMC, KORZELIUS JP, VAN PELT JA, SHULAEV V, SILVERMAN P & RASKIN I MUELLER MJ, BUCHALA AJ, MÉTRAUX (1997) Airborne signalling by methyl sali- JP, BROWN R, KAZAN K, VAN LOON cylate in plant pathogen resistance. LC, DONG XN & PIETERSE CMJ (2003) Nature, 385, 718-721. NPR1 modulates cross-talk between sali- SIVASANKAR S, SHELDRICK B & ROTH- cylate- and jasmonate-dependent defense STEIN SJ (2000) Expression of allene pathways through a novel function in the oxide synthase determines defense gene cytosol. Plant Cell, 15, 760-770. activation in tomato. Plant Physiology, STANJEK V, HERHAUS C, RITGEN U, 122, 1335-1342. BOLAND W & STÄDLER E (1997) SKIRI HT, GALIZIA CG & MUSTAPARTA H Changes in the leaf surface chemistry of (2004) Representation of primary plant Apium graveolens (Apiaceae) stimulated odorants in the antennal lobe of the moth by jasmonic acid and perceived by a spe- Heliothis virescens using calcium imaging. cialist insect. Helvetica Chimica Acta, 80, Chemical Senses, 29, 253-267. 1408-1420. SMITH MR (1947) A generic and subgeneric STAPEL JO, CORTESERO AM, DEMORAES synopsis of the United-States ants, based CM, TUMLINSON JH & LEWIS WJ (1997) on the workers (Hymenoptera, Formici- Extrafloral nectar, honeydew, and sucrose dae). American Midland Naturalist, 37, effects on searching behavior and effi- 521-647. ciency of Microplitis croceipes (Hymenop- SOBRINHO TG, SCHOEREDER JH, RODRI- tera: Braconidae) in cotton. Environmental GUES LL & COLLEVATTI RG (2002) Ant Entomology, 26, 617-623. visitation (Hymenoptera: Formicidae) to STASWICK PE & TIRYAKI I (2004) The extrafloral nectaries increases seed set oxylipin signal jasmonic acid is activated and seed viability in the tropical weed by an enzyme that conjugates it to isoleu- Triumfetta semitriloba. Sociobiology, 39, cine in Arabidopsis. Plant Cell, 16, 2117- 353-368. 2127. SOLANO R, STEPANOVA A, CHAO QM & STEIDLE JLM & VAN LOON JJA (2002) ECKER JR (1998) Nuclear events in Chemoecology of parasitoid and predator ethylene signaling: A transcriptional cas- oviposition behaviour. Pages 291-317 in cade mediated by ethylene-insensitive3 HILKER M & MEINERS T (eds.)

152 References

Chemoecology of insect eggs and egg ted by herbivore-infested plants: Relative deposition. Blackwell, London. influence of plant and herbivore. STEPHENSON AG (1982) The role of the Chemoecology, 2, 1-6. extrafloral nectaries of Catalpa speciosa TAKABAYASHI J, DICKE M & POSTHUMUS in limiting herbivory and increasing fruit MA (1994a) Volatile herbivore-induced production. Ecology, 63, 663-669. terpenoids in plant-mite interactions - STOUT MJ, WORKMAN J & DUFFEY SS Variation caused by biotic and abiotic fac- (1994) Differential induction of tomato tors. Journal of Chemical Ecology, 20, foliar proteins by arthropod herbivores. 1329-1354. Journal of Chemical Ecology, 20, 2575- TAKABAYASHI J, TAKAHASHI S, DICKE M & 2594. POSTHUMUS MA (1995) Developmental STOUT MJ, FIDANTSEF AL, DUFFEY SS & stage of herbivore Pseudaletia separata BOSTOCK RM (1999) Signal interactions affects production of herbivore-induced in pathogen and insect attack: Systemic synomone by corn plants. Journal of plant-mediated interactions between Chemical Ecology, 21, 273-287. pathogens and herbivores of the tomato, TAKABAYASHI J, DICKE M, TAKAHASHI S, Lycopersicon esculentum. Physiological POSTHUMUS MA & VANBEEK TA and Molecular Plant Pathology, 54, 115- (1994b) Leaf age affects composition of 130. herbivore-induced synomones and attrac- STRÄßNER M (1999) Klimadiagramme der tion of predatory mites. Journal of Chemi- Erde - Teil 2: Asien, Lateinamerika, Afrika, cal Ecology, 20, 373-386. Australien und Ozeanien, Polarländer. TAKAHASHI Y, BERBERICH T, MIYAZAKI A, Dortmunder Vertrieb für Bau- und SEO S, OHASHI Y & KUSANO T (2003) Planungsliteratur, Dortmund. Spermine signalling in tobacco: Activation STRAUSS SY, RUDGERS JA, LAU JA & IR- of mitogen-activated protein kinases by WIN RE (2002) Direct and ecological spermine is mediated through mitochon- costs of resistance to herbivory. Trends in drial dysfunction. Plant Journal, 36, 820- Ecology and Evolution, 17, 278-285. 829. STRONG DR & LARSSON S (1994) Is the TAMAYO MC, RUFAT M, BRAVO JM & SAN evolution of herbivore resistance influ- SEGUNDO B (2000) Accumulation of a enced by parasitoids? Pages 261-276 in maize proteinase inhibitor in response to HAWKINS BA & SHEEHAN W (eds.) wounding and insect feeding and charac- Parasitoid community ecology. Oxford terization of its activity toward digestive University Press, Oxford. proteinases of Spodoptera littoralis larvae. SZEKERES M (2003) Brassinosteroid and Planta, 211, 62-71. systemin: Two hormones perceived by the TEMPEL AS (1983) Bracken fern (Pteridium same receptor. Trends in Plant Science, aquilinum) and nectar-feeding ants: A 8, 102-104. nonmutualistic interaction. Ecology, 64, TAKABAYASHI J & DICKE M (1996) Plant- 1411-1422. carnivore mutualism through herbivore- THALER JS (1999) Jasmonate-inducible plant induced carnivore attractants. Trends in defences cause increased parasitism of Plant Science, 1, 109-113. herbivores. Nature, 399, 686-688. TAKABAYASHI J, DICKE M & POSTHUMUS THALER JS, FIDANTSEF AL, DUFFEY SS & MA (1991) Variation in composition of BOSTOCK RM (1999) Trade-offs in plant predator-attracting allelochemicals emit- defense against pathogens and herbi-

References 153

vores: A field demonstration of chemical induced plant odors by host-seeking para- elicitors of induced resistance. Journal of sitic wasps. Science, 250, 1251-1253. Chemical Ecology, 25, 1597-1609. TURLINGS TCJ, WÄCKERS FL, VET LEM, THISTLE HW, PETERSON H, ALLWINE G, LEWIS JM & TUMLINSON JH (1993) LAMB B, STRAND T, HOLSTEN EH & Learning of host-finding cues by hymen- SHEA PJ (2004) Surrogate pheromone opterous parasitoids. Pages 51-78 in PA- plumes in three forest trunk spaces: Com- PAJ DR & LEWIS AC (eds.) Insect learn- posite statistics and case studies. Forest ing - Ecology and evolutionary perspec- Science, 50, 610-625. tives. Chapman & Hall, New York. THOMA I, LOEFFLER C, SINHA AK, GUPTA TURLINGS TCJ, LOUGHRIN JH, MCCALL PJ, M, KRISCHKE M, STEFFAN B, ROITSCH RÖSE USR, LEWIS WJ & TUMLINSON T & MUELLER MJ (2003) Cyclopente- JH (1995) How caterpillar-damaged plants none isoprostanes induced by reactive protect themselves by attracting parasitic oxygen species trigger defense gene acti- wasps. Proceedings of the National Acad- vation and phytoalexin accumulation in emy of Sciences of the United States of plants. Plant Journal, 34, 363-375. America, 92, 4169-4174. TILMAN D (1978) Cherries, ants and tent cat- TURNER JG, ELLIS C & DEVOTO A (2002) erpillars - Timing of nectar production in The jasmonate signal pathway. Plant Cell, relation to susceptibility of caterpillars to 14, S153-S164. ant predation. Ecology, 59, 686-692. ULLOA RM, RAICES M, MACINTOSH GC, TOMITA A, MORI S & TAMAI Y (1971) Cata- MALDONADO S & TELLEZ-INON MT lytic behavior of carbon on oxidation of (2002) Jasmonic acid affects plant mor- cyclohexene. Carbon, 9, 224-225. phology and calcium-dependent protein TRELEASE W (1881) The foliar nectar gland kinase expression and activity in Solanum of Populus. Botanical Gazette, 6, 284- tuberosum. Physiologia Plantarum, 115, 290. 417-427. TRUITT CL, WEI HX & PARÉ PW (2004) A VAN ALPHEN JJM & JERVIS MA (1996). For- plasma membrane protein from Zea mays aging behaviour in JERVIS M & KIDD N binds with the herbivore elicitor volicitin. (eds.) Insect natural enemies. Chapman & Plant Cell, 16, 523-532. Hall, London. TSCHARNTKE T, THIESSEN S, DOLCH R & VAN DEN BOOM CEM, VAN BEEK TA, BOLAND W (2001) Herbivory, induced POSTHUMUS MA, DE GROOT A & resistance, and interplant signal transfer DICKE M (2004) Qualitative and quantita- in Alnus glutinosa. Biochemical Systemat- tive variation among volatile profiles in- ics and Ecology, 29, 1025-1047. duced by Tetranychus urticae feeding on TURLINGS TCJ & TUMLINSON JH (1991) Do plants from various families. Journal of parasitoids use herbivore-induced plant Chemical Ecology, 30, 69-89. chemical defences to locate hosts? VAN DER FITS L & MEMELINK J (2000) Florida Entomologist, 74, 42-50. ORCA3, a jasmonate-responsive tran- TURLINGS TCJ & BENREY B (1998) Effects scriptional regulator of plant primary and of plant metabolites on the behavior and secondary metabolism. Science, 289, development of parasitic wasps. Eco- 295-297. science, 5, 321-333. VAN DER MEIJDEN E & KLINKHAMER PGL TURLINGS TCJ, TUMLINSON JH & LEWIS (2000) Conflicting interests of plants and WJ (1990) Exploitation of herbivore-

154 References

the natural enemies of herbivores. Oikos, genic potato plants leads to an increase in 89, 202-208. aphid performance. Proceedings of the VAN DER PUTTEN WH, VET LEM, HARVEY National Academy of Sciences of the JA & WÄCKERS FL (2001) Linking United States of America, 98, 8139-8144. above- and belowground multitrophic VANDER MEER R, BREED M, ESPELIE K & interactions of plants, herbivores, patho- WINSTON M (1998) Pheromone commu- gens, and their antagonists. Trends in nication in social insects: Ants, wasps, Ecology and Evolution, 16, 547-554. bees, and termites. Westview Press, VAN DORT HM, JAGERS PP, TERHEIDE R & Boulder. VANDERWEERDT AJA (1993) Narcissus VET LEM & DICKE M (1992) Ecology of trevithian and Narcissus geranium - infochemical use by natural enemies in a Analysis and synthesis of compounds. tritrophic context. Annual Review of Journal of Agricultural and Food Chemis- Entomology, 37, 141-172. try, 41, 2063-2075. VET LEM, LEWIS WJ & CARDÉ RT (1995) VAN LOON JJA, DE BOER JG & DICKE M Parasitoid foraging and learning. Pages (2000) Parasitoid-plant mutualism: Parasi- 65-101 in CARDÉ R & BELL WJ (eds.) toid attack of herbivore increases plant Chemical ecology of insects. Vol. 2. reproduction. Entomologia Experimentalis Chapman & Hall, New York. et Applicata, 97, 219-227. VIJAYAN P, SHOCKEY J, LEVESQUE CA, VAN POECKE RMP & DICKE M (2004) Indi- COOK RJ & BROWSE J (1998) A role for rect defence of plants against herbivores: jasmonate in pathogen defense of Arabi- Using Arabidopsis thaliana as a model dopsis. Proceedings of the National plant. Plant Biology, 6, 387-401. Academy of Sciences of the United States VAN POECKE RMP, POSTHUMUS MA & of America, 95, 7209-7214. DICKE M (2001) Herbivore-induced vola- VISSER JH (1986) Host odor perception in tile production by Arabidopsis thaliana phytophagous insects. Annual Review of leads to attraction of the parasitoid Cote- Entomology, 31, 121-144. sia rubecula: Chemical, behavioral, and VISSER JH & AVÉ DA (1978) General green gene-expression analysis. Journal of leaf volatiles in the olfactory orientation of Chemical Ecology, 27, 1911-1928. the Colorado beetle, Leptinotarsa decem- VAN RIJN PCJ & TANIGOSHI LK (1999) The lineata. Entomologia Experimentalis et contribution of extrafloral nectar to sur- Applicata, 24, 538-549. vival and reproduction of the predatory VOELCKEL C & BALDWIN IT (2004) General- mite Iphiseius degenerans on Ricinus ist and specialist lepidopteran larvae elicit communis. Experimental & Applied different transcriptional responses in Nico- Acarology, 23, 281-296. tiana attenuata, which correlate with larval VAN TOL R, VAN DER SOMMEN ATC, BOFF FAC profiles. Ecology Letters, 7, 770-775. MIC, VAN BEZOOIJEN J, SABELIS MW VOELCKEL C, SCHITTKO U & BALDWIN IT & SMITS PH (2001) Plants protect their (2001) Herbivore-induced ethylene burst roots by alerting the enemies of grubs. reduces fitness costs of jasmonate- and Ecology Letters, 4, 292-294. oral secretion-induced defenses in Nico- VANCANNEYT G, SANZ C, FARMAKI T, tiana attenuata. Oecologia, 127, 274-280. PANEQUE M, ORTEGO F, CASTANERA VÖLKL W & SULLIVAN DJ (2000) Foraging P & SÁNCHEZ-SERRANO JJ (2001) behaviour, host plant and host location in Hydroperoxide lyase depletion in trans- the aphid hyperparasitoid Euneura auga-

References 155

rus. Entomologia Experimentalis et Appli- WASTERNACK C & PARTHIER B (1997) cata, 97, 47-56. Jasmonate-signalled plant gene expres- VON DAHL CC & BALDWIN IT (2004) Methyl sion. Trends in Plant Science, 2, 302-307. jasmonate and cis-jasmone do not dis- WEISSBECKER B, VAN LOON JJA, POST- pose of the herbivore-induced jasmonate HUMUS MA, BOUWMEESTER HJ & burst in Nicotiana attenuata. Physiologia DICKE M (2000) Identification of volatile Plantarum, 120, 474-481. potato sesquiterpenoids and their olfac- VON WETTSTEIN R (1889) Über die Compo- tory detection by the two-spotted stinkbug siten der österreichisch-ungarischen Flora Perillus bioculatus. Journal of Chemical mit zuckerabscheidenden Hüllschuppen. Ecology, 26, 1433-1445. Sitzungsberichte der Kaiserlichen Aka- WILDON DC, THAIN JF, MINCHIN PEH, demie der Wissenschaften, Mathema- GUBB IR, REILLY AJ, SKIPPER YD, tisch-Naturwissenschaftliche Classe, Ser. DOHERTY HM, O'DONNELL PJ & 1, 97, 570-589. BOWLES DJ (1992) Electrical signaling VOS M, BERROCAL SM, KARAMAOUNA F, and systemic proteinase-inhibitor induc- HEMERIK L & VET LEM (2001) Plant- tion in the wounded plant. Nature, 360, mediated indirect effects and the persis- 62-65. tence of parasitoid-herbivore communi- WINZ RA & BALDWIN IT (2001) Molecular ties. Ecology Letters, 4, 38-45. interactions between the specialist herbi- WÄCKERS FL & BEZEMER TM (2003) Root vore Manduca sexta (Lepidoptera, herbivory induces an above-ground indi- Sphingidae) and its natural host Nicotiana rect defence. Ecology Letters, 6, 9-12. attenuata. IV. Insect-induced ethylene WÄCKERS FL & BONIFAY C (2004) How to reduces jasmonate-induced nicotine ac- be sweet? Extrafloral nectar allocation by cumulation by regulating putrescine Gossypium hirsutum fits optimal defense N-methyltransferase transcripts. Plant theory predictions. Ecology, 85, 1512- Physiology, 125, 2189-2202. 1518. XU Y, CHANG PFL, LIU D, NARASIMHAN WÄCKERS FL, ZUBER D, WUNDERLIN R & ML, RAGHOTHAMA KG, HASEGAWA KELLER F (2001) The effect of herbivory PM & BRESSAN RA (1994) Plant defense on temporal and spatial dynamics of foliar genes are synergistically induced by nectar production in cotton and castor. ethylene and methyl jasmonate. Plant Annals of Botany, 87, 365-370. Cell, 6, 1077-1085. WANG CX, ZIEN CA, AFITLHILE M, WELTI R, XU YH, WANG JW, WANG S, WANG JY & HILDEBRAND DF & WANG XM (2000) CHEN XY (2004) Characterization of Involvement of phospholipase D in GaWRKY1, a cotton transcription factor wound-induced accumulation of jasmonic that regulates the sesquiterpene synthase acid in Arabidopsis. Plant Cell, 12, 2237- gene (+)-δ-cadinene synthase-A. Plant 2246. Physiology, 135, 507-515. WANG KLC, LI H & ECKER JR (2002) Ethyl- YAMAKAWA H, KAMADA H, SATOH M & ene biosynthesis and signaling networks. OHASHI Y (1998) Spermine is a salicy- Plant Cell, 14, S131-S151. late-independent endogenous inducer for WANG ZY & HE JX (2004) Brassinosteroid both tobacco acidic pathogenesis-related signal transduction - Choices of signals proteins and resistance against tobacco and receptors. Trends in Plant Science, 9, mosaic virus infection. Plant Physiology, 91-96. 118, 1213-1222.

156 References

YAMAKAWA H, KATOU S, SEO S, MITSU- SIPK. Proceedings of the National Aca- HARA I, KAMADA H & OHASHI Y (2004) demy of Sciences of the United States of Plant MAPK phosphatase interacts with America, 95, 7225-7230. calmodulins. Journal of Biological Chem- ZHU ZH, FINNERTY J, LU GQ & YANG RT istry, 279, 928-936. (2002) A comparative study of carbon

YANG K & JOHNSON MA (1977) Novel car- gasification with O2 and CO2 by density bon catalysis - Oxidation in basic solution. functional theory calculations. Energy & Journal of Organic Chemistry, 42, 3754- Fuels, 16, 1359-1368. 3754. ZIMMERMANN JG (1932) Über die extraflo- ZACHARIA R (2004). Desorption of gases ralen Nektarien der Angiospermen. Bota- from graphitic and porous carbon sur- nisches Zentrallblatt Beiheft, 49, 99-196. faces. PhD thesis, Fachbereich Biologie, ZIMMERMANN S, EHRHARDT T, PLESCH G Chemie, Pharmazie, Berlin. & MÜLLER-RÖBER B (1999) Ion chan- ZANGERL AR (2003) Evolution of induced nels in plant signaling. Cellular and Mo- plant responses to herbivores. Basic and lecular Life Sciences, 55, 183-203. Applied Ecology, 4, 91-103. ZORRO BI I, MAQUET A & BAUDOIN JP ZHANG SQ & KLESSIG DF (1998) The (2005) Mating system of wild Phaseolus tobacco wounding-activated mitogen- lunatus L and its relationship to population activated protein kinase is encoded by size. Heredity, 94, 153-158.

Selbständigkeitserklärung 157

12. Selbständigkeitserklärung oder am Ende jedes Manuskriptes Entsprechend der Promotionsordnung der (Acknowledgements) angegeben. Ich ha- Biologisch-Pharmazeutischen Fakultät der be nicht die Hilfe eines Promotionsbera- Friedrich-Schiller-Universität (Nr. III, § 5, ters in Anspruch genommen und Dritte Ziff. 3) erkläre ich, dass mir die geltende haben weder unmittelbar noch mittelbar Promotionsordnung der Fakultät bekannt geldwerte Leistungen von mir für Arbeiten ist. Die vorliegende Arbeit habe ich selb- erhalten, die im Zusammenhang mit dem ständig und nur unter Verwendung der Inhalt der vorliegenden Dissertation ste- angegebenen Hilfsmittel, persönlichen hen. Ich habe die Dissertation noch nicht Mitteilungen, Quellen und Literatur ange- als Prüfungsarbeit für eine staatliche oder fertigt habe. Personen, die an der experi- andere wissenschaftliche Prüfung einge- mentellen Durchführung, Auswertung des reicht. Ferner habe ich nicht versucht, die- Datenmaterials oder bei der Verfassung se Arbeit oder eine in wesentlichen Teilen der Manuskripte beteiligt waren, sind am ähnliche oder eine andere Abhandlung bei Beginn der Arbeit (2. Thesis outline – List einer anderen Hochschule als Dissertation of manuscripts and author’s contribution) einzureichen.

Jena, den . .2006 …………………………… Christian Kost

158 Curriculum vitae

13. Curriculum vitae

Personal data Name: Christian Kost Date of birth: 09.09.1975 Place of birth: Bad Sobernheim, Germany Home address: Franziskastr. 28, 55569 Monzingen, Germany marital status: unmarried E-mail: [email protected], [email protected]

Scientific career since 04/2003 PhD student at the Max Planck Institute for Chemical Ecology in Jena and the Friedrich-Schiller-University Jena.

08/01 – 03/03 Research project at the University of Würzburg in the Department of Animal Ecology and Tropical Biology of Prof. K. E. Linsenmair: The influence of driver ants (Dorylus (Anomma) nigricans on soil-living arthropod communities in Côte d’Ivoire, West-Africa.

07/00 – 07/01 Diploma thesis: Foraging strategies and diversity of the symbiotic Streptomycete in leaf-cutting ants (Formicidae: Attini) supervised by Dr. Rainer Wirth (Department of General Botany) and Dr. habil. Mat- thias Redenbach (Department of Genetics).

10/95 – 07/00 University of Kaiserslautern, Diploma in biology; Major: botany, minors: genetics, ecology and biochemistry

Education 01/87 - 08/95 Emanuel Felke Gymnasium Bad Sobernheim (Abitur) 07/83 - 06/86 Grundschule Monzingen

Practical training and stays abroad 10/04 - 12/04 Field work at sites close to Puerto Escondido, Oaxaca, Mexico. and 10/03 - 12/03

05/02 - 09/02 Field work in the Comoé National Park, Côte d’Ivoire, West-Africa. and 09/01 - 11/01

12/99 - 03/00 Field work in Paracou, French Guiana.

07/99 - 09/99 Volunteer on Utila, Honduras. 03/99 - 04/99 Field work in the Sahara, Tunisia.

Curriculum vitae 159

Publications

ARIMURA GI, KOST C & BOLAND W (2005) Herbivore-induced, indirect plant defences. Biochimica et Biophysica Acta: Lipids and Lipid Metabolism, 1734, 91-111.

KOST C, DE OLIVEIRA EG, KNOCH TA & WIRTH R (2005) Spatio-temporal permanence and plasticity of foraging trail systems in young and mature leaf-cutting ant colonies (Atta spp). Journal of Tropical Ecology, 21, 1-12.

KOST C & HEIL M (2005) Increased availability of extrafloral nectar reduces herbivory in Lima beans (Phaseolus lunatus, Fabaceae). Basic and Applied Ecology, 6, 237-248.

KOST C & HEIL M (2005) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants. Journal of Ecology, accepted.

DIETRICH R, PLOSS K, KOST C & HEIL M (2005) Pathogen resistance induction acceler- ates senescence in local and systemic tissues of Arabidopsis thaliana. submitted to Eu- ropean Journal of Plant Pathology.

LEAL IR, FISCHER A, KOST C, TABARELLI M & WIRTH R (2005) Ant-mediated protection against herbivores and nectar thieves in Passiflora coccinea flowers. submitted to Eco- science.

MEKEM SONWA M, KOST C, BIEDERMANN A, WEGENER R, SCHULZ S & BOLAND W (2005) Dehydrogenation of ocimene by active carbon: Artefact formation during headspace sampling from leaves of Phaseolus lunatus. submitted to Tetrahedron.

Oral presentations

KOST C, WEISSER W, BOLAND W & HEIL M (2005) Lima bean neighbourhood watch - Herbivore-induced volatiles induce an indirect defence in neighbouring plants. Kurt- Mothes-Doktoranden-Workshop, Leibniz Institut für Pflanzenbiochemie, Halle, Germany.

KOST C, BOLAND W & HEIL M (2005) Two induced defences benefit Lima bean in nature. 17. Irseer Naturstofftage der DECHEMA e.V., Irsee, Germany.

160 Curriculum vitae

KOST C & HEIL M (2004) Indirekte Verteidigung der Limabohne im Freiland. Multitrophische Interaktionen, Göttingen, Germany.

KOST C (2004) Duft oder Nektar? - Wie sich die Limabohne im Freiland gegen Fraßfeinde verteidigt. 27. Fränkisch-Mitteldeutsches Naturstoffchemiker-Treffen, Bayreuth, Ger- many.

KOST C, MODY K & LINSENMAIR KE (2002) Determinants of local biodiversity II: Driver ants. 1st International BIOTA-West Workshop, Abidjan, Côte d'Ivoire, West-Africa.

KOST C & WIRTH R (2001) Symbiotic soil bacteria as broadband fungicides? High diversity of Streptomycetes in leaf-cutting ant colonies in French Guiana. Gesellschaft für Tropenökologie, Bremen, Germany.

Poster presentations

KOST C & HEIL M (2005) Lima bean neighbourhood watch - Herbivore-induced volatiles induce an indirect defence in neighbouring plants. 21st annual meeting of the Inter- national Society for Chemical Ecology, Washington, United States of America.

KOST C, BOLAND W & HEIL M (2005) Two induced defences benefit Lima bean in nature. 17. Irseer Naturstofftage der DECHEMA e.V., Irsee, Germany.

KOST C & HEIL M (2004) Two induced indirect defences benefit Lima bean plants in nature. 12th Symposium on Insect-Plant Relationships, Berlin, Germany.

MODY K, KOST C & LINSENMAIR KE (2002) Small-scale mosaics of arthropod communities in natural habitats: patterns and determinants. 1st International BIOTA-West Work- shop, Abidjan, Côte d'Ivoire, West Africa.

MODY K, KOST C & LINSENMAIR KE (2001) Determinants of small-scale mosaics of arthropod communities in natural and anthropogenically disturbed habitats. Annual Status Seminar of BIOLOG, Bonn, Germany.

Curriculum vitae 161

WIRTH R, KOST C & SCHMIDT S (2001) Costs of trail construction and maintenance may affect the pattern of herbivory in leaf-cutting-ants. Verhandlungen der Gesellschaft für Ökologie, Band 31; Basel, Switzerland.

BÖTTCHER IG, KOST C, WIRTH R & REDENBACH M (2000) Identification and analysis of actinomycetes derived from fungus growing leaf cutting ants. Vereinigung für Allgemeine und Angewandte Mikrobiologie: Biologie bakterieller Naturstoffproduzenten, Bonn, Ger- many.

Jena, den . .2006 …………………………… Christian Kost