the online version of this article doi:10.1007/s10886 - 007 - 9404 - 0 contains supplementary material , which is available to authorized users .
plants respond to herbivore attack with an array of changes in plant chemistry , morphology , and physiology , which frequently result in the increased resistance of plants to further attack ( karban and baldwin 1997 ) .
induced resistance may be caused by direct effects on the herbivore through plant - derived toxic metabolites or anti - digestive and anti - nutritive compounds .
furthermore , plants may utilize indirect defenses that facilitate top - down control of herbivore populations by the herbivore s predators and parasitoids ( price et al .
one way a plant may attract beneficial arthropods is by providing suitable food sources such as extrafloral nectar ( efn ; koptur 1992 ) .
efn is an aqueous solution , with sugars and amino acids being the most abundant solutes ( ruffner and clark 1986 ; galetto and bernardello 1992 ; heil et al .
in addition , plants may increase the emission rate of volatile organic compounds ( vocs ) in response to herbivore attack .
vocs can serve as a cue that guides foraging parasitoids and predators to the feeding herbivore ( turlings et al .
the composition of the herbivore - induced volatile blend depends not only on the plant species or cultivar but also varies with the species and even the larval stage of the herbivore ( for review , see arimura et al .
2005 ) , thus providing highly reliable signals to the members of the third trophic level .
in many cases , plants do not rely on a single defense strategy but employ a complex array of different defensive mechanisms . for example , several plant species feature both voc emission and efn secretion ( arimura et al .
the presence of two indirect defenses within one individual plant gives rise to the questions whether both defenses contribute to plant fitness and how they interact in attracting beneficial arthropods ( price et al .
studies on the benefit of indirect defense traits in nature are generally rare ( but see thaler 1999 and kessler and baldwin 2001 ) .
no study to date has tried to study the defensive roles of two indirect defenses within one plant species simultaneously .
the lima bean ( phaseolus lunatus ) is a common model system in studies on induced indirect defenses .
heil ( 2004b ) showed that plants growing in the wild , which had been induced by exogenous application of the phytohormone jasmonic acid ( ja ) , responded by increasing both their voc emission and efn secretion .
the application of ja repeatedly ( i.e. , every 3  days ) resulted in benefits for the treated plants as reflected by a decreased herbivory rate and an increased seed set .
however , ja also affects fitness - relevant processes such as fruit development and ripening ( creelman and mullet 1997 ) .
thus , the multiple effects constrain the interpretation of the observed morphological changes as being exclusively attributable to induced defenses .
furthermore , kost and heil ( 2005 ) demonstrated that an artificial increase of the amount of available efn benefits lima bean in nature by attracting predacious and parasitoid arthropods .
these two pilot studies represented a starting point for unraveling whether both induced defense traits contribute to plant defense or whether efn secretion solely is responsible for lima bean protection .
we chose an experimental approach similar to the one used in heil ( 2004b ) : groups of five tendrils were used as experimental units ( fig .  1 ; table  1 ) .
the inclusion of two additional bean tendrils with artificially increased amounts of either vocs or efn enabled us to study the protective effect of the two defenses .
five groups of lima bean tendrils ( p. lunatus ) served as experimental unit with c no treatment , tc application of lanolin paste , ja spraying with jasmonic acid , v application of a synthetic volatile blend dissolved in lanolin paste , and n application of a synthetic mixture of efn . see table 1 for detailstable  1five chemical treatments and their effect on treated tendrilsgroup name ( abbreviation)treatmentdefensive traits ( i  =  induced , e  =  experimentally increased)effectcontrol ( c)untreatednoneuntreated controltreatment control ( tc)lanolin pastenonelanolin paste onlyja - treatment ( ja)javocs ( i ) efn ( i ) putative direct resistance ( i)induction of vocs , efn , and putative ja - dependent direct defenses ( heil 2004b)volatile treatment ( v)volatile mixture dissolved in lanolin pastevocs ( e ) efn ( i)induction of efn , attraction of arthropods to vocs and efn ( kost and heil 2006)nectar treatment ( n)synthetic mixture of efnefn ( e)attraction of arthropods to efn ( kost and heil 2005)ja jasmonic acid , efn extrafloral nectar , voc volatile organic compound experimental design .
five groups of lima bean tendrils ( p. lunatus ) served as experimental unit with c no treatment , tc application of lanolin paste , ja spraying with jasmonic acid , v application of a synthetic volatile blend dissolved in lanolin paste , and n application of a synthetic mixture of efn .
see table 1 for details five chemical treatments and their effect on treated tendrils ja jasmonic acid , efn extrafloral nectar , voc volatile organic compound we repeatedly applied these five treatments to address the following questions : ( 1 ) what is the relative contribution of the two indirect defenses  volatile emission and extrafloral nectar secretion to the overall herbivore defense of the lima bean ? ( 2 ) is there an additional influence of a putative ja - dependent direct defense ? and ( 3 )
what kind of arthropods ( including putative defenders ) are attracted to the treated tendrils ?
study site and species this study was conducted in the coastal area near puerto escondido in the state of oaxaca , mexico . the climate in the study area
is characterized by one main rainy season from june to october , which follows a bimodal distribution peaking in july and september .
annual rainfall averages between 1 and 1.4  m , and the mean annual temperature is 28c ( strssne 1999 ) . the two sites were used in previous studies ( heil 2004b ; kost and heil 2005 ) and located 15  km northwest of puerto escondido and about 3  km apart ( 1555.596n/09709.118w and 1555.357n/9708.336w ) . in this study ,
lima bean grows naturally along dirt roads that lead to extensively used pastures or plantations .
all experiments were performed on this native population of lima bean plants in 2003 and 2004 , during the transition from wet to dry season ( october to december ) .
design of the long - term experiment to reduce environmental and genotypic variability , 23 groups of five lima bean tendrils were selected as experimental units . within a unit ,
the maximum distance between the two outer tendrils was less than 3  m , and tendrils forming one group usually were part of the same plant individual , although this could not always be ensured because of the tangled growth of the lima bean .
all selected tendrils were trained along supporting ropes , and the tendrils of each group assigned randomly to one of five treatments ( for details , see the next section ) : tendrils were either left untreated ( control group ) or treated every 34  days with lanolin paste only ( treatment control group ) , sprayed with an aqueous solution of ja ( ja group ) , or supplied with a synthetic blend of vocs ( volatile group ) or efn ( efn group ; fig .  1 ; table  1 ) .
application of pure lanolin served as a procedural control to test for any effect caused by lanolin alone .
starting 27th october 2003 , the experiment lasted 25  days and comprised six applications . during this time
, the initial number of tendril groups was reduced to a final sample size of 17 because of cattle and human impact .
treatment of tendril groups tendrils of the ja group were sprayed with an aqueous solution of 1  mmol ja until runoff ( i.e. , approximately 10  l per cm leaf area ) .
levels of ja were adjusted to those of previous experiments in which similar concentrations were applied without phytotoxic effects becoming apparent ( heil 2004b ) .
the synthetic volatile blend and the synthetic mixture of efn were adjusted to mimic their natural models within 3  days post - induction according to previous experiments ( kost and heil 2005 , 2006 ) . however , the amount of vocs and efn cumulatively applied ranged largely below the physiological limit of a plant for producing the two indirect defenses within this period ( kost and heil 2005 , 2006 ) . the synthetic volatile blend consisted of 0.12  g ( r)-()-linalool , 0.13  g -caryophyllene , 0.19  g methyl salicylate , 0.26  g ( z)-jasmone ( all purchased from sigma - aldrich ) , 0.02  g ( 3z)-hexen-1-yl acetate ( avocado research chemicals ltd . ,
leysham , lancaster , uk ) , 0.85  g ( e , z)--ocimene ( mixture of e / z - isomers approximately 70:30 ; kindly provided by roger snowden , firmenich , geneva , switzerland ) , 0.63  g ( 3e)-4,8-dimethyl - nona-1,3,7-triene ( dmnt ) , and 0.9  g ( 3e,7e)-4,8,12-trimethyltrideca-1,3,7,11-tetraene ( tmtt ; synthesized by standard methods ; pattenden and weedon 1968 ) per microliter lanolin .
pure lanolin and lanolin paste that contained volatiles were spotted on green plastic strips attached to tendrils to prevent any diffusion of compounds into the treated plant .
a total of 120  l of paste were applied per five leaves of either the volatile- or the treatment - control group .
the synthetic efn consisted of an aqueous solution of 4.01  g l sucrose and 24.24  g l of each fructose and glucose . of this blend ,
40  l were applied with an eppendorf pipette directly to the extrafloral nectaries of every trifoliate leaf of the efn group .
comparison of treatments to ensure comparability between experimental groups of the long - term experiment , we quantified voc and efn levels of differently treated tendrils in preceding experiments .
the ability of our voc mixture to mimic the volatile emission of herbivore - damaged and ja - induced tendrils was examined by quantifying the vocs present in the headspace of tendrils that had received one of five treatments .
tendrils were either left untreated or treated with lanolin paste , infested for 48  h with a representative mixture of natural herbivores of lima bean ( six ensifera or caelifera and ten beetles of various species , i.e. , cerotoma ruficornis , gynandrobrotica guerreroensis , epilachna varivestis , and a curculionid species ; for details , see kost and heil 2006 ) , treated with a blend of synthetic vocs , or sprayed with a 1-mmol aqueous solution of ja .
groups of five tendrils that had received the different treatments originated from plants that were spaced apart at a maximum distance of approximately 3  m , had five leaves , matched each other in terms of leaf age and visual appearance , and were collected at the same time for simultaneous volatile collection .
treated tendrils were detached , immediately supplied with a water reservoir , and bagged in a pet foil (  bratenschlauch , toppits , minden , germany ) that does not emit detectable amounts of volatiles by itself .
emitted vocs were collected continuously on charcoal traps ( 1.5  mg charcoal , clsa filters , le ruisseau de montbrun , france ) by using air circulation , as described by donath and boland ( 1995 ) .
after 24  h , volatiles were eluted from the carbon trap with dichloromethane ( 40  l ) that contained 1-bromodecane ( 200  ng l ) as an internal standard .
samples were transferred to glass capillaries , sealed by melting the open end , and stored at < 5c for transport .
samples were analyzed on a gas chromatography  trace mass spectrometer ( thermo finnigan , http://www.thermofinnigan.com ) according to koch et al .
individual compounds ( peak areas ) were quantified with respect to the peak area of the internal standard . to compare quantitatively
the amount of efn secreted from tendrils after exposure to airborne vocs or induction with ja , 11 groups of three tendrils spread across the two sites were selected ; these groups were located < 1  m apart .
the first tendril within each group was treated with lanolin paste , the second with lanolin paste that contained volatiles , and the third was sprayed with ja .
the three tendrils were then placed in gauze bags ( mesh size , 0.5  mm ) , and a ring of sticky resin ( tangletrap , tanglefoot company , grand rapids , mi , usa ) was applied at the base as protection against flying and crawling nectar consumers .
after 24  h , the amount of newly produced efn was measured as the amount of secreted soluble solids ( i.e. , sugars , amino acids ; see heil et al .
2000 ) by quantifying the nectar volume with micro - capillaries and the nectar concentration with a portable , temperature - compensated refractometer ( heil et al .
both the amount of vocs emitted and efn secreted were referenced to the dry weight of the measured tendrils .
fitness - relevant plant parameters to assess the effect of treatments on the fitness of the study tendrils , the following fitness - relevant plant parameters were considered : number of leaves , inflorescences , living and dead shoot tips , and herbivory rate .
herbivory rate was estimated as percent leaf loss , as described in kost and heil ( 2005 ) , and the remaining parameters were quantified by counting .
all five parameters were assessed at the beginning of the experiment and after 25  days .
differences between these two values were calculated to determine the development of the respective parameter in the course of the study period .
insect counts and sticky traps the insect community that visited the treated bean tendrils was assessed by counting and with sticky traps .
two series of insect countings were performed , in which 20 tendril groups of the long - term experiment located at both sites were visited repeatedly .
the first series of countings started on day  7 and the second on day  18 after the onset of the experiment .
ten groups of tendrils were selected at each study site , and the number of ants , wasps , or flies present on the plants was recorded , as these insects represented the most abundant groups .
the first census was performed before tendril treatment at 8:00  a.m. thereafter , all tendrils were treated , and insects on all experimental tendrils were counted repeatedly every 2 or 3  h until midnight .
two additional censuses were performed at 9:00 and 10:00  a.m. on the following 2  days , resulting in a total of 14 monitorings .
the number of all insects counted per tendril was summed up to test for an effect of the treatment on the total number of insects observed . to assess the functional groups of insects attracted to the experimental tendrils ,
two sticky traps were attached with plastic strings to each tendril of 14 groups that were equally distributed between the study sites .
the sticky traps consisted of 100  cm pieces of green plastic foil that had been coated with a thin layer of a trapping adhesive ( tangletrap ) .
after 24  h of exposure , traps were collected and the insects transferred to 75% ethanol .
insects were identified to order or family level with keys and information provided by arnett ( 2000 ) , schaefer et al .
( 1994 ) , and noyes ( 2003 ) . on the basis of the natural history information provided by kelsey ( 1969 , 1981 ) , honomichl et al .
( 1998 ) , the collected arthropods were assigned to the following guilds according to nutritional or functional aspects : predator / entomophaga ( r ) , parasitoid ( p ) , utilization of plant - derived resources including floral or extrafloral nectar , pollen and honeydew ( s ) , frugivore ( f ) , herbivore and flower feeder ( h ) , detrivore including phytosaprophage and zoosaprophage ( d ) , blood - sucking and ectoparasitic ( b ) , and fungivore ( m ) .
statistical analysis amounts of volatiles emitted from differentially treated tendrils were compared with a kruskal  wallis rank sum test and a subsequent nonparametrical multiple test procedure of the behrens  fisher type ( munzel and hothorn 2001 ) by using the open source software r 2.3.1 ( http://www.r-project.org ) .
our randomized complete block design allowed us to analyze the data of the efn - induction experiment , the fitness - relevant plant parameters , and the cumulative insect numbers with a mixed - effect model ( univariate glm procedure ) with 
the following variables were transformed to meet the assumption of homogeneity of variances ( transformation in brackets ) : number of living shot tips and number of wasps ( square root ) , number of inflorescences and dead shoot tips ( log ) , number of leaves , cumulative number of ants and flies ( ln ) .
post hoc comparisons ( least significant difference , lsd ) were performed to test for statistically significant differences among treatments .
these statistical analyses were done using statistical package for the social sciences 13.0 ( spss inc . , chicago , usa ) .
comparison of treatments the headspace of lima bean tendrils , which had been treated with either a blend of synthetic vocs or with ja , largely resembled the volatile blend typically emitted from herbivore - damaged bean tendrils in terms of quantity and quality ( table  2 ) .
only ( 3z)-hexen-1-yl acetate was emitted in significantly lower amounts from the synthetic voc mixture than from herbivore - induced tendrils . also , ja- and herbivore - treated tendrils showed a marked quantitative similarity in the composition of their emitted volatile bouquets .
however , ja - treated tendrils emitted significantly lower amounts of ( r)-()-linalool and higher amounts of dmnt compared to herbivore - damaged tendrils .
in general , volatile blends emitted from herbivore- , volatile- , and ja - treated plants were characterized by high quantitative variability ( table  2 ) . 

table  2quantitative comparison of five - leaved lima bean tendrils , which were left untreated ( c ) ; treated with lanolin paste ( tc ) ; exposed to a mixture of herbivores , which are characteristic for the lima bean for 48  h ( h ) ; treated with the artificial volatile blend dissolved in lanolin paste ( v ) ; or sprayed with jacompoundemission ( avocais g 24  h)c ( n  =  8)tc ( n  =  7)h ( n  =  6)vocs ( n  =  7)ja ( n  =  7)kw test(3z)-hexen-1-yl acetate0.03    0.030.01    0.010.15    0.210.03    0.060.34    0.35=0.07(z)--ocimene0.00    0.000.00    0.000.03    0.050.13    0.100.07    0.12<0.001(e)--ocimene0.01    0.010.02    0.040.42    0.640.23    0.190.86    1.60<0.01(r)-(-)-linalool0.05    0.070.06    0.060.64    0.550.39    0.410.06    0.09<0.01dmnt0.04    0.050.07    0.070.76    0.911.18   
0.860.96    1.56<0.001c10h140.03    0.060.18    0.221.43    0.811.16    0.970.32    0.42<0.001methyl salicylate0.02    0.020.13    0.100.34    0.310.78    0.580.07    0.12<0.01c10h16o0.08 
  0.110.15    0.280.45    1.010.45    0.670.68    1.03=0.64(z)-jasmone0.01    0.020.00    0.000.21    0.221.21    1.210.35    0.44<0.01-caryophyllene0.01    0.010.03    0.051.23    2.100.75    0.660.26    0.28<0.001tmtt0.02 
  0.020.09    0.080.43    0.340.85    0.710.69    1.21<0.01the dominant emitted volatiles are given as mean peak area ( 95% confidence interval ) relative to the peak area of an internal standard per 24  h and per gram dry weight .
significant treatment effects are indicated by different superscript letters ( nonparametrical multiple test procedure of the behrens  fisher type , p  <  0.05 ) .
quantitative comparison of five - leaved lima bean tendrils , which were left untreated ( c ) ; treated with lanolin paste ( tc ) ; exposed to a mixture of herbivores , which are characteristic for the lima bean for 48  h ( h ) ; treated with the artificial volatile blend dissolved in lanolin paste ( v ) ; or sprayed with ja the dominant emitted volatiles are given as mean peak area ( 95% confidence interval ) relative to the peak area of an internal standard per 24  h and per gram dry weight .
significant treatment effects are indicated by different superscript letters ( nonparametrical multiple test procedure of the behrens  fisher type , p  <  0.05 ) .
a similar trend became obvious when efn secretion rates were compared among treatments . both the application of the synthetic volatile mixture and the ja treatment increased the efn secretion rate within 24  h compared to controls treated with lanolin only ( univariate analysis of variance , anova , p < 0.01 , n  =  11 ) .
the efn secretion rate in both treatments was twice that of the treatment control ( fig .  2 )
however , no significant difference was detected between the volatile- and the ja - treated group ( lsd post hoc test , p  >  0.05 ) . 

2effect of different treatments on the secretion rate of extrafloral nectar ( efn ) given in milligrams soluble solids per gram leaf dry mass per 24  h. comparisons between tendrils treated with lanolin paste ( tc ) , the synthetic volatile blend dissolved in lanolin ( v ) , and jasmonic acid ( ja ) are shown .
different letters indicate significant differences among treatments ( univariate anova , p  <  0.05 according to lsd post hoc test ) .
sample size was eleven groups of tendrils effect of different treatments on the secretion rate of extrafloral nectar ( efn ) given in milligrams soluble solids per gram leaf dry mass per 24  h. comparisons between tendrils treated with lanolin paste ( tc ) , the synthetic volatile blend dissolved in lanolin ( v ) , and jasmonic acid ( ja ) are shown .
different letters indicate significant differences among treatments ( univariate anova , p  <  0.05 according to lsd post hoc test ) .
sample size was eleven groups of tendrils effect of the treatments on fitness - relevant plant parameters the three treatments , ja , volatiles , and efn , significantly affected vegetative and reproductive plant traits after 25  days as compared to the controls .
tendril groups treated with volatiles or efn showed a significant increase in the number of newly produced leaves , shoot tips , and inflorescences , bore fewer dead shoot tips , and had suffered less herbivore damage than the controls ( fig . 
ja treatment also significantly decreased the number of dead shoot tips and the herbivory rate , whereas no difference could be detected for the number of living shoot tips , leaves , and inflorescences . according to a lsd post hoc test , the tendrils of the ja group ranked between the volatile and efn group on one hand and the two controls on the other ( fig .  3 ) . 

each tendril within groups of five lima bean tendrils received one of five treatments : no treatment ( c ) , treatment with lanolin paste ( tc ) , spraying with jasmonic acid ( ja ) , application of a synthetic volatile blend dissolved in lanolin paste ( v ) , and application of a synthetic mixture of efn ( n ) .
values above zero ( dashed line ) indicate an increase and values below a decrease in comparison to the starting situation .
asterisks indicate significant treatment effects ( univariate anova ; * p  <  0.05 , * * p 
<  0.01 , * * * p  <  0.001 ) , and different letters indicate significant differences among treatments ( lsd post hoc test , p  <  0.05 ) .
sample size was 20 groups of tendrils effect of five treatments on fitness - relevant plant parameters .
each tendril within groups of five lima bean tendrils received one of five treatments : no treatment ( c ) , treatment with lanolin paste ( tc ) , spraying with jasmonic acid ( ja ) , application of a synthetic volatile blend dissolved in lanolin paste ( v ) , and application of a synthetic mixture of efn ( n ) .
values above zero ( dashed line ) indicate an increase and values below a decrease in comparison to the starting situation .
asterisks indicate significant treatment effects ( univariate anova ; * p  <  0.05 , * * p  <  0.01 , * * * p  <  0.001 ) , and different letters indicate significant differences among treatments ( lsd post hoc test , p  <  0.05 ) .
sample size was 20 groups of tendrils effect of the treatments on insect abundance ants , wasps , and flies were the most abundant groups observed visiting the study tendrils , with ants being by far the dominant group .
their numeric abundance on the experimental tendrils exceeded that of flies 5- to 15-fold and that of wasps 20- to 30-fold ( fig . 
treatments were no treatment ( c ) , treatment with lanolin paste ( tc ) , spraying with jasmonic acid ( ja ) , application of a synthetic volatile blend dissolved in lanolin paste ( v ) , and application of a synthetic mixture of efn ( n ) . see table 1 for details .
fourteen censuses were performed within 3  days at two sites ( site 1 on day  7 and site 2 on day  18 after the beginning of the experiment ) .
<  0.05 , * * p  <  0.01 , * * * p  <  0.001 ) , and different letters indicate significant differences among treatments ( lsd post hoc test , p  <  0.05 ) .
sample size was 20 tendril groups effect of the five treatments on insect numbers visiting the lima bean .
treatments were no treatment ( c ) , treatment with lanolin paste ( tc ) , spraying with jasmonic acid ( ja ) , application of a synthetic volatile blend dissolved in lanolin paste ( v ) , and application of a synthetic mixture of efn ( n ) . see table 1 for details .
fourteen censuses were performed within 3  days at two sites ( site 1 on day  7 and site 2 on day  18 after the beginning of the experiment ) .
asterisks indicate significant treatment effects ( univariate anova ; * p  <  0.05 , * * p  <  0.01 , * * * p  <  0.001 ) , and different letters indicate significant differences among treatments ( lsd post hoc test , p  <  0.05 ) .
sample size was 20 tendril groups the five treatments significantly affected the insect visitation rate of the experimental tendrils .
the mean cumulative number of ants was increased significantly on tendrils treated with ja , volatiles , and efn relative to the two control tendrils ( fig .  4 , lsd post hoc test after univariate anova , p < 0.05 ) .
volatile treatment doubled the median cumulative number of ants on the experimental tendrils , whereas the ja and nectar treatment led to a threefold increase over the number on the untreated state .
correspondingly , the median wasp number ranged between 1.5 ( ja group ) , 2 ( volatile group ) , and 2.5 ( efn group ) , whereas these insects were significantly less frequently encountered on control tendrils ( fig .  4 ; lsd post hoc test after univariate anova , p < 0.05 ) .
flies responded less to the five treatments . despite the significantly increased visitation rate of flies to the ja - treated tendrils as compared to the controls ( fig .  4 , lsd post hoc test after univariate anova , p < 0.05 ) ,
no such effect was observed for voc- and efn - treated tendrils ( fig . 
the two latter groups were statistically indistinguishable from the ja - treated tendrils and both control groups ( fig .  4 , lsd post hoc test after univariate anova , p  >  0.05 ) .
community composition of arthropods visiting lima bean in total , 899 arthropods were caught on sticky traps , and > 94% were identified to the order or family level . among them , diptera ( 55% ) and hymenoptera ( 26% ) were the most abundant ( table  1 ; fig . 
other groups trapped included coleoptera ( 6% ) , araneida ( 5% ) , and thysanoptera ( 3% ) .
based on information derived from the literature , the trapped arthropods were assigned to feeding guilds , whereas multiple affiliations per taxon were allowed .
this analysis indicated that 73% of all trapped arthropods were characterized by parasitoid or predacious life habits and may , thus , be classified as potentially beneficial to the lima bean ( fig .  5 ) .
on the other hand , only 16% of the trapped arthropods were assigned to herbivorous or flower - feeding groups , which potentially could have had detrimental effects on the plant .
other plant - derived food sources , such as efn , pollen , or honeydew , are known to be used by 67% of all trapped arthropod groups , and 19% of all trapped taxa additionally rely on other food sources such as fungi , fruits , or detritus .
tourists and are likely to have a neutral effect on the lima bean ( fig .  5 ; table s1 ) . 

insert affiliation of trapped taxa to different guilds : predator / entomophaga ( r ) ; parasitoid ( p ) ; utilization of plant - derived resources including floral or extrafloral nectar , pollen and honeydew ( s ) ; frugivore ( f ) ; herbivore and flower feeder ( h ) ; detrivore including phytosaprophage and zoosaprophage ( d ) ; blood - sucking and ectoparasitic ( b ) ; and fungivore ( m ) .
arthropod groups were assigned to guilds according to nutritional or functional aspects whenever larval or adult stages feature the respective trait .
sample size was two traps per 14 tendril groups arthropods taxa trapped on sticky traps .
insert affiliation of trapped taxa to different guilds : predator / entomophaga ( r ) ; parasitoid ( p ) ; utilization of plant - derived resources including floral or extrafloral nectar , pollen and honeydew ( s ) ; frugivore ( f ) ; herbivore and flower feeder ( h ) ; detrivore including phytosaprophage and zoosaprophage ( d ) ; blood - sucking and ectoparasitic ( b ) ; and fungivore ( m ) .
arthropod groups were assigned to guilds according to nutritional or functional aspects whenever larval or adult stages feature the respective trait .
sample size was two traps per 14 tendril groups a closer look at the trapped arthropod taxa revealed that dolichopodidae ( 34% of all trapped diptera ) , phoridae ( 24% ) , and chloropidae ( 7% ) were the most abundantly trapped dipterans ( table s1 ) .
all three groups share parasitoid and predacious life habits and are occasionally known to feed on efn ( table s1 ) .
the individuals trapped by this superfamily of parasitoid wasps belonged to 16 different families , with eulopidae ( 13% of all trapped hymenoptera ) , encyrtidae ( 12% ) , and pteromalidae ( 8% ) being most frequently trapped . among the hymenopterans ,
formicidae ( 12% ) and braconidae ( 6% ) were the most often captured non - chalcid families .
comparison of treatments the aim of the present study was to study simultaneously the protective effect of the two indirect defenses , efn secretion and voc emission , on the lima bean under field conditions .
the performance of plants that were induced by spraying with ja and , thus , had increased amounts of vocs and efn , was monitored and compared to plants of which the amount of either vocs or efn was increased experimentally . an important prerequisite for any conclusion that can be drawn from such an experimental design is knowledge of the performance of the two indirect defenses under investigation .
a quantitative and qualitative comparison of the voc blends emitted from herbivore - damaged , volatile - treated , and ja - sprayed lima bean tendrils revealed  besides subtle differences  a pronounced similarity among these three groups with respect to the 13 dominant emitted compounds ( table  2 ) .
however , the volatile blends emitted from these three groups were characterized by a high degree of quantitative variation , an observation well known from literature ( kessler and baldwin 2001 ; fritsche - hoballah et al . 2002 ; rse and tumlinson 2004 ) . the question whether such differences hamper the ability of predators and parasitoids to locate
the voc - emitting plant has been studied intensively under laboratory conditions , where it has been shown that carnivorous arthropods can discriminate even minor differences in volatile blends offered ( e.g. , changes in the enantiomer ratios ; dicke et al .
1990 ) . a species of curculionid beetles , for example , which also was observed feeding on lima bean in this study ( c. kost , personal observation ) , in laboratory experiments , used vocs of slightly induced plants as a host - location cue , yet avoided plants with high induction levels ( heil 2004a ) .
in the field , plants generally show a higher quantitative and qualitative variability of their emitted vocs than under constant laboratory conditions ( gouinguene et al .
foraging predators should respond to this situation by relaxing the specificity of their search patterns ( dicke et al .
2003 ) . indeed , field studies on the attractiveness of vocs suggest that predatory and predacious insects are also attracted to jasmonate - induced plants ( thaler 1999 ; kessler and baldwin 2001 ) , even if the emitted volatiles differ substantially from blends emitted by herbivore - damaged plants ( farag and par 2002 ) .
moreover , field experiments indicate that the presence of single compounds can be sufficient to attract carnivorous insects ( james 2003 , 2005 ; james and price 2004 ) , which then act as indirect plant defenders ( kessler and baldwin 2001 ) . consequently , under our experimental conditions , the vocs of both ja- and volatile - treated tendrils likely have attracted plant defenders .
2 ) , thus , confirming recent results obtained for the same plant species under laboratory ( choh et al .
2006 ) and field conditions ( heil and kost 2006 ; kost and heil 2006 ) .
hence , the tendrils of the volatile group experienced the combined defensive effect of both vocs and efn to an extent comparable to the tendrils of the ja group .
these two defenses are not only connected by the shared signaling molecule ja ( heil 2004b ) but are also airborne vocs implicated in the induction of efn secretion within one ( heil and silva bueno 2007 ) or between two conspecific plant individuals ( kost and heil 2006 ) . consequently , this physiological linkage precludes an experimental separation of the two indirect defenses .
treatment effects on the plant tendrils of the ja , the volatile , and the efn group generally benefited from the respective treatments , as these tendrils suffered less herbivory by leaf - chewing herbivores and bore fewer dead shoot tips than the two control groups ( fig . 
the picture , however , changes when other fitness - relevant parameters are considered : the numbers of living shoot tips , leaves , and inflorescences differed significantly from the controls only in tendrils treated with vocs and efn ( fig . 
a possible explanation for the weaker effect experienced by the ja - treated tendrils could be that induction with ja incurred allocation costs to the treated tendrils , which were greater than costs imposed by the voc - induced efn secretion and absent in efn - treated tendrils ( heil 2002 ; strauss et al .
2002 ) . beyond eliciting stress - related responses , such as insect and disease resistance ,
ja is known to be involved in various physiological or morphological changes not necessarily related to resistance ( creelman and mullet 1997 ) .
given that these are costly in terms of metabolic resources , the induction of such processes may have affected the measured fitness parameters and could explain the weaker growth and reproductive status of ja - treated tendrils ( agrawal et al .
ja may also directly affect processes such as fruit development and ripening ( creelman and mullet 1997 ) .
an increased seed set of ja - treated plants , therefore , does not necessarily reflect an enhancement of the plant s defense status .
furthermore , external application of ja in elevated concentrations can , in principle , have detrimental effects on a plant and cause chlorosis , necrosis , or abscission of leaves ( husain et al .
in contrast , the leaves of ja - treated tendrils looked even healthier than those of both control tendrils .
additionally , ja application may have induced putative direct defenses ( halitschke and baldwin 2004 ) .
however , no such alternative defense strategy has yet been described for lima bean . in its close relative , p. vulgaris
, english - loeb and karban ( 1991 ) did not find evidence for induced direct resistance to spider mites .
however , the protective effect of a direct defense , which in our long - term experiment could have been induced after ja application , can not be excluded . in this case
, the direct defense did not significantly contribute to plant protection because tendrils with increased amounts of efn only ( efn group ) or volatiles and efn combined ( volatile group ) performed better than tendrils with volatiles , efn , and the putative direct defense ( ja group ; fig . 
the development of the fitness - relevant plant parameters measured in the ja - treated group and the two controls confirmed a preceding study ( heil 2004b ) . in both studies ,
ja treatment increased the number of newly produced leaves and decreased both the number of dead shoot tips , as well as the herbivory rate .
the induction of these two indirect defenses was shown to benefit lima bean plants in two independent studies performed in two consecutive years . by applying synthetic vocs and efn externally
, we could trace the observed effects back to these two kinds of defensive metabolites and corroborate the importance of efn secretion and voc emission for lima bean defense in nature .
treatment effects on the insect community ants were by far the dominant insect group observed on experimental tendrils , and they were significantly attracted to tendrils that experienced the ja , volatile , and efn treatment ( fig . 
although chemical cues are generally important for ants ( vander meer et al . 1998 ; keeling et al .
2004 ) , little is known about whether plant - derived volatiles also influence ant behavior .
some reports are available on the role of yet unidentified plant chemicals that orient ants to their host plant ( fiala and maschwitz 1990 ; agrawal and dubin - thaler 1999 ; djieto - lordon and dejean 1999a , b ) or facilitate within - host plant patrolling behavior ( brouat et al . 2000 ) . in lima bean
ants could use herbivore - induced volatiles as long - distance cues to detect patches of increased availability of efn , which simultaneously are characterized by the increased presence of herbivores ( i.e. , potential prey ) . however
, preliminary experiments with camponotus novogranadensis , one of the three dominant ant species that visit lima bean at the two study sites ( kost and heil 2005 ) did not support this hypothesis .
given the choice in a y - olfactometer , workers of c. novogranadensis neither preferred lanolin paste that contained volatiles over pure lanolin paste .
they also did not discriminate between ja - induced vs water - sprayed control plants .
however , in previous trials they significantly chose an arm with mashed banana ( c. kost , unpublished data ) .
more experiments of this kind will clarify the role of plant volatiles in ant foraging behavior .
in contrast to vocs , efn is a known attractant to ants ( koptur 1992 ) that has been reported to translate into enhanced plant protection in the vast majority of studies ( for review , see bentley 1977 ; koptur 1992 ; heil and mckey 2003 ; oliveira and freitas 2004 ) , although some studies did not detect a defensive effect ( odowd and catchpole 1983 ; boecklen 1984 ; becerra and venable 1989 ; rashbrook et al .
also , wasps were more attracted to the tendrils of the ja , volatile , and efn groups than to the control tendrils , yet in much smaller numbers than ants .
the majority of wasps trapped on experimental tendrils belonged to predacious or parasitoid families ( table s1 ) .
these observations suggest that not only ants but also wasps likely contributed to the protection of the treated tendrils .
because of the intrinsic physiological linkage between volatiles and efn , however , it remains unclear which indirect defense was mainly responsible for wasp attraction . as the emission of vocs from a lima bean plant
is correlated with its amount of efn secreted , it appears reasonable to assume that wasps may have used vocs as long - distance cues to detect patches with an increased herbivore density and/or efn availability .
comprehensive evidence for the volatile - mediated attraction of wasp is available from many laboratory - based studies ( e.g. , turlings et al . 1990 ; takabayashi et al .
1996 ) , yet relatively few field studies . among them , james ( 2005 ) identified methyl salicylate as an attractant for parasitic wasps such as encyrtidae and mymaridae . furthermore , braconid wasps were attracted to ( 3z)-hexen-1-yl acetate and ( z)-jasmone ( james 2005 ) .
these three compounds were also constituents of the synthetic volatile blend used in our study , and the above - mentioned parasitic wasps were also trapped on our experimental tendrils . in another study ,
dmnt ( another constituent of the lima bean s induced voc blend ) emitted from molasses grass ( melinis minutiflora ) significantly attracted parasitoid wasps in the y - tube olfactometer and was likely involved in increasing the parasitation rate of stem - borer larvae that were feeding on nearby growing maize plants ( khan et al .
wasps feeding on efn have been reported for several different plant species ( for review , see koptur 1992 ) , yet so far , only one study has also demonstrated a fitness - benefit for the efn - secreting plant ( cuautle and rico - gray 2003 ) . a more detailed analysis of the attractive effects of vocs and efn on wasps
4 ) , suggesting that flies were more attracted to synthetic efn than to airborne vocs .
the community of trapped diptera covered a diverse spectrum of feeding habits ranging from predacious or parasitoid over herbivorous to detritus- or fungi - feeding ( table s1 ) .
this heterogeneous composition complicates a clear functional assignment of the caught flies . in most cases
, the offered efn seems to have been exploited by the flies as an additional food source rather than having contributed to a large extent to plant protection . in this case
, the consumption of efn without providing plant protection would cause ecological costs because the efn - producing plant would be less protected against herbivores ( heil 2002 ; heil et al . 2004 ) . in summary , ecological studies on the benefit of indirect defenses have generally focused on the protective role of a single defensive trait . while this simplification is easy to understand from the viewpoint of experimental feasibility , such univariate approaches may be inappropriate because they do not appreciate the complex interplay of several plant defenses that can co - occur within one plant species ( duffey and stout 1996 ) .
the mere application of synthetic efn ( nectar group ) resulted in a fitness benefit that was always stronger or quantitatively similar to that experienced by tendrils of the ja and voc treatments ( fig . 
moreover , the number of ants observed on the experimental tendrils ( i.e. , typical efn feeders but less known to respond to vocs ) overwhelmingly exceeded the number of all other arthropod groups ( fig . 
these observations suggest that , under our experimental conditions , the presence of efn was more important for plant defense than was the voc - mediated attraction of arthropods .
however , the inducing effect of vocs on efn impeded an experimental separation of vocs and efn and , thus , the exclusion of an attractive effect of airborne vocs on flying or crawling arthropods .
this finding underlines the necessity of studying different plant traits such as indirect defenses simultaneously in their ecological context . studying them separately may provide a distorted picture of their true function and likely cause an under- or overestimation of their true effect
this issue needs to be addressed in future studies , which should focus especially on the role of volatiles and efn for short- and long - distance attraction of herbivores and plant defenders .
several of the arthropod taxa that were identified in this study could serve as possible targets for such analyses .
further laboratory and field - based experimentation is needed to study whether inductive situations exist , in which either the volatile emission or the efn secretion is differentially up- or down - regulated or if both defenses always respond similarly to herbivore attack .
below is the link to the electronic supplementary material : 
  arthropod taxa trapped on the experimental tendrils with sticky traps ( doc 168 kb ) .
 lima bean ( phaseolus lunatus ) features two indirect anti - herbivore defenses  emission of volatile organic compounds ( vocs ) and secretion of extrafloral nectar ( efn)which are both inducible upon herbivore damage . in a previous field study , 
 lima bean benefited from the simultaneous induction of the two defenses , yet it remained unclear whether both had contributed to plant protection . 
 our experimental approach aimed at studying the defensive role of both indirect defenses simultaneously . 
 tendrils were sprayed with jasmonic acid ( ja ) to induce both defenses , and performance was compared to that of others that were treated with a synthetic blend of either efn or vocs . confirming earlier results , ja treatment and application of the voc mixture induced efn secretion in treated tendrils in quantitatively similar amounts . 
 the composition of the applied synthetic blend of efn was adjusted to match the concentration of efn secreted from ja- and voc - treated tendrils . 
 repeated application of either enhanced the performance of several fitness - relevant plant parameters such as growth rate and flower production . 
 tendrils treated with ja showed a similar trend , yet some fitness - related parameters responded less to this treatment . 
 this suggests a minor importance of any putative ja - dependent direct defense traits or higher costs of ja - elicited responses as compared to vocs and efn , as otherwise ja - treated tendrils should have outperformed voc- and efn - treated tendrils . 
 moreover , the beneficial effect of applying synthetic efn alone equaled or exceeded that of vocs and ja . 
 ants were by far the dominant group among the arthropods that was attracted to ja- , voc- , or efn - treated tendrils . 
 the results suggest that efn plays a more important role as an indirect defense of lima bean than vocs or any other ja - responsive trait.electronic supplementary materialthe online version of this article doi:10.1007/s10886 - 007 - 9404 - 0 contains supplementary material , which is available to authorized users . 
Electronic supplementary material
Introduction
Methods and Materials
Results
Discussion
Electronic Supplementary Material