BACKGROUND
numerous microorganisms and arthropods are capable of transforming plant tissues into galls.
the galling habit is especially common among insects with more than  <dig>  known gall-forming species from several orders  <cit> .
gall induction has evolved convergently among and within various insect lineages and the multiple, independent origins of gall-formation indicate that this phenomenon is highly adaptive.
although the molecular mechanisms of gall induction remain to be unveiled, numerous ecological studies and phylogenetic analyses strongly suggest that the insects are in control of the gall traits, which they exploit for their own benefit  <cit> .
galls are thus considered as an extended phenotype of the inducer’s genes  <cit> .
serving as “incubators” that promote the development of the insects within, galls may have more than a single adaptive function  <cit> .
the proposed advantages of the galling habit fall into three main categories  <cit> :  microclimatic stabilization: galls protect the insects from unfavourable abiotic conditions such as high temperature and low humidity  nutrition: gall tissue provides an abundance of high quality nutrients and  defence: morphology and chemistry of the gall tissue protect the inducing insect from various natural enemies, including predators, parasitoids, pathogens and other herbivores.
this notion has been termed the enemy hypothesis  <cit> .
improved nutrition and defence is achieved by the insect’s ability to considerably manipulate its host plant’s morphology and physiology  <cit> .
primary and secondary plant compounds are not randomly distributed in the galled tissue as the inner layers on which the insects feed are enhanced sinks for photosynthates and may have low concentrations of secondary compounds, thus providing better nutrition for the gall insect  <cit> .
the outer, non-nutritive gall layers, on the other hand, may contain increased amounts of potentially defensive chemicals that could deter antagonists  <cit> .
phylogenetic evidence exists that supports the enemy hypothesis, in particular with regard to gall morphology  <cit> .
however, the adaptive value of gall chemicals remains uncertain  <cit>  and we are unaware of any study that has experimentally confirmed the role of gall secondary metabolites in reducing mortality by natural enemies, which includes vertebrate herbivores although not explicitly mentioned.
volatile organic compounds such as terpenoids are emitted by many plant species and mediate a wide array of interactions.
these volatiles are generally released in response to insect attack and can be exploited as signals by natural enemies of herbivorous arthropods.
induced volatile emission is therefore regarded as an indirect plant defence mechanism  <cit>  although the net benefit for the plant still needs to be shown  <cit> .
in contrast to other herbivorous feeding guilds, only few studies have examined how gall-inducers affect the emission of volatiles  <cit> .
however, these have provided interesting insights, suggesting that in some cases gall-inducing insects can take control over the plant’s defence.
like in many non-galling herbivores, plant volatiles may serve as host location cues for parasitoids of the gall insect antistrophus rufus <cit> .
interestingly, this gall wasp is also known to alter the ratios of monoterpenes in its host plant which then serve as a sex pheromone  <cit> .
in another case, gelechiid moths were found to suppress the host plant’s ability to produce volatiles which may help these gall insects to avoid predation or parasitism  <cit> .
in this study we have focused on the role of volatile terpenes in the conspicuous cauliflower-shaped galls that are induced by the aphid slavum wertheimae  on the lateral buds of pistacia atlantica  trees   <cit> .
inside this structure, the fundatrix and her offspring reproduce parthenogenetically and feed on the phloem sap until autumn when galls turn red.
by then, the gall may contain thousands of aphids that eventually disperse.
unlike many other gall insects, but similar to north american species in the related genus pemphigus, s. wertheimae are not known to be attacked by parasitoids  <cit> .
however, anecdotal reports of predation by bulbuls and dipteran and lepidopteran larvae exist  <cit> .
members of the genus pistacia are widespread in central asia and the middle east and serve as obligate hosts for several specialized gall-forming aphid species   <cit> .
the galls of these species show several morphological and chemical traits with postulated defensive functions  <cit>  that may contribute to protection from natural enemies.
one of the risks these galls face is inadvertent predation by various mammalian herbivores such as cattle  <cit> , mountain gazelles  <cit> , camels  <cit>  or goats  <cit>  that browse the leaves of pistacia trees and can also reach galls on large parts of the tree.
to further assess the functions of plant volatiles in tritrophic interactions with galls, we explored the following questions:  does gall formation by s. wertheimae lead to enhanced concentrations of volatile compounds in the gall?
does increased storage and emission of volatile compounds protect the gall and the insects inside from damage?
individuals of capra hircus hircus  were used to explore the defensive role of gall volatiles.
this species was chosen as a model for a common, non-selective, intensive browser of many plant species including p. atlantica.
the goat was domesticated from its wild ancestor c. hircus aegagrus in the fertile crescent region about  <dig>  years ago  <cit>  and became an integral and dominant component of local habitats.
our results demonstrate a new function for plant volatiles by showing that s. wertheimae galls are chemically well defended against herbivorous mammals.
we speculate that this trait could be adaptive.
RESULTS
total tannin concentrations
to obtain a more comprehensive picture of the defence chemistry of s. wertheimae, we assessed levels of non-volatile tannins in galls and compared these with levels in leaves and fruits of p. atlantica.
aphid galls contained nearly four times higher amounts of total tannins than leaves .
no tannins were detected in any of the p. atlantica fruit samples.
stored terpene concentrations
comparative measurements of extracted terpenes from gall, leaf and fruit tissues were carried out.
the analyses revealed large differences in numbers and quantities of detected compounds .
twenty mono- and  <dig> sesquiterpenes were identified from s. wertheimae galls with the three monoterpenes α-pinene, sabinene, and limonene accounting for 67% of the total compounds.
these monoterpenes were also the main components in the tissues of leaves and fruits.
sesquiterpenes made up for 11% of total terpenes in galls, 36% in leaves and only 2% in fruits.
in total, the concentration of terpenes was almost two magnitudes higher in galls than in leaves or in fruits .
a principal component analysis clearly separated galls from leaves and fruits.
the terpenes strongly correlated with the galls, yet substance profiles of individual galls were variable .
*compound identified by comparison with authenticated standard.
tentative identification of other compounds by comparison of retention indices and mass spectra with wiley  <dig> and massfinder/terpenoids library databanks.
m monoterpene or derivative, s sesquiterpene or derivative, n.d. not detected.
mean values ± standard errors are given.
volatile emission
the amount of volatile terpenes emitted by intact galls of s. wertheimae was found to be significantly higher than the emission by leaves or infructescences .
sixteen monoterpenes and the sesquiterpene germacrene d were identified from the headspaces of galls with limonene, 3-carene, sabinene and α-pinene as dominant compounds.
in leaves and infructescences only five and seven monoterpenes, respectively, were above the detection limit.
volatile release rates were approximately the same in leaves and infructescences of p. atlantica.both plant parts, however, emitted ca.
<dig>  times less terpenes than galls .
limonene, sabinene and α-pinene were the main compounds in the infructescences, as well.
principal component analysis separated galls from leaves and infructescences.
most measured terpenes correlated with the galls, however, camphene  was associated with infructenscences.
the emission of -β-ocimene  and -ocimene  was characteristic for leaves .
*compound identified by comparison with authenticated standard.
tentative identification of other compounds by comparison of retention indices and mass spectra with wiley  <dig> and massfinder/terpenoids library databanks.
m monoterpene or derivative, s sesquiterpene or derivative, n.d. not detected.
mean values ± standard errors are given.
few needle pricks significantly increased the total amount of emitted gall terpenes by 80%.
qualitative changes in the compound blend were observed as not all monoterpenes were emitted in larger amounts following mechanical damage .
significantly increased emission rates were found in α-pinene, β-pinene, β-myrcene, β-phellandrene, limonene and -β-ocimene.
goat behaviour
individual goats that were offered a single branch of p. atlantica completely consumed all of the leaves but none of the galls.
two out of ten goats briefly tasted a single gall but were quick to let it drop.
otherwise, galls were assessed without contact.
in the dual-choice olfaction tests, goats significantly preferred the scent of p. atlantica leaves .
on average, animals spent at least twice as much time assessing leaf samples than s. wertheimae galls.
this was the case for intact as well as wounded leaves and galls.
however, wounding had no significant effect on the duration of sniffing.
goats that were given a choice between food pellets treated with the three main gall monoterpenes and untreated pellets significantly consumed more than twice as much of the latter .
discussion
the galls induced by s. wertheimae on wild pistachio trees contained and emitted large amounts of volatile terpenes that deterred goats from feeding.
this presents a new function for plant volatiles where the insect uses a host defence trait for its own protection.
our findings also support the frequently debated, but to our knowledge, unproven hypothesis that gall secondary metabolites can confer defence against natural enemies.
trees and shrubs in the genus pistacia are known to produce both tannins and terpene-containing resins and therefore have a long history in traditional folk medicine  <cit> .
our data confirmed the existence of high levels of tannins in the leaves of p. atlantica and furthermore show that their concentrations in aphid galls were four-fold higher.
increased accumulation of tannins and other phenolics have previously been reported from a range of other gall insects  <cit> .
likewise, high amounts of mono- and sesquiterpenes were found in gall tissues of which a subset was measured in the leaves and fruits of p. atlantica.
the dominant components in all assessed structures and the headspaces of galls were the three monoterpenes α-pinene, sabinene and limonene.
terpene concentrations measured in the tissues of galls, leaves and fruits/infructescences differed considerably more than terpene levels in the headspaces of these structures.
we conclude that this was due to employing different sampling methods  that do not allow for direct comparison of stored and emitted terpenes.
in pistacia spp., terpenes are mainly present in the resin ducts of the plant and destructive gall tissue sampling will therefore yield large quantities.
however, it was unequivocal that intact galls constitutively emitted larger amounts of mono- and sesquiterpenes than the surrounding plant tissues and thus should be chemically more apparent.
terpenes are known to have strong biological activities and they are involved in plant defences  <cit> .
the analysed gall chemicals influenced the feeding behaviour of the damascus goats: animals that were offered leaves and galls attached to a twig of p. atlantica completely rejected the galls but readily consumed every leaf.
in this food selection process, initial olfactory assessment of the potential food items had played an essential role because volatiles emanating from galls signalled that galls were unpalatable.
increasing the amount of released volatiles by 80% as a result of minor mechanical damage, however, did not further reduce the animals’ interest.
we speculate that stronger emission is necessary to see dose-dependent effects.
our data further suggest that the presence of the three main terpenes could readily explain the avoidance response of the mammals.
food pellets treated with these volatiles  in concentrations found inside gall tissues significantly reduced their palatability .
the observed rejection after olfactory and gustatory evaluation was not as strong as in intact galls because all food was initially sampled and therefore it can be conceived that other terpenes and/or the high tannin content of galls may have contributed to their low attractiveness.
whether goats had an innate aversion against galls or whether their choices were affected by negative experience in the past remains unknown.
however, short-term learning can be excluded as each goat was used only once per experiment.
the defensive role of tannins awaits further exploration and it is possible that the high tannin levels found in galls have also contributed to their unpalatability.
however, goats are known to be more tolerant to tannin-rich diets than other mammalian herbivores as, for example, sheep.
it has been suggested that this is because goat saliva contains increased amounts of inducible proteins that can precipitate tannins  <cit> .
to date, numerous studies have generated a detailed picture of the multiple roles that volatile plant components like terpenes play in the interaction with insects or even birds  <cit> .
these volatiles are an important part of the plant’s defensive arsenal and act directly by reducing herbivore damage or indirectly by attracting the herbivore’s natural enemies  <cit> .
surprisingly, the relationship between plant volatiles and gall insects, which are highly evolved herbivores with sophisticated biology and physiology  <cit> , has been explored rather rudimentarily.
limited knowledge suggests that the advantage in the co-evolutionary arms race can be on either side, the plant’s or the insect’s, depending on the specific interaction.
while it has been shown in at least one case that plants emit volatiles in response to a galling herbivore that can help parasitoids to locate their host  <cit> , it has also been demonstrated that gall insects are capable of suppressing the treacherous response  <cit> .
adding to this, our findings suggest a different strategy where volatile emission is largely increased in the gall and thus repels mammal herbivores that can inadvertently destroy a whole aphid colony.
it can be speculated that the emission of volatiles from the galls of s. wertheimae may promote easy detection by predators but such a trade-off has yet to be investigated.
given that galls on the top of trees face a low risk of browsing, one could speculate that these may contain fewer defence compounds.
however, gall chemicals may also protect against frugivorous and insectivorous birds that can prey on the aphids  <cit> .
while this needs further clarification, we have found that bulbuls were deterred by gall compounds when mixed into artificial diet .
because galls are sessile, long lasting, and often conspicuous, there has been strong selection for gall defences against a variety of natural enemies including pathogens, parasitoids, predators and also herbivores.
inquilines that feed on internal gall tissues can play an important role as they have been shown to directly or indirectly kill the gall-forming insect  <cit> .
protection can be achieved by defensive behaviors of the gall inducer  <cit> , physical gall traits  or secondary metabolites  <cit> .
the notion that gall chemicals are an adaptive defence is supported, for instance, by the positive correlation between tannin content in oak leaves and the density of cynipid galls on these trees  <cit> .
adding to this, tannin content correlated negatively with mortality due to fungal infection in the cynipid wasp dryocosmus dubiosus <cit> .
in our study we show that insect galls create a distinct headspace which is different from the surrounding plant tissues and hence push the borders of their extended phenotype.
volatiles emanating from galls may serve as a first line in the insect’s defence and constitute an honest signal as they can warn potential predators or herbivores before damaging the gall which would result in exposure to high levels of stored chemicals.
recently, the adaptive nature of gall conspicuousness has been addressed  <cit>  and, among other hypotheses  <cit> , it has been suggested that some galls may be aposematic.
galls heavily protected by defensive metabolites could advertise this feature by using warning signals.
the strong release of volatiles in conjunction with the gall’s red colouration supports the aposematic gall hypothesis as both increase gall conspicuity.
the combination of multimodal warning displays may stimulate several sensory cascades that can promote learning and memory efficiencies in potential enemies  <cit> .
CONCLUSIONS
unlike in free-living aphids that actively escape goat predation  <cit> , the protection of the gall-inducing aphids studied here depends on plant traits.
we unequivocally demonstrated that galls accumulate large amounts of defensive secondary metabolites that protect the aphid colony within from a generalist herbivore.
therefore, it seems plausible that this trait is adaptive.
however, further evidence is necessary to support the idea that browsers exert enough selection pressure to have this influence on the gall’s phenotype.
alternative explanations of why s. wertheimae galls contain considerably higher concentrations of well-known defence compounds than their host plants are possible.
for instance, secondary metabolite accumulation could be an unavoidable physiological side-effect of gall formation that also happens to be defensive.
so far, the molecular and biochemical mechanisms that lead to gall formation are not understood in enough detail to either refute or support this notion.
comparing gall defences that have been subjected to different selection pressures from natural enemies over time may answer the question of adaptiveness.
recent evidence suggests that herbivore-driven evolution of plant defences may need only few generations  <cit> .
in s. wertheimae volatile emission serves as an antagonistic signal and may be regarded as the outer boundary of the gall inducer’s extended defensive phenotype.
our findings thus add a new direction to the growing body of evidence that illustrate the multiple ecological functions accomplished by plant volatiles.

 numerous microorganisms and arthropods are capable of transforming plant tissues into galls. the galling habit is especially common among insects with more than known gall-forming species from several orders. gall induction has evolved convergently among and within various insect lineages and the multiple, independent origins of gall-formation indicate that this phenomenon is highly adaptive. although the molecular mechanisms of gall induction remain to be unveiled, numerous ecological studies and phylogenetic analyses strongly suggest that the insects are in control of the gall traits, which they exploit for their own benefit. galls are thus considered as an extended phenotype of the inducer’s genes. serving as “incubators” that promote the development of the insects within, galls may have more than a single adaptive function. the proposed advantages of the galling habit fall into three main categories: microclimatic stabilization: galls protect the insects from unfavourable abiotic conditions such as high temperature and low humidity nutrition: gall tissue provides an abundance of high quality nutrients and defence: morphology and chemistry of the gall tissue protect the inducing insect from various natural enemies, including predators, parasitoids, pathogens and other herbivores. this notion has been termed the enemy hypothesis. improved nutrition and defence is achieved by the insect’s ability to considerably manipulate its host plant’s morphology and physiology. primary and secondary plant compounds are not randomly distributed in the galled tissue as the inner layers on which the insects feed are enhanced sinks for photosynthates and may have low concentrations of secondary compounds, thus providing better nutrition for the gall insect. the outer, non-nutritive gall layers, on the other hand, may contain increased amounts of potentially defensive chemicals that could deter antagonists. phylogenetic evidence exists that supports the enemy hypothesis, in particular with regard to gall morphology. however, the adaptive value of gall chemicals remains uncertain and we are unaware of any study that has experimentally confirmed the role of gall secondary metabolites in reducing mortality by natural enemies, which includes vertebrate herbivores although not explicitly mentioned. volatile organic compounds such as terpenoids are emitted by many plant species and mediate a wide array of interactions. these volatiles are generally released in response to insect attack and can be exploited as signals by natural enemies of herbivorous arthropods. induced volatile emission is therefore regarded as an indirect plant defence mechanism although the net benefit for the plant still needs to be shown. in contrast to other herbivorous feeding guilds, only few studies have examined how gall-inducers affect the emission of volatiles. however, these have provided interesting insights, suggesting that in some cases gall-inducing insects can take control over the plant’s defence. like in many non-galling herbivores, plant volatiles may serve as host location cues for parasitoids of the gall insect antistrophus rufus. interestingly, this gall wasp is also known to alter the ratios of monoterpenes in its host plant which then serve as a sex pheromone. in another case, gelechiid moths were found to suppress the host plant’s ability to produce volatiles which may help these gall insects to avoid predation or parasitism. in this study we have focused on the role of volatile terpenes in the conspicuous cauliflower-shaped galls that are induced by the aphid slavum wertheimae on the lateral buds of pistacia atlantica trees. inside this structure, the fundatrix and her offspring reproduce parthenogenetically and feed on the phloem sap until autumn when galls turn red. by then, the gall may contain thousands of aphids that eventually disperse. unlike many other gall insects, but similar to north american species in the related genus pemphigus, s. wertheimae are not known to be attacked by parasitoids. however, anecdotal reports of predation by bulbuls and dipteran and lepidopteran larvae exist. members of the genus pistacia are widespread in central asia and the middle east and serve as obligate hosts for several specialized gall-forming aphid species. the galls of these species show several morphological and chemical traits with postulated defensive functions that may contribute to protection from natural enemies. one of the risks these galls face is inadvertent predation by various mammalian herbivores such as cattle, mountain gazelles, camels or goats that browse the leaves of pistacia trees and can also reach galls on large parts of the tree. to further assess the functions of plant volatiles in tritrophic interactions with galls, we explored the following questions: does gall formation by s. wertheimae lead to enhanced concentrations of volatile compounds in the gall? does increased storage and emission of volatile compounds protect the gall and the insects inside from damage? individuals of capra hircus hircus were used to explore the defensive role of gall volatiles. this species was chosen as a model for a common, non-selective, intensive browser of many plant species including p. atlantica. the goat was domesticated from its wild ancestor c. hircus aegagrus in the fertile crescent region about  years ago and became an integral and dominant component of local habitats. our results demonstrate a new function for plant volatiles by showing that s. wertheimae galls are chemically well defended against herbivorous mammals. we speculate that this trait could be adaptive.  total tannin concentrations to obtain a more comprehensive picture of the defence chemistry of s. wertheimae, we assessed levels of non-volatile tannins in galls and compared these with levels in leaves and fruits of p. atlantica. aphid galls contained nearly four times higher amounts of total tannins than leaves. no tannins were detected in any of the p. atlantica fruit samples. stored terpene concentrations comparative measurements of extracted terpenes from gall, leaf and fruit tissues were carried out. the analyses revealed large differences in numbers and quantities of detected compounds. twenty mono- and sesquiterpenes were identified from s. wertheimae galls with the three monoterpenes α-pinene, sabinene, and limonene accounting for 67% of the total compounds. these monoterpenes were also the main components in the tissues of leaves and fruits. sesquiterpenes made up for 11% of total terpenes in galls, 36% in leaves and only 2% in fruits. in total, the concentration of terpenes was almost two magnitudes higher in galls than in leaves or in fruits. a principal component analysis clearly separated galls from leaves and fruits. the terpenes strongly correlated with the galls, yet substance profiles of individual galls were variable. *compound identified by comparison with authenticated standard. tentative identification of other compounds by comparison of retention indices and mass spectra with wiley and massfinder/terpenoids library databanks. m monoterpene or derivative, s sesquiterpene or derivative, n.d. not detected. mean values ± standard errors are given. volatile emission the amount of volatile terpenes emitted by intact galls of s. wertheimae was found to be significantly higher than the emission by leaves or infructescences. sixteen monoterpenes and the sesquiterpene germacrene d were identified from the headspaces of galls with limonene, 3-carene, sabinene and α-pinene as dominant compounds. in leaves and infructescences only five and seven monoterpenes, respectively, were above the detection limit. volatile release rates were approximately the same in leaves and infructescences of p. atlantica.both plant parts, however, emitted ca. times less terpenes than galls. limonene, sabinene and α-pinene were the main compounds in the infructescences, as well. principal component analysis separated galls from leaves and infructescences. most measured terpenes correlated with the galls, however, camphene was associated with infructenscences. the emission of -β-ocimene and -ocimene was characteristic for leaves. *compound identified by comparison with authenticated standard. tentative identification of other compounds by comparison of retention indices and mass spectra with wiley and massfinder/terpenoids library databanks. m monoterpene or derivative, s sesquiterpene or derivative, n.d. not detected. mean values ± standard errors are given. few needle pricks significantly increased the total amount of emitted gall terpenes by 80%. qualitative changes in the compound blend were observed as not all monoterpenes were emitted in larger amounts following mechanical damage. significantly increased emission rates were found in α-pinene, β-pinene, β-myrcene, β-phellandrene, limonene and -β-ocimene. goat behaviour individual goats that were offered a single branch of p. atlantica completely consumed all of the leaves but none of the galls. two out of ten goats briefly tasted a single gall but were quick to let it drop. otherwise, galls were assessed without contact. in the dual-choice olfaction tests, goats significantly preferred the scent of p. atlantica leaves. on average, animals spent at least twice as much time assessing leaf samples than s. wertheimae galls. this was the case for intact as well as wounded leaves and galls. however, wounding had no significant effect on the duration of sniffing. goats that were given a choice between food pellets treated with the three main gall monoterpenes and untreated pellets significantly consumed more than twice as much of the latter. discussion the galls induced by s. wertheimae on wild pistachio trees contained and emitted large amounts of volatile terpenes that deterred goats from feeding. this presents a new function for plant volatiles where the insect uses a host defence trait for its own protection. our findings also support the frequently debated, but to our knowledge, unproven hypothesis that gall secondary metabolites can confer defence against natural enemies. trees and shrubs in the genus pistacia are known to produce both tannins and terpene-containing resins and therefore have a long history in traditional folk medicine. our data confirmed the existence of high levels of tannins in the leaves of p. atlantica and furthermore show that their concentrations in aphid galls were four-fold higher. increased accumulation of tannins and other phenolics have previously been reported from a range of other gall insects. likewise, high amounts of mono- and sesquiterpenes were found in gall tissues of which a subset was measured in the leaves and fruits of p. atlantica. the dominant components in all assessed structures and the headspaces of galls were the three monoterpenes α-pinene, sabinene and limonene. terpene concentrations measured in the tissues of galls, leaves and fruits/infructescences differed considerably more than terpene levels in the headspaces of these structures. we conclude that this was due to employing different sampling methods that do not allow for direct comparison of stored and emitted terpenes. in pistacia spp., terpenes are mainly present in the resin ducts of the plant and destructive gall tissue sampling will therefore yield large quantities. however, it was unequivocal that intact galls constitutively emitted larger amounts of mono- and sesquiterpenes than the surrounding plant tissues and thus should be chemically more apparent. terpenes are known to have strong biological activities and they are involved in plant defences. the analysed gall chemicals influenced the feeding behaviour of the damascus goats: animals that were offered leaves and galls attached to a twig of p. atlantica completely rejected the galls but readily consumed every leaf. in this food selection process, initial olfactory assessment of the potential food items had played an essential role because volatiles emanating from galls signalled that galls were unpalatable. increasing the amount of released volatiles by 80% as a result of minor mechanical damage, however, did not further reduce the animals’ interest. we speculate that stronger emission is necessary to see dose-dependent effects. our data further suggest that the presence of the three main terpenes could readily explain the avoidance response of the mammals. food pellets treated with these volatiles in concentrations found inside gall tissues significantly reduced their palatability. the observed rejection after olfactory and gustatory evaluation was not as strong as in intact galls because all food was initially sampled and therefore it can be conceived that other terpenes and/or the high tannin content of galls may have contributed to their low attractiveness. whether goats had an innate aversion against galls or whether their choices were affected by negative experience in the past remains unknown. however, short-term learning can be excluded as each goat was used only once per experiment. the defensive role of tannins awaits further exploration and it is possible that the high tannin levels found in galls have also contributed to their unpalatability. however, goats are known to be more tolerant to tannin-rich diets than other mammalian herbivores as, for example, sheep. it has been suggested that this is because goat saliva contains increased amounts of inducible proteins that can precipitate tannins. to date, numerous studies have generated a detailed picture of the multiple roles that volatile plant components like terpenes play in the interaction with insects or even birds. these volatiles are an important part of the plant’s defensive arsenal and act directly by reducing herbivore damage or indirectly by attracting the herbivore’s natural enemies. surprisingly, the relationship between plant volatiles and gall insects, which are highly evolved herbivores with sophisticated biology and physiology, has been explored rather rudimentarily. limited knowledge suggests that the advantage in the co-evolutionary arms race can be on either side, the plant’s or the insect’s, depending on the specific interaction. while it has been shown in at least one case that plants emit volatiles in response to a galling herbivore that can help parasitoids to locate their host, it has also been demonstrated that gall insects are capable of suppressing the treacherous response. adding to this, our findings suggest a different strategy where volatile emission is largely increased in the gall and thus repels mammal herbivores that can inadvertently destroy a whole aphid colony. it can be speculated that the emission of volatiles from the galls of s. wertheimae may promote easy detection by predators but such a trade-off has yet to be investigated. given that galls on the top of trees face a low risk of browsing, one could speculate that these may contain fewer defence compounds. however, gall chemicals may also protect against frugivorous and insectivorous birds that can prey on the aphids. while this needs further clarification, we have found that bulbuls were deterred by gall compounds when mixed into artificial diet. because galls are sessile, long lasting, and often conspicuous, there has been strong selection for gall defences against a variety of natural enemies including pathogens, parasitoids, predators and also herbivores. inquilines that feed on internal gall tissues can play an important role as they have been shown to directly or indirectly kill the gall-forming insect. protection can be achieved by defensive behaviors of the gall inducer, physical gall traits or secondary metabolites. the notion that gall chemicals are an adaptive defence is supported, for instance, by the positive correlation between tannin content in oak leaves and the density of cynipid galls on these trees. adding to this, tannin content correlated negatively with mortality due to fungal infection in the cynipid wasp dryocosmus dubiosus. in our study we show that insect galls create a distinct headspace which is different from the surrounding plant tissues and hence push the borders of their extended phenotype. volatiles emanating from galls may serve as a first line in the insect’s defence and constitute an honest signal as they can warn potential predators or herbivores before damaging the gall which would result in exposure to high levels of stored chemicals. recently, the adaptive nature of gall conspicuousness has been addressed and, among other hypotheses, it has been suggested that some galls may be aposematic. galls heavily protected by defensive metabolites could advertise this feature by using warning signals. the strong release of volatiles in conjunction with the gall’s red colouration supports the aposematic gall hypothesis as both increase gall conspicuity. the combination of multimodal warning displays may stimulate several sensory cascades that can promote learning and memory efficiencies in potential enemies.  unlike in free-living aphids that actively escape goat predation, the protection of the gall-inducing aphids studied here depends on plant traits. we unequivocally demonstrated that galls accumulate large amounts of defensive secondary metabolites that protect the aphid colony within from a generalist herbivore. therefore, it seems plausible that this trait is adaptive. however, further evidence is necessary to support the idea that browsers exert enough selection pressure to have this influence on the gall’s phenotype. alternative explanations of why s. wertheimae galls contain considerably higher concentrations of well-known defence compounds than their host plants are possible. for instance, secondary metabolite accumulation could be an unavoidable physiological side-effect of gall formation that also happens to be defensive. so far, the molecular and biochemical mechanisms that lead to gall formation are not understood in enough detail to either refute or support this notion. comparing gall defences that have been subjected to different selection pressures from natural enemies over time may answer the question of adaptiveness. recent evidence suggests that herbivore-driven evolution of plant defences may need only few generations. in s. wertheimae volatile emission serves as an antagonistic signal and may be regarded as the outer boundary of the gall inducer’s extended defensive phenotype. our findings thus add a new direction to the growing body of evidence that illustrate the multiple ecological functions accomplished by plant volatiles. 
to defend against insects, for example, damaged plants emit volatile organic compounds that attract the herbivore’s natural enemies. increased emission of plant volatiles in response to insect activity is commonly looked upon as a “cry for help” by the plant to attract the insect’s natural enemies. our data support the enemy hypothesis insofar that high levels of gall secondary metabolites confer protection against natural enemies. chemical analyses of galls triggered by the gregarious aphid slavum wertheimae on wild pistachio trees showed that these structures contained and emitted considerably higher quantities of plant terpenes than neighbouring leaves and fruits. behavioural assays using goats as a generalist herbivore confirmed that the accumulated terpenes acted as olfactory signals and feeding deterrents, thus enabling the gall-inducers to escape from inadvertent predation by mammals. so far, plant volatile responses have been studied extensively in conjunction with leaf chewing and sap sucking insects, yet little is known about the relationship between plant volatiles and gall-inducers, the most sophisticated herbivores.
 plants have evolved an astonishing array of survival strategies. to defend against insects, for example, damaged plants emit volatile organic compounds that attract the herbivore’s natural enemies. so far, plant volatile responses have been studied extensively in conjunction with leaf chewing and sap sucking insects, yet little is known about the relationship between plant volatiles and gall-inducers, the most sophisticated herbivores. here we describe a new role for volatiles as gall-insects were found to benefit from this plant defence.  chemical analyses of galls triggered by the gregarious aphid slavum wertheimae on wild pistachio trees showed that these structures contained and emitted considerably higher quantities of plant terpenes than neighbouring leaves and fruits. behavioural assays using goats as a generalist herbivore confirmed that the accumulated terpenes acted as olfactory signals and feeding deterrents, thus enabling the gall-inducers to escape from inadvertent predation by mammals.  increased emission of plant volatiles in response to insect activity is commonly looked upon as a “cry for help” by the plant to attract the insect’s natural enemies. in contrast, we show that such volatiles can serve as a first line of insect defences that extends the ‘extended phenotype’ represented by galls, beyond physical boundaries. our data support the enemy hypothesis insofar that high levels of gall secondary metabolites confer protection against natural enemies. capra hircusenemy hypothesisextended phenotypeherbivoryintraguild predationplant defencetanninsterpenesvolatile organic compounds 