BACKGROUND
plants have evolved to defend themselves against biotic stressors such as insects and pathogens.
various insect secretions including oviposition fluids, oral secretions , and insect excreta are known to act as elicitors of induced plant defenses against insect herbivory  <cit> .
plant defense signaling cascades induced by insects begin with plant recognition of insect-derived elicitors followed by plasma trans-membrane potential  depolarization  <cit> , the rise in cytosolic calcium ions  <cit>  and a burst of reactive oxygen species , including hydrogen peroxide  and nitric oxide   <cit> .
these cascades lead to a rise in production of the phytohormone, jasmonic acid  and salicylic acid   <cit>  that regulate the transcript level of defense-related genes  <cit> , and end with metabolic changes including release of volatile organic compounds   <cit>  and production of toxic compounds in the plants  <cit> .
plasma trans-membrane potential  depolarization, rise in cytosolic calcium ions and a burst of reactive oxygen species  which occurs from seconds to hour/s after insect damage referred to as early plant defense responses, while production of the phytohormone, change in transcript level of defense-related genes and metabolic changes including release of vocs and production of toxic compounds which occurs from hour/s to day/s after insect damage referred to as late plant defense responses  <cit> .
foliar feeding insects ingest leaves by snipping plant material continuously.
this process causes a series of mechanical injury, usually supplemented with introduction of oral secretions into the damaged tissue  <cit> .
it is vital for plants to differentiate mechanical injury from herbivore damage and change these different biotic stress signals into suitable physiological responses.
studies have shown that plants are able to differentiate simple mechanical injury from herbivore injury  <cit> .
investigations at the molecular level have revealed different gene expression patterns of defense-related genes in plants with mechanical injury versus plants damaged by insects  <cit> .
application of insect os to mechanical injury can mimic most plant responses to herbivory  <cit> , showing that the os constitute elicitors by which plants recognize insect attack  <cit> .
indeed, several elicitors have been isolated from insect os that trigger plant defenses against herbivory, such as β-glucosidase  <cit> , volicitin, a fatty acid–amino acid conjugate  <cit> , caeliferins  <cit> , and inceptins  <cit> .
lepidopteran os consists of saliva from mandibular and labial secretions, and regurgitant from digestive tract  <cit> .
the os deposited on herbivore fed plant material also contains secretions from the ventral eversible gland   <cit> .
despite the discovery of several elicitors, studies that show whether these elicitors originated from salivary glands or from other organs associated with feeding, such as the ventral eversible gland  are limited.
volicitin originated from the gut tissues of spodoptera litura larvae  <cit>  and inceptins are partially digested chloroplast protein formed when spodoptera frugiperda attack cowpea  <cit> .
the veg is a secretory structure found on the ventral surface of the thorax of caterpillars .
it consists of two regions with different functions: a non-eversible glandular sac lined with secretory cells and an eversible cuticular tube.
eversion of the cuticular tube forms a visible papilla, whereas secretions from the secondary gland area on the cuticular tube are transferred to the apex of the papilla and released  <cit> .
since the tip of the everted veg can reach the mandibles  <cit> , its secretions are deposited onto the food substrate with the os  <cit> .
secretions from the veg of caterpillars have been associated with defense against predators and the production of anti-aggregation pheromones  <cit> .
however, the role of veg secretions in plant-insect interactions remains unclear.
recently, zebelo and maffei  <cit>  demonstrated that secretions from the veg of spodoptera littoralis caterpillars trigger early defense signaling events in arabidopsis thaliana.
in the present study, we investigated possible involvement of veg secretions from s. exigua caterpillars in the induction of late defense signaling in tomato.
we quantified and compared the activity of defense-related enzymes, transcript levels of terpene synthase genes and other defense-related genes, and voc emission in tomato plants damaged by s. exigua caterpillars with the veg intact  versus plants damaged by caterpillars with the veg ablated  as well as mechanically injured plants treated with os from vegi caterpillars  versus mechanically injured plants treated with os from vega caterpillars .
RESULTS
veg ablation didn’t affect s. exigua feeding activity
before we started to assess the impact of veg secretions on triggering plant defense, we evaluated whether veg ablation affects feeding activity of s. exigua caterpillars.
there were no significant differences between vega  and vegi  caterpillars on leaf area consumption after 24 h .
veg secretions activate defense-related enzymes in tomato
the selected defense-related enzymes, peroxidase , polyphenol oxidase , and lipoxygenase , were expressed in significantly higher amounts in plants damaged by vegi caterpillars and mi + osvegi than in plants damaged by vega caterpillars, mechanically injured  plants, mi + osvega plants, and untreated  plants.
activity of pod was significantly higher in vegi-damaged and mi + osvegi tomato plants than in vega-damaged, mi, mi + osvega or undamaged plants, starting as early as 24 h after treatment .
activity of ppo 48 h after treatment was  <dig> ,  <dig> ,  <dig>  and  <dig> % higher in plants damaged by vegi caterpillars than in plants damaged by vega caterpillars, mi, mi + osvega, or undamaged plants, respectively .
a significant increase in lox-specific activity levels was detected as early as 24 h after treatment in plants damaged by vegi caterpillars and md + osvegi plants compared to the other treatments.
activity of lox 72 h after treatment was  <dig> ,  <dig>   <dig>  and  <dig> %, higher in plants damaged by vegi caterpillars than in plants damaged by vega caterpillars, mi, mi + osvega, or undamaged plants, respectively .
in general, no significant differences were recorded in enzymatic activity between plants damaged by vegi caterpillars and mi + osvegi plants .
table shows activity  of three defense-related enzymes, peroxidase , polyphenol oxidase , and lipoxygenase , in tomato plants damaged by caterpillars with the veg intact , plants damaged by caterpillars with the veg ablated , mechanically injured  plants, mechanically injured plants treated with oral secretion  from vegi caterpillars , mechanically injured plants treated with os from vega caterpillars , and undamaged  plants, at  <dig>   <dig>   <dig> and 72 h after caterpillar feeding.
data were collected from three plants  per treatment.
means  within the same column and time period having different letters are significantly different .
veg secretions induce vocs emission in tomato
key differences were recorded in the headspace voc profiles of tomato plants from the different treatments .
increased emission of vocs was detected in the headspace of plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars, mechanically injured  plants, and untreated  plants.
specifically, green leaf volatiles  and certain monoterpenes were emitted in higher amounts by plants damaged by vegi caterpillars than in the other treatments .
in particular, the glvs, -2-hexenal, -3-hexenal, -3-hexenyl acetate and -2-hexenol were emitted in 7-fold, 5-fold, 7-fold and 10-fold, respectively, in plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars .
table shows emission of vocs  by tomato plants damaged by caterpillars with the veg intact , plants damaged by caterpillars with the veg ablated , mechanically injured  plants, and undamaged  plants.data were collected from three plants  per treatment.
means  within the same row having different letters are significantly different .
kovats retention index  is indicated for each compound.
nd = not detected.
the monoterpenes, β-linalool and γ-terpinene, were emitted in significantly higher amounts by plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars and the other treatments .
however, no significant differences were recorded among the treatments in the emission of α-pinene, β-phellendrene, β-pinene and β-ocimene.
the sesquiterpenes, -β-caryophyllene, α-humulene and β-elemene, were also emitted in significantly higher amounts by plants damaged by vegi caterpillars compared to the other treatments.
furthermore, emission of the fatty acids  and organic ester , was higher in vegi-damaged plants .
veg secretions increase transcript levels of defense-related genes in tomato
we used quantitative rt-pcr to quantify the transcript levels of six defense-related genes including genes encoding lipoxygenase , allene oxide synthase , and four genes involved in terpene biosynthesis .
lox and aos are key enzymes in the jasmonic acid  biosynthesis pathway.
most of defense-related genes were found up-regulated in plants damaged by vegi caterpillars and in mi + osvegi plants compared to plants damaged by vega caterpillars, mechanically injured , mi + osvega, or untreated  plants .
in particular, the transcript levels of the terpene synthase genes, tps <dig>   <cit> , and tps <dig> -β-caryophyllene and α-humulene)  <cit> , were significantly higher in plants damaged by vegi caterpillars and mi + osvegi plants compared to the other treatments.
differential expression of genes involved in jasmonic acid  and terpene biosynthesis in tomato plants damaged by caterpillars with the veg intact , plants damaged by caterpillars with the veg ablated , mechanically injured  plants, mechanically injured plants treated with oral secretion  from vegi caterpillars , mechanically injured plants treated with os from vega caterpillars , and undamaged  plants.
qrt-pcr analyses are shown as fold change in expression.
means  within the same row having different letters are significantly different .
discussion
tomato plants damaged by s. exigua caterpillars with intact ventral eversible gland  expressed significantly higher amounts of defense-related enzymes and genes, and headspace vocs than plants damaged by caterpillars with ablated veg .
these results suggest that secretions from the veg of s. exigua caterpillars contain elicitors of late defense signaling in tomato.
to our knowledge, this is the first report on the role of caterpillar veg secretions as an elicitor of late defense signaling in plants.
a previous study by zebelo and maffei  <cit>  showed that veg secretions of spodoptera littoralis caterpillars induced early defense signaling in arabidopsis thaliana.
the three defense-related enzymes, peroxidase , polyphenol oxidase , and lipoxigenase , were expressed in significantly higher amounts in plants damaged by vegi caterpillars or mechanically injured plants treated with oral secretion  from vegi caterpillars  than in plants damaged by vega caterpillars, mechanically injured  plants, mechanically injured plants treated with oral secretion  from vega caterpillars , or untreated  plants.
all three enzymes are components of the octadecanoid signal transduction pathway, which regulates the production of the phytohormone, jasmonic acid   <cit> .
peroxidases  are a group of plant defense-related enzymes, which limit plant nutritional quality to insect herbivores through quinone and reactive oxygen species generation with subsequent inhibition of insect digestion of plant material  <cit> .
over-expression of pods can enhance plant resistance to insects  <cit>  and limit plant nutritional quality to insect herbivores  <cit> .
suzuki et al.
<cit>  reported that herbivory by caterpillars and high pod activity resulted in the up-regulation of several tomato genes including genes encoding proteinase inhibitors.
polyphenol oxidase  is an inducible enzyme that is found throughout the plant kingdom and known to have defensive role against herbivores  <cit>  and pathogens  <cit> .
production of ppo is induced by mechanical injury, methyl jasmonate  and herbivory  <cit> .
similar to our results, chung et al.
<cit>  reported high ppo levels in tomato plants wounded mechanically and treated with oral secretions  from colorado potato beetle, leptinotarsa decemlineata, suggesting that insect os contain elicitors of ppo activity  <cit> .
lipoxygenases  are another group of anti-oxidative enzymes involved in plant defense against herbivory and pathogens through the octadecanoid pathway  <cit> .
one of the most important functions of lox in plant defense is the oxidation of linolenic acid in the ja signaling pathway  <cit> .
allene oxide synthase  catalyzes the first step of the lox pathway that leads to ja biosynthesis  <cit> .
in the present study, we observed an early induction of lox-specific activity within 24 h of feeding by caterpillars with intact veg .
likewise, the transcript levels of lox and aos genes were higher in plants damaged by vegi caterpillars compared to plants damaged by caterpillars with ablated veg .
these results are consistent with previous studies which demonstrate that caterpillar feeding up-regulates the expression of lox genes in tomato  <cit> .
our results also showed increased emission of vocs in tomato plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars or mechanically injured plants.
among the common vocs induced by herbivory are those that are lox-derived, such as green leaf volatiles , terpenoids and methyl salicylate  <cit> .
numerous plants emit glvs and other vocs as an indirect defense strategy against herbivory, as these volatiles can attract predacious and parasitic natural enemies of herbivores  <cit> .
in this study, glvs and certain monoterpenes were emitted in higher amounts by plants damaged by vegi caterpillars, suggesting the involvement of the veg in the induction of plant vocs.
for instance, most glvs including -2-hexenal, -3-hexenal, -2-hexenol and -3-hexenyl acetate were detected in higher amounts in plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars.
interestingly, many of these glvs are used as host location cues by caterpillar parasitoids  <cit> , suggesting that veg secretions may impact tritrophic interactions.
glvs have also been reported to play a role in plant-plant interactions  <cit> .
another highly diverse group of plant compounds are the terpenoids, which are synthesized by a group of enzymes called terpene synthases  to produce mono-, sesqui- and diterpenes  <cit> .
terpenes are more costly to synthesize per gram than most other primary and secondary plant compounds  <cit> .
studies have shown that a single mechanical plant tissue injury event may not elicit induced defense related volatile organic compounds   <cit> .
however, application of os to mechanically wounded site could elicit the release of inducible volatile compounds and thereby mimic herbivory  <cit> .
in the present study, we observed significantly higher emission of the monoterpenes, β-linalool and γ-terpinene, in plants damaged by vegi caterpillars compared to those damaged by vega caterpillars.
however, there were no significant differences recorded among the treatments in the emission of other monoterpenes such as α-pinene, β-phellendrene, β-pinene and β-ocimene, suggesting that not all vocs are inducible by veg secretions.
like monoterpenes, sesquiterpenes are phytoalexins which play a pivotal role in direct and indirect defenses against herbivores  <cit> .
in the present study, several sesquiterpenes -β-caryophyllene, α-humulene and β-elemene), some fatty acids  and an organic ester  were emitted in significantly higher amounts by plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars.
results from gene expression studies showed that most terpene synthase genes -β-caryophyllene and α-humulene) and the genes involved in the biosynthesis of glvs and jasmonic acid  were up-regulated in plants damaged by vegi caterpillars as well as in mechanically injured plants treated with oral secretion from vegi caterpillars .
however, the transcript levels of these genes were not up-regulated in plants damaged by vega caterpillars, mechanically injured plants treated with oral secretion from vega caterpillars , or mechanically injured  plants.
these results suggest that an intact veg in s. exigua caterpillars is crucial for eliciting late defense signaling via the expression of defense-related genes.
these findings are in agreement with those of bricchi et al.
<cit>  which showed that mechanical injury alone failed to increase the transcript levels of terpene synthase and ja biosynthesis genes in arabidobsis thaliana, but mechanical injury treated with spodoptera littoralis oral secretion activated the genes.
in a recent review of the role of caterpillar secretions on induced plant defenses, felton  <cit>  suggested that the veg may play an important role in secretion during feeding by caterpillars in the family noctuidae.
the structure and proximity of the veg to the caterpillar mouthparts lend credence to this proposal.
when a caterpillar feeds on a plant material the veg is distended from its eversible position on the ventral surface of the caterpillar thorax and reaches the injured plant surface  <cit> .
furthermore, because the tip of the everted veg can reach the mandibles during feeding  <cit> , the veg secretions are usually deposited onto the food substrate with the os  <cit> .
our results confirm that the veg secretions, which are deposited along with oral secretions or regurgitate onto plants during caterpillar feeding can induce late defense signaling in tomato.
further studies are needed to identify the bioactive components of the veg secretions that trigger plant defense signaling.
CONCLUSIONS
the veg was first reported in  <dig>  <cit> , but very little is known about its role in plant-insect interactions.
our current results suggest that the veg of s. exigua contain elicitors of late plant defense signaling which may trigger defense-related enzymatic activity, regulate expression of terpene synthase genes and other defense-related genes, and induce plant vocs, with potential ramifications for plant-insect and tritrophic interactions.
studies are underway in our lab to investigate whether the veg secretion alone or in combination with other labial gland secretions and gut regurgitates trigger plant responses against insect herbivory.
further studies are needed to comprehend the complexity of plant signaling networks and the role of insect oral secretions in mediating plant-insect and trititrophic interactions.

 plants have evolved to defend themselves against biotic stressors such as insects and pathogens. various insect secretions including oviposition fluids, oral secretions, and insect excreta are known to act as elicitors of induced plant defenses against insect herbivory. plant defense signaling cascades induced by insects begin with plant recognition of insect-derived elicitors followed by plasma trans-membrane potential depolarization, the rise in cytosolic calcium ions and a burst of reactive oxygen species, including hydrogen peroxide and nitric oxide. these cascades lead to a rise in production of the phytohormone, jasmonic acid and salicylic acid that regulate the transcript level of defense-related genes, and end with metabolic changes including release of volatile organic compounds and production of toxic compounds in the plants. plasma trans-membrane potential depolarization, rise in cytosolic calcium ions and a burst of reactive oxygen species which occurs from seconds to hour/s after insect damage referred to as early plant defense responses, while production of the phytohormone, change in transcript level of defense-related genes and metabolic changes including release of vocs and production of toxic compounds which occurs from hour/s to day/s after insect damage referred to as late plant defense responses. foliar feeding insects ingest leaves by snipping plant material continuously. this process causes a series of mechanical injury, usually supplemented with introduction of oral secretions into the damaged tissue. it is vital for plants to differentiate mechanical injury from herbivore damage and change these different biotic stress signals into suitable physiological responses. studies have shown that plants are able to differentiate simple mechanical injury from herbivore injury. investigations at the molecular level have revealed different gene expression patterns of defense-related genes in plants with mechanical injury versus plants damaged by insects. application of insect os to mechanical injury can mimic most plant responses to herbivory, showing that the os constitute elicitors by which plants recognize insect attack. indeed, several elicitors have been isolated from insect os that trigger plant defenses against herbivory, such as β-glucosidase, volicitin, a fatty acid–amino acid conjugate, caeliferins, and inceptins. lepidopteran os consists of saliva from mandibular and labial secretions, and regurgitant from digestive tract. the os deposited on herbivore fed plant material also contains secretions from the ventral eversible gland. despite the discovery of several elicitors, studies that show whether these elicitors originated from salivary glands or from other organs associated with feeding, such as the ventral eversible gland are limited. volicitin originated from the gut tissues of spodoptera litura larvae and inceptins are partially digested chloroplast protein formed when spodoptera frugiperda attack cowpea. the veg is a secretory structure found on the ventral surface of the thorax of caterpillars. it consists of two regions with different functions: a non-eversible glandular sac lined with secretory cells and an eversible cuticular tube. eversion of the cuticular tube forms a visible papilla, whereas secretions from the secondary gland area on the cuticular tube are transferred to the apex of the papilla and released. since the tip of the everted veg can reach the mandibles, its secretions are deposited onto the food substrate with the os. secretions from the veg of caterpillars have been associated with defense against predators and the production of anti-aggregation pheromones. however, the role of veg secretions in plant-insect interactions remains unclear. recently, zebelo and maffei demonstrated that secretions from the veg of spodoptera littoralis caterpillars trigger early defense signaling events in arabidopsis thaliana. in the present study, we investigated possible involvement of veg secretions from s. exigua caterpillars in the induction of late defense signaling in tomato. we quantified and compared the activity of defense-related enzymes, transcript levels of terpene synthase genes and other defense-related genes, and voc emission in tomato plants damaged by s. exigua caterpillars with the veg intact versus plants damaged by caterpillars with the veg ablated as well as mechanically injured plants treated with os from vegi caterpillars versus mechanically injured plants treated with os from vega caterpillars.  veg ablation didn’t affect s. exigua feeding activity before we started to assess the impact of veg secretions on triggering plant defense, we evaluated whether veg ablation affects feeding activity of s. exigua caterpillars. there were no significant differences between vega and vegi caterpillars on leaf area consumption after 24 h. veg secretions activate defense-related enzymes in tomato the selected defense-related enzymes, peroxidase, polyphenol oxidase, and lipoxygenase, were expressed in significantly higher amounts in plants damaged by vegi caterpillars and mi + osvegi than in plants damaged by vega caterpillars, mechanically injured plants, mi + osvega plants, and untreated plants. activity of pod was significantly higher in vegi-damaged and mi + osvegi tomato plants than in vega-damaged, mi, mi + osvega or undamaged plants, starting as early as 24 h after treatment. activity of ppo 48 h after treatment was,, and % higher in plants damaged by vegi caterpillars than in plants damaged by vega caterpillars, mi, mi + osvega, or undamaged plants, respectively. a significant increase in lox-specific activity levels was detected as early as 24 h after treatment in plants damaged by vegi caterpillars and md + osvegi plants compared to the other treatments. activity of lox 72 h after treatment was, and %, higher in plants damaged by vegi caterpillars than in plants damaged by vega caterpillars, mi, mi + osvega, or undamaged plants, respectively. in general, no significant differences were recorded in enzymatic activity between plants damaged by vegi caterpillars and mi + osvegi plants. table shows activity of three defense-related enzymes, peroxidase, polyphenol oxidase, and lipoxygenase, in tomato plants damaged by caterpillars with the veg intact, plants damaged by caterpillars with the veg ablated, mechanically injured plants, mechanically injured plants treated with oral secretion from vegi caterpillars, mechanically injured plants treated with os from vega caterpillars, and undamaged plants, at and 72 h after caterpillar feeding. data were collected from three plants per treatment. means within the same column and time period having different letters are significantly different. veg secretions induce vocs emission in tomato key differences were recorded in the headspace voc profiles of tomato plants from the different treatments. increased emission of vocs was detected in the headspace of plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars, mechanically injured plants, and untreated plants. specifically, green leaf volatiles and certain monoterpenes were emitted in higher amounts by plants damaged by vegi caterpillars than in the other treatments. in particular, the glvs, -2-hexenal, -3-hexenal, -3-hexenyl acetate and -2-hexenol were emitted in 7-fold, 5-fold, 7-fold and 10-fold, respectively, in plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars. table shows emission of vocs by tomato plants damaged by caterpillars with the veg intact, plants damaged by caterpillars with the veg ablated, mechanically injured plants, and undamaged plants.data were collected from three plants per treatment. means within the same row having different letters are significantly different. kovats retention index is indicated for each compound. nd = not detected. the monoterpenes, β-linalool and γ-terpinene, were emitted in significantly higher amounts by plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars and the other treatments. however, no significant differences were recorded among the treatments in the emission of α-pinene, β-phellendrene, β-pinene and β-ocimene. the sesquiterpenes, -β-caryophyllene, α-humulene and β-elemene, were also emitted in significantly higher amounts by plants damaged by vegi caterpillars compared to the other treatments. furthermore, emission of the fatty acids and organic ester, was higher in vegi-damaged plants. veg secretions increase transcript levels of defense-related genes in tomato we used quantitative rt-pcr to quantify the transcript levels of six defense-related genes including genes encoding lipoxygenase, allene oxide synthase, and four genes involved in terpene biosynthesis. lox and aos are key enzymes in the jasmonic acid biosynthesis pathway. most of defense-related genes were found up-regulated in plants damaged by vegi caterpillars and in mi + osvegi plants compared to plants damaged by vega caterpillars, mechanically injured, mi + osvega, or untreated plants. in particular, the transcript levels of the terpene synthase genes, tps, and tps -β-caryophyllene and α-humulene), were significantly higher in plants damaged by vegi caterpillars and mi + osvegi plants compared to the other treatments. differential expression of genes involved in jasmonic acid and terpene biosynthesis in tomato plants damaged by caterpillars with the veg intact, plants damaged by caterpillars with the veg ablated, mechanically injured plants, mechanically injured plants treated with oral secretion from vegi caterpillars, mechanically injured plants treated with os from vega caterpillars, and undamaged plants. qrt-pcr analyses are shown as fold change in expression. means within the same row having different letters are significantly different. discussion tomato plants damaged by s. exigua caterpillars with intact ventral eversible gland expressed significantly higher amounts of defense-related enzymes and genes, and headspace vocs than plants damaged by caterpillars with ablated veg. these results suggest that secretions from the veg of s. exigua caterpillars contain elicitors of late defense signaling in tomato. to our knowledge, this is the first report on the role of caterpillar veg secretions as an elicitor of late defense signaling in plants. a previous study by zebelo and maffei showed that veg secretions of spodoptera littoralis caterpillars induced early defense signaling in arabidopsis thaliana. the three defense-related enzymes, peroxidase, polyphenol oxidase, and lipoxigenase, were expressed in significantly higher amounts in plants damaged by vegi caterpillars or mechanically injured plants treated with oral secretion from vegi caterpillars than in plants damaged by vega caterpillars, mechanically injured plants, mechanically injured plants treated with oral secretion from vega caterpillars, or untreated plants. all three enzymes are components of the octadecanoid signal transduction pathway, which regulates the production of the phytohormone, jasmonic acid. peroxidases are a group of plant defense-related enzymes, which limit plant nutritional quality to insect herbivores through quinone and reactive oxygen species generation with subsequent inhibition of insect digestion of plant material. over-expression of pods can enhance plant resistance to insects and limit plant nutritional quality to insect herbivores. suzuki et al. reported that herbivory by caterpillars and high pod activity resulted in the up-regulation of several tomato genes including genes encoding proteinase inhibitors. polyphenol oxidase is an inducible enzyme that is found throughout the plant kingdom and known to have defensive role against herbivores and pathogens. production of ppo is induced by mechanical injury, methyl jasmonate and herbivory. similar to our results, chung et al. reported high ppo levels in tomato plants wounded mechanically and treated with oral secretions from colorado potato beetle, leptinotarsa decemlineata, suggesting that insect os contain elicitors of ppo activity. lipoxygenases are another group of anti-oxidative enzymes involved in plant defense against herbivory and pathogens through the octadecanoid pathway. one of the most important functions of lox in plant defense is the oxidation of linolenic acid in the ja signaling pathway. allene oxide synthase catalyzes the first step of the lox pathway that leads to ja biosynthesis. in the present study, we observed an early induction of lox-specific activity within 24 h of feeding by caterpillars with intact veg. likewise, the transcript levels of lox and aos genes were higher in plants damaged by vegi caterpillars compared to plants damaged by caterpillars with ablated veg. these results are consistent with previous studies which demonstrate that caterpillar feeding up-regulates the expression of lox genes in tomato. our results also showed increased emission of vocs in tomato plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars or mechanically injured plants. among the common vocs induced by herbivory are those that are lox-derived, such as green leaf volatiles, terpenoids and methyl salicylate. numerous plants emit glvs and other vocs as an indirect defense strategy against herbivory, as these volatiles can attract predacious and parasitic natural enemies of herbivores. in this study, glvs and certain monoterpenes were emitted in higher amounts by plants damaged by vegi caterpillars, suggesting the involvement of the veg in the induction of plant vocs. for instance, most glvs including -2-hexenal, -3-hexenal, -2-hexenol and -3-hexenyl acetate were detected in higher amounts in plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars. interestingly, many of these glvs are used as host location cues by caterpillar parasitoids, suggesting that veg secretions may impact tritrophic interactions. glvs have also been reported to play a role in plant-plant interactions. another highly diverse group of plant compounds are the terpenoids, which are synthesized by a group of enzymes called terpene synthases to produce mono-, sesqui- and diterpenes. terpenes are more costly to synthesize per gram than most other primary and secondary plant compounds. studies have shown that a single mechanical plant tissue injury event may not elicit induced defense related volatile organic compounds. however, application of os to mechanically wounded site could elicit the release of inducible volatile compounds and thereby mimic herbivory. in the present study, we observed significantly higher emission of the monoterpenes, β-linalool and γ-terpinene, in plants damaged by vegi caterpillars compared to those damaged by vega caterpillars. however, there were no significant differences recorded among the treatments in the emission of other monoterpenes such as α-pinene, β-phellendrene, β-pinene and β-ocimene, suggesting that not all vocs are inducible by veg secretions. like monoterpenes, sesquiterpenes are phytoalexins which play a pivotal role in direct and indirect defenses against herbivores. in the present study, several sesquiterpenes -β-caryophyllene, α-humulene and β-elemene), some fatty acids and an organic ester were emitted in significantly higher amounts by plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars. results from gene expression studies showed that most terpene synthase genes -β-caryophyllene and α-humulene) and the genes involved in the biosynthesis of glvs and jasmonic acid were up-regulated in plants damaged by vegi caterpillars as well as in mechanically injured plants treated with oral secretion from vegi caterpillars. however, the transcript levels of these genes were not up-regulated in plants damaged by vega caterpillars, mechanically injured plants treated with oral secretion from vega caterpillars, or mechanically injured plants. these results suggest that an intact veg in s. exigua caterpillars is crucial for eliciting late defense signaling via the expression of defense-related genes. these findings are in agreement with those of bricchi et al. which showed that mechanical injury alone failed to increase the transcript levels of terpene synthase and ja biosynthesis genes in arabidobsis thaliana, but mechanical injury treated with spodoptera littoralis oral secretion activated the genes. in a recent review of the role of caterpillar secretions on induced plant defenses, felton suggested that the veg may play an important role in secretion during feeding by caterpillars in the family noctuidae. the structure and proximity of the veg to the caterpillar mouthparts lend credence to this proposal. when a caterpillar feeds on a plant material the veg is distended from its eversible position on the ventral surface of the caterpillar thorax and reaches the injured plant surface. furthermore, because the tip of the everted veg can reach the mandibles during feeding, the veg secretions are usually deposited onto the food substrate with the os. our results confirm that the veg secretions, which are deposited along with oral secretions or regurgitate onto plants during caterpillar feeding can induce late defense signaling in tomato. further studies are needed to identify the bioactive components of the veg secretions that trigger plant defense signaling.  the veg was first reported in, but very little is known about its role in plant-insect interactions. our current results suggest that the veg of s. exigua contain elicitors of late plant defense signaling which may trigger defense-related enzymatic activity, regulate expression of terpene synthase genes and other defense-related genes, and induce plant vocs, with potential ramifications for plant-insect and tritrophic interactions. studies are underway in our lab to investigate whether the veg secretion alone or in combination with other labial gland secretions and gut regurgitates trigger plant responses against insect herbivory. further studies are needed to comprehend the complexity of plant signaling networks and the role of insect oral secretions in mediating plant-insect and trititrophic interactions. 
these results suggest that the veg of s. exigua caterpillars contains elicitors of late plant defense signaling in tomato which trigger defense-related enzymatic activity, regulate expression of defense-related genes, and induce emission of plant vocs. however, studies that shows whether these elicitors originated from salivary glands or from other organs associated with feeding, such as the ventral eversible gland are limited. studies have shown that oral secretions from caterpillars contain elicitors of induced plant responses. the quantified defense-related enzymes were expressed in significantly higher amounts in plants damaged by vegi caterpillars than in plants damaged by vega caterpillars. similarly, the genes that encode for the key enzymes involved in the biosynthesis of jasmonic acid and terpene synthase genes that regulate production of terpene vocs, were up-regulated in plants damaged by vegi caterpillars. increased emissions of vocs were detected in the headspace of plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars. therefore, it is essential that plants are capable of sensing and differentiating mechanical injury from herbivore injury. to test this hypothesis, we quantified and compared the activity of defense-related enzymes, transcript levels of defense-related genes and voc emission in tomato plants damaged by s. exigua caterpillars with the veg intact versus plants damaged by caterpillars with the veg ablated. moreover, the os of vega caterpillars were less active in inducing the expression of defense genes in tomato plants. these signaling cascades may have important ramifications for plant-insect and tritrophic interactions. here, we tested the hypothesis that the secretions from the veg gland of spodoptera exigua caterpillars contain elicitors that induce plant defenses by regulating the expression of genes involved in the biosynthesis of volatile organic compounds and other defense-related genes.
 plant induced defense against herbivory are generally associated with metabolic costs that result in the allocation of photosynthates from growth and reproduction to the synthesis of defense compounds. therefore, it is essential that plants are capable of sensing and differentiating mechanical injury from herbivore injury. studies have shown that oral secretions from caterpillars contain elicitors of induced plant responses. however, studies that shows whether these elicitors originated from salivary glands or from other organs associated with feeding, such as the ventral eversible gland are limited. here, we tested the hypothesis that the secretions from the veg gland of spodoptera exigua caterpillars contain elicitors that induce plant defenses by regulating the expression of genes involved in the biosynthesis of volatile organic compounds and other defense-related genes. to test this hypothesis, we quantified and compared the activity of defense-related enzymes, transcript levels of defense-related genes and voc emission in tomato plants damaged by s. exigua caterpillars with the veg intact versus plants damaged by caterpillars with the veg ablated.  the quantified defense-related enzymes were expressed in significantly higher amounts in plants damaged by vegi caterpillars than in plants damaged by vega caterpillars. similarly, the genes that encode for the key enzymes involved in the biosynthesis of jasmonic acid and terpene synthase genes that regulate production of terpene vocs, were up-regulated in plants damaged by vegi caterpillars. moreover, the os of vega caterpillars were less active in inducing the expression of defense genes in tomato plants. increased emissions of vocs were detected in the headspace of plants damaged by vegi caterpillars compared to plants damaged by vega caterpillars.  these results suggest that the veg of s. exigua caterpillars contains elicitors of late plant defense signaling in tomato which trigger defense-related enzymatic activity, regulate expression of defense-related genes, and induce emission of plant vocs. these signaling cascades may have important ramifications for plant-insect and tritrophic interactions. vegenzymatic activityvocsdefense-related genes 