Systemic Induction of Terpenoid Aldehydes in Cotton Pigment Glands by Feeding of Larval Spodoptera exigua

Pigment glands in cotton contain terpenoid aldehydes that are toxic and deterrent to feeding of several generalist lepidopteran insects. We hypothesized that previously observed systemically induced feeding deterrence may be associated with pigment glands. We conducted experiments to determine the dynamics and chemical nature of inducible feeding deterrents in leaves of cotton, Gossypium hirsutum L, to larvae of the beet armyworm, Spodoptera exigua. Production and/or filling of pigment glands was influenced by physiological age of Deltapine 90 cotton plants. In undamaged plants, successively formed leaves contained more pigment glands, up to the seventh or eighth true-leaf developmental stage. Feeding choice tests conducted one or seven days after initial feeding damage revealed that third instars of S. exigua consumed more of the two youngest leaves from control cotton plants than from plants whose two oldest leaves had been fed on previously for 24 hr by S. exigua. The preference for leaves from control plants was significant one day after initial damage and highly significant seven days after damage. Consumption of mature foliage (leaf immediately above initially damaged leaves) from control plants and damaged plants did not differ. More pigment glands were counted on the youngest leaf of damaged plants than on the youngest leaf of control plants one day after initial damage. HPLC analysis revealed greater amounts of hemigossypolone, heliocides 1 and 2 (H₁ and H₂), and total terpenoid aldehydes per gland in young foliage of damaged plants than control plants one day after initial injury. By seven days after initial injury, greater quantities of hemigossypolone and all heliocides except H₄ were detected in young foliage from damaged plants compared to control plants. Concentrations of H₁ per gland in young leaves from damaged plants increased the most of all terpenoid aldehydes measured (3.4× the amount found in leaves from control plants). Mature leaves from damaged plants did not contain more terpenoid aldehydes than mature leaves from control plants. We suggest that systemically induced feeding deterrence to S. exigua in young leaves of glanded cotton was due to increased amounts of terpenoid aldehydes in pigment glands.     

Specificity of Systemically Released Cotton Volatiles as Attractants for Specialist and Generalist Parasitic Wasps

Cotton plants under herbivore attack release volatile semiochemicals that attract natural enemies of the herbivores to the damaged plant. The volatiles released in response to herbivory are not only released from the damaged leaves but from the entire cotton plant. We found that cotton plants that released myrcene, (Z)-3-hexenyl acetate, (E)--ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene, (E)--farnesene, and (E, E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene systemically from undamaged leaves of caterpillar damaged plants were attractive to the generalist parasitoid Cotesia marginiventris and the specialist parasitoid Microplitis croceipes. Plants from which the caterpillar damaged leaves were removed and that released those compounds systemically were significantly preferred over undamaged control plants in two-choice experiments in a flight tunnel. Artificially damaged cotton plants that released green leafy volatiles and constitutive terpenoids were less attractive for M. croceipes and C. marginiventris. Only C. marginiventris preferred artificially damaged plants over undamaged control plants, whereas M. croceipes showed no preference. The apparent lack of specificity of systemically released compounds in response to different herbivores feeding on the lower leaves is discussed.     

Systemic Induction of Allelochemicals in Glanded and Glandless Isogenic Cotton by Spodoptera exigua Feeding

Induction of systemic resistance to feeding of beet armyworm, Spodoptera exigua, was investigated in two isogenic lines of Stoneville 213 cotton, Gossypium hirsutum, that differed in the presence of pigment glands. In laboratory bioassays, larvae strongly preferred to feed on glandless cotton plants when presented a choice between undamaged terminal leaves of undamaged glanded and glandless plants. Feeding damage inflicted by S. exigua larvae on the two oldest leaves of glanded plants seven days prior to feeding bioassays caused larvae to prefer by 33-fold the undamaged terminal foliage from undamaged plants compared to that from damaged plants. Feeding damage on glandless plants caused only a 2.6-fold greater preference for terminal foliage from undamaged plants over foliage from previously damaged plants. Extracts of terminal foliage from glanded cotton damaged seven days earlier had significantly greater quantities of terpenoid aldehydes (hemigossypolone, gossypol, and heliocides) than did foliage from undamaged glanded plants. Terpenoid aldehydes were undetectable in extracts of both undamaged and previously damaged glandless plants. The profile of volatile compounds collected from the headspace of mechanically damaged terminal leaves of undamaged glanded and glandless plants differed. Both cotton isolines released large quantities of lipoxygenase products (hexenyl alcohols, acetates, and butyrates), but glandless plants released only small amounts of mono- and sesquiterpenes compared to glanded plants. Glandless plants damaged seven days prior to volatile collection released significantly greater quantities of lipoxygenase products, -ocimene, and - and -farnesene than did undamaged glandless plants. Previously damaged glanded plants released significantly greater quantities of all mono- and sesquiterpenes and hexenyl acetates and butyrates, but not alcohols. The relative importance of volatile compounds versus terpenoid aldehydes in induced feeding deterrence in cotton to S. exigua larvae is still unclear.     

Comparative GC-EAD Responses of A Specialist (<Emphasis Type="Italic">Microplitis croceipes</Emphasis>) and A Generalist (<Emphasis Type="Italic">Cotesia marginiventris</Emphasis>) Parasitoid to Cotton Volatiles Induced by Two Caterpillar Species

Plants emit volatile blends that may be quantitatively and/or qualitatively different in response to attack by different herbivores. These differences may convey herbivore-specific information to parasitoids, and are predicted to play a role in mediating host specificity in specialist parasitoids. Here, we tested the above prediction by using as models two parasitoids (Hymenoptera: Braconidae) of cotton caterpillars with different degree of host specificity: Microplitis croceipes, a specialist parasitoid of Heliothis spp., and Cotesia marginiventris, a generalist parasitoid of caterpillars of several genera including Heliothis spp. and Spodoptera spp. We compared GC-EAD (coupled gas chromatography electroantennogram detection) responses of both parasitoid species to headspace volatiles of cotton plants damaged by H. virescens (a host species for both parasitoids) vs. S. exigua (a host species for C. marginiventris). Based on a recent study in which we reported differences in the EAG responses of both parasitoids to different types of host related volatiles, we hypothesized that M. croceipes (specialist) would show relatively greater GC-EAD responses to the herbivore-induced plant volatile (HIPV) components of cotton headspace, whereas C. marginiventris (generalist) would show greater response to the green leaf volatile (GLV) components. Thirty volatile components were emitted by cotton plants in response to feeding by either of the two caterpillars, however, 18 components were significantly elevated in the headspace of H. virescens damaged plants. Sixteen consistently elicited GC-EAD responses in both parasitoids. As predicted, C. marginiventris showed significantly greater GC-EAD responses than M. croceipes to most GLV components, whereas several HIPV components elicited comparatively greater responses in M. croceipes. These results suggest that differences in the ratios of identical volatile compounds between similar volatile blends may be used by specialist parasitoids to discriminate between host-plant and non-host-plant complexes.     

Increased Terpenoid Accumulation in Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) Foliage is a General Wound Response

The subepidermal pigment glands of cotton accumulate a variety of terpenoid products, including monoterpenes, sesquiterpenes, and terpenoid aldehydes that can act as feeding deterrents against a number of insect herbivore species. We compared the effect of herbivory by Spodoptera littoralis caterpillars, mechanical damage by a fabric pattern wheel, and the application of jasmonic acid on levels of the major representatives of the three structural classes of terpenoids in the leaf foliage of 4-week-old Gossypium hirsutum plants. Terpenoid levels increased successively from control to mechanical damage, herbivory, and jasmonic acid treatments, with E-β-ocimene and heliocide H₁ and H₄ showing the highest increases, up to 15-fold. Herbivory or mechanical damage to older leaves led to terpenoid increases in younger leaves. Leaf-by-leaf analysis of terpenes and gland density revealed that higher levels of terpenoids were achieved by two mechanisms: (1) increased filling of existing glands with terpenoids and (2) the production of additional glands, which were found to be dependent on damage intensity. As the relative response of individual terpenoids did not differ substantially among herbivore, mechanical damage, and jasmonic acid treatments, the induction of terpenoids in cotton foliage appears to represent a non-specific wound response mediated by jasmonic acid. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Prey and Non-prey Arthropods Sharing a Host Plant: Effects on Induced Volatile Emission and Predator Attraction

It is well established that plants infested with a single herbivore species can attract specific natural enemies through the emission of herbivore-induced volatiles. However, it is less clear what happens when plants are simultaneously attacked by more than one species. We analyzed volatile emissions of lima bean and cucumber plants upon multi-species herbivory by spider mites (Tetranychus urticae) and caterpillars (Spodoptera exigua) in comparison to single-species herbivory. Upon herbivory by single or multiple species, lima bean and cucumber plants emitted volatile blends that comprised mostly the same compounds. To detect additive, synergistic, or antagonistic effects, we compared the multi-species herbivory volatile blend with the sum of the volatile blends induced by each of the herbivore species feeding alone. In lima bean, the majority of compounds were more strongly induced by multi-species herbivory than expected based on the sum of volatile emissions by each of the herbivores separately, potentially caused by synergistic effects. In contrast, in cucumber, two compounds were suppressed by multi-species herbivory, suggesting the potential for antagonistic effects. We also studied the behavioral responses of the predatory mite Phytoseiulus persimilis, a specialized natural enemy of spider mites. Olfactometer experiments showed that P. persimilis preferred volatiles induced by multi-species herbivory to volatiles induced by S. exigua alone or by prey mites alone. We conclude that both lima bean and cucumber plants effectively attract predatory mites upon multi-species herbivory, but the underlying mechanisms appear different between these species.     

Host Suitability Affects Odor Association in <Emphasis Type="Italic">Cotesia marginiventris</Emphasis>: Implications in Generalist Parasitoid Host-Finding

Insect herbivores often induce plant volatile compounds that can attract natural enemies. Cotesia marginiventris (Hymenoptera: Braconidae) is a generalist parasitoid wasp of noctuid caterpillars and is highly attracted to Spodoptera exigua-induced plant volatiles. The plasticity of C. marginiventris associative learning to volatile blends of various stimuli, such as host presence, also has been shown, but little is known about how this generalist parasitoid distinguishes between host species of varying suitability. Spodoptera exigua is an excellent host that yields high parasitoid emergence, while Trichoplusia ni serves as a sub-optimal host species due to high pre-imaginal wasp mortality. We have found that S. exigua and T. ni induce different volatile blends while feeding on cotton. Here, wind tunnel flight assays were used to determine the importance of differentially induced volatiles in host-finding by C. marginiventris. We found that, while this generalist parasitoid wasp can distinguish between the two discrete volatile blends when presented concurrently, a positive oviposition experience on the preferred host species (S. exigua) is more important than host-specific volatile cues in eliciting flight behavior towards plants damaged by either host species. Furthermore, wasps with oviposition experience on both host species did not exhibit a deterioration in positive flight behavior, suggesting that oviposition in the sub-optimal host species (T. ni) does not cause aversive odor association.     

Exogenous Application of Jasmonic Acid Induces Volatile Emissions in Rice and Enhances Parasitism of <Emphasis Type="Italic">Nilaparvata lugens</Emphasis> Eggs by theParasitoid <Emphasis Type="Italic">Anagrus nilaparvatae</Emphasis>

Jasmonate signaling pathway plays an important role in induced plant defense against herbivores and pathogens, including the emission of volatiles that serve as attractants for natural enemies of herbivores. We studied the volatiles emitted from rice plants that were wounded and treated with jasmonic acid (JA) and their effects on the host-searching behavior of the rice brown planthopper, Nilaparvata lugens (Stål), and its mymarid egg parasitoid Anagrus nilaparvatae Pang et Wang. Female adults of N. lugens significantly preferred to settle on JA-treated rice plants immediately after release. The parasitoid A. nilaparvatae showed a similar preference and was more attracted to the volatiles emitted from JA-treated rice plants than to volatiles from control plants. This was also evident from greenhouse and field experiments in which parasitism of N. lugens eggs by A. nilaparvatae on plants that were surrounded by JA-treated plants was more than twofold higher than on control plants. Analyses of volatiles collected from rice plants showed that JA treatment dramatically increased the release of volatiles, which included aliphatic aldehydes and alcohols, monoterpenes, sesquiterpenes, methyl salicylate, n-heptadecane, and several as yet unidentified compounds. These results confirm an involvement of the JA pathway in induced defense in rice plants and demonstrate that the egg parasitoid A. nilaparvatae exploits plant-provided cues to locate hosts. We explain the use of induced plant volatiles by the egg parasitoid by a reliable association between planthopper feeding damage and egg presence.     

Plant–herbivore–carnivore Interactions in Cotton, <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>: Linking Belowground and Aboveground

Most studies on plant–herbivore interactions focus on either root or shoot herbivory in isolation, but above- and belowground herbivores may interact on a shared host plant. Cotton (Gossypium spp.) produces gossypol and a variety of other gossypol-like terpenoids that exhibit toxicity to a wide range of herbivores and pathogens. Cotton plants also can emit herbivore-induced volatile compounds at the site of damage and systemically on all tissues above the site of damage. As these volatile compounds attract natural enemy species of the herbivore, they are thought to represent an indirect plant defense. Our study quantified gossypol and gossypol-like compounds in cotton plants with foliage feeding (Heliocoverpa zea), root feeding (Meloidogyne incognita), or their combination. Cotton plants with these treatments were studied also with respect to induced local and systemic volatile production and the attraction of the parasitic wasp Microplitis croceipes to those plants. We also evaluated whether foliage or root feeding affected foliar nitrogen levels in cotton. After 48 hr of leaf feeding and 5 wk of root feeding, local and systemic induction of volatiles (known to attract parasitoids such as M. croceipes) occurred with herbivore damage to leaves, and it increased in levels when root herbivory was added. Nevertheless, M. croceipes were equally attracted to plants with both leaf and root damage and leaf damage only. In contrast to previous studies in cotton, production of gossypol and gossypol-like compounds was not induced in leaf and root tissue following foliage or root herbivory, or their combination. We conclude that root feeding by M. incognita has little influence on direct and indirect defenses of Gossypium hirsutum against insect herbivory.     

Volatile Emissions Triggered by Multiple Herbivore Damage: Beet Armyworm and Whitefly Feeding on Cotton Plants

Plants are commonly attacked by more than one species of herbivore, potentially causing the induction of multiple, and possibly competing, plant defense systems. In the present paper, we determined the interaction between feeding by the phloem feeder silverleaf whitefly (SWF), Bemisia tabaci Gennadius (B-biotype = B. argentifolii Bellows and Perring), and the leaf-chewing beet armyworm (BAW), Spodoptera exigua Hübner, with regard to the induction of volatile compounds from cotton plants. Compared to undamaged control plants, infestation with SWF did not induce volatile emissions or affect the number and density of pigment glands that store volatile and nonvolatile terpenoid compounds, whereas infestation by BAW strongly induced plant volatile emission. When challenged by the two insect herbivores simultaneously, volatile emission was significantly less than for plants infested with only BAW. Our results suggest that tritrophic level interactions between cotton, BAW, and natural enemies of BAW, that are known to be mediated by plant volatile emissions, may be perturbed by simultaneous infestation by SWF. Possible mechanisms by which the presence of whiteflies may attenuate volatile emissions from caterpillar-damaged cotton plants are discussed.     

Nitrogen Deficiency Affects Bottom-Up Cascade Without Disrupting Indirect Plant Defense

Nitrogen (N) is an important macronutrient for plants and insects alike, and the availability of this critical element may considerably modify bottom-up effects in tritrophic systems. By using hydroponically cultured Glycine max, we investigated the impact of N deficiency on plant growth, photosynthetic efficiency, primary metabolism, and herbivore-induced volatile (VOC) emission. Cascading effects of N deficiency on higher trophic levels were assessed by measuring the performances of the herbivore Spodoptera frugiperda and its parasitoid Cotesia marginiventris. In addition, we studied the volatile-guided foraging behavior of C. marginiventris to explore whether nutrient stress affects the plant’s indirect defense. Our results show that photosynthetic efficiency, leaf N, and soluble protein content were significantly reduced in N deficient plants whereas root biomass was increased. Nitrogen starved plants emitted the same range of herbivore-induced VOCs as control plants, but quantitative changes occurred in the release of the main compound and two other volatiles. Herbivore growth and the performance of parasitoids developing inside the affected hosts were attenuated when caterpillars fed on N deficient plants. The behavioral response of C. marginiventris to induced VOCs from N deficient hosts, however, remained unaffected. In summary, N stress had strong bottom-up effects over three trophic levels, but the plant’s indirect defense remained intact.     

Herbivore-Induced Plant Volatiles Mediate In-Flight Host Discrimination by Parasitoids

Herbivore feeding induces plants to emit volatiles that are detectable and reliable cues for foraging parasitoids, which allows them to perform oriented host searching. We investigated whether these plant volatiles play a role in avoiding parasitoid competition by discriminating parasitized from unparasitized hosts in flight. In a wind tunnel set-up, we used mechanically damaged plants treated with regurgitant containing elicitors to simulate and standardize herbivore feeding. The solitary parasitoid Cotesia rubecula discriminated among volatile blends from Brussels sprouts plants treated with regurgitant of unparasitized Pieris rapae or P. brassicae caterpillars over blends emitted by plants treated with regurgitant of parasitized caterpillars. The gregarious Cotesia glomerata discriminated between volatiles induced by regurgitant from parasitized and unparasitized caterpillars of its major host species, P. brassicae. Gas chromatography-mass spectrometry analysis of headspace odors revealed that cabbage plants treated with regurgitant of parasitized P. brassicae caterpillars emitted lower amounts of volatiles than plants treated with unparasitized caterpillars. We demonstrate (1) that parasitoids can detect, in flight, whether their hosts contain competitors, and (2) that plants reduce the production of specific herbivore-induced volatiles after a successful recruitment of their bodyguards. As the induced volatiles bear biosynthetic and ecological costs to plants, downregulation of their production has adaptive value. These findings add a new level of intricacy to plant–parasitoid interactions.     

The Effects of Arbuscular Mycorrhizal Fungi on Direct and Indirect Defense Metabolites of Plantago lanceolata L.

Arbuscular mycorrhizal fungi can strongly influence the metabolism of their host plant, but their effect on plant defense mechanisms has not yet been thoroughly investigated. We studied how the principal direct defenses (iridoid glycosides) and indirect defenses (volatile organic compounds) of Plantago lanceolata L. are affected by insect herbivory and mechanical wounding. Volatile compounds were collected and quantified from mycorrhizal and non-mycorrhizal P. lanceolata plants that underwent three different treatments: 1) insect herbivory, 2) mechanical wounding, or 3) no damage. The iridoids aucubin and catalpol were extracted and quantified from the same plants. Emission of terpenoid volatiles was significantly higher after insect herbivory than after the other treatments. However, herbivore-damaged mycorrhizal plants emitted lower amounts of sesquiterpenes, but not monoterpenes, than herbivore-damaged non-mycorrhizal plants. In contrast, mycorrhizal infection increased the emission of the green leaf volatile (Z)-3-hexenyl acetate in untreated control plants, making it comparable to emission from mechanically wounded or herbivore-damaged plants whether or not they had mycorrhizal associates. Neither mycorrhization nor treatment had any influence on the levels of iridoid glycosides. Thus, mycorrhizal infection did not have any effect on the levels of direct defense compounds measured in P. lanceolata. However, the large decline in herbivore-induced sesquiterpene emission may have important implications for the indirect defense potential of this species.     

Ozone Degrades Common Herbivore-Induced Plant Volatiles: Does This Affect Herbivore Prey Location by Predators and Parasitoids?

Inducible terpenes and lipoxygenase pathway products, e.g., green-leaf volatiles (GLVs), are emitted by plants in response to herbivory. They are used by carnivorous arthropods to locate prey. These compounds are highly reactive with atmospheric pollutants. We hypothesized that elevated ozone (O₃) may affect chemical communication between plants and natural enemies of herbivores by degrading signal compounds. In this study, we have used two tritrophic systems (Brassica oleracea–Plutella xylostella–Cotesia plutellae and Phaseolus lunatus–Tetranychus urticae–Phytoseiulus persimilis) to show that exposure of plants to moderately enhanced atmospheric O₃ levels (60 and 120 nl l⁻¹) results in complete degradation of most herbivore-induced terpenes and GLVs, which is congruent with our hypothesis. However, orientation behavior of natural enemies was not disrupted by O₃ exposure in either tritrophic system. Other herbivore-induced volatiles, such as benzyl cyanide, a nitrile in cabbage, and methyl salicylate in lima bean, were not significantly reduced in reactions with O₃. We suggest that more atmospherically stable herbivore-induced volatile compounds can provide important long-distance plant-carnivore signals and may be used by natural enemies of herbivores to orientate in O₃-polluted environments.     

Attractiveness of Constitutive and Herbivore-Induced Sesquiterpene Blends of Maize to the Parasitic Wasp <Emphasis Type="Italic">Cotesia marginiventris</Emphasis> (Cresson)

Plant volatile compounds induced by herbivore attack have been demonstrated to provide a signal to herbivore enemies such as parasitic wasps that use these volatiles to locate their hosts. However, in addition to herbivore-induced volatiles, plants often release volatiles constitutively. We assessed the interaction between herbivore-induced and constitutively released volatiles of maize in the attraction of the wasp Cotesia marginiventris that parasitizes herbivorous lepidopteran larvae feeding on maize. Experiments were carried out with olfactometers in which the sources of volatiles were transgenic Arabidopsis thaliana plants overexpressing maize sesquiterpene synthases that produce blends of herbivore-induced or constitutive compounds. We found that the constitutive volatiles of maize terpene synthase 8 (TPS8) were attractive to C. marginiventris, just like the herbivore-induced volatiles of TPS10 studied earlier. A mixture of both the TPS8 and TPS10 volatile blends, however, was more effective in parasitoid attraction, indicating that constitutively released sesquiterpenes enhance the attraction of those induced by herbivores. While C. marginiventris did not distinguish among the volatiles of TPS8, TPS10, nor those of another maize sesquiterpene synthase (TPS5), when these blends were combined, their attractiveness to the wasp appeared to increase with the complexity of the blend.     

Herbivore-induced volatile emissions from cotton (Gossypium hirsutum L.) seedlings

The effect of herbivory on the composition of the volatile blends released by cotton seedlings was investigated by collecting volatiles from undamaged, freshly damaged (0–2 hr after initiation of feeding), and old damaged (16–19 hr after initiation of feeding) plants on which corn earworm caterpillars (Helicoverpa zea Boddie) were actively feeding. A blend of 22 compounds was consistently observed to be emitted by the old damaged plants with nine occurring either only in, or in significantly greater amounts in old damaged, as compared with freshly damaged plants. These were (Z)-3-hexenyl acetate, hexyl acetate, (E)--ocimene, (3E)-4,8-dimethyl-1,3,7-nonatriene, (Z)-3-hexenyl butyrate, (E)-2-hexenyl butyrate, (Z)-3-hexenyl 2-methylbutyrate, (E)-2-hexenyl 2-methylbutyrate, and indole. The nature of this response is compared with other studies where herbivore-induced volatile responses are also known. The presence of large amounts of terpenes and aldehydes seen at the onset of feeding and the appearance of other compounds hours later suggest that cotton defense mechanisms may consist of a constitutive repertoire that is augmented by an induced mechanism mobilized in response to attack. A number of the induced compounds are common to many plants where, in addition to an immediate defensive function, they are known to be involved in the attraction of natural enemies.     

Above- and Below-Ground Terpenoid Aldehyde Induction in Cotton, Gossypium herbaceum, Following Root and Leaf Injury

Studies on induced defenses have predominantly focused on foliar induction by above-ground herbivores and pathogens. However, roots are attacked by as many if not more phytophages than shoots, so in reality plants are exposed to above- and below-ground attack. Here, we report effects of foliar and/or root damage on terpenoid aldehyde accumulation in cotton (Gossypium herbaceum). Using HPLC, we analyzed concentrations of individual terpenoid aldehydes in foliage and root tissue. In undamaged plants, terpenoid aldehydes were concentrated in young immature main leaves. Concentrations in side leaves, branching from the main leaves, did not differ among leaf position. Above-ground feeding by Spodopterta exigua larvae on a mature leaf enhanced terpenoid concentrations in immature leaves but not in the damaged leaf. In particular, concentrations of hemigossypolone and the heliocides 1 and 4 were enhanced following herbivory. Root herbivory by wireworms (Agriotes lineatus) also resulted in an increase in terpenoid levels in the foliage. In contrast with foliar herbivory, both immature and mature leaves were induced. However, the level of induction after root herbivory was much lower compared to foliar herbivory. Plants exposed to root herbivory also had significantly higher levels of terpenoid aldehydes in root tissue, while no such effect was found following foliar herbivory. Plants exposed to both root and foliar herbivory appeared to induce primarily above-ground at the cost of below-ground defense. The implications for above- and below-ground Mutitrophic interactions are discussed.     

Volicitin, An Elicitor of Maize Volatiles in Oral Secretion of Spodoptera Exigua: Isolation and Bioactivity

Plants respond to insect-inflicted injury by systemically releasing relatively large amounts of several volatile compounds, mostly terpenoids and indole. As a result, the plants become highly attractive to natural enemies of the herbivorous insects. In maize, this systemic response can be induced by the uptake via the stem of an elicitor present in the oral secretions of caterpillars. Such an elicitor was isolated from the regurgitant of Spodoptera exigua larvae, identified as N-(17-hydroxylinolenoyl)-L-glutamine, and named volicitin. Here we present details on the procedure that was used to isolate volicitin and the biosasays that demonstrate its potency as an elicitor of maize volatiles that attract parasitoids. With a series of liquid chromatography purification steps, volicitin was separated from all other inactive substances in the regurgitant of larvae of the noctuid moth S. exigua. Maize seedlings that were incubated in very low concentrations of pure natural volicitin released relatively large amounts of terpenoids and became highly attractive to the parasitoid Microplitis croceipes. The identification of this and other insect-derived elicitors should allow us to determine their precise source and function, and better understand the evolutionary history of the phenomenon of herbivore-induced volatile emissions in plants.     

Fungal Infection Reduces Herbivore-Induced Plant Volatiles of Maize but does not Affect Naïve Parasitoids

Plants attacked by insects release volatile compounds that attract the herbivores' natural enemies. This so-called indirect defense is plastic and may be affected by an array of biotic and abiotic factors. We investigated the effect of fungal infection as a biotic stress agent on the emission of herbivore-induced volatiles and the possible consequences for the attraction of two parasitoid species. Maize seedlings that were simultaneously attacked by the fungus Setosphaeria turcica and larvae of Spodoptera littoralis emitted a blend of volatiles that was qualitatively similar to the blend emitted by maize that was damaged by only the herbivore, but there was a clear quantitative difference. When simultaneously challenged by fungus and herbivore, the maize plants emitted in total 47% less of the volatiles. Emissions of green leaf volatiles were unaffected. In a six-arm olfactometer, the parasitoids Cotesia marginiventris and Microplitis rufiventris responded equally well to odors of herbivore-damaged and fungus- and herbivore-damaged maize plants. Healthy and fungus-infected plants were not attractive. An additional experiment showed that the performance of S. littoralis caterpillars was not affected by the presence of the pathogen, nor was there an effect on larvae of M. rufiventris developing inside the caterpillars. Our results confirm previous indications that naïve wasps may respond primarily to the green leaf volatiles.     

Differential attractiveness of induced odors emitted by eight maize varieties for the parasitoid cotesia marginiventris: is quality or quantity important?

Herbivore-induced plant volatiles can function as indirect defense signals that attract natural enemies of herbivores. Several parasitoids are known to exploit these plant-provided cues to locate their hosts. One such parasitoid is the generalist Cotesia marginiventris, which is, among others, attracted to maize volatiles induced by caterpillar damage. Maize plants can be induced to produce the same blend of attractive volatiles by treating them with regurgitant of Spodoptera species. We collected and analyzed the regurgitant-induced emissions of two plant species (cowpea and maize) and of eight Mexican maize varieties and found significant differences among their volatile emissions, both in terms of total quantity and the quality of the blends. In a Y-tube olfactometer, the odors of the same artificially induced plant species and Mexican varieties were offered in dual choice experiments to naïve mated females of C. marginiventris. Wasps preferred cowpea over maize odor and, in 3 of 12 combinations with the maize varieties, they showed a preference for the odors of one of the varieties. A comparison of the odor collection with results from the behavioral assays indicates that not only the quantity of the volatile emissions, but also the quality composition of the volatile blends is important for attraction of C. marginiventris. The results are discussed in the context of the possibility of breeding crop varieties that are particularly attractive to parasitoids.