Feeding stimulatory and inhibitory chemicals from an acceptable nonhost plant forManduca sexta: Improved detection by larvae deprived of selected chemosensory organs

The chemical basis of feeding responses to the acceptable nonhost plantVigna sinensis (cowpea) by larvae ofManduca sexta was investigated using chemical isolation techniques directed by a novel chemosensory-based bioassay. The presence of feeding stimulatory and inhibitory compounds in leaves or leaf extracts was determined in a two-choice preference test using leaf disks or glass fiber filter paper disks laced with leaf extract as test substrate and filter paper disks laced with water as control. Larvae strongly prefer the control disks over leaf disks, indicating the presence of feeding inhibitory compounds in the leaf. An ethanol extract of both fresh and dried leaves neither stimulated nor inhibited feeding. The cause of this inactivity was examined by using larvae that respond strongly to either feeding stimulatory or inhibitory compounds due to selective chemosensory deprivation. Larvae having chemosensory organs remaining only on the maxillary palps are stimulated to feed by whole leaf disks and by the ethanol extracts. In contrast, larvae having only the medial and lateral maxillary sensilla styloconica and the epipharyngeal sensilla remaining are strongly inhibited by whole leaf disks and the ethanol extract of fresh leaves. Thus, the ethanol extract contains both feeding stimulatory and inhibitory compounds, which elicit opposite behavioral effects in unoperated larvae, therefore nullifying any stimulatory and inhibitory activity. These compounds can only be demonstrated by using discrimination-enhanced larvae in the choice tests. Further isolation of the feeding stimulatory principle inV. sinensis yielded two separate fractions of neutral compounds, suggesting at least two different chemicals belonging to two different classes: nonpolar and polar lipids. Feeding inhibitory chemicals have apparently polar properties because strong activity was found in the ethyl acetate and methanol extracts of dried leaves. The role of feeding stimulatory and inhibitory compounds in food selection ofM. sexta larvae is discussed.     

The role of leaf lipids in food selection by larvae of the tobacco hornworm,Manduca sexta

The role of leaf lipids in food plant selection by larvae ofManduca sexta was investigated by measuring preference responses in a two-choice preference test using glass fiber filter paper disks laced with extract (test) or water (control). The larvae respond to the petroleum ether extract of whole leaves of the host-plantLycopersicon esculentum (tomato) extract in a concentration-dependent manner. At natural concentration it is the most strongly stimulating extract or compound yet tested using the disk test. This response is affected by food plant experience of the larvae, suggesting stimulation by plant-specific compounds in the extract. The extract contains volatile compounds that attract the larvae. In contrast, it does not promote continued feeding on an agar-cellulose diet that incorporates the extract. Also stimulating are the extracts of leaf surfaces of two hosts,L. esculentum andSolanum pseudocapsicum, and two acceptable nonhosts,Brassicae napus andVigna sinensis, indicating the presence of nonpolar feeding stimulants at the leaf surface. However, similar leaf-surface extracts of the unacceptable plantCanna generalis were inactive, although the surface extraction process renders this plant acceptable. Leaf-surface extracts ofL. esculentum, S. pseudocapsicum, andB. napus evoke feeding responses that are qualitatively comparable to those of their corresponding leaves. However, no such parallel is found for surface extracts ofV. sinensis andC. generalis. Thus, nonpolar compounds at the leaf surface of host and some acceptable non-host-plant species strongly stimulate feeding and hence must play an important role in food selection by the tobacco hornworm.     

Leaf surface extracts ofSolanum berthaultii hawkes deter colorado potato beetle feeding

Leaf rinses ofS. berthaultii PI 473334 with methylene chloride were deterrent to feeding by the Colorado potato beetle when applied toS. tuberosum tuber and leaf disks. When the leaf rinse was separated into its nonvolatile and volatile fractions and applied to tuber disks, the nonvolatile fraction was highly deterrent, while the volatile fraction reduced consumption, but not significantly compared to the controls. A hexane leaf rinse was not deterrent to feeding, while an acetone rinse was approximately twofold more deterrent than the methylene chloride rinse when applied to leaf disks. Three cycles of bioassay-guided, reversed-phase open-column fractionation of an acetone leaf rinse yielded a relatively polar fraction with low deterrent activity, and two nonpolar fractions exhibiting higher specific activity. Reversed-phase preparative HPLC of these fractions yielded seven active fractions among the 10 assayed. Subsequent analytical HPLC indicated that two fractions each contained a single UV-absorbing compound, while another represented a mixture of at least four compounds. The remaining fractions were composed of complex mixtures of possibly ionic or polymeric compounds that were poorly resolved by HPLC.     

Effects ofneo-clerodane diterpenes fromTeucrium on feeding behavior of colorado potato beetle larvae

Treatment of potato leaf disks with fourneo-clerodane diterpenes fromTeucrium (eriocephalin, teucrin-A, teucvin, and teuscorolide) significantly reduced feeding by larvae ofLeptinotarsa decemlineata (Colorado potato beetle). Choice and no-choice tests suggest that teuscorolide acts as a feeding deterrent, whereas the antifeedant activity of teucrin-A, teucvin, and eriocephalin is likely associated with a toxic mode of action. Nutritional tests, antifeedant simulation assays, and posttreatment studies confirmed that teucrin-A can be categorized by its mode of action as a toxin, rather than as a feeding deterrent.     

Chemical Analysis of Squirt-gun Defense in Bursera and Counterdefense by Chrysomelid Beetles

The genus Bursera produces resin stored in canals in the leaf. When leaves are damaged, some, but not all, species release abundant resin. Species of Blepharida are specialized herbivores of Bursera, and they exhibit variation in their counterdefensive behavior. Species feeding on resin-releasing plants cut the leaf veins before feeding, which often makes them more prone to predation. They also adorn their backs with their feces and may regurgitate and release an anal secretion when attacked or disturbed by predators. Species that feed on Bursera species that release no fluids do not sever the leaf veins prior to feeding, and they do not carry their feces on their backs. Instead, they face their predators, raise their heads in a "boxing-like" display, and rapidly swing their abdomens from side to side. We performed a comparative chemical analysis of the compounds found in Bursera schlechtendalii, a species that releases abundant resins, and B. biflora, a species that does not. We also analyzed the frass, enteric discharges, and larvae of the two species of Blepharida that feed on each of these plants. The compounds found in the body, feces, and discharges of the Blepharida species that adorns itself with feces match the chemical mixture of its host plant, suggesting that this beetle species can compensate its higher risk of predation by using the compounds present in the plant for defense. The chemical mixture of B. biflora is more complex and does not match the compounds found in the body or frass of its beetle herbivore, suggesting that the defensive strategy of this insect is behavioral and does not rely on its host's constituents.     

Host-plant acceptance by geographic populations of the colorado potato beetle,Leptinotarsa decemlineata

We used a detailed analysis of feeding behavior to investigate the role of solanaceous alkaloids as sensory-based feeding deterrents for the Colorado potato beetle,Leptinotarsa decemlineata (Say). Experiments were conducted on three geographic, host-adapted populations of beetles to determine whether evolutionary changes in host use have been accompanied by behavioral adaptations to alkaloids. Solanine and tomatine, steroidal glycoalkaloids found in two regional host plants, did not reduce leaf consumption or significantly alter behavior patterns of newly emerged beetles, including those from populations that normally will not feed on plants containing the compounds. Atropine, a tropane alkaloid found in several taxonomically related nonhost species, caused a significant increase in sampling behavior (indicating direct action on the sensory system) and reduced acceptance of treated potato leaves. We propose that variable acceptance of host plants among regional populations ofL. decemlineata has evolved independently of adaptations to alkaloids at the sensory level. To establish that secondary compounds such as atropine influence host choice in nature, field observations are needed to confirm that beetles routinely encounter, and sample, nonhost species.     

Effects ofHymenaea courbaril leaf resin on the generalist herbivoreSpodoptera exigua (beet armyworm)

The hypothesis that leaf resin ofHymenaea courbaril (Leguminosae) functions as a defence against herbivory was tested in a series of experiments with the generalist herbivore, beet armyworm (Spodoptera exigua Hübn). Pure leaf resin obtained by steam distillation was incorporated into an artificial diet at 0.0%, 0.16%, 0.50%, and 1.60% (dry weight) in one experiment and 0.0%, 1.0%, and 3.2% (dry weight) in a second experiment;S. exigua larvae were reared on these diets. In four palatability experiments the amount of leaf area eaten was compared between pairs of different leaf material. Mortality due to viral infection of larvae in the first feeding experiment showed a dose-response to leaf resin concentration. In the second experiment larvae showed a dose-response in the reduction of pupal weight and delay of time to pupation. These are interpreted as a dose-related stress onS. exigua which occurs with increased resin concentration in the diet. In the palatability tests,S. exigua strongly preferred untreated to resintreated bean leaf disks, bean disks toH. courbaril immature leaf disks,H. courbaril immature leaf toH. courbaril mature leaf, and bean leaf disks toH. courbaril newly emerged leaf disks. From these results, we conclude thatH. courbaril leaf resins possess toxic and feeding-deterrent properties which make them effective as an herbivore defense.     

Chemical and experiential basis for rejection ofTropaeolum majus byPieris rapae larvae

Rejection of nasturtium,Tropaeolum majus, by cabbage-reared larvae ofPieris rapae has been explained by the presence of feeding deterrents in the nastrutium foliage. Sensitivity to the deterrents develops as neonate larvae feed on cabbage. The most prominent deterrent compound, which is present in nasturtium at a concentration of 40 mg/100 g fresh leaves, was identified as chlorogenic acid. When neonate larvae were fed on a cabbage leaf treated with high concentrations of deterrent-containing extracts of nasturtium foliage, they remained insensitive to the deterrents, so they accepted nasturtium when transferred as second instars. When neonate larvae were reared on a cabbage leaf treated with 0.1 mg chlorogenic acid, ca. 35% of the second instars accepted nasturtium. Similar dietary exposure of neonates to the subunits of chlorogenic acid, caffeic acid and quinic, acid resulted in much less or no effect on the rejection behavior of second instars. The results suggest that the combined effects of specific chemical constituents of nasturtium can explain the rejection of this plant by larvae ofP. rapae, but if larvae are continuously exposed to these compounds immediately after hatching, they apparently become habituated to the feeding deterrents. The lack of activity of the subunits of chlorogenic acid suggests that specific structural features are necessary for a dietary constituent to cause such habituation or suppression of sensitivity development.     

Phenolic compounds on the pod-surface of pigeonpea,Cajanus cajan,mediate feeding behavior of Helicoverpa armigera larvae

A methanol extract of the pod surfaces of Cajanus cajan, a feeding stimulant for fifth-instar Helicoverpa armigera, was shown to contain four main phenolic compounds. Three of these were identified as isoquercitrin, quercetin, and quercetin-3-methyl ether, by comparing UV spectra and HPLC retention times with authentic standards. The fourth compound was isolated by semipreparative HPLC and determined to be 3-hydroxy-4-prenyl-5- methoxystilbene-2-carboxylic acid (stilbene) by NMR spectroscopy and mass spectrometry. Quercetin, isoquercitrin, and quercetin-3-methyl did not affect the selection-behavior of fifth-instar H. armigera. However, larvae were deterred from feeding on glass-fiber disks impregnated with the stilbene. Furthermore, larvae exposed to quercetin-3-methyl ether consumed significant amounts of both disks. In a binary-choice bioassay, a combination of quercetin-3-methyl ether and the stilbene on one disk and pure quercetin-3-methyl ether on the other disk resulted in increased consumption of both glass-fiber disks by larvae. In contrast, consumption was reduced if the combination was presented to larvae on one disk with purified stilbene on the other disk. Cajanus cajan cultivars that varied in their susceptibility to H. armigera were surveyed for the presence of the four phenolic compounds. An absence of quercetin and higher concentrations of isoquercitrin than the cultivated variety characterized pod surface extracts of pod-borer-resistant cultivars. In addition, the ratio of the stilbene to quercetin-3-methyl ether was greater in the pod-borer-resistant cultivars. These findings are discussed in relation to the identification of chemical characters that can be used for crop improvement.     

Larval Feeding Stimulants for a Rutaceae-Feeding Swallowtail Butterfly, Papilio xuthus L. in Citrus unshiu Leaves

Larvae of a swallowtail butterfly, Papilio xuthus L., feed exclusively on plants of the family Rutaceae, including various Citrus crops. Larvae were strongly stimulated to feed on paper strips impregnated with ethanolic extracts of host-plant leaves. Stimulation of feeding on extracts of Citrus unshiu leaves required a mixture of chemicals including sugars (d-glucose, d-fructose, and d-sucrose), a betaine [(−)-stachydrine], a cyclic peptide (citrusin I), a polymethoxyflavone (isosinensetin), and the lipids 1-linolenoylglycerol, 1-linoleoylglycerol, 1-octadecenoylglycerol, 1-stearoylglycerol, and 1,2-dilinolenoyl-3-galactosyl-sn-glycerol. When these compounds were assayed individually, few larvae consumed test strips. However, larvae readily chewed the test strips treated with a mixture of all compounds, indicating that host recognition by P. xuthus larvae is mediated by a specific combination of both primary and secondary substances. Comparison of 11 stimulant components with 10 compounds from C. unshiu leaves previously reported as stimulant components for oviposition by P. xuthus adult females revealed only one compound, stachydrine, as an ingredient in common. While the larval feeding-stimulant mixture is dominated by nutrients and other compounds of general significance for primary metabolism, the component oviposition stimulants are mostly secondary substances, including flavonoid glycosides, protoalkaloids, a cyclitol, and a betaine, that have restricted distributions in plants. Reliance by adult females on unique profiles of secondary compounds presumably reflects the need to locate and recognize specific host-plant species within a diverse flora. Since the initial host choice for the larvae is made typically by the ovipositing female, however, unique secondary compounds may be less important for larval feeding than are compounds useful for indicating food and microhabitat quality once on the host plant.     

Insect antifeedant activity of clerodane diterpenoids against larvae ofSpodoptera Littoralis (Boisd.) (Lepidoptera)

Antifeedant activities of nine clerodane diterpenoids, isolated in this laboratory from different species ofAjuga plants, have been studied against larvae of Egyptian cotton leafwormSpodoptera littoralis (Boisd.) (Lepidoptera) by application of the leaf disk method. Evaluation of activity was carried out by calculating, at different time intervals, the feeding ratio (FR) from the relationship between the consumed areas of treated disks (CTD) and control disks (CCD); for comparison purposes a FR₅₀ defined as the FR at a CCD of 50% was established. Some compounds exhibited activity at a 0.01 g/cm² dose (0.3 ppm). Structure-activity relationships are discussed     

Sexual Maturation and Temporal Variation of Neural Responses in Adult Colorado Potato Beetles to Volatiles Emitted by Potato Plants

Neural responses of the Colorado potato beetle (CPB), Leptinotarsa decemlineata to volatiles emitted by potato plants, Solanum tuberosum L were investigated. Amplitudes of electroantennograms to measured amounts of a standard odorant, (Z)-3-hexenyl acetate, increased from day of emergence through at least six to eight days of adulthood. Among 20 potato volatiles examined, several constitutive compounds, e.g., the green leaf volatiles (Z)-3-hexen-1-ol, (E)-2-hexen-1-ol, and (Z)-3-hexenyl butyrate, and systemic volatiles released primarily in response to insect feeding, e.g., (±)-linalool, nonanal, methyl salicylate, and indole, were the most effective stimuli. A statistic called linear age-skew (linear orthogonal polynomial) was used to examine differences in responses to potato volatiles between young and mature CPB. Based on plots of linear age-skew and overall neural responsiveness, 10 volatiles could be identified for which responses increased at a rate similar to or greater than the standard. The results are discussed with regard to the relationship of the CPB to its host plant and developmental studies of insect sensory responses to chemical signals.     

Chemical Stimuli from Apple Influence the Behavior of Neonate Caterpillars of the Generalist Herbivore, Epiphyas postvittana

When introduced into a wind tunnel with low windspeeds (0.2–0.3 m/sec), neonate E. postvittana larvae were more likely to walk in a downwind rather than upwind direction. This tendency to walk downwind did not change when odors from apple leaves or fruit were introduced into the wind tunnel. In a second assay that measured travel times of larvae as they walked from the center to the edge of filter paper disks, larvae moved more slowly on disks treated with extracts of apple leaves or extracts of apple fruit, but did not slow their movement on disks treated with extracts of a nonhost, Coprosma repens. Analysis of videorecords revealed that larvae on disks treated with a dichloromethane extract of apple leaves took more circuitous routes, walked more slowly, and stopped more frequently than larvae walking on solvent-treated disks. When the dosage of this dichloromethane apple leaf extract was increased or when larvae were held without food prior to testing, differences between travel times on solvent- and extract-treated disks did not increase significantly. The dichloromethane apple leaf extract, when tested in the wind tunnel with low windspeeds, also caused larvae to delay spinning down on a silken thread after reaching the edge of the disk, but had no effect on spin-down times when tested in still-air conditions. Testing of rotary evaporated apple leaf extracts and fractionation of these extracts indicated that a number of both volatile and relatively involatile chemicals contribute to the behavioral responses of E. postvittana larvae.     

Chemical Defense in <Emphasis Type="Italic">Elodea nuttallii</Emphasis> Reduces Feeding and Growth of Aquatic Herbivorous Lepidoptera

The submersed macrophyte Elodea nuttallii (Hydrocharitaceae) is invasive in Europe and frequently found in aquatic plant communities. Many invertebrate herbivores, such as larvae of the generalist aquatic moth, Acentria ephemerella (Lepidoptera, Pyralidae), avoid feeding on E. nuttallii and preferably consume native species. First instar larvae exhibited a high mortality on E. nuttallii compared to the native macrophyte Potamogeton perfoliatus. Mortality of older larvae was also high when fed E. nuttallii exposed to high light intensities. Growth of older larvae was strongly reduced on E. nuttallii compared to pondweeds (Potamogeton lucens). Neither differences in nitrogen nor phosphorus content explained the different performance on these submerged macrophytes, but plants differed in their flavonoid content. To investigate whether plant-derived allelochemicals from E. nuttallii affect larval performance in the same way as live plants, we developed a functional bioassay, in which Acentria larvae were reared on artificial diets. We offered larvae Potamogeton leaf disks coated with crude Elodea extracts and partially purified flavonoids. Elodea extracts deterred larvae from feeding on otherwise preferred Potamogeton leaves, and yet, unknown compounds in the extracts reduced growth and survival of Acentria. The flavonoid fraction containing luteolin-7-O-diglucuronide, apigenin-7-O-diglucuronide, and chrysoeriol-7-O-diglucuronide strongly reduced feeding of larvae, but did not increase mortality. The concentrations of these compounds in our assays were 0.01–0.09% of plant dry mass, which is in the lower range of concentrations found in the field (0.02–1.2%). Chemical defense in E. nuttallii thus plays an ecologically relevant role in this aquatic plant–herbivore system.     

Chemical constituents of an unacceptable crucifer,Erysimum cheiranthoides,deter feeding byPieris rapae

The wild cruciferErysimum cheiranthoides was found to contain extractable constituents that deterred feeding by larvae of the crucifer specialistPieris rapae when applied to cabbage leaf disks in both choice and nochoice bioassays. High-performance liquid chromatography was used to separate the extract into several fractions, two of which retained the feeding deterrent activity of the extract. UV-absorption spectra of the fractions suggested that one contained cardenolides similar or identical to those reported to deter oviposition byP. rapae onE. cheiranthoides. The other active fraction evidently contains a compound that deters larval feeding but not adult oviposition. The results suggest that the chemical defense ofE. cheiranthoides depends on two types of compounds acting on separate developmental stages of the insect.     

Potato Expressing Beetle-Specific Bacillus thuringiensis Cry3Aa Toxin Reduces Performance of a Moth

Expression of the Bacillus thuringiensis beetle-specific toxin Cry3Aa, which renders a genetically modified potato cultivar resistant to the Colorado potato beetle Leptinotarsa decemlineata, exerts a deleterious effect on the polyphagous moth Spodoptera littoralis. The caterpillars of S. littoralis feed less and produce smaller pupae on the genetically modified cultivar (NewLeaf Superior) than on the parental nontransgenic cultivar (Superior). The conversion efficiencies of total dry matter, combustion heat, carbon, and nitrogen from leaves to insect biomass are similar on both cultivars. In spite of similar food utilization and a relatively small difference in the body mass at pupation, female adults that developed from caterpillars fed on NewLeaf Superior lay a mean of 309 eggs compared to a mean of 713 eggs deposited by females that developed from caterpillars fed on Superior. Because of this difference and a simultaneous reduction in fertility (egg hatchability) from 78 to 48%, a pair of adults that fed as larvae on NewLeaf Superior produces only 148 larvae, whereas a pair of adults that fed as larvae on Superior produces 556 larvae. We suggest that small amounts of Cry3Aa that accumulate in insect tissue and persist until the adult stage are responsible for the decline in reproduction.     

Female sex pheromone of the pickleworm,Diaphania nitidalis (Lepidoptera: Pyralidae)

Heptane extracts of the ovipositors from pickleworm adults (Diaphania nitidalis) were found to contain (E)-11-hexadecenal along with proportionally smaller amounts of (Z)-11-hexadecenal, (E)- and (Z)-11-hexadecen-1-ol, hexadecanol, hexadecanal, and a trace amount of (E,Z)-10,12-hexadecadienal. Assays conducted in a flight tunnel and in the field showed that a synthetic mixture of the five unsaturated compounds elicited behavioral responses from pickleworm males that were indistinguishable from those elicited by extracts of the female or by mate-calling females. When any component was deleted from the set of five unsaturated compounds, the intensity and extent of male responses to the resulting mixtures were significantly attenuated. The female sex pheromone of the pickleworm resembles the pheromone of a congeneric species,D. hyalinata, but bioassays indicated that (E,E)-10,12-hexadecadienal, produced byD. hyalinata but not by the pickleworm, plays a role in pheromonal specificity.     

A quantitative feeding bioassay forPissodes strobi Peck (Coleoptera: Curculionidae)

A feeding bioassay forPissodes strobi Peck was developed that consisted of plastic petri dishes containing paired agar disks immersed in paraffin wax. Lens paper covering the top surface of the disks provided 1, 2, or 3 weevils in each replicate with a surface through which they made regular feeding punctures which could be easily counted. The number of feeding punctures was correlated with the amount of agar ingested. Candidate feeding stimulants or deterrents were applied to the paper covering one of the disks in the dishes, while the other served as a solvent control. Feeding stimulants were tested using disks of pure agar, and deterrents were assayed on agar disks that contained 2% ground, dried Sitka spruce bark,Picea sitchensis Carr. The weevils exhibited a concentration-dependent response to the amount of spruce bark in the agar disk. Feeding stimulation by Sitka spruce bark extracts, and deterrency by leaf oil of western red cedar,Thuja plicata Donn, was demonstrated. The bioassay would be useful in chemical isolation programs aimed at identification of feeding stimulants and deterrents.Research supported by National Research Council of Canada Cooperative Grant A0243 and Operating Grants A3881 and A3706.     

Tryptamine-induced resistance in tryptophan decarboxylase transgenic poplar and tobacco plants against their specific herbivores

The presence of amines and their derivatives in plant tissues is known to influence insect feeding and reproduction. The enzyme tryptophan decarboxylase (TDC) catalyzes the decarboxylation of tryptophan to tryptamine, which is both a bioactive amine and a precursor of other indole derivatives. Transgenic poplar and tobacco plants ectopically expressing TDC1 accumulated elevated levels of tryptamine without affecting plant growth and development. This accumulation was consistently associated with adverse effects on feeding behavior and physiology of Malacosoma disstria Hub. (forest tent caterpillar, FTC) and Manduca sexta L. (tobacco hornworm, THW). Behavior studies with FTC and THW larvae showed that acceptability of the leaf tissue to larvae was inversely related to foliar tryptamine levels. Physiological studies with FTC and THW larvae showed that consumption of leaf tissue from the transgenic lines is deleterious to larvae growth, apparently due to a postingestive mechanism. Thus, ectopic expression of TDC1 can allow sufficient tryptamine to accumulate in poplar and tobacco leaf tissue to suppress significantly the growth of insect pests that normally feed on these plants.     

Volatile Emissions from Aesculus hippocastanum Induced by Mining of Larval Stages of Cameraria ohridella Influence Oviposition by Conspecific Females

Larval stages of the horse chestnut leafminer Cameraria ohridella can completely destroy the surface of horse chestnut leaves, Aesculus hippocastanum. This study investigated the effect of the degree of leaf browning caused by the insect’s larvae on olfactory detection, aggregation, and oviposition of C. ohridella adults. The influence of A. hippocastanum flower scent on oviposition of the first generation was also evaluated. Utilizing gas chromatography coupled with parallel detection by mass spectrometry and electroantennography (GC-MS/EAD), more than 30 compounds eliciting responses from antennae of C. ohridella were detected. Oviposition and mining by C. ohridella caused significant changes in the profile of leaf volatiles of A. hippocastanum. After oviposition and subsequent mining by early larval stages (L1–L3), substances such as benzaldehyde, 1,8-cineole, benzyl alcohol, 2-phenylethanol, methyl salicylate, (E)-β-caryophyllene, and (E,E)-α-farnesene were emitted in addition to the compounds emitted by uninfested leaves. Insects were able to detect these compounds. The emitted amount of these substances increased with progressive larval development. During late larval stages (L4, L5) and severe loss of green leaf area, (E,E)-2,4-hexadienal, (E/Z)-linalool oxide (furanoid), nonanal, and decanal were also released by leaves. These alterations of the profile of volatiles caused modifications in aggregation of C. ohridella on leaves. In choice tests, leaves in early infestation stages showed no significant effect on aggregation, whereas insects avoided leaves in late infestation stages. Further choice tests with leaves treated with single compounds led to the identification of substances mediating an increase or decrease in oviposition.