Analysis of volatiles from black pine (Pinus nigra): significance of wounding and egg deposition by a herbivorous sawfly

The composition of headspace volatiles of black pine (Pinus nigra) was analysed by coupled gas chromatography-mass spectrometry (GC-MS). It has been shown in a previous study that egg deposition of the sawfly Diprion pini on P. sylvestris induces a quantitative change of the pine volatile blend . Chrysonotomyia ruforum, an egg parasitoid of D. pini, is known to be attracted by volatiles from egg-carrying P. sylvestris, but not by odour from egg-laden P. nigra. Therefore, the present study focused on the question whether also P. nigra as another host plant of this sawfly responds to egg deposition by change of its volatile blend. The headspace of untreated, egg-carrying, and artificially wounded P. nigra twigs were compared. The artificial damage inflicted to the twigs mimicked the damage by the sawfly female prior to egg deposition. Thirty five mainly terpenoid compounds that were identified in more than 50% of the egg-carrying P. nigra twigs could also be detected in the headspace of untreated and artificially wounded twigs. Quantitative differences of the blends of volatiles from differently treated P. nigra twigs were compared by multivariate data analyses. PLS-DA (projection to latent structures-discriminant analysis) revealed that blends of volatiles from differently treated P. nigra differed significantly. When comparing volatiles from egg-carrying and artificially wounded P. nigra with respective P. sylvestris samples, qualitative and quantitative differences were detected. The differences in volatile composition of P. nigra and P. sylvestris are discussed with special respect to the egg parasitoid's response to odours of egg-carrying pine twigs.     

A plant notices insect egg deposition and changes its rate of photosynthesis

Scots pine (Pinus sylvestris) is known to change its terpenoid metabolism in response to egg deposition by the sawfly Diprion pini (Hymenoptera, Diprionidae). Three days after egg deposition, parts of the pine twig adjacent to the egg-laden one are induced to emit volatiles, which attract egg parasitoids. In this study, we investigated whether egg deposition by this sawfly affects pine photosynthesis. Measurements of photosynthesis were taken from untreated control twigs and from pine twigs adjacent to egg-laden ones (i.e. systemically oviposition-induced twigs) for a period of 3 d starting after egg deposition. The net photosynthetic rate of oviposition-induced pine twigs was lower than that of untreated control twigs, whereas the respiration rate of pine twigs was not affected by egg deposition. CO2 response curves of oviposition-induced twigs tended to be lower than those of controls. The potential rate of electron transport (J(max)) and the maximum rate of Rubisco activity (V(cmax)) were calculated from the data of the CO2 response curves. J(max) of oviposition-induced twigs was significantly lower than that of controls at day 1 after egg deposition, while the difference diminished from day 2 to day 3. A similar pattern was observed for V(cmax). Light response curves of oviposition-induced twigs were significantly lower than those of untreated ones during 3 d of measurements. Stomatal conductance was slightly lowered by egg deposition. When considering photosynthetic activity as a physiological currency to measure costs of induction of plant defense, the effects of insect egg deposition on gas exchange of pine are discussed with respect to known effects of insect feeding on the photosynthesis activity of plants.     

Choosy egg parasitoids: Specificity of oviposition-induced pine volatiles exploited by an egg parasitoid of pine sawflies

Generalist parasitoids are well‐known to be able to cope with the high genotypic and phenotypic plasticity of plant volatiles by learning odours during their host encounters. In contrast, specialised parasitoids often respond innately to host‐specific cues. Previous studies have shown that females of the specialised egg parasitoid Chrysonotomyia ruforum Krausse (Hymenoptera: Eulophidae) are attracted to volatiles from Pinus sylvestris L. induced by the egg deposition of its host Diprion pini L. (Hymenoptera: Diprionidae), when they have previously experienced pine twigs with host eggs. In this study we investigated by olfactometer bioassays how specifically C. ruforum responded to oviposition‐induced plant volatiles. Furthermore, we studied whether parasitoids show an innate response to oviposition‐induced pine volatiles. Naïve parasitoids were not attracted to oviposition‐induced pine volatiles. The attractiveness of volatiles from pines carrying eggs was shown to be specific for the pine and herbivore species, respectively (species specificity). We also tested whether not only oviposition, but also larval feeding, induces attractive volatiles (developmental stage specificity). The feeding of D. pini larvae did not induce the emission of P. sylvestris volatiles attractive to the egg parasitoid. Our results show that a specialist egg parasitoid does not innately show a positive response to oviposition‐induced plant volatiles, but needs to learn them. Furthermore, the results show that C. ruforum as a specialist does not learn a wide range of volatiles as some generalists do, but instead learns only a very specific oviposition‐induced plant volatile pattern, i.e., a pattern induced by the most preferred host species laying eggs on the most preferred food plant.     

Reduction of ethylene emission from Scots pine elicited by insect egg secretion

Pinus sylvestris L. is known to activate indirect defence in response to attack by an herbivorous sawfly. Egg deposition by the sawfly Diprion pini L. induces pine to release, three days after egg laying, locally and systemically terpenoid volatiles that attract parasitoids to kill the eggs. The elicitor of the pine's response is located in the sawfly's oviduct secretion enveloping the eggs after deposition. Application of this secretion on twigs with artificially conducted ovipositional woundings mimics the effects of egg deposition. Furthermore, jasmonic acid (JA) induces a volatile pattern similar, but not identical, to the one induced by egg deposition. To gain deeper insight into the transduction of plant signals induced by herbivore egg deposition, it was investigated whether ethylene emission from pine is affected by sawfly egg deposition. Systemically induced ethylene emission from differently treated pine twigs was monitored for a period of 3 d after treatment. Ethylene emissions from untreated control twigs were compared with those from twigs treated as follows: (i) sawfly egg secretion [=oviduct secretion (OVI)] was transferred on artificially wounded pine needles (attractive volatiles), (ii) needles were artificially wounded (non-attractive volatiles), and (iii) the twig was supplied with JA (attractive volatiles). Ethylene emission from systemically OVI-induced twigs was significantly lower than from untreated controls, whereas artificial wounding had no detectable effect. JA-treated twigs released much more ethylene and showed higher variability of ethylene emission than artificially wounded twigs and OVI-treated ones. Ethylene emissions from pine after the various treatments studied here are discussed with respect to known effects of insect feeding on ethylene release from plants.     

The influence of intact-plant and excised-leaf bioassay designs on volicitin- and jasmonic acid-induced sesquiterpene volatile release in Zea mays

Induced plant responses to insect attack include the release of volatile chemicals. These volatiles are used as host-location signals by foraging parasitoids, which are natural enemies of insect herbivores. A plant's response to herbivory can be influenced by factors present in insect oral secretions. Volicitin (N-(17-hydroxylinolenoyl)-L-glutamine), identified in beet armyworm (Spodoptera exigua) oral secretions, stimulates volatile release in corn (Zea mays L.) seedlings in a manner similar to beet armyworm herbivory. Volicitin is hypothesized to trigger release of induced volatiles, at least in part, by modulating levels of the wound hormone, jasmonic acid (JA). We compare the sesquiterpene volatile release of damaged leaves treated with aqueous buffer only or with the same buffer containing volicitin or JA. Leaves were damaged by scratching with a razor and test solutions were applied to the scratched area. The leaves were either excised from the plant or left intact shortly after this treatment. Plants were treated at three different times (designated as Evening, Midnight, and Morning) and volatiles were collected in the subsequent photoperiod. JA and volicitin treatments stimulated the release of volatile sesquiterpenes, namely beta-caryophyllene, (E)-alpha-bergamotene, and (E)-beta-farnesene. In all cases, JA stimulated significant sesquiterpene release above mechanical damage alone. Volicitin induced an increase in sesquiterpene volatiles for all excised-leaf bioassays and the Midnight intact plants. Volicitin treatments in the Evening and Morning intact plants produced more sesquiterpenes than the untreated controls, while mechanical damage alone produced an intermediate response that did not differ from either treatment group. Excised leaves produced a 2.5- to 8.0-fold greater volatile response than similarly treated intact plants. Excision also altered the ratio of JA-and volicitin-induced sesquiterpene release by preferentially increasing (E)-beta-farnesene levels relative to beta-caryophyllene. The inducibility of volatile release varied with time of treatment. On average, sesquiterpene release was highest in the Midnight excised leaves and lowest in the Morning intact plants. The duration of induced volatile release also differed between treatments. On average, JA produced a sustained release of sesquiterpenes over time, with over 20% of the combined sesquiterpenes released in the third and final volatile collection period. In contrast, less than 8% of the combined sesquiterpenes induced by volicitin were emitted during this period. The large quantitative differences between intact plants and detached leaves suggest that the results of assays using excised tissues should be cautiously interpreted when considering intact-plant models.     

Maize landraces recruit egg and larval parasitoids in response to egg deposition by a herbivore

Natural enemies respond to herbivore-induced plant volatiles (HIPVs), but an often overlooked aspect is that there may be genotypic variation in these 'indirect' plant defence traits within plant species. We found that egg deposition by stemborer moths (Chilo partellus) on maize landrace varieties caused emission of HIPVs that attract parasitic wasps. Notably, however, the oviposition-induced release of parasitoid attractants was completely absent in commercial hybrid maize varieties. In the landraces, not only were egg parasitoids (Trichogramma bournieri) attracted but also larval parasitoids (Cotesia sesamiae). This implies a sophisticated defence strategy whereby parasitoids are recruited in anticipation of egg hatching. The effect was systemic and caused by an elicitor, which could be extracted from egg materials associated with attachment to leaves. Our findings suggest that indirect plant defence traits may have become lost during crop breeding and could be valuable in new resistance breeding for sustainable agriculture.     

Hyperparasitoids Use Herbivore-Induced Plant Volatiles to Locate Their Parasitoid Host

Plants respond to herbivory with the emission of induced plant volatiles. These volatiles may attract parasitic wasps (parasitoids) that attack the herbivores. Although in this sense the emission of volatiles has been hypothesized to be beneficial to the plant, it is still debated whether this is also the case under natural conditions because other organisms such as herbivores also respond to the emitted volatiles. One important group of organisms, the enemies of parasitoids, hyperparasitoids, has not been included in this debate because little is known about their foraging behaviour. Here, we address whether hyperparasitoids use herbivore-induced plant volatiles to locate their host. We show that hyperparasitoids find their victims through herbivore-induced plant volatiles emitted in response to attack by caterpillars that in turn had been parasitized by primary parasitoids. Moreover, only one of two species of parasitoids affected herbivore-induced plant volatiles resulting in the attraction of more hyperparasitoids than volatiles from plants damaged by healthy caterpillars. This resulted in higher levels of hyperparasitism of the parasitoid that indirectly gave away its presence through its effect on plant odours induced by its caterpillar host. Here, we provide evidence for a role of compounds in the oral secretion of parasitized caterpillars that induce these changes in plant volatile emission. Our results demonstrate that the effects of herbivore-induced plant volatiles should be placed in a community-wide perspective that includes species in the fourth trophic level to improve our understanding of the ecological functions of volatile release by plants. Furthermore, these findings suggest that the impact of species in the fourth trophic level should also be considered when developing Integrated Pest Management strategies aimed at optimizing the control of insect pests using parasitoids.     

Differential electroantennogram response of females and males of two parasitoid species to host-related green leaf volatiles and inducible compounds

Parasitoids employ different types of host-related volatile signals for foraging and host-location. Host-related volatile signals can be plant-based, originate from the herbivore host or produced from an interaction between herbivores and their plant host. In order to investigate potential sex- and species-related differences in the antennal response of parasitoids to different host-related volatiles, we compared the electroantennogram (EAG) responses of both sexes of the specialist parasitoid, Microplitis croceipes (Cresson), and the generalist, Cotesia marginiventris (Cresson), to varying doses of selected plant-based host-related volatiles: two green leaf volatiles (cis-3-hexenol and hexanal) and three inducible compounds (cis-3-hexenyl acetate, linalool, and (E,E)-alpha-farnesene). Mating had no significant effect on EAG response. Females of both species showed significantly greater EAG responses than conspecific males to green leaf volatiles, which are released immediately after initiation of herbivore feeding damage. In contrast, males showed greater responses than conspecific females to inducible compounds released much later after initial damage. Cotesia marginiventris females and males showed greater EAG responses than counterpart M. croceipes to the tested compounds at various doses, suggesting that the generalist parasitoid shows greater antennal sensitivity than the specialist to the tested host-plant volatiles. These results are discussed in relation to the possible roles of green leaf volatiles and inducible compounds in the ecology of female and male parasitoids.     

The piercing-sucking herbivores Lygus hesperus and Nezara viridula induce volatile emissions in plants

Plant volatiles induced by herbivory are often used as olfactory cues by foraging herbivores and their natural enemies, and thus have potential for control of agricultural pests. Compared to chewing insects and mites, little is known about plant volatile production following herbivory by insects with piercing-sucking mouthparts. Here, we studied factors (insect life stage, gender, the role of salivary glands, and type of bioassay used for volatile induction) that influence the induction of plant volatiles by two agriculturally important hemipterans, Lygus hesperus and Nezara viridula. Feeding on intact cotton by virgin females of L. hesperus induced 2.6-fold greater volatile response compared to that induced by mated females, possibly due to increased feeding activity by virgin females. This plant volatile response was associated with elicitors present in the insect's salivary glands as well as to the degree of mechanical injury. Feeding injury by N. viridula females also increased volatile emissions in intact maize by approximately 2-fold compared to control plants. Maize seedlings injured by N. viridula emitted higher amounts of the monoterpene linalool, the sesquiterpenes (E)-beta-caryophyllene, alpha-trans-bergamotene, and (E,E)-beta-farnesene, and the homoterpene (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, but not amounts of green leaf volatiles, compared to uninjured plants. Emissions from intact maize injured by adult males were lower than those emitted by adult females of the same age and did not differ from those emitted by uninjured plants. Similarly, feeding by virgin female N. viridula followed by excision led to 64% higher quantities of volatiles compared to untreated plants. Volatile emission in excised plants, however, was considerably greater than in intact plants, suggesting that careful consideration must be given to bioassay design in studies of herbivore-induced plant volatiles. Salivary gland extracts of N. viridula led to sesquiterpene emissions approximately 2.5-fold higher than for controls, although no significant differences were observed for green leaf volatiles, monoterpenes, and homoterpenes. These results indicate that L. hesperus and female N. viridula feeding induce volatile production in plants, and that volatile production is affected by gender and life stage of the bug. Although oviposition and mechanical injury by stylets may increase release of volatiles, elicitors from salivary glands of L. hesperus and N. viridula also seem to play a role in the emission of plant volatiles.     

Habitats as Complex Odour Environments: How Does Plant Diversity Affect Herbivore and Parasitoid Orientation?

Plant diversity is known to affect success of host location by pest insects, but its effect on olfactory orientation of non-pest insect species has hardly been addressed. First, we tested in laboratory experiments the hypothesis that non-host plants, which increase odour complexity in habitats, affect the host location ability of herbivores and parasitoids. Furthermore, we recorded field data of plant diversity in addition to herbivore and parasitoid abundance at 77 grassland sites in three different regions in Germany in order to elucidate whether our laboratory results reflect the field situation. As a model system we used the herb Plantago lanceolata, the herbivorous weevil Mecinus pascuorum, and its larval parasitoid Mesopolobus incultus. The laboratory bioassays revealed that both the herbivorous weevil and its larval parasitoid can locate their host plant and host via olfactory cues even in the presence of non-host odour. In a newly established two-circle olfactometer, the weeviĺs capability to detect host plant odour was not affected by odours from non-host plants. However, addition of non-host plant odours to host plant odour enhanced the weeviĺs foraging activity. The parasitoid was attracted by a combination of host plant and host volatiles in both the absence and presence of non-host plant volatiles in a Y-tube olfactometer. In dual choice tests the parasitoid preferred the blend of host plant and host volatiles over its combination with non-host plant volatiles. In the field, no indication was found that high plant diversity disturbs host (plant) location by the weevil and its parasitoid. In contrast, plant diversity was positively correlated with weevil abundance, whereas parasitoid abundance was independent of plant diversity. Therefore, we conclude that weevils and parasitoids showed the sensory capacity to successfully cope with complex vegetation odours when searching for hosts.     

Influence of host log age and refuge from natural enemies on colonization and survival of Phoracantha semipunctata

The pine cone weevil, Pissodes validirostris Gyll. (Coleoptera: Curculionidae), attacks seed cones of most Eurasian pine species, except these of Swiss stone pine (Pinus cembra L.). Behavioural responses of adult weevils to cone volatile emissions of Swiss stone pine and to those of a common host, mountain pine (Pinus uncinata Ram.), were compared in an olfactometer. Weevils were significantly attracted by the volatile blend emitted by mountain pine, but Swiss stone pine volatiles elicited an inverse response, with most weevils moving in the opposite direction to the odour source. However, the majority of second instar weevil larvae that were extracted from mountain pine cones and transferred into Swiss stone pine cones were capable of developing to the adult stage. This suggests that Swiss stone pine cones do not contain strong feeding deterrents that could prevent larval development. The possible factors involved in the absence of colonization of Swiss stone pine cones by cone weevils are discussed.     

Synergism and redundancy in a plant volatile blend attracting grapevine moth females

A flight tunnel study was done to decipher the behavioral effect of grape odor in grapevine moth Lobesia botrana. A blend of 10 volatile compounds, which all elicit a strong antennal response, attracts mated grapevine moth females from a distance, by upwind orientation flight. These 10 grape volatiles are in part behaviorally redundant, since attraction to a 3-component blend of beta-caryophyllene, (E)-beta-farnesene and (E)-4,8-dimethyl-1,3,7-nonatriene was not significantly different from the 10-component blend. Blending these three compounds had a strong synergistic effect on female attraction, and omission of any one compound from this 3-component blend almost abolished attraction. It was nonetheless possible to substitute the three compounds with the other grape volatiles which are perceived by the female antenna, to partly restore attraction. Several blends, of varying composition, elicited significant attraction. The observed behavioral plasticity in response to grape volatile blends probably reflects the variation of the natural plant signal, since females oviposit on different grape varieties, in different phenological stages.     

Induced volatiles in soybean and pigeon pea plants artificially infested with the neotropical brown stink bug, Euschistus heros, and their effect on the egg parasitoid, Telenomus podisi

Herbivory is known to increase the emission of volatiles, which attract natural enemies to herbivore‐damaged plants in laboratory and agricultural systems. We report on signalling through volatiles induced by Euschistus heros (F.) (Heteroptera: Pentatomidae) in two legumes that influence the attraction and retention of the egg parasitoid Telenomus podisi (Ashmead) (Hymenoptera: Scelionidae). Air‐borne extracts obtained from two host plants of E. heros, soybean, Glycine max, and pigeon pea, Cajanus cajan (Leguminosae), produce a different blend of emitted volatiles when attacked by adult males or females and nymphs of the pest species, compared with the undamaged plants. The same results were obtained when the plants were treated with extracted saliva of E. heros which had been mechanically introduced into the plants. This indicates that some substance in the saliva contributed to the release of the volatiles. Bioassays in a Y‐tube olfactometer with female T. podisi and treated plants confirmed the significant preference of the egg parasitoid for plants attacked by either males, females, or fourth instar nymphs of the pest species. On the other hand, volatile extracts obtained from soybean subjected to the velvetbean caterpillar, Anticarsia gemmatalis, a non‐host species for T. podisi, showed a different blend of volatiles compared to those obtained from plants subjected to E. heros. Additionally, the volatiles obtained from this plant–host complex were not attractive to T. podisi. These results indicate that E. heros causes an increase in the emission of specific plant volatiles, and that the induction is possibly caused by an elicitor present in the pest saliva. The possibility that these plant volatiles play an important role in the attraction and retention of the egg parasitoid T. podisi is discussed.     

Host plants alter their volatiles to help a solitary egg parasitoid distinguish habitats with parasitized hosts from those without

When attacked by herbivores, plants emit volatiles to attract parasitoids and predators of herbivores. However, our understanding of the effect of plant volatiles on the subsequent behaviour of conspecific parasitoids when herbivores on plants are parasitized is limited. In this study, rice plants were infested with gravid females of the brown planthopper (BPH) Nilaparvata lugens for 24 hr followed by another 24 hr in which the BPH eggs on plants were permitted to be parasitized by their egg parasitoid, Anagrus nilaparvatae; volatiles from rice plants that underwent such treatment were less attractive to subsequent conspecific parasitoids compared to the volatiles from plants infested with gravid BPH females alone. Chemical analysis revealed that levels of JA and JA-Ile as well as of four volatile compounds—linalool, MeSA, α-zingiberene and an unknown compound—from plants infested with BPH and parasitized by wasps were significantly higher than levels of these compounds from BPH-infested plants. Laboratory and field bioassays revealed that one of the four increased chemicals—α-zingiberene—reduced the plant's attractiveness to the parasitoid. These results suggest that host plants can fine-tune their volatiles to help egg parasitoids distinguish host habitats with parasitized hosts from those without.     

Role of volatile semiochemicals in host location by the egg parasitoid Anagrus breviphragma

Recent investigations conducted on several tritrophic systems have demonstrated that egg parasitoids, when searching for host eggs, may exploit plant synomones that have been induced as a consequence of host oviposition. In this article we show that, in a system characterized by host eggs embedded in the plant tissue, naïve females of the egg parasitoid Anagrus breviphragma Soyka (Hymenoptera: Mymaridae) responded in a Y‐tube olfactometer to volatiles from leaves of Carex riparia Curtis (Cyperaceae) containing eggs of one of its hosts, Cicadella viridis (L.) (Hemiptera: Cicadellidae). The wasp did not respond to host eggs or to clean leaves from non‐infested plants compared with clean air, whereas it showed a strong preference for the olfactometer arm containing volatiles of leaves with embedded host eggs, compared with the arm containing volatiles of leaves from a non‐infested plant or host eggs extracted from the plant. When the eggs were removed from an infested leaf, the parasitoid preference was observed only if eggs were added aside, suggesting a synergistic effect of a local plant synomone and an egg kairomone. The parasitoid also responded to clean leaves from an egg‐infested plant when compared with leaves from a non‐infested plant, indicating a systemic effect of volatile induction.     

Attraction of <Emphasis Type="Italic">Telenomus podisi</Emphasis> to volatiles induced by <Emphasis Type="Italic">Euschistus heros</Emphasis> in three different plant species

Specialized natural enemies that forage for polyphagous hosts need to locate hosts on different plants. Telenomus podisi (Hymenoptera: Platygastridae) is a stink bug egg parasitoid with a preference for Euschistus heros (Hemiptera, Pentatomidae), a polyphagous species. The aim of this study was to evaluate the induction of defences in three E. heros host plants: maize (Zea mays), sunflower (Helianthus annuus) and pigeon pea (Cajanus cajan). We hypothesized that E. heros damage to these three plants enhances the attraction of the parasitoid T. podisi as has been observed in other systems. Using Y-tube olfactometer bioassays, we tested parasitoid responses to combinations of the following odour sources: clean air, undamaged plants and plants damaged by stink bug feeding. Volatiles were collected by means of dynamic headspace collection and analysed by gas chromatography coupled to mass spectrometry. T. podisi did not distinguish odours from undamaged plants against air for any of the three plant species. For maize, the parasitoid preferred the odour from herbivore-damaged plants over both clean air and undamaged plants. For sunflower, the parasitoid only preferred the odour of herbivore-damaged plants over the odour of undamaged plants. For pigeon pea, no preferences were observed. Quantitative differences in the volatile profile of damaged and undamaged plants were observed in each plant species. We conclude that sunflower and maize plants, when damaged by E. heros, release volatiles that attract the parasitoid T. podisi; the parasitoid appears to use a different blend composition to distinguish herbivore-damaged plants of each species.     

半闭弯尾姬蜂寄主搜索中的学习行为

研究了半闭弯尾姬蜂寄主搜索过程中的学习行为。结果表明,成虫期之前的饲养寄主所取食的寄主植物对成蜂行为没有影响,而雌蜂早期的短暂经历可对其随后的行为反应产生显著影响,从而对已经历的植物气味表现出显著的嗜好,但这种通过学习所表现出的嗜好又可因新的经历而改变。雌成蜂不仅能对其所经历的虫伤寄主植物释放的信息化合物进行学习,而且对其所经历的寄主幼虫的信息化合物也能进行学习。