Attraction of the Air Potato Leaf Beetle, Lilioceris Cheni, (Coleoptera: Chrysomelidae) to leaf Volatiles of the Air Potato, Dioscorea bulbifera.

Air potato, Dioscorea bulbifera L., is an invasive vine found in the southeastern United States and is native to Asia and Africa. The air potato leaf beetle Lilioceris cheni (Coleoptera: Chrysomelidae), is a host specific biological control agent introduced for D. bulbifera control. In this study, odor cues that control the attraction of L. cheni to D. bulbifera were investigated. The first experiment investigated the response of L. cheni to D. bulbifera leaves versus no leaves in the presence or absence of air flow. The experiment showed a significant response of L. cheni to D. bulbifera leaves in the presence of air flow with leaves placed upwind. When air flow and/or leaves were absent, L. cheni dispersed randomly between the upwind and downwind targets, indicating L. cheni uses volatiles from D. bulbifera in host selection. The second experiment investigated L. cheni response to undamaged, larval-damaged, and adult-damaged plants. Lilioceris cheni showed preference to move towards conspecific damaged plants compared to undamaged plants but did not discriminate between larvae-damaged or adult-damaged plants. The third experiment investigated volatile profiles of damaged D. bulbifera plants using gas chromatography coupled with mass spectroscopy. We found significant differences in volatile profiles between adult and larval damaged plants compared to mechanically damaged and undamaged plants, with increases in 11 volatile compounds. However, larval and adult-damaged volatile profiles did not differ. The information acquired during this study could be used to develop strategies to monitor for L. cheni and improve its biological control program.

The Asian corn borer Ostrinia furnacalis feeding increases the direct and indirect defence of mid-whorl stage commercial maize in the field.

The Asian corn borer (Ostrinia furnacalis Guenée) is a destructive pest of maize (Zea mays L.). Despite large-scale commercial maize production, little is known about the defensive responses of field-grown commercial maize to O. furnacalis herbivory, and how these responses result in direct and indirect defence against this pest. To elucidate the maize transcriptome response to O. furnacalis feeding, leaves of maize hybrid Jingke968 were infested with O. furnacalis for 0, 2, 4, 12 and 24 h. Ostrinia furnacalis feeding elicited stronger and more rapid changes in the defence-related gene expression (i.e. after 2 h), and more differentially expressed genes (DEGs) were up-regulated than down-regulated at all times post-induction (i.e. 2, 4, 12 and 24 h) in the O. furnacalis pre-infested maize plants. KEGG pathway analysis indicated that the DEGs in the O. furnacalis pre-infested maize are involved in benzoxazinoids, phytohormones, volatiles, and other metabolic pathways related to maize resistance to herbivores. In addition, the maize leaves previously infested by O. furnacalis for 24 h showed an obvious inhibition of the subsequent O. furnacalis performance, and maize volatiles induced by O. furnacalis feeding for 24 and 48 h attracted the parasitic wasp, Macrocentrus cingulum Brischke. The increased direct and indirect defences induced by O. furnacalis feeding were correlated with O. furnacalis-induced phytohormones, benzoxazinoids, and volatiles. Together, our findings provide new insights into how commercial maize orchestrates its transcriptome and metabolome to directly and indirectly defend against O. furnacalis at the mid-whorl stage in the field.

Specificity of Herbivore Defense Responses in a Woody Plant, Black Poplar (Populus nigra).

The specificity of woody plant defense responses to different attacking herbivores is poorly known. We investigated the responses of black poplar (Populus nigra) to leaf feeding by three lepidopteran species (Lymantria dispar, Laothoe populi and Amata mogadorensis) and two leaf beetle species (Phratora vulgatissima and Chrysomela populi). Of the direct defenses monitored, increases in trypsin protease inhibitor activity and the salicinoid salicin were triggered by herbivore damage, but this was not herbivore-specific. Moreover, the majority of leaf salicinoid content was present constitutively and not induced by herbivory. On the other hand, volatile emission profiles did vary among herbivore species, especially between coleopterans and lepidopterans. Monoterpenes and sesquiterpenes were induced in damaged and adjacent undamaged leaves, while the emission of green leaf volatiles, aromatic and nitrogen-containing compounds (known to attract herbivore enemies) was restricted to damaged leaves. In conclusion, indirect defenses appear to show more specific responses to attacking herbivores than direct defenses in this woody plant.

Maize Chlorotic Mottle Virus Induces Changes in Host Plant Volatiles that Attract Vector Thrips Species.

Maize lethal necrosis is one of the most devastating diseases of maize causing yield losses reaching up to 90% in sub-Saharan Africa. The disease is caused by a combination of maize chlorotic mottle virus (MCMV) and any one of cereal viruses in the Potyviridae group such as sugarcane mosaic virus. MCMV has been reported to be transmitted mainly by maize thrips (Frankliniella williamsi) and onion thrips (Thrips tabaci). To better understand the role of thrips vectors in the epidemiology of the disease, we investigated behavioral responses of F. williamsi and T. tabaci, to volatiles collected from maize seedlings infected with MCMV in a four-arm olfactometer bioassay. Volatile profiles from MCMV-infected and healthy maize plants were compared by gas chromatography (GC) and GC coupled mass spectrometry analyses. In the bioassays, both sexes of F. williamsi and male T. tabaci were significantly attracted to volatiles from maize plants infected with MCMV compared to healthy plants and solvent controls. Moreover, volatile analysis revealed strong induction of (E)-4,8-dimethyl-1,3,7-nonatriene, methyl salicylate and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene in MCMV-infected maize seedlings. Our findings demonstrate MCMV induces changes in volatile profiles of host plants to elicit attraction of thrips vectors. The increased vector contact rates with MCMV-infected host plants could enhance virus transmission if thrips feed on the infected plants and acquire the pathogen prior to dispersal. Uncovering the mechanisms mediating interactions between vectors, host plants and pathogens provides useful insights for understanding the vector ecology and disease epidemiology, which in turn may contribute in designing integrated vector management strategies.

The Fungus Raffaelea lauricola Modifies Behavior of Its Symbiont and Vector, the Redbay Ambrosia Beetle (Xyleborus Glabratus), by Altering Host Plant Volatile Production.

The redbay ambrosia beetle Xyleborus glabratus is the vector of the symbiotic fungus, Raffaelea lauricola that causes laurel wilt, a highly lethal disease to members of the Lauraceae family. Pioneer X. glabratus beetles infect live trees with R. lauricola, and only when tree health starts declining more X. glabratus are attracted to the infected tree. Until now this sequence of events was not well understood. In this study, we investigated the temporal patterns of host volatiles and phytohormone production and vector attraction in relation to laurel wilt symptomology. Following inoculations with R. lauricola, volatile collections and behavioral tests were performed at different time points. Three days after infection (DAI), we found significant repellency of X. glabratus by leaf odors of infected swamp bay Persea palustris as compared with controls. However, at 10 and 20 DAI, X. glabratus were more attracted to leaf odors from infected than non-infected host plants. GC-MS analysis revealed an increase in methyl salicylate (MeSA) 3 DAI, whereas an increase of sesquiterpenes and leaf aldehydes was observed 10 and 20 DAI in leaf volatiles. MeSA was the only behaviorally active repellent of X. glabratus in laboratory bioassays. In contrast, X. glabratus did not prefer infected wood over healthy wood, and there was no associated significant difference in their volatile profiles. Analyses of phytohormone profiles revealed an initial increase in the amount of salicylic acid (SA) in leaf tissues following fungal infection, suggesting that the SA pathway was activated by R. lauricola infection, and this activation caused increased release of MeSA. Overall, our findings provide a better understanding of X. glabratus ecology and underline chemical interactions with its symbiotic fungus. Our work also demonstrates how the laurel wilt pathosystem alters host defenses to impact vector behavior and suggests manipulation of host odor by the fungus that attract more vectors.

The Role of Leaf Volatiles of Ludwigia octovalvis (Jacq.) Raven in the Attraction of Altica cyanea (Weber) (Coleoptera: Chrysomelidae).

Larvae and adults of Altica cyanea (Weber) (Coleoptera: Chrysomelidae) feed on the rice-field weed Ludwigia octovalvis (Jacq.) Raven (Onagraceae), commonly known as willow primrose, which is considered a biocontrol agent of the weed. Volatile organic compounds from undamaged plants, plants after 4, 12, and 36 h of continuous feeding by A. cyanea larvae or adult females and after mechanical damaging were identified by GC-MS and GC-FID analyses. Twenty nine compounds were identified from undamaged plants. 2Z-Penten-1-ol, geraniol, and 1-tridecanol were present in all plants damaged by larvae. In contrast, feeding by adults caused the release of 2Z-penten-1-ol only after 12 and 36 h; whereas geraniol and 1-tridecanol appeared only after 36 h. Farnesyl acetone was detected after 12 and 36 h of feeding by larvae and after 36 h of feeding by adults. Farnesene was detected after 36 h of feeding by larvae and adults. Linalool was unique after 36 h of feeding by larvae. In Y-shaped glass tube olfactometer bioassays, A. cyanea females were attracted to volatiles after 36 h of feeding by larvae or adults compared to volatiles released by undamaged plants. The insects were attracted to five synthetic compounds: 3-hexanol, α-pinene, linalool oxide, geraniol, and phytol. Synthetic blends were more attractive than individual compounds. Compared to undamaged plants, volatiles released by plants, damaged by conspecific individuals, were more attractive to A. cyanea females, due to elevated emissions of 3-hexanol, α-pinene, linalool oxide, geraniol, and phytol.

Cytochrome P450 CYP71AT96 catalyses the final step of herbivore-induced phenylacetonitrile biosynthesis in the giant knotweed, Fallopia sachalinensis.

The giant knotweed Fallopia sachalinensis (Polygonaceae) synthesizes phenylacetonitrile (PAN) from L-phenylalanine when infested by the Japanese beetle Popillia japonica or treated with methyl jasmonate (MeJA). Here we identified (E/Z)-phenylacetaldoxime (PAOx) as the biosynthetic precursor of PAN and identified a cytochrome P450 that catalysed the conversion of (E/Z)-PAOx to PAN. Incorporation of deuterium-labelled (E/Z)-PAOx into PAN emitted from the leaves of F. sachalinensis was detected using gas chromatography-mass spectrometry. Further, using liquid chromatography-tandem mass spectrometry, we detected the accumulation of (E/Z)-PAOx in MeJA-treated leaves. These results showed that (E/Z)-PAOx is the biosynthetic precursor of PAN. MeJA-induced mRNAs were analysed by differential expression analysis using a next-generation sequencer. Of the 74,329 contigs obtained from RNA-seq and de novo assembly, 252 contigs were induced by MeJA treatment. Full-length cDNAs encoding MeJA-induced cytochrome P450s CYP71AT96, CYP82AN1, CYP82D125 and CYP715A35 were cloned using 5'- and 3'-RACE and were expressed using a baculovirus expression system. Among these cytochrome P450s, CYP71AT96 catalysed the conversion of (E/Z)-PAOx to PAN in the presence of NADPH and a cytochrome P450 reductase. It also acted on (E/Z)-4-hydroxyphenylacetaldoxime and (E/Z)-indole-3-acetaldoxime. The broad substrate specificity of CYP71AT96 was similar to that of aldoxime metabolizing cytochrome P450s. Quantitative RT-PCR analysis showed that CYP71AT96 expression was highly induced because of treatment with MeJA as well as feeding by the Japanese beetle. These results indicate that CYP71AT96 likely contributes the herbivore-induced PAN biosynthesis in F. sachalinensis.

Volatile Organic Compounds Induced by Herbivory of the Soybean Looper Chrysodeixis includens in Transgenic Glyphosate-Resistant Soybean and the Behavioral Effect on the Parasitoid, Meteorus rubens.

Transgenic soybean plants (RR) engineered to express resistance to glyphosate harbor a variant of the enzyme EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) involved in the shikimic acid pathway, the biosynthetic route of three aromatic amino acids: phenylalanine, tyrosine, and tryptophan. The insertion of the variant enzyme CP4 EPSPS confers resistance to glyphosate. During the process of genetic engineering, unintended secondary effects are likely to occur. In the present study, we quantified volatile organic compounds (VOCs) emitted constitutively or induced in response to herbivory by the soybean looper Chrysodeixis includens in transgenic soybean and its isogenic (untransformed) line. Since herbivore-induced plant volatiles (HIPVs) are known to play a role in the recruitment of natural enemies, we assessed whether changes in VOC profiles alter the foraging behavior of the generalist endoparasitic larval parasitoid, Meteorus rubens in the transgenic line. Additionally, we assessed whether there was a difference in plant quality by measuring the weight gain of the soybean looper. In response to herbivory, several VOCs were induced in both the conventional and the transgenic line; however, larger quantities of a few compounds were emitted by transgenic plants. Meteorus rubens females were able to discriminate between the odors of undamaged and C. includens-damaged plants in both lines, but preferred the odors emitted by herbivore-damaged transgenic plants over those emitted by herbivore-damaged conventional soybean plants. No differences were observed in the weight gain of the soybean looper. Our results suggest that VOC-mediated tritrophic interactions in this model system are not negatively affected. However, as the preference of the wasps shifted towards damaged transgenic plants, the results also suggest that genetic modification affects that tritrophic interactions in multiple ways in this model system.

Emission of Volatile Compounds from Apple Plants Infested with Pandemis heparana Larvae, Antennal Response of Conspecific Adults, and Preliminary Field Trial.

This study investigated the volatile emission from apple (Malus x domestica Borkh., cv. Golden Delicious) foliage that was either intact, mechanically-damaged, or exposed to larval feeding by Pandemis heparana (Denis and Schiffermüller) (Lepidoptera: Tortricidae). Volatiles were collected by closed-loop-stripping-analysis and characterized by gas chromatography-mass spectrometry in three time periods: after 1 h and again 24 and 48 h later. Volatiles for all treatments also were monitored continuously over a 72-h period by the use of proton transfer reaction - time of flight-mass spectrometry (PTR-ToF-MS). In addition, the volatile samples were analyzed by gas chromatography-electroantennographic detection (GC-EAD) using male and female antennae of P. heparana. Twelve compounds were detected from intact foliage compared with 23 from mechanically-damaged, and 30 from P. heparana-infested foliage. Interestingly, six compounds were released only by P. heparana-infested foliage. The emission dynamics of many compounds measured by PTR-ToF-MS showed striking differences according to the timing of herbivory and the circadian cycle. For example, the emission of green leaf volatiles began shortly after the start of herbivory, and increased over time independently from the light-dark cycle. Conversely, the emission of terpenes and aromatic compounds showed a several-hour delay in response to herbivory, and followed a diurnal rhythm. Methanol was the only identified volatile showing a nocturnal rhythm. Consistent GC-EAD responses were found for sixteen compounds, including five aromatic ones. A field trial in Sweden demonstrated that benzyl alcohol, 2-phenylethanol, phenylacetonitrile, and indole lures placed in traps were not attractive to Pandemis spp. adults, but 2-phenylethanol and phenylacetonitrile when used in combination with acetic acid were attractive to both sexes.

Herbivory and floral signaling: phenotypic plasticity and tradeoffs between reproduction and indirect defense.

Plant defense against herbivores may compromise attraction of mutualists, yet information remains limited about the mechanisms underlying such signaling tradeoffs. Here, we investigated the effects of foliar herbivory by two herbivore species on defense compounds, floral signaling, pollinator and parasitoid attraction, and seed production. Herbivory generally reduced the quantity of many floral volatile organic compounds VOCs) in Brassica rapa. By contrast, floral color, flower diameter, and plant height remained unaffected. The decreased amounts of floral volatiles led to reduced attractiveness of flowers to pollinators, but increased the attractiveness of herbivore-infested plants to parasitoids. Plants infested with the native butterfly Pieris brassicae produced more flowers during early flowering, effectively compensating for the lower olfactory attractiveness. Herbivory by the invasive Spodoptera littoralis increased the amounts of glucobrassicanapin, and led to delayed flowering. These plants tended to attract fewer pollinators and to produce fewer seeds. Our study indicates a tradeoff between pollinator attraction and indirect defense (parasitoid attraction), which can be mitigated by reduced floral VOC emission and production of more early flowers. We suggest that this compensatory mechanism is specific to plant-herbivore associations with a coevolutionary history.

Plant volatiles in polluted atmospheres: stress responses and signal degradation.

Plants emit a plethora of volatile organic compounds, which provide detailed information on the physiological condition of emitters. Volatiles induced by herbivore feeding are among the best studied plant responses to stress and may constitute an informative message to the surrounding community and further function in plant defence processes. However, under natural conditions, plants are potentially exposed to multiple concurrent stresses with complex effects on the volatile emissions. Atmospheric pollutants are an important facet of the abiotic environment and can impinge on a plant's volatile-mediated defences in multiple ways at multiple temporal scales. They can exert changes in volatile emissions through oxidative stress, as is the case with ozone pollution. The pollutants, in particular, ozone, nitrogen oxides and hydroxyl radicals, also react with volatiles in the atmosphere. These reactions result in volatile breakdown products, which may themselves be perceived by community members as informative signals. In this review, we demonstrate the complex interplay among stresses, emitted signals, and modification in signal strength and composition by the atmosphere, collectively determining the responses of the biotic community to elicited signals.

Herbivore-induced volatile emission in black poplar: regulation and role in attracting herbivore enemies.

After herbivory, plants release volatile organic compounds from damaged foliage as well as from nearby undamaged leaves that attract herbivore enemies. Little is known about what controls the volatile emission differences between damaged and undamaged tissues and how these affect the orientation of herbivore enemies. We investigated volatile emission from damaged and adjacent undamaged foliage of black poplar (Populus nigra) after herbivory by gypsy moth (Lymantria dispar) caterpillars and determined the compounds mediating the attraction of the gypsy moth parasitoid Glyptapanteles liparidis (Braconidae). Female parasitoids were more attracted to gypsy moth-damaged leaves than to adjacent non-damaged leaves. The most characteristic volatiles of damaged versus neighbouring undamaged leaves included terpenes, green leaf volatiles and nitrogen-containing compounds, such as aldoximes and nitriles. Electrophysiological recordings and olfactometer bioassays demonstrated the importance of nitrogenous volatiles. Under field conditions, parasitic Hymenoptera were more attracted to traps baited with these substances than most other compounds. The differences in volatile emission profiles between damaged and undamaged foliage appear to be regulated by jasmonate signalling and the local activation of volatile biosynthesis. We conclude that characteristic volatiles from damaged black poplar foliage are essential cues enabling parasitoids to find their hosts.

Profiles of Volatiles Emitted from Orange Fruits Infested by Ceratitis capitata (Diptera: Tephritidae).

The profiles of volatile organic compounds (VOCs) emitted from orange fruits infested by Ceratitis capitata were examined. VOCs were collected from the headspace of oranges immediately, 24 h, 5 days, 8 days, and 13 days after oviposition and when larvae exit the fruit by applying either static or dynamic sampling techniques. A total of 32 and 47 compounds were detected in infested orange fruits when using static and dynamic techniques, respectively. Differences in the volatile profile of oranges were observed. Classification models were employed, showing that infested fruits emitted a chemical profile distinct from that of non-infested ones. Limonene was associated with cell disruption. (E)-ß-Ocimene, (E)-4,8-dimethyl-1,3,7-nonatriene, hexyl butanoate, butyl hexanoate, and hexyl hexanoate were the VOCs that were correlated with the infestation of sweet oranges by C. capitata.

Review: Trait plasticity during plant-insect interactions: From molecular mechanisms to impact on community dynamics.

Phenotypic plasticity, prevalent in all domains of life, enables organisms to cope with unpredictable or novel changes in their growing environment. Plants represent an interesting example of phenotypic plasticity which also directly represents and affects the dynamics of biological interactions occurring in a community. Insects, which interact with plants, manifest phenotypic plasticity in their developmental, physiological, morphological or behavioral traits in response to the various host plant defenses induced upon herbivory. However, plant-insect interactions are generally more complex and multidimensional because of their dynamic association with their respective microbiomes and macrobiomes. Moreover, these associations can alter plant and insect responses towards each other by modulating the degree of phenotypic plasticity in their various traits and studying them will provide insights into how plants and insects reciprocally affect each other's evolutionary trajectory. Further, we explore the consequences of phenotypic plasticity on relationships and interactions between plants and insects and its impact on their development, evolution, speciation and ecological organization. This overview, obtained after exploring and comparing data obtained from several inter-disciplinary studies, reveals how genetic and molecular mechanisms, underlying plasticity in traits, impact species interactions at the community level and also identifies mechanisms that could be exploited in breeding programs.

SpitWorm, a Herbivorous Robot: Mechanical Leaf Wounding with Simultaneous Application of Salivary Components.

Induction of jasmonate-mediated plant defense against insect herbivory is initiated by a combination of both mechanical wounding and chemical factors. In order to study both effects independently on plant defense induction, SpitWorm, a computer-controlled device which mimics the damage pattern of feeding insect larvae on leaves and, in addition, can apply oral secretions (OS) or other solutions to the 'biting site' during 'feeding,' was developed and evaluated. The amount of OS left by a Spodoptera littoralis larva during feeding on Phaseolus lunatus (lima bean) leaves was estimated by combining larval foregut volume, biting rate, and quantification of a fluorescent dye injected into the larvae's foregut prior to feeding. For providing OS amounts by SpitWorm equivalent to larval feeding, dilution and delivery rate were optimized. The effectiveness of SpitWorm was tested by comparing volatile organic compounds (VOC) emissions of P. lunatus leaves treated with either SpitWorm, MecWorm, or S. littoralis larvae. Identification and quantification of emitted VOCs revealed that SpitWorm induced a volatile bouquet that is qualitatively and quantitatively similar to herbivory. Additionally, RT-qPCR of four jasmonic acid responsive genes showed that SpitWorm, in contrast to MecWorm, induces the same regulation pattern as insect feeding. Thus, SpitWorm mimics insect herbivory almost identically to real larvae feeding.

Light-induced alterations of pineapple (Ananas comosus [L.] Merr.) juice volatiles during accelerated ageing and mass spectrometric studies into their precursors.

Alterations of volatiles during accelerated light-induced ageing of pineapple juice were assessed by HS-SPME-GC-MS in a non-targeted profiling analysis over a 16-week period. Multivariate statistics permitted to reveal substantial chemical markers generally describing the effect of light storage. Volatiles generated comprised phenylpropenes, carbonyls, 2-methylthiophene, toluene, and furfural, while concentrations of methyl and ethyl esters, terpenes, and furanones decreased. In addition, the qualitative composition of phenolic compounds and glycoside-bound volatiles in selected samples was characterized by HPLC-DAD-ESI-MSn as well as HR-ESI-MS. The fresh juice contained unique pineapple metabolites such as S-p-coumaryl, S-coniferyl, S-sinapylglutathione, and structurally related derivatives. Among others, the presence of p-coumaroyl, feruloyl, and caffeoylisocitrate as well as three 4-hydroxy-2,5-dimethyl-3(2H)-furanone glycosides in pineapples could be substantiated by the HR-ESI-MS experiment. Mass spectrometric assignments of selected metabolites are presented, and putative linkages between volatiles and their precursors are established.

Off-season biogenic volatile organic compound emissions from heath mesocosms: responses to vegetation cutting.

Biogenic volatile organic compounds (BVOCs) affect both atmospheric processes and ecological interactions. Our primary aim was to differentiate between BVOC emissions from above- and belowground plant parts and heath soil outside the growing season. The second aim was to assess emissions from herbivory, mimicked by cutting the plants. Mesocosms from a temperate Deschampsia flexuosa-dominated heath ecosystem and a subarctic mixed heath ecosystem were either left intact, the aboveground vegetation was cut, or all plant parts (including roots) were removed. For 3-5 weeks, BVOC emissions were measured in growth chambers by an enclosure method using gas chromatography-mass spectrometry. CO2 exchange, soil microbial biomass, and soil carbon and nitrogen concentrations were also analyzed. Vegetation cutting increased BVOC emissions by more than 20-fold, and the induced compounds were mainly eight-carbon compounds and sesquiterpenes. In the Deschampsia heath, the overall low BVOC emissions originated mainly from soil. In the mixed heath, root, and soil emissions were negligible. Net BVOC emissions from roots and soil of these well-drained heaths do not significantly contribute to ecosystem emissions, at least outside the growing season. If insect outbreaks become more frequent with climate change, ecosystem BVOC emissions will periodically increase due to herbivory.