Developing Oak Buds Produce Volatile Emissions in Response to Herbivory by Freshly Hatched Caterpillars.

Plant responses to damage by insectivorous herbivores are well-documented in mature leaves. The resulting herbivore-induced plant volatiles (HIPVs) protect the plant by attracting carnivorous arthropods and even some insectivorous vertebrates, to parasitize or consume the plant invaders. However, very little is known about plant production of HIPVs in developing buds, particularly when herbivorous insects are too small to be considered a prey item. It is additionally unclear whether plants respond differently to generalist and specialist chewing insects that overlap in distribution. Therefore, we compared HIPV production of Downy oak (Quercus pubescens Willd.) buds infested with freshly hatched caterpillars of Tortrix viridana (specialist) and Operophtera brumata (generalist), against uninfested buds. Of the compounds identified in both years of the experiment, we found that (Z)-hex-3-enyl acetate, (E)-ß-ocimene, acetophenone, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), methyl salicylate, α-copaene, α-humulene, (E)-caryophyllene, and (E,E)-α-farnesene appeared to be higher in infested buds compared to controls. We found no difference in HIPV production between the specialist and the generalist herbivores. Production of HIPVs was also associated with leaf damage, with higher HIPV production in more severely attacked buds. Thus, our study shows that oak trees already start responding to insect herbivory before leaves are developed, by producing compounds similar to those found in damaged mature leaves. Future work should focus on how Downy oak may benefit from initiating alarm cues at a time when carnivorous arthropods and insectivorous vertebrates are unable to use herbivorous insects as host or food.

Identification of Semiochemical Candidates Involved in Glossina Palpalis Gambiensis Larviposition Site Selection and Behavioural Responses of Adult Gravid Females.

Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of human and animal trypanosomes. This viviparous insect develops and produces a single larva at 10-day intervals deposited in specific sites. In some species aggregation of larvae has been shown and seems to be mediated by both physical factors and volatile semiochemicals of larval origin. In this context, this study aims to identify chemicals emitted during the pupariation process in Glossina palpalis gambiensis. Volatile Organic Compounds (VOCs) emitted by larvae were identified using static headspace solid-phase microextraction and gas-chromatography mass-spectrometry (GC-MS) analysis. Electrophysiology and behavioural assays were performed on gravid females to confirm VOCs behavioural activity and attractiveness. GC-MS results revealed ten chemicals emitted during the pupariation process of G. p. gambiensis larvae. Among these chemicals, gravid females were shown to detect nine of them during coupled gas chromatography - electroantennographic detection tests. Behavioural assays highlighted two compounds were as attractive as pupae and one compound and a blend of four compounds were more attractive than pupae. Although the larval origin of some of them needs to be confirmed as they may also likely produced by micro-organisms, these compounds induced significant behavioural responses in the laboratory. Further experiments have to explore the biological activity and competitiveness of these compounds in the field. This work opens interesting opportunities for behavioural manipulation and control of tsetse flies.

The influence of plant odours on sexual readiness in an insectivorous songbird.

Many organisms rely on environmental cues to predict and anticipate the annual optimal timing of reproduction. In insectivorous birds, preparation for breeding often coincides with the time vegetation starts to develop in spring. Whether there is a direct relationship between the two, and through which mechanisms this link could come about, has rarely been investigated. Plants release herbivore-induced plant volatiles (HIPVs) when they are attacked by insects, and recent studies have shown that birds can detect and orient to those odours when searching for food. Whether those volatiles also stimulate sexual reproductive development and timing of reproduction remains to be discovered. We tested this hypothesis by monitoring gonadal growth in pairs of blue tits (Cyanistes caeruleus) exposed to air from caterpillar-infested oak trees or from a control, in spring. We found that while males and females grew their gonads over time, gonads grew at the same rate in both odour treatments. More exploratory (i.e. a proxy of personality) females did, however, have larger ovarian follicle sizes when exposed to the HIPVs than to the control air, which is consistent with earlier results showing that fast explorers have larger gonads in spring and are more sensitive to HIPVs. If HIPVs constitute powerful attractants in foraging birds, their influence on gonadal development prior to breeding appears to be relatively subtle and to only enhance reproductive readiness in some individuals. These results are nevertheless important as they set olfaction as a new player in the seasonal timing of reproduction in birds.

Insights into the Intraspecific Variability of the above and Belowground Emissions of Volatile Organic Compounds in Tomato.

The in-vivo monitoring of volatile organic compound (VOC) emissions is a potential non-invasive tool in plant protection, especially in greenhouse cultivation. We studied VOC production from above and belowground organs of the eight parents of the Multi-Parent Advanced Generation Intercross population (MAGIC) tomato population, which exhibits a high genetic variability, in order to obtain more insight into the variability of constitutive VOC emissions from tomato plants under stress-free conditions. Foliage emissions were composed of terpenes, the majority of which were also stored in the leaves. Foliage emissions were very low, partly light-dependent, and differed significantly among genotypes, both in quantity and quality. Soil with roots emitted VOCs at similar, though more variable, rates than foliage. Soil emissions were characterized by terpenes, oxygenated alkanes, and alkenes and phenolic compounds, only a few of which were found in root extracts at low concentrations. Correlation analyses revealed that several VOCs emitted from foliage or soil are jointly regulated and that above and belowground sources are partially interconnected. With respect to VOC monitoring in tomato crops, our results underline that genetic variability, light-dependent de-novo synthesis, and belowground sources are factors to be considered for successful use in crop monitoring.

Floral trait differentiation in Anacamptis coriophora: Phenotypic selection on scents, but not on colour.

Current divergent selection may promote floral trait differentiation among conspecific populations in flowering plants. However, whether this applies to complex traits such as colour or scents has been little studied, even though these traits often vary within species. In this study, we compared floral colour and odour as well as selective pressures imposed upon these traits among seven populations belonging to three subspecies of the widespread, generalist orchid Anacamptis coriophora. Colour was characterized using calibrated photographs, and scents were sampled using dynamic headspace extraction and analysed using gas chromatography-mass spectrometry. We then quantified phenotypic selection exerted on these traits by regressing fruit set values on floral trait values. We showed that the three studied subspecies were characterized by different floral colour and odour, with one of the two predominant floral volatiles emitted by each subspecies being taxon-specific. Plant size was positively correlated with fruit set in most populations, whereas we found no apparent link between floral colour and female reproductive success. We detected positive selection on several taxon-specific compounds in A. coriophora subsp. fragrans, whereas no selection was found on floral volatiles of A. coriophora subsp. coriophora and A. coriophora subsp. martrinii. This study is one of the first to document variation in phenotypic selection exerted on floral scents among conspecific populations. Our results suggest that selection could contribute to ongoing chemical divergence among A. coriophora subspecies.

Pigeon odor varies with experimental exposure to trace metal pollution.

Trace metals are chemical pollutants that have well-known noxious effects on wildlife and that are current major environmental issues in urban habitats. Previous studies have demonstrated their negative (e.g. lead) or positive (e.g. zinc) effects on body condition, immunity and reproductive success. Because of their effects on condition, trace metals are likely to influence the production of condition-dependent ornaments. The last decade has revealed that bird odors, like mammal odors, can convey information on individual quality and might be used as secondary sexual ornaments. Here, we used solid-phase microextraction headspace sampling with gas chromatography-mass spectrometry to investigate whether plumage scent varied with experimental supplementation in lead and/or zinc in feral pigeons. Zinc supplementation (alone or in combination with lead) changed the proportion of several volatiles, including an increase in the proportion of hydroxy-esters. The production of these esters, that most likely originate from preen gland secretions, may be costly and might thus be reduced by stress induced by zinc deficiency. Although lead is known to negatively impact pigeon condition, it did not statistically affect feather scent, despite most of the volatiles that increased with zinc exposure tended to be decreased in lead-supplemented pigeons. Further studies should evaluate the functions of plumage volatiles to predict how trace metals can impact bird fitness.

Integrative taxonomy of the fly orchid group: insights from chemical ecology.

Several authors have recently stressed the need to develop an integrative approach in taxonomy, but studies applying such an approach to Mediterranean orchids are scarce. In sexually deceptive orchids from the taxonomically difficult genus Ophrys, pollination is specific and performed by male insects attracted to the flowers by sex pheromone-mimicking floral scents. Floral compounds are therefore of primary importance for reproductive isolation and species delimitations in this genus. In the fly orchid group, molecular, morphological, and ecological characters have been extensively studied, but a comprehensive survey of floral scents is still lacking. In the present study, the blends of floral compounds of its three members, Ophrys insectifera, Ophrys aymoninii, and Ophrys subinsectifera, were extracted and analyzed by gas chromatography-mass spectrometry. A total of 107 compounds were found, with a majority of saturated and unsaturated hydrocarbons. Significant differentiation, both qualitative and quantitative, was found among the three taxa. This result, pooled with those from the literature, forms a comprehensive and congruent dataset that allows us to elucidate the taxonomic rank of the three members of the fly orchid group.

Evolution of a genetic polymorphism with climate change in a Mediterranean landscape.

Many species show changes in distribution and phenotypic trait variation in response to climatic warming. Evidence of genetically based trait responses to climate change is, however, less common. Here, we detected evolutionary variation in the landscape-scale distribution of a genetically based chemical polymorphism in Mediterranean wild thyme (Thymus vulgaris) in association with modified extreme winter freezing events. By comparing current data on morph distribution with that observed in the early 1970s, we detected a significant increase in the proportion of morphs that are sensitive to winter freezing. This increase in frequency was observed in 17 of the 24 populations in which, since the 1970s, annual extreme winter freezing temperatures have risen above the thresholds that cause mortality of freezing-sensitive morphs. Our results provide an original example of rapid ongoing evolutionary change associated with relaxed selection (less extreme freezing events) on a local landscape scale. In species whose distribution and genetic variability are shaped by strong selection gradients, there may be little time lag associated with their ecological and evolutionary response to long-term environmental change.

Earthworms and plants can decrease soil greenhouse gas emissions by modulating soil moisture fluctuations and soil macroporosity in a mesocosm experiment.

Earthworms can stimulate microbial activity and hence greenhouse gas (GHG) emissions from soils. However, the extent of this effect in the presence of plants and soil moisture fluctuations, which are influenced by earthworm burrowing activity, remains uncertain. Here, we report the effects of earthworms (without, anecic, endogeic, both) and plants (with, without) on GHG (CO2, N2O) emissions in a 3-month greenhouse mesocosm experiment simulating a simplified agricultural context. The mesocosms allowed for water drainage at the bottom to account for the earthworm engineering effect on water flow during two drying-wetting cycles. N2O cumulative emissions were 34.6% and 44.8% lower when both earthworm species and only endogeic species were present, respectively, and 19.8% lower in the presence of plants. The presence of the endogeic species alone or in combination with the anecic species slightly reduced CO2 emissions by 5.9% and 11.4%, respectively, and the presence of plants increased emissions by 6%. Earthworms, plants and soil water content interactively affected weekly N2O emissions, an effect controlled by increased soil dryness due to drainage via earthworm burrows and mesocosm evapotranspiration. Soil macroporosity (measured by X-ray tomography) was affected by earthworm species-specific burrowing activity. Both GHG emissions decreased with topsoil macropore volume, presumably due to reduced moisture and microbial activity. N2O emissions decreased with macropore volume in the deepest layer, likely due to the presence of fewer anaerobic microsites. Our results indicate that, under experimental conditions allowing for plant and earthworm engineering effects on soil moisture, earthworms do not increase GHG emissions, and endogeic earthworms may even reduce N2O emissions.

Urinary VOCs as biomarkers of early stage lung tumour development in mice.

BACKGROUND: Lung cancer is the primary cause of cancer-induced death. In addition to prevention and improved treatment, it has increasingly been established that early detection is critical to successful remission. OBJECTIVE: The aim of this study was to identify volatile organic compounds (VOCs) in urine that could help diagnose mouse lung cancer at an early stage of its development. METHODS: We analysed the VOC composition of urine in a genetically engineered lung adenocarcinoma mouse model with oncogenic EGFR doxycycline-inducible lung-specific expression. We compared the urinary VOCs of 10 cancerous mice and 10 healthy mice (controls) before and after doxycycline induction, every two weeks for 12 weeks, until full-blown carcinomas appeared. We used SPME fibres and gas chromatography - mass spectrometry to detect variations in cancer-related urinary VOCs over time. RESULTS: This study allowed us to identify eight diagnostic biomarkers that help discriminate early stages of cancer tumour development (i.e., before MRI imaging techniques could identify it). CONCLUSION: The analysis of mice urinary VOCs have shown that cancer can induce changes in odour profiles at an early stage of cancer development, opening a promising avenue for early diagnosis of lung cancer in other models.

Ozone alters the chemical signal required for plant - insect pollination: The case of the Mediterranean fig tree and its specific pollinator.

Tropospheric ozone (O3) is likely to affect the chemical signal emitted by flowers to attract their pollinators through its effects on the emission of volatile organic compounds (VOCs) and its high reactivity with these compounds in the atmosphere. We investigated these possible effects using a plant-pollinator interaction where the VOCs responsible for pollinator attraction are known and which is commonly exposed to high O3 concentration episodes: the Mediterranean fig tree (Ficus carica) and its unique pollinator, the fig wasp (Blastophaga psenes). In controlled conditions, we exposed fig trees bearing receptive figs to a high-O3 episode (5 h) of 200 ppb and analyzed VOC emission. In addition, we investigated the chemical reactions occurring in the atmosphere between O3 and pollinator-attractive VOCs using real-time monitoring. Finally, we tested the response of fig wasps to the chemical signal when exposed to increasing O3 mixing ratios (0, 40, 80, 120 and 200 ppb). The exposure of the fig tree to high O3 levels induced a significant decrease in leaf stomatal conductance, a limited change in the emission by receptive figs of VOCs not involved in pollinator attraction, but a major change in the relative abundances of the compounds among pollinator-attractive VOCs in O3-enriched atmosphere. Fig VOCs reacted with O3 in the atmosphere even at the lowest level tested (40 ppb) and the resulting changes in VOC composition significantly disrupted the attraction of the specific pollinator. These results strongly suggest that current O3 episodes are probably already affecting the interaction between the fig tree and its specific pollinator.

An experimental test of the hypothesis of non-homeostatic consumer stoichiometry in a plant litter-microbe system.

Stoichiometric homeostasis of heterotrophs is a common, but not always well-examined premise in ecological stoichiometry. We experimentally evaluated the relationship between substrate (plant litter) and consumer (microorganisms) stoichiometry for a tropical terrestrial decomposer system. Variation in microbial C : P and N : P ratios tracked that of the soluble litter fraction, but not that of bulk leaf litter material. Microbial N and P were not isometrically related, suggesting higher rates of P than N sequestration in microbial biomass. Shifts in microbial stoichiometry were related to changes in microbial community structure. Our results indicate that P in dissolved form is a major driver of terrestrial microbial stoichiometry, similar to aquatic environments. The demonstrated relative plasticity in microbial C : P and N : P and the critical role of P have important implications for theoretical modelling and contribute to a process-based understanding of stoichiometric relationships and the flow of elements across trophic levels in decomposer systems.

Volatile organic compounds influence prey composition in Sarracenia carnivorous plants.

Sarracenia pitcher plants display interspecific differences in prey, so far only explained by pitcher morphology. We hypothesized that pitcher odours play a role in prey composition. We first compared odour and prey compositions among Sarracenia taxa grown together, forming a kinship gradient from S. purpurea known to capture primarily ants towards S. leucophylla known to capture many flying insects: S. purpurea, S. X mitchelliana, and S. X Juthatip soper & S. X leucophylla horticultural hybrids. We then measured several pitcher traits to disentangle the contributions of morphology and odour to prey variation. The pitcher odours were as diverse as those of generalist-pollinated flowers but with notable differences among taxa, reflecting their relatedness. VOC similarity analyses revealed taxon specificities, that mirrored those revealed by prey similarity analyses. S. X leucophylla stood out by being more specialised in flying insects like bees and moths and by releasing more monoterpenes known to attract flower visitors. S. X Juthatip soper trapped as many bees but fewer moths, sesquiterpenes contributing less to its scent. Ants and Diptera were the main prey of the other two with fatty-acid-derivative-dominated scents. Quantities of the different prey groups can be inferred 98% from quantities of the odour classes and pitcher dimensions. Two syndromes were revealed: ants associated with fatty-acid-derivatives and short pitchers; flying insects associated with monoterpenes, benzenoids and tall pitchers. In S. X leucophylla, emission rate of fatty-acid-derivatives and pitcher length explained most variation in ant captures; monoterpenes and pitcher length explained most variation in bee and moth captures; monoterpenes alone explained most variation in Diptera and wasp captures. Our results suggest that odours are key factors of the diet composition of pitcher plants. They support the hypothesis of perceptual exploitation of insect biases in carnivorous plants and provide new insights into the olfactory preferences of insect groups.

Floral scent of the Mediterranean fig tree: significant inter-varietal difference but strong conservation of the signal responsible for pollinator attraction.

For thousands of years, humans have domesticated different plants by selecting for particular characters, often affecting less-known traits, including the volatile organic compounds (VOCs) emitted by these plants for defense or reproduction. The fig tree Ficus carica has a very wide range of varieties in the Mediterranean region and is selected for its traits affecting fruits, including pollination, but the effect of human-driven diversification on the VOCs emitted by the receptive figs to attract their pollinator (Blastophaga psenes) is not known. In the present study, VOCs from receptive figs of eight varieties in northern Morocco, were collected at different times within the manual pollination period and analyzed by gas chromatography-mass spectrometry. Genetic analyses using microsatellite loci were performed on the same varieties. Despite strong inter-varietal differences in the quantity and relative proportions of all VOCs, the relative proportions of the four pollinator-attractive VOCs showed limited variation among varieties. There was no significant correlation between genetic markers and chemical profiles of the different varieties. While diversification driven by humans has led to differences between varieties in VOC profiles, this paper suggests that throughout the process of domestication and varietal diversification, stabilizing selection has maintained a strong signal favoring pollinator attraction.

Odours of cancerous mouse congeners: detection and attractiveness.

Chemical communication plays a major role in social interactions. Cancer, by inducing changes in body odours, may alter interactions between individuals. In the framework of research targeting non-invasive methods to detect early stages of cancer development, this study asked whether untrained mice could detect odour changes in cancerous congeners. If yes, were they able to detect cancer at an early developmental stage? Did it influence female preference? Did variations in volatile organic components of the odour source paralleled mice behavioural responses? We used transgenic mice strains developing or not lung cancer upon antibiotic ingestion. We sampled soiled bedding of cancerous mice (CC) and not cancerous mice (NC), at three experimental conditions: before (T0), early stage (T2) and late stage (T12) of cancer development. Habituation/generalisation and two-way preference tests were performed where soiled beddings of CC and NC mice were presented to wild-derived mice. The composition and relative concentration of volatile organic components (VOC) in the two stimuli types were analysed. Females did not show directional preference at any of the experimental conditions, suggesting that cancer did not influence their choice behaviour. Males did not discriminate between CC and NC stimuli at T0 but did so at T2 and T12, indicating that wild-derived mice could detect cancer at an early stage of development. Finally, although the VOC bouquet differed between CC and NC it did not seem to parallel the observed behavioural response suggesting that other types of odorant components might be involved in behavioural discrimination between CC and NC mice.

No evidence for long-range male sex pheromones in two malaria mosquitoes.

Cues involved in mate seeking and recognition prevent hybridization and can be involved in speciation processes. In malaria mosquitoes, females of the two sibling species Anopheles gambiae s.s. and An. coluzzii mate in monospecific male swarms and hybrids are rare. Long-range sex pheromones driving this behaviour have been debated in literature but so far, no study has proven their existence or their absence. Here, we attempted to bring to light their existence. To put all the odds in our favour, we used different chemical ecology methods such as behavioural and electrophysiological assays as well chemical analyses, and we worked with mosquitoes at their optimal physiological mating state that is with swarming males during their natural swarming windows. Despite all our efforts, our results support the absence of long-range sex pheromones involved in swarm detection and recognition by females. We briefly discuss the implications of this finding in ecology, evolution and for control strategies.

Volatile organic compound emissions induced by the aphid Myzus persicae differ among resistant and susceptible peach cultivars and a wild relative.

Little is known on aphid-induced emissions of volatile organic compounds (VOCs) from trees and particularly on their intraspecific variability in association with resistance traits. We compared VOC emissions from five peach cultivars (Prunus persica (L.) Batsch) and a wild relative (Prunus davidiana (Carrière) Franch) that differ in their level (susceptible/resistant) and type (antixenosis, antibiosis) of resistance to the green peach aphid, Myzus persicae (Sulzer). Additionally, the kinetics of VOC induction in response to aphids was compared with that by mechanical wounding. Qualitative and overall quantitative differences among peach genotypes were found in VOC emissions that were mainly composed of methyl-salicylate, farnesenes, (E)-ß-ocimene and (E)-4,8-dimethyl-1,3,7-nonatriene. Irrespective of the type of resistance, all resistant genotypes had increased VOC emissions upon aphid attack, while in susceptible genotypes emissions remained low. Emission increases were highest in the genotypes that express increased aphid resistance during second infestations, which had also the highest proportions of methyl-salicylate in their emissions. VOC induction by aphids proceeded slowly with a delay of several hours. Artificial wounding of leaves did not result in emissions of aphid-induced VOCs but caused an immediate burst of green leaf volatiles and benzaldehyde. We conclude that VOC induction in resistant peach cultivars is part of a general defence syndrome that is being avoided or suppressed by M. persicae in the susceptible genotypes. The induction likely involves an aphid-specific elicitor and (methyl)-salicylate in the subsequent signalling and regulation processes that should include gene activation due to the marked delay in the emission response. The results are compared with those of the literature and discussed in view of their ecological and environmental significance.

Higher tree diversity increases soil microbial resistance to drought.

Predicted increases in drought frequency and severity may change soil microbial functioning. Microbial resistance and recovery to drought depend on plant community characteristics, among other factors, yet how changes in plant diversity modify microbial drought responses is uncertain. Here, we assessed how repeated drying-rewetting cycles affect soil microbial functioning and whether tree species diversity modifies these effects with a microcosm experiment using soils from different European forests. Our results show that microbial aerobic respiration and denitrification decline under drought but are similar in single and mixed tree species forests. However, microbial communities from mixed forests resist drought better than those from mono-specific forests. This positive tree species mixture effect is robust across forests differing in environmental conditions and species composition. Our data show that mixed forests mitigate drought effects on soil microbial processes, suggesting greater stability of biogeochemical cycling in mixed forests should drought frequency increase in the future.

Author Correction: Chemical signal is in the blend: bases of plant-pollinator encounter in a highly specialized interaction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chemical signal is in the blend: bases of plant-pollinator encounter in a highly specialized interaction.

In several highly specialized plant-insect interactions, scent-mediated specificity of pollinator attraction is directed by the emission and detection of volatile organic compounds (VOCs). Although some plants engaged in such interactions emit singular compounds, others emit mixtures of VOCs commonly emitted by plants. We investigated the chemical ecological bases of host plant recognition in the nursery pollination mutualism between the dioecious Ficus carica and its specific pollinator Blastophaga psenes. Using Y-tube olfactometer tests, we show that B. psenes females are attracted by VOCs of receptive figs of both sexes and do not exhibit preference for VOCs of either male or female figs. Electrophysiological tests and chemical analysis revealed that of all the VOCs emitted by receptive figs, only five were found to be active on female antennae. Behavioural tests show that, in contrast to VOCs presented alone, only a blend with a particular proportion of four of these VOCs is as attractive as the odour of receptive figs, and that if there is a very small change in this blend proportion, the pollinator is no longer attracted. This study revealed that in highly specialized mutualistic interactions specificity could be mediated by a particular blend of common compounds emitted by plants.