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.

Ozone exposure induces metabolic stress and olfactory memory disturbance in honey bees.

Human activities, urbanization, and industrialization contribute to pollution that affects climate and air quality. A main atmospheric pollutant, the tropospheric ozone (O3), can damage living organisms by generating oxidative radicals, causing respiratory problems in humans and reducing yields and growth in plants. Exposure to high concentrations of O3 can result in oxidative stress in plants and animals, eventually leading to substantial ecological consequences. Plants produce volatile organic compounds (VOCs) emitted in the environment and detected by pollinators (mainly by their antennae), foraging for nutritious resources. Several pollinators, including honey bees, recognize and discriminate flowers through olfactory cues and memory. Exposure to different concentrations of O3 was shown to alter the emission of floral VOCs by plants as well as their lifetime in the atmosphere, potentially impacting plant-pollinator interactions. In this report, we assessed the impacts of exposure to field-realistic concentrations of O3 on honey bees' antennal response to floral VOCs, on their olfactory recall and discriminative capacity and on their antioxidant responses. Antennal activity is altered depending on VOCs structure and O3 concentrations. During the behavioral tests, we first check consistency between olfactory learning rates and memory scores after 15 min. Then bees exposed to 120 and 200 ppb of ozone do not exert specific recall responses with rewarded VOCs 90 min after learning, compared to controls whose specific recall responses were consistent between time points. We also report for the first time in honey bees how the superoxide dismutase enzyme, an antioxidant defense against oxidative stress, saw its enzymatic activity rate decreases after exposure to 80 ppb of ozone. This work tends to demonstrate how hurtful can be the impact of air pollutants upon pollinators themselves and how this type of pollution needs to be addressed in future studies aiming at characterizing plant-insect interactions more accurately.

Attractant Activity of Host-Related Chemical Blends on the Poultry Red Mite at Different Spatial Scales.

Many blood-feeding arthropods use volatile organic compounds (VOCs) to detect their vertebrate hosts. The role of chemical interactions in mediating the behavior of hematophagous insects and ticks has been investigated before but remains poorly understood in hematophagous mesostigmatic mites. The poultry red mite Dermanyssus gallinae is an obligatory blood-sucking mesostigmatic mite that feeds on birds and causes damage in poultry farms. We characterized the attractive response of D. gallinae to candidate VOCs previously reported from the odor emitted by living hens. We performed in-vitro choice-test bioassays as well as semi-field and field trials using baited and unbaited traps, in the presence and absence of hens. Among different tested combinations of VOCs, a blend of 5 VOCs (mix1.0) was significantly attractive to our reference population of D. gallinae in vitro, whereas the same individual compounds tested alone were not attractive. Ammonia was attractive on its own and increased the mix1.0 attractiveness. The attractiveness of mix1.0 was confirmed at 'natural' spatial scales in the absence of hens both at the lab and on the farm that provided the reference population. The presence of hens inhibited the mix1.0 attractiveness. The attractive power of mix1.0 was not found in other farms. This research is an important step to advance our understanding of host-parasite interactions in hematophagous mesostigmatic mites and paves the way for developing alternative control tools against D. gallinae by interfering with chemical interactions. Moreover, it underlines the importance of assessing kairomonal activity on different pest populations when developing attract-and-kill systems.

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.

Acute ozone exposure impairs detection of floral odor, learning, and memory of honey bees, through olfactory generalization.

Air pollution stemming from human activities affects the environment in which plant and animal species live and interact. Similar to primary air pollutants which are emitted, secondary air pollutants, such as tropospheric ozone (O3) formed from nitrogen oxides, are also harmful to human health and plant physiology. Yet, few reports studied the effects of O3 on pollinators' physiology, despite that this pollutant, with its high oxidative potential, likely affects pollinators behaviors, especially the perception of signals they rely on to navigate their environment. Volatile Organic Compounds (VOCs) released by plants are used as signals by different animals. For pollination services, VOCs attract different insects to the flowers and strengthen these interactions. Here, we used the honey bee Apis mellifera as a model to characterize the effects of acute exposure to different realistic mixing ratios of O3 (80-, 120-, and 200-ppb) on two crucial aspects: first, how exposed honey bees detect VOCs; and second, how O3 affects these pollinators' learning and memory processes. With electroantennogram (EAG) recordings, we showed that increasing O3 mixing ratios had a biphasic effect: an initial 25% decrease of the antennal activity when bees were tested directly after exposure (O3 direct effect), followed by a 25% increase in activity and response when bees were allowed a two-hour rest after exposure (O3 delayed effect). In parallel, during olfactory conditioning, increasing O3 mixing ratios in both exposure protocols scarcely affected olfactory learning, followed by a decrease in recall of learned odors and an increase of response to new odors, leading to a higher generalization rate (i.e., discrimination impairment). These results suggest a link between O3-related oxidative stress and olfactory coding disturbance in the honey bee brain. If ozone affects the pollinators' olfaction, foraging behaviors may be modified, in addition with a possible long-term harmful effect on pollination services.

Ozone Induces Distress Behaviors in Fig Wasps with a Reduced Chance of Recovery.

Among anthropogenic environmental risks, air pollution has the potential to impact animal and plant physiology, as well as their interactions and the long-term survival of populations, which could threaten the functioning of ecosystems. What is especially alarming is that the concentration of tropospheric ozone (O3) has dramatically increased since pre-industrial times. However, the direct effects of O3 on the behavior of pollinators themselves have not been investigated so far even though insect behavior is key to their ecological interactions, which underpin the stability of ecological networks responsible for species biodiversity in ecosystems. In this study, we aim to determine the potential effects of O3 episodes at different field-realistic concentrations (0, 40, 80, 120, and 200 ppb for 60 min) on the behavior of the fig wasp Blastophaga psenes by monitoring exposed individuals hourly for 5 h after exposure. We found that ozone episodes induced major changes in insect behavior, which were already significant at 80 ppb with individuals displaying abnormal motility. The tracking over time clearly showed that exposed individuals might only have a reduced chance of recovery, with a decreasing proportion of active fig wasps despite the cessation of an O3 episode. These findings illustrate that O3 episodes can affect pollinator behavior, which may have detrimental implications for pollination systems. It is, therefore, of importance to assess the effects of O3 on insect behavior in order to predict how it could modify ecological interactions and species biodiversity in ecosystems.

Ozone Pollution Alters Olfaction and Behavior of Pollinators.

Concentration of air pollutants, particularly ozone (O3), has dramatically increased since pre-industrial times in the troposphere. Due to the strong oxidative potential of O3, negative effects on both emission and lifetime in the atmosphere of plant volatile organic compounds (VOCs) have already been highlighted. VOCs alteration by O3 may potentially affect the attraction of pollinators that rely on these chemical signals. Surprisingly, direct effects of O3 on the olfaction and the behavioral response of pollinators have not been investigated so far. We developed a comprehensive experiment under controlled conditions to assess O3 physiological and behavioral effects on two pollinator species, differing in their ecological traits. Using several realistic concentrations of O3 and various exposure times, we investigated the odor antennal detection and the attraction to VOCs present in the floral scents of their associated plants. Our results showed, in both species, a clear effect of exposure to high O3 concentrations on the ability to detect and react to the floral VOCs. These effects depend on the VOC tested and its concentration, and the O3 exposure (concentration and duration) on the pollinator species. Pollination systems may, therefore, be impaired in different ways by increased levels of O3, the effects of which will likely depend on whether the exposure is chronic or, as in this study, punctual, likely causing some pollination systems to be more vulnerable than others. While several studies have already shown the negative impact of O3 on VOCs emission and lifetime in the atmosphere, this study reveals, for the first time, that this impact alters the pollinator detection and behavior. These findings highlight the urgent need to consider air pollution when evaluating threats to pollinators.

Chemical detection triggers honey bee defense against a destructive parasitic threat.

Invasive species events related to globalization are increasing, resulting in parasitic outbreaks. Understanding of host defense mechanisms is needed to predict and mitigate against the consequences of parasite invasion. Using the honey bee Apis mellifera and the mite Varroa destructor, as a host-parasite model, we provide a comprehensive study of a mechanism of parasite detection that triggers a behavioral defense associated with social immunity. Six Varroa-parasitization-specific (VPS) compounds are identified that (1) trigger Varroa-sensitive hygiene (VSH, bees' key defense against Varroa sp.), (2) enable the selective recognition of a parasitized brood and (3) induce responses that mimic intrinsic VSH activity in bee colonies. We also show that individuals engaged in VSH exhibit a unique ability to discriminate VPS compounds from healthy brood signals. These findings enhance our understanding of a critical mechanism of host defense against parasites, and have the potential to apply the integration of pest management in the beekeeping sector.

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.

Modulation of feed composition is able to make hens less attractive to the poultry red mite Dermanyssus gallinae.

The poultry red mite (PRM) is an obligatory haematophagous pest that causes substantial economic losses in poultry worldwide. The PRM does not live on the host but in the bird's environment and must find its host remotely. Hence, manipulating chicken odours is of interest. Several crude plant-originating volatile organic compounds (VOCs) have already been shown as repellent to Dermanyssus gallinae. We aimed to test whether these VOCs can interfere with PRM host-seeking behaviour by their oral administration to the poultry. The objectives were to determine (1) if hen odours are modified by supplemented feed ingestion and (2) if such treatment makes hens less attractive to the PRM. Chemical characterization by gas chromatography-mass spectrometry of the hen odour was conducted before and after the hens ingested the supplemented feed. The chromatograms obtained show that hen odour was substantially modified after the hens consumed it. Among the molecules recurrently detected from the supplemented hens, 26% were nearly absent in the unsupplemented hens. Behavioural choice tests to compare the effect of the modified and unmodified-host odours on the PRM show that some of the plant-originating emitted VOCs and the modified whole-hen odours were repellent to the PRM.

Olfactory pathway of the hornet Vespa velutina: New insights into the evolution of the hymenopteran antennal lobe.

In the course of evolution, eusociality has appeared several times independently in Hymenoptera, within different families such as Apidae (bees), Formicidae (ants), and Vespidae (wasps and hornets), among others. The complex social organization of eusocial Hymenoptera relies on sophisticated olfactory communication systems. Whereas the olfactory systems of several bee and ant species have been well characterized, very little information is as yet available in Vespidae, although this family represents a highly successful insect group, displaying a wide range of life styles from solitary to eusocial. Using fluorescent labeling, confocal microscopy, and 3D reconstructions, we investigated the organization of the olfactory pathway in queens, workers, and males of the eusocial hornet Vespa velutina. First, we found that caste and sex dimorphism is weakly pronounced in hornets, with regard to both whole-brain morphology and antennal lobe organization, although several male-specific macroglomeruli are present. The V. velutina antennal lobe contains approximately 265 glomeruli (in females), grouped in nine conspicuous clusters formed by afferent tract subdivisions. As in bees and ants, hornets display a dual olfactory pathway, with two major efferent tracts, the medial and the lateral antennal lobe tracts (m- and l-ALT), separately arborizing two antennal lobe hemilobes and projecting to partially different regions of higher order olfactory centers. Finally, we found remarkable anatomical similarities in the glomerular cluster organizations among hornets, ants, and bees, suggesting the possible existence of homologies in the olfactory pathways of these eusocial Hymenoptera. We propose a common framework for describing AL compartmentalization across Hymenoptera and discuss possible evolutionary scenarios. J. Comp. Neurol. 524:2335-2359, 2016. © 2016 Wiley Periodicals, Inc.