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Terpene synthases (TPSs) have diverse biological functions in plants. Though the roles of TPSs in herbivore defense are well established in many plant species, their role in bacterial defense has been scarce and is emerging. Through functional genomics, here we report the in planta role of potato (Solanum tuberosum) terpene synthase (StTPS18) in bacterial defense. Expression of StTPS18 was highest in leaves and was induced in response to Pseudomonas syringae and methyl jasmonate treatments. The recombinant StTPS18 exhibited bona fide (E,E)-farnesol synthase activity forming a sesquiterpenoid, (E,E)-farnesol as the sole product, utilising (E,E)-farnesyl diphosphate (FPP). Subcellular localization of GFP fusion protein revealed that StTPS18 is localized to the cytosol. Silencing and overexpression of StTPS18 in potato resulted in reduced and enhanced tolerance, respectively, to bacterial pathogens P. syringae and Ralstonia solanacearum. Bacterial growth assay using medium containing (E,E)-farnesol significantly inhibited P. syringae growth. Moreover, StTPS18 overexpressing transgenic potato and Nicotiana tabacum leaves, and (E,E)-farnesol and P. syringae infiltrated potato leaves exhibited elevated expression of sterol pathway and members of pathogenesis-related genes with enhanced phytosterol accumulation. Interestingly, enhanced phytosterols in

Insects perform a wide array of intricate behaviors over large spatial and temporal scales in complex natural environments. A mechanistic understanding of insect cognition has direct implications on how brains integrate multimodal information and can inspire bio-based solutions for autonomous robots. Virtual Reality (VR) offers an opportunity assess insect neuroethology while presenting complex, yet controlled, stimuli. Here, we discuss the use of insects as inspiration for artificial systems, recent advances in different VR technologies, current knowledge gaps, and the potential for application of insect VR research to bio-inspired robots. Finally, we advocate the need to diversify our model organisms, behavioral paradigms, and embrace the complexity of the natural world. This will help us to uncover the proximate and ultimate basis of brain and behavior and extract general principles for common challenging problems.

Contemporary losses of biodiversity, sometimes referred to as the sixth mass extinction, continue to mount (1, 2). A recent assessment by the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services (IPBES) estimates that one million of approximately 10 million species that exist now are threatened with extinction along with the ecosystems they inhabit (3). Yet, in the post-coronavirus disease 2019 (COVID-19) world, investments in conservation are likely to decline further. To arrest biodiversity losses, much of the recent debate advocates two traditional approaches: Put more land under wilderness (4, 5), and mitigate drivers of change through improved governance and policies (2). India and other nations need new frameworks that integrate science with policies to enhance human well-being, restore and conserve nature, and build capacity. Image credit: Sandesh Kadur (photographer). However, in the populous tropics, where most biodiversity resides, conservation of our ecosystems will remain elusive until there is a clear demonstration of the relevance of biodiversity to people’s well-being, most of whom live below the poverty line. Although nature-based solutions for addressing our most pressing challenges such as climate change, water scarcity, agricultural intensification, and health are well known (3), the link between biodiversity and human well-being has remained elusive. A major constraint, at least in the tropics, has been the lack of investment in biodiversity science that can build human resources and demonstrate the potential of biodiversity in 1) tackling climate change, natural disasters, stagnating agricultural productivity, water shortages, polluted air, and newly emerging infectious diseases such as COVID-19, 2) increasing human well-being, and 3) fostering future socioeconomic development. Here, we outline a National Mission on Biodiversity and Human Well-being for India that calls for a considerable increase in investment for this new framework for integrating biodiversity science with human well-being. This will demonstrate the linkages … [↵][1]1To whom correspondence may be addressed. Email: kamal.bawa{at}gmail.com. [1]: #xref-corresp-1-1

Terpene synthases (TPSs) produce a variety of terpenoids that play numerous functional roles in primary and secondary metabolism, as well as in ecological interactions. Here, we report the functional characterization of an inducible potato TPS gene encoding bulnesol/elemol synthase (StBUS/ELS). The expression of StBUS/ELS in potato leaves was significantly induced in response to both bacterial (Pseudomonas syringae) and fungal (Alternaria solani) infection as well as methyl jasmonate treatment, indicating its role in defense. The leaves exhibited the highest StBUS/ELS expression followed by the stem with least and similar expression in tuber, sprout and root. Recombinant StBUS/ELS catalyzed the formation of different sesquiterpenes by utilizing farnesyl diphosphate as substrate, and the monoterpene geraniol from geranyl diphosphate. Among the sesquiterpenes formed by StBUS/ELS, elemol was the predominant product followed by α-bulnesene, bulnesol and β-elemene. Further gas chromatography-mass spectrometry (GC-MS) analysis of StBUS/ELS assay products at different injection temperatures revealed elemol and bulnesol as the major products at 275 and 200/150°C, respectively, without much change in the levels of minor products. This indicated thermal rearrangement of bulnesol into elemol at higher temperatures. Transient overexpression of StBUS/ELS in potato leaves conferred tolerance against the growth of bacteria P. syringae and Ralstonia solanacearum, and the fungus A. solani. Further, expression analysis of pathogenesis-related (PR) genes in StBUS/ELS overexpressing leaves showed no significant change in comparison to control, indicating a direct involvement of StBUS/ELS enzymatic products against pathogens. Overall, our study suggested that StBUS/ELS is a pathogen-inducible TPS encoding bulnesol/elemol synthase and could provide a direct role in defense against biotic stress in potato.

Significance India is the world’s largest fruit producer and second most populous country. Pollinators are therefore important for India’s food security. India also contains 9 of the world’s 10 most polluted cities, but the impact of air pollution on plant and animal systems is largely unknown. We performed a multiyear study in the megacity of Bangalore to correlate the mechanistic effects of air pollution on a major Indian pollinator, the Giant Asian honey bee, Apis dorsata. Wild honey bees and lab-reared Drosophila exposed to air pollution exhibited differences in survival, behavior, heart rate, blood cell count, and/or the expression of genes related to stress, immunity, and metabolism. Our study indicates the urgency for more studies on wild systems to better inform international air quality guidelines., While the impact of air pollution on human health is well studied, mechanistic impacts of air pollution on wild systems, including those providing essential ecosystem services, are largely unknown, but directly impact our health and well-being. India is the world’s largest fruit producer, second most populous country, and contains 9 of the world’s 10 most polluted cities. Here, we sampled Giant Asian honey bees, Apis dorsata, at locations with varying air pollution levels in Bangalore, India. We observed significant correlations between increased respirable suspended particulate matter (RSPM) deposition and changes in bee survival, flower visitation, heart rate, hemocyte levels, and expression of genes related to lipid metabolism, stress, and immunity. Lab-reared Drosophila melanogaster exposed to these same sites also exhibited similar molecular and physiological differences. Our study offers a quantitative analysis on the current impacts of air pollution on insects, and indicates the urgency for more nonhuman studies to accurately assess the effects of pollution on our natural world.

Significance Flying insects are Earth’s most diverse taxonomic class. They exhibit remarkable abilities to locate resources while flying through complex 3D environments. However, we still have not deciphered how flying insects integrate multiple stimuli and extract depth to locate distant targets. Here, we assessed long-range search behavior in tethered flies using virtual reality. We first show that insects of multiple species can navigate to virtual 3D objects. We then show flies can employ perspective and motion parallax to distinguish size and distance amidst a complex background, and can incorporate directional airflow and odor to navigate toward distant targets. These results provide the foundation to elucidate search processes for pest management, pollination, and dispersal, and apply them to robotics and search algorithms., The exemplary search capabilities of flying insects have established them as one of the most diverse taxa on Earth. However, we still lack the fundamental ability to quantify, represent, and predict trajectories under natural contexts to understand search and its applications. For example, flying insects have evolved in complex multimodal three-dimensional (3D) environments, but we do not yet understand which features of the natural world are used to locate distant objects. Here, we independently and dynamically manipulate 3D objects, airflow fields, and odor plumes in virtual reality over large spatial and temporal scales. We demonstrate that flies make use of features such as foreground segmentation, perspective, motion parallax, and integration of multiple modalities to navigate to objects in a complex 3D landscape while in flight. We first show that tethered flying insects of multiple species navigate to virtual 3D objects. Using the apple fly Rhagoletis pomonella, we then measure their reactive distance to objects and show that these flies use perspective and local parallax cues to distinguish and navigate to virtual objects of different sizes and distances. We also show that apple flies can orient in the absence of optic flow by using only directional airflow cues, and require simultaneous odor and directional airflow input for plume following to a host volatile blend. The elucidation of these features unlocks the opportunity to quantify parameters underlying insect behavior such as reactive space, optimal foraging, and dispersal, as well as develop strategies for pest management, pollination, robotics, and search algorithms.

It has been established that smell and taste loss are frequent symptoms during COVID-19 onset. Most evidence stems from medical exams or self-reports. The latter is particularly confounded by the common confusion of smell and taste. Here, we tested whether practical smelling and tasting with household items can be used to assess smell and taste loss. We conducted an online survey and asked participants to use common household items to perform a smell and taste test. We also acquired generic information on demographics, health issues including COVID-19 diagnosis, and current symptoms. We developed several machine learning models to predict COVID-19 diagnosis. We found that the random forest classifier consistently performed better than other models like support vector machines or logistic regression. The smell and taste perception of self-administered household items were statistically different for COVID-19 positive and negative participants. The most frequently selected items that also discriminated between COVID-19 positive and negative participants were clove, coriander seeds, and coffee for smell and salt, lemon juice, and chillies for taste. Our study shows that the results of smelling and tasting household items can be used to predict COVID-19 illness and highlight the potential of a simple home-test to help identify the infection and prevent the spread.

Host shifts are considered a key generator of insect biodiversity. For insects, adaptation to new host plants often requires changes in larval/pupal development and behavioural preference towards new hosts. Neurochemicals play key roles in both development and behaviour, and therefore provide a potential source for such synchronization. Here, we correlated life history timing, brain development, and corresponding levels of 14 neurochemicals in Rhagoletis pomonella (Diptera: Tephritidae), a species undergoing ecological speciation through an ongoing host shift from hawthorn to apple fruit. These races exhibit differences in pupal diapause timing as well as adult behavioural preference with respect to their hosts. This difference in behavioral preference is coupled with differences in neurophysiological response to host volatiles. We found that apple race pupae exhibited adult brain morphogenesis three weeks faster after an identical simulated winter than the hawthorn race, which correlated with significantly lower titres of several neurochemicals. In some cases, particularly biogenic amines, differences in titres were reflected in the mature adult stage, when host preference is exhibited. In summary, life history timing, neurochemical titre, and brain development can be coupled in this speciating system, providing new hypotheses for the origins of new species through host shifts.

Changes in behaviour often drive rapid adaptive evolution and speciation. However, the mechanistic basis for behavioural shifts is largely unknown. The tephritid fruit fly Rhagoletis pomonella is an example of ecological specialization and speciation in action via a recent host plant shift from hawthorn to apple. These flies primarily use specific odours to locate fruit, and because they mate only on or near host fruit, changes in odour preference for apples versus hawthorns translate directly to prezygotic reproductive isolation, initiating speciation. Using a variety of techniques, we found a reversal between apple and hawthorn flies in the sensory processing of key odours associated with host fruit preference at the first olfactory synapse, linking changes in the antennal lobe of the brain with ongoing ecological divergence. Indeed, changes to specific neural pathways of any sensory modality may be a broad mechanism for changes in animal behaviour, catalysing the genesis of new biodiversity.

Changes in behaviour often drive rapid adaptive evolution and speciation. However, the mechanistic basis for behavioural shifts is largely unknown. The tephritid fruit fly Rhagoletis pomonella is an example of ecological specialization and speciation in action via a recent host plant shift from hawthorn to apple. These flies primarily use specific odours to locate fruit, and because they mate only on or near host fruit, changes in odour preference for apples versus hawthorns translate directly to prezygotic reproductive isolation, initiating speciation. Using a variety of techniques, we found a reversal between apple and hawthorn flies in the sensory processing of key odours associated with host fruit preference at the first olfactory synapse, linking changes in the antennal lobe of the brain with ongoing ecological divergence. Indeed, changes to specific neural pathways of any sensory modality may be a broad mechanism for changes in animal behaviour, catalysing the genesis of new biodiversity.

Significance The coeveolution of flowers and pollinators is well known, but how generalist pollinators identify suitable flowers across environments and flower species is not well understood. Hoverflies, which are found across the globe, are one of the most important alternative pollinators after bees and bumblebees. Here we measured, predicted, and finally recreated multimodal cues from individual flowers visited by hoverflies in three different environments (hemiboreal, alpine, and tropical). We found that although “flower signatures” were unique for each environment, some cues were ubiquitously attractive, despite not resembling cue combinations from real flowers. Our results provide unique insights into how a cosmopolitan pollinator identifies flower objects across environments, which has important implications for our understanding of pollination as a global ecological service., With more than 80% of flowering plant species specialized for animal pollination, understanding how wild pollinators utilize resources across environments can encourage efficient planting and maintenance strategies to maximize pollination and establish resilience in the face of environmental change. A fundamental question is how generalist pollinators recognize “flower objects” in vastly different ecologies and environments. On one hand, pollinators could employ a specific set of floral cues regardless of environment. Alternatively, wild pollinators could recognize an exclusive signature of cues unique to each environment or flower species. Hoverflies, which are found across the globe, are one of the most ecologically important alternative pollinators after bees and bumblebees. Here, we have exploited their cosmopolitan status to understand how wild pollinator preferences change across different continents. Without employing any a priori assumptions concerning the floral cues, we measured, predicted, and finally artificially recreated multimodal cues from individual flowers visited by hoverflies in three different environments (hemiboreal, alpine, and tropical) using a field-based methodology. We found that although “flower signatures” were unique for each environment, some multimodal lures were ubiquitously attractive, despite not carrying any reward, or resembling real flowers. While it was unexpected that cue combinations found in real flowers were not necessary, the robustness of our lures across insect species and ecologies could reflect a general strategy of resource identification for generalist pollinators. Our results provide insights into how cosmopolitan pollinators such as hoverflies identify flowers and offer specific ecologically based cues and strategies for attracting pollinators across diverse environments.

In their Letter, Negri et al. (1) point out four concerns with our recent publication (2). The first is that the study is correlative, and the next two that we did not measure the toxicity of air pollution directly, nor how the particulate matter (PM) measured on the surface of the cuticle and internal organs could be impacting health. Finally, the authors are concerned with the identity of the elements we found using scanning electron microscopy coupled with X-ray spectroscopy (SEM-EDX). First, some specific responses. The assertion that we did not investigate the contact between PM and the internal organs is erroneous and is already shown in figure 2 (crop and midgut … [↵][1]1To whom correspondence may be addressed. Email: shannon{at}nice.ncbs.res.in or geetha{at}nice.ncbs.res.in. [1]: #xref-corresp-1-1

Antagonism of plant pathogenic fungi by endophytic fungi is a well-known phenomenon. In plate assays, the antagonism could be due to mycoparasitism, competition for space or antibiosis, involving a chemical diffusate, or a volatile organic compound (VOC). In this study, we demonstrate that besides mycoparasitism, VOCs play a major role in antagonism of pathogenic fungi by four endophytic fungi belonging to the genus Trichoderma. Using a double-plate assay, we show that all the four endophytic Trichoderma species significantly inhibited mycelial growth of three of the four pathogens, (Sclerotinia sclerotiorum-TSS, Sclerotium rolfsii-CSR and Fusarium oxysporum-CFO), while that of Macrophomina phaseolina-CMP was not affected. GC-MS analysis of the pure cultures of one of the endophytic fungi studied, namely, Trichoderma longibrachiatum strain 2 (Acc. No. MK751758) and the pathogens, F. oxysporum-CFO and M. phaseolina-CMP revealed the presence of several VOCs including hydrocarbons, alcohols, ketones, aldehydes, esters, acids, ethers and different classes of terpenes. In mixed double plates, where the endophyte was grown along with either of the two plant pathogens, F. oxysporum-CFO or M. phaseolina-CMP, there was an induction of a number of new VOCs that were not detected in the pure cultures of either the endophyte or the pathogens. Several of these new VOCs are reported to possess antifungal and cytotoxic activity. We discuss these results and highlight the importance of such interactions in endophyte-pathogen interactions.

Chemical signaling is ubiquitous and employs a variety of receptor types to detect the cacophony of molecules relevant for each living organism. Insects, our most diverse taxon, have evolved unique olfactory receptors with as little as 10% sequence identity between receptor types. We have identified a promiscuous volatile, 2-methyltetrahydro-3-furanone (coffee furanone), that elicits chemosensory and behavioral activity across multiple insect orders and receptors. In vivo and in vitro physiology showed that coffee furanone was detected by roughly 80% of the recorded neurons expressing the insect-specific olfactory receptor complex in the antenna of Drosophila melanogaster, at concentrations similar to other known, and less promiscuous, ligands. Neurons expressing specialized receptors, other chemoreceptor types, or mutants lacking the complex entirely did not respond to this compound. This indicates that coffee furanone is a promiscuous ligand for the insect olfactory receptor complex itself and did not induce non-specific cellular responses. In addition, we present homology modeling and docking studies with selected olfactory receptors that suggest conserved interaction regions for both coffee furanone and known ligands. Apart from its physiological activity, this known food additive elicits a behavioral response for several insects, including mosquitoes, flies, and cockroaches. A broad-scale behaviorally active molecule non-toxic to humans thus has significant implications for health and agriculture. Coffee furanone serves as a unique tool to unlock molecular, physiological, and behavioral relationships across this diverse receptor family and animal taxa.

A mixture of behaviorally active volatiles was identified from the fruit of snowberry, Symphoricarpos albus laevigatus, for Rhagoletis zephyria flies reared from snowberry fruit. A nine-component blend containing 3-methylbutan-1-ol (3%), dimethyl trisulfide (1%), 1-octen-3-ol (40%), myrcene (8%), nonanal (9%), linalool (13%), (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT, 6%), decanal (15%), and β-caryophyllene (5%) was identified that gave consistent electroantennogram activity and was behaviorally active in flight tunnel tests. In other flight tunnel assays, snowberry flies from two sites in Washington state, USA, displayed significantly greater levels of upwind oriented flight to sources with the snowberry volatile blend compared with previously identified volatile blends from domestic apple (Malus domestica) and downy hawthorn (Crataegus mollis) fruit from the eastern USA, and domestic apple, black hawthorn (C. douglasii) and ornamental hawthorn (C. monogyna) from Washington state. Selected subtraction assays showed that whereas removal of DMNT or 1-octen-3-ol significantly reduced the level of upwind flight, removal of myrcene and β-caryophyllene, or dimethyl trisulfide alone did not significantly affect the proportion of upwind flights. Our findings add to previous studies showing that populations of Rhagoletis flies infesting different host fruit are attracted to unique mixtures of volatile compounds specific to their respective host plants. Taken together, the results support the hypothesis that differences among flies in their behavioral responses to host fruit odors represent key adaptations involved in sympatric host plant shifts, contributing to host specific mating and generating prezygotic reproductive isolation among members of the R. pomonella sibling species complex.

Highly populated tropical countries face tremendous pressures in reconciling the needs for improved economic security and the protection of declining biodiversity. India is no exception and its biodiversity is under severe pressure due to complex interactions among land use change, other human economic activities, and climate change. Preservation and restoration of biodiversity is perhaps the cheapest and least risky way to mitigate the impacts of threats such as climate change, diminishing food and nutritional security, declining economy, absence of affordable healthcare, rising zoonotic diseases and lack of capacity to address these issues. Here we describe a framework for biodiversity conservation – the National Mission on Biodiversity and Human Well-Being (NMBHWB) for India – which integrates biodiversity, ecosystem services, climate change, agriculture, health, bio-economy and capacity building in the realm of biodiversity science. We provide an overview of the seven Programs of the Mission which make it interdisciplinary, integrative, and comprehensive in its approach. The Mission explicitly links research with policy-making and implementation for effective management of biodiversity with sustainable development. With its emphasis on convergence and synergies among various goals, themes and project sites, the Mission will further develop new models for stakeholder consultations and co-production of knowledge. We posit that the NMBHWB will enable India to realize the United Nations Sustainable Development Goals and Targets while advancing India's commitments to the Paris Agreement on climate change and other international environmental conventions and treaties.

Recent studies have shown that flies from sympatric populations of Rhagoletis pomonella infesting hawthorn, apple, and flowering dogwood fruit can distinguish among unique volatile blends identified from each host. Analysis of peripheral chemoreception in Rhagoletis flies suggests that changes in receptor specificity and/or receptor neuron sensitivity could impact olfactory preference among the host populations and their hybrids. In an attempt to validate these claims, we have combined flight tunnel analyses and single sensillum electrophysiology in F(2) and backcross hybrids displaying a variety of behavioral phenotypes. Results show that differences in peripheral chemoreception among second-generation adults do not provide a direct correlation between peripheral coding and olfactory behavior. We conclude that either the plasticity of the central nervous system in Rhagoletis can compensate for significant alterations in peripheral coding or that peripheral changes present subtle effects on behavior not easily detectable with current techniques. The results of this study imply that the basis for olfactory behavior in Rhagoletis has a complicated genetic and neuronal basis, even for populations with a recent divergence in preference.

In this issue of Chemical Senses, Baker et al. propose a provocative and intriguing explanation for a commonly observed phenomenon in moth chemocommunication. Sex pheromones in moths typically consist of mixtures of long-chain unsaturated compounds in specific ratios. These ratios are correspondingly detected by male moths using separate olfactory sensory neurons for each pheromone component housed singly or multiply in long trichoid sensilla on the antennal surface. These neurons are often present in different proportions, typically with the neuron responding to the highest ratio component present in greatest abundance or with the largest dendritic diameter. In their article, Baker et al. postulate that these physical differences in neuron magnitudes arise to compensate for the higher molecular flux present with the most abundant pheromone components. Such a suggestion raises several questions concerning the physiological and behavioral nature of pheromone communication. Specifically, is the flux in a natural pheromone plume high enough to warrant increased flux detection for the most abundant components? Second, how can changes in neuronal number or size lead to increased flux detection? And finally, how would this increased flux detection be accomplished at molecular, cellular, and ultimately network scales? We address each of these questions and propose future experiments that could offer insight into the stimulating proposition raised by Baker et al.

Recent insights into insect olfactory signaling based on in vitro analyses have created an urgent need for equivalent in vivo analyses using living organisms. Here, we present a microinjection system that establishes a "virtual petri dish" within sensory structures for the application of agents to sensory neurons. Our system uses a series of pumps to inject chemical agents via air pressure into the surrounding lymph. We show using tetrodotoxin and forskolin application that robust effects on response dynamics of Drosophila melanogaster olfactory sensory neurons could be observed within 200 s, and suggest data analysis techniques to improve estimation of pharmacological effects on response kinetics. This approach provides an improved in vivo method to investigate questions in sensory neuron physiology as a complement to heterologous expression systems.

There is an ever-increasing demand for data to be embedded in our environment at ever-decreasing temporal and spatial scales. Whilst current communication and storage technologies generally exploit the electromagnetic properties of media, chemistry offers us a new alternative for nanoscale signaling using molecules as messengers with high information content. Biological systems effectively overcome the challenges of chemical communication using highly specific biosynthetic pathways for signal generation together with specialized protein receptors and nervous systems. Here we consider a new approach for information transmission based upon nature's quintessential example of infochemical communication, the moth pheromone system. To approach the sensitivity, specificity and versatility of infochemical communication seen in nature, we describe an array of biologically-inspired technologies for the production, transmission, detection, and processing of molecular signals. We show how it is possible to implement each step of the moth pheromone pathway for biosynthesis, transmission, receptor protein binding/transduction, and antennal lobe processing of monomolecular and multimolecular signals. For each implemented step, we discuss the value, current limitations, and challenges for the future development and integration of infochemical communication technologies. Together, these building blocks provide a starting point for future technologies that can utilize programmable emission and detection of multimolecular information for a new and robust means of communicating chemical information.

SUMMARY The Rhagoletis pomonella species complex is one of the foremost examples supporting the occurrence of sympatric speciation. A recent study found that reciprocal F1 hybrid offspring from different host plant-infesting populations in the complex displayed significantly reduced olfactory host preference in flight-tunnel assays. Behavioral and electrophysiological studies indicate that olfactory cues from host fruit are important chemosensory signals for flies to locate fruit for mating and oviposition. The reduced olfactory abilities of hybrids could therefore constitute a significant post-mating barrier to gene flow among fly populations. The present study investigated the source of changes in the hybrid olfactory system by examining peripheral chemoreception in F1 hybrid flies, using behaviorally relevant volatiles from the parent host fruit. Single-sensillum electrophysiological analyses revealed significant changes in olfactory receptor neuron (ORN) response specificities in hybrid flies when compared to parent ORN responses. We report that flies from F1 crosses of apple-, hawthorn- and flowering dogwood-origin populations of R. pomonella exhibited distinct ORN response profiles absent from any parent population. These peripheral alterations in ORN response profiles could result from misexpression of multiple receptors in hybrid neurons as a function of genomic incompatibilities in receptor-gene pathways in parent populations. We conclude that these changes in peripheral chemoreception could impact olfactory host preference and contribute directly to reproductive isolation in the Rhagoletis complex, or could be genetically coupled to other host-associated traits.

Every decision an animal makes is the result of the brain’s complex processing of input from the environment to produce a specific neural output. Yet, the world is a cacophony of information. How, then, has the nervous system evolved to sort through this massive influx of data and reach a particular outcome? Our research explores how complex sensory information can lead to decision making in one of nature’s most efficient “microbrains”: the insect. To locate an odor source, an olfactory system must be able to simultaneously discriminate both the identity and location of the odor source from a noisy chemical background. Flying insects, in particular, have tremendous capabilities to localize odor sources while challenged by the high-speed performance of flight. We find that insects make olfactory decisions through stepwise spatiotemporal feature extraction of complex “odor objects” from the antenna to the brain. Our work in moths suggests that complex odors can be considered “objects” in much the same way we consider objects as composite images in the visual sense. The sensory repertoire of the antenna acts as a filter for ecologically-relevant information, while the antennal lobe of the moth brain extracts odor objects from multicomponent chemical blends. Our work in Drosophila melanogaster also finds that the signal transduction cascades of insect sensory neurons are ideally suited to localize turbulent odor object plumes during flight. Finally, our comparative analyses across species also imply that insects have evolved to define odor objects at different levels of the brain depending on their ecological niche. Taken together, these analyses provide both mechanistic insight as well as an experimental database to understand how flying insects have evolved to make decisions in a complex natural odor space.

Electrophysiology is an invaluable technique to quickly and quantitatively assess the response of the olfactory system to odor stimuli. For measuring the response of the insect antenna, two basic techniques exist, electroantennography and single sensillum recording. Here, we describe the general practice of both methods in terms of equipment used, insect preparation, recording technique, and basic analysis.

The reproductive success of herbivorous insects largely depends on the mother’s oviposition preference. In nocturnal insects, olfaction is arguably the most important sensory modality mediating mate finding, foraging, and host location. In most habitats, gravid females select among a number of plants of varying suitability, yet assessment of the neuroethological mechanisms underlying odor-guided choice between host plants is rare. Using a series of behavioral, electrophysiological, and chromatographic analyses in the Hawk moth, Manduca sexta, we show that gravid females perform a hierarchical choice among host plants of different species and qualities using olfactory cues. Both relevant plant species and qualities can be distinguished by volatile profiles collected from the headspace of these plants, and olfactory sensilla on female antennae detect more than half of the about 120 analytically detected volatiles in host plant headspace samples. Although olfactory sensory neurons present in antennal sensilla are mainly broadly tuned to multiple host compounds, some sensilla exhibit species and condition-specific responses. In fact, species and quality can be distinguished by the physiologically active components alone. Our findings thus suggest that distinguishing characteristics of both host species and quality are already represented at the sensory periphery.

Electrophysiology is an invaluable technique to quickly and quantitatively assess the response of the olfactory system to odor stimuli. For measuring the response of the insect antenna, two basic techniques exist, electroantennography and single sensillum recording. Here, we describe the general practice of both methods in terms of equipment used, insect preparation, recording technique, and basic analysis.

Behavioural changes in habitat or mate choice can trigger population divergence, leading to speciation. However, little is known about the neurological bases for such changes. Rhagoletis pomonella (Diptera: Tephritidae) is a model for ecological speciation via host plant shifts. Within the past 180 years, Rhagoletis flies infesting hawthorn ( Crataegus spp.) shifted to attack domesticated apple ( Malus pumila ). The two populations differ in their olfactory preferences for apple versus hawthorn fruit. Here, we looked for patterns of sensory organization that may have contributed to this shift by characterizing the morphology, specificity and distribution of olfactory sensory neurons (OSNs) on the antennae of Rhagoletis responding to host fruit and non-host volatiles. Of 28 OSN classes identified, two colocalized OSN pairs were found that specifically responded to the major behavioural attractant and antagonist volatiles for each fly population. A reversal in the response of these OSNs to fruit volatiles, either through a switch in receptor expression between these paired neurons or changes in neuronal projections in the brain, could therefore account for the behavioural difference between apple and hawthorn flies. The finding supports the hypothesis that relatively minor changes in olfactory sensory pathways may contribute to rapid host shifting and divergence in Rhagoletis .

Animals typically perceive their olfactory environment as a complex blend of natural odor cues. In insects, the initial processing of odors occurs in the antennal lobe (AL). Afferent peripheral input from olfactory sensory neurons (OSNs) is modified via mostly inhibitory local interneurons (LNs) and transferred by projection neurons (PNs) to higher brain centers. Here we performed optophysiological studies in the AL of the moth,Manduca sexta, and recorded odor-evoked calcium changes in response to antennal stimulation with five monomolecular host volatiles and their artificial mixture. In a double staining approach, we simultaneously measured OSN network input in concert with PN output across the glomerular array. By comparing odor-evoked activity patterns and response intensities between the two processing levels, we show that host mixtures could generally be predicted from the linear summation of their components at the input of the AL, but output neurons established a unique, nonlinear spatial pattern separate from individual component identities. We then assessed whether particularly high levels of signal modulation correspond to behavioral relevance. One of our mixture components, phenyl acetaldehyde, evoked significant levels of nonlinear input-output modulation in observed spatiotemporal activation patterns that were unique from the other individual odorants tested. This compound also accelerated behavioral activity in subsequent wind tunnel tests, whereas another compound that did not exhibit high levels of modulation also did not affect behavior. These results suggest that the high degree of input-output modulation exhibited by the AL for specific odors can correlate to behavioral output.

Insect odorant receptors (ORs) have a unique design of heterodimers formed by an olfactory receptor protein and the ion channel Orco. Heterologously expressed insect ORs are activated via an ionotropic and a metabotropic pathway that leads to cAMP production and activates the Orco channel. The contribution of metabotropic signaling to the insect odor response remains to be elucidated. Disruption of the Gq protein signaling cascade reduces the odor response (Kain et al., 2008). We investigated this phenomenon in HEK293 cells expressing Drosophila Orco and found that phospholipase C (PLC) inhibition reduced the sensitivity of Orco to cAMP. A similar effect was seen upon inhibition of protein kinase C (PKC), whereas PKC stimulation activated Orco even in the absence of cAMP. Mutation of the five PKC phosphorylation sites in Orco almost completely eliminated sensitivity to cAMP. To test the impact of PKC activity in vivo we combined single sensillum electrophysiological recordings with microinjection of agents affecting PLC and PKC function and observed an altered response of olfactory sensory neurons (OSNs) to odorant stimulation. Injection of the PLC inhibitor U73122 or the PKC inhibitor Gö6976 into sensilla reduced the OSN response to odor pulses. Conversely, injection of the PKC activators OAG, a diacylglycerol analog, or phorbol myristate acetate (PMA) enhanced the odor response. We conclude that metabotropic pathways affecting the phosphorylation state of Orco regulate OR function and thereby shape the OSN odor response.

Olfactory studies have expanded beyond the study of single compound odor perception to explore the processing of complex mixtures and blends. The spatiotemporal presentation of blend stimuli is a challenging task requiring volatiles with diverse chemical and physical properties to be presented as a unified stimulus. This not only necessitates accurate control of the timing and homogeneity of the odor stream, but requires attention to the concentration of each blend component presented. We have developed a novel, multicomponent stimulus system for use in olfactory experiments that is capable of presenting up to 8 different odors simultaneously or in sequence at defined concentrations and time scales. Each odor is separated to minimize physical or chemical interactions, and stimulations are performed from a saturated headspace of the odor solution. Stimulus concentrations can be measured empirically or estimated using common gas laws. Photoionization detector measurements show that stimuli could be presented as cohesive blends or single components at frequencies of at least 10Hz without leakage or contamination. Solid phase microextraction measurements also show that the concentration of each component could be equilibrated through regulation of each component line's flow rate based on the different partial vapor pressures of the odorants. This device provides a unique method for introducing complex volatile mixtures for olfactory studies in a variety of animal taxa and allows for accurate control of odor intensities in both time and space.

By exploiting recent advances in both our understanding of pheromone biosynthesis pathways and the detection of molecules in the insect nervous system, we are developing a novel communication system based on functional equivalents of the cellular, sub-cellular and molecular biological machinery. This will form the basis of a new branch of information technology employing infochemical ligands for communication over space and time. In order to achieve this objective, both chemical signal generation and reception are replicated by taking a systematic approach that implements reaction steps as biosynthetic modules, which are then hierarchically integrated as a technological solution towards realising ‘infochemical communication’.

Although several studies have examined the effect of human odor on kin recognition and mate choice, few have focused on the impact of familiarity on recognition of nonrelatives by olfactory cues. As part of a program designed to engage students in scientific research, 53 high school students researched, planned, and implemented a project to analyze the effect of odor on human recognition of, and preference for, friends, sex, and self. A total of 37 students, including friends of their choosing, wore T-shirts for three consecutive nights. During that time, subjects were controlled for exposure to extraneous perfumes, household odors, and other humans. The students were then asked to smell a series of five shirts and evaluate them with respect to pleasantness. Students were also asked to identify the shirts belonging to themselves and their friend, and determine the sex of the person who wore each shirt. Although subjects were unable to distinguish sex by olfactory cues alone, a significant percentage of subjects were able to identify their own odor (51.6%), as well as distinguish the odor cue of their friend (38.7%). Additionally, subjects who could not identify their friend's cue were apt to choose the odor of a member of the opposite sex as their friend. This result was not believed to rely on odor preference as neither individual, friend, nor stranger odors were rated significantly different with respect to odor pleasantness. The ability to recognize friends via odor cues lends credence to the hypothesis that association and familiarity are important aspects of conspecific olfactory recognition in humans. Furthermore, this study augments evidence that olfaction may supplement visual and auditory cues used in human conspecific and kin recognition.

The Rhagoletis species complex has been a key player in the sympatric speciation debate for much of the last 50 years. Studies indicate that differences in olfactory preference for host fruit volatiles could be important in reproductively isolating flies infesting each type of fruit via premating barriers to gene flow. Single sensillum electrophysiology was used to compare the response characteristics of olfactory receptor neurons from apple, hawthorn, and flowering dogwood-origin populations of R. pomonella, as well as from the blueberry maggot, R. mendax (an outgroup). Eleven volatiles were selected as stimuli from behavioral/electroantennographic studies of the three R. pomonella host populations. Previously, we reported that differences in preference for host fruit volatile blends are not a function of alterations in the general class of receptor neurons tuned to key host volatiles. In the present study, population comparisons involving dose–response trials with the key volatiles revealed significant variability in olfactory receptor neuron sensitivity and temporal firing pattern both within and among Rhagoletis populations. It is concluded that such variability in peripheral sensitivity and temporal firing pattern could influence host preference and contribute to host fidelity and sympatric host shifts in the Rhagoletis complex.

Neural responses to odor blends often exhibit non-linear interactions to blend components. The first olfactory processing center in insects, the antennal lobe (AL), exhibits a complex network connectivity. We attempt to determine if non-linear blend interactions can arise purely as a function of the AL network connectivity itself, without necessitating additional factors such as competitive ligand binding at the periphery or intrinsic cellular properties. To assess this, we compared blend interactions among responses from single neurons recorded intracellularly in the AL of the moth Manduca sexta with those generated using a population-based computational model constructed from the morphologically based connectivity pattern of projection neurons (PNs) and local interneurons (LNs) with randomized connection probabilities from which we excluded detailed intrinsic neuronal properties. The model accurately predicted most of the proportions of blend interaction types observed in the physiological data. Our simulations also indicate that input from LNs is important in establishing both the type of blend interaction and the nature of the neuronal response (excitation or inhibition) exhibited by AL neurons. For LNs, the only input that significantly impacted the blend interaction type was received from other LNs, while for PNs the input from olfactory sensory neurons and other PNs contributed agonistically with the LN input to shape the AL output. Our results demonstrate that non-linear blend interactions can be a natural consequence of AL connectivity, and highlight the importance of lateral inhibition as a key feature of blend coding to be addressed in future experimental and computational studies.