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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.

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.

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

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.

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.

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.

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.

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.

As part of a project to develop a novel class of technology for infochemical communication, we investigated olfactory processing in the insect brain to form the basis for a multicomponent detector system able to recover ratiometric odor information deployed in the world. By unraveling neuronal network processing, we support the generation of a tuned detector capable of deciphering chemical signals produced by a biosynthetic chemoemmitter. This chemoemitter/receiver complex establishes an entirely new communication system based on the chemical signaling of insects. Our main challenge is to reveal how complex odor information is encoded in the insect olfactory system. In insects, the initial percept of odors detected by the antenna occurs in the first olfactory center of the brain, the antennal lobe (AL), the insect analog of the mammalian olfactory bulb Afferent input is modified via interneuronal connections (LNs) and the resultant representation is carried by projection neurons (PNs, Output) to higher-order brain centers. Accordingly, the antennal lobe representation of an odor mixture may either retain the single-odor information of blend components, or reveal non-linear interactions due to processing in the AL network. The phenomenon of a non-linear response to a mixture that is not predictable from its component responses is called a “mixture interaction”. Combining intracellular electrophysiology, optical imaging, and 3-D morphological reconstruction, we report a highly combinatorial, non-linear process for coding complex host blends in moths that is presumably shaped by the AL network: Blend responses exhibited an array of interactions including suppression, hypoadditivity, and synergism that established a unique blend representation. Our results indicate that each neuron utilizes several different elements to produce the signal representing the entire blend, including the spatial location, rate, latency, and temporal pattern of the response. This suggests that a biomimetic system based on the moth brain can, with a minimum of detectors and processing power, decipher complex odor information on both temporal and spatial scales.

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.