Search

Plants have evolved many defenses against insect herbivores, including numerous chemicals that can reduce herbivore growth, performance, and fitness. One group of chemicals, the tropane alkaloids, is commonly found in the nightshade family (Solanaceae) and has been thought to reduce performance and fitness in insects. We examined the effects of the tropane alkaloid scopolamine, the alkaloid constituent of Datura wrightii , which is the most frequent host plant for the abundant and widespread insect herbivore Manduca sexta in the southwestern United States. We exposed caterpillars of two different species to scopolamine: M. sexta , which has a shared evolutionary history with Datura and other solanceous plants, and Galleria mellonella , which does not. We showed that the addition of ecologically-realistic levels of scopolamine to both the diet and the hemolymph of these two caterpillar species ( M. sexta and G. mellonella ) had no effect on the growth of either species. We also showed that M. sexta has no behavioral preference for or against scopolamine incorporated into an artificial diet. These results are contrary to other work showing marked differences in performance for other insect species when exposed to scopolamine, and provide evidence that scopolamine might not provide the broad-spectrum herbivore resistance typically attributed to it. It also helps to clarify the coevolutionary relationship between M. sexta and one of its main host plants, as well as the physiological mechanism of resistance against scopolamine.

The Asian longhorned beetle Anoplophora glabripennis (Motchulsky) is an exotic forest pest that has repeatedly invaded North America and Europe from Asia, and has the potential to kill millions of trees and cause billions of dollars in damage. Traps baited with an attractive mixture of volatile organic compounds from hosts have been of limited success in monitoring invasion sites. We propose that lures might be improved through studying the olfactory system of adult beetles, especially the gene family of odorant receptors (ORs) and the structure of the antennal lobes of the brain. Here, we report identification of 132 ORs in the genome of A. glabripennis (inclusive of one Orco gene and 11 pseudogenes), some of which are orthologous to known pheromone receptors of other cerambycid beetles. We also identified three ORs that are strongly biased toward expression in the female transcriptome, and a single OR strongly biased toward males. Three-dimensional reconstruction of the antennal lobes of adults suggested a male-specific macroglomerulus and several enlarged glomeruli in females. We predict that functional characterization of ORs and glomeruli will lead to identification of key odorants in the life history of A. glabripennis that may aid in monitoring and controlling future invasions.

Herbivory is a key innovation in insects, yet has only evolved in one-third of living orders. The evolution of herbivory likely involves major behavioral changes mediated by remodeling of canonical chemosensory modules. Herbivorous flies in the genus Scaptomyza (Drosophilidae) are compelling species in which to study the genomic architecture linked to the transition to herbivory because they recently evolved from microbe-feeding ancestors and are closely related to Drosophila melanogaster. We found that Scaptomyza flava, a leaf-mining specialist on plants in the family (Brassicaceae), was not attracted to yeast volatiles in a four-field olfactometer assay, whereas D. melanogaster was strongly attracted to these volatiles. Yeast-associated volatiles, especially short-chain aliphatic esters, elicited strong antennal responses in D. melanogaster, but weak antennal responses in electroantennographic recordings from S. flava. We sequenced the genome of S. flava and characterized this species' odorant receptor repertoire. Orthologs of odorant receptors, which detect yeast volatiles in D. melanogaster and mediate critical host-choice behavior, were deleted or pseudogenized in the genome of S. flava. These genes were lost step-wise during the evolution of Scaptomyza. Additionally, Scaptomyza has experienced gene duplication and likely positive selection in paralogs of Or67b in D. melanogaster. Olfactory sensory neurons expressing Or67b are sensitive to green-leaf volatiles. Major trophic shifts in insects are associated with chemoreceptor gene loss as recently evolved ecologies shape sensory repertoires.

Olfactory stimuli that are essential to an animal's survival and reproduction are often complex mixtures of volatile organic compounds in characteristic proportions. Here, we investigated how these proportions are encoded in the primary olfactory processing center, the antennal lobe, of male Manduca sexta moths. Two key components of the female's sex pheromone, present in an approximately 2:1 ratio, are processed in each of two neighboring glomeruli in the macroglomerular complex (MGC) of males of this species. In wind-tunnel flight experiments, males exhibited behavioral selectivity for ratios approximating the ratio released by conspecific females. The ratio between components was poorly represented, however, in the firing-rate output of uniglomerular MGC projection neurons (PNs). PN firing rate was mostly insensitive to the ratio between components, and individual PNs did not exhibit a preference for a particular ratio. Recording simultaneously from pairs of PNs in the same glomerulus, we found that the natural ratio between components elicited the most synchronous spikes, and altering the proportion of either component decreased the proportion of synchronous spikes. The degree of synchronous firing between PNs in the same glomerulus thus selectively encodes the natural ratio that most effectively evokes the natural behavioral response to pheromone.

Male Manduca sexta moths are attracted to a mixture of two components of the female's sex pheromone at the natural concentration ratio. Deviation from this ratio results in reduced attraction. Projection neurons innervating prominent male-specific glomeruli in the male's antennal lobe produce maximal synchronized spiking activity in response to synthetic mixtures of the two components centering around the natural ratio, suggesting that behaviorally effective mixture ratios are encoded by synchronous neuronal activity. We investigated the physiological activity and morphology of downstream protocerebral neurons that responded to antennal stimulation with single pheromone components and their mixtures at various concentration ratios. Among the tested neurons, only a few gave stronger responses to the mixture at the natural ratio whereas most did not distinguish among the mixtures that were tested. We also found that the population response distinguished among the two pheromone components and their mixtures, prior to the peak population response. This observation is consistent with our previous finding that synchronous firing of antennal-lobe projection neurons reaches its maximum before the firing rate reaches its peak. Moreover, the response patterns of protocerebral neurons are diverse, suggesting that the representation of olfactory stimuli at the level of protocerebrum is complex.

Pollinators exhibit a range of innate and learned behaviors that mediate interactions with flowers, but the olfactory bases of these responses in a naturalistic context remain poorly understood. The hawkmoth Manduca sexta is an important pollinator for many night-blooming flowers but can learn--through olfactory conditioning--to visit other nectar resources. Analysis of the flowers that are innately attractive to moths shows that the scents all have converged on a similar chemical profile that, in turn, is uniquely represented in the moth's antennal (olfactory) lobe. Flexibility in visitation to nonattractive flowers, however, is mediated by octopamine-associated modulation of antennal-lobe neurons during learning. Furthermore, this flexibility does not extinguish the innate preferences. Such processing of stimuli through two olfactory channels, one involving an innate bias and the other a learned association, allows the moths to exist within a dynamic floral environment while maintaining specialized associations.

Triatomine insects are a problem for human health in southwestern United States because of the moderate-to-severe allergic reactions their bites can cause and because they are potential vectors of Chagas Disease. Although both infected insects and wild mammalian reservoirs are plentiful in southern U.S., only seven cases of autochthonous transmission (plus 16 new presumed cases) of this disease have been reported to date. Therefore, the purpose of this study was to investigate triatomine distribution and abundance in a metropolitan area in southern Arizona. Species, life-stage, locality, and date of collection were recorded for 1,878 triatomine insects collected during 4 yr inside and around houses. For both sexes of the most abundant species, Triatoma rubida (Uhler) (>95% of triatomines collected), dispersal followed a typical year-to-year pattern: dispersal started at the beginning of May and peaked during the first-second week of June. T. rubida was found widely distributed in suburban areas. Triatomines of the two less abundant species, T. recurva (Stal) and T. protracta (Uhler), were collected in all suburban areas throughout the 4-yr survey. All of these population characteristics were observed both at a large (i.e., all collection sites pooled) and a small (i.e., single collection sites) scale. In total, approximately 55-60% of the triatomines were collected inside houses, and 30-35% of those were found in or near beds; thus, it is likely that they fed on humans. To our knowledge, this study is the first comprehensive multi-year analysis of triatomine distribution and abundance in the U.S., providing data that allow inferences about risks to human health.

We developed microfabricated flexible neural probes (FNPs) to provide a bi-directional electrical link to the moth Manduca sexta. These FNPs can deliver electrical stimuli to, and capture neural activity from, the insect's central nervous system. They are comprised of two layers of polyimide with gold sandwiched in between in a split-ring geometry that incorporates the bi-cylindrical anatomical structure of the insect's ventral nerve cord. The FNPs provide consistent left and right abdominal stimulation both across animals and within an individual animal. The features of the stimulation (direction, threshold charge) are aligned with anatomical features of the moth. We also have used these FNPs to record neuronal activity in the ventral nerve cord of the moth. Finally, by integrating carbon nanotube (CNT)-Au nanocomposites into the FNPs we have reduced the interfacial impedance between the probe and the neural tissue, thus reducing the magnitude of stimulation voltage. This in turn allows use of the FNPs with a wireless stimulator, enabling stimulation and flight biasing of freely flying moths. Together, these FNPs present a potent new platform for manipulating and measuring the neural circuitry of insects, and for other nerves in humans and other animals with similar dimensions as the ventral nerve cord of the moth., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Insect antennae are sensory organs involved in a variety of behaviors, sensing many different stimulus modalities. As mechanosensors, they are crucial for flight control in the hawkmoth Manduca sexta. One of their roles is to mediate compensatory reflexes of the abdomen in response to rotations of the body in the pitch axis. Abdominal motions, in turn, are a component of the steering mechanism for flying insects. Using a radio controlled, programmable, miniature stimulator, we show that ultra-low-current electrical stimulation of antennal muscles in freely-flying hawkmoths leads to repeatable, transient changes in the animals' pitch angle, as well as less predictable changes in flight speed and flight altitude. We postulate that by deflecting the antennae we indirectly stimulate mechanoreceptors at the base, which drive compensatory reflexes leading to changes in pitch attitude.

The simplicity and accessibility of the olfactory systems of insects underlie a body of research essential to understanding not only olfactory function but also general principles of sensory processing. As insect olfactory neurobiology takes advantage of a variety of species separated by millions of years of evolution, the field naturally has yielded some conflicting results. Far from impeding progress, the varieties of insect olfactory systems reflect the various natural histories, adaptations to specific environments, and the roles olfaction plays in the life of the species studied. We review current findings in insect olfactory neurobiology, with special attention to differences among species. We begin by describing the olfactory environments and olfactory-based behaviors of insects, as these form the context in which neurobiological findings are interpreted. Next, we review recent work describing changes in olfactory systems as adaptations to new environments or behaviors promoting speciation. We proceed to discuss variations on the basic anatomy of the antennal (olfactory) lobe of the brain and higher-order olfactory centers. Finally, we describe features of olfactory information processing including gain control, transformation between input and output by operations such as broadening and sharpening of tuning curves, the role of spiking synchrony in the antennal lobe, and the encoding of temporal features of encounters with an odor plume. In each section, we draw connections between particular features of the olfactory neurobiology of a species and the animal's life history. We propose that this perspective is beneficial for insect olfactory neurobiology in particular and sensory neurobiology in general., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Chagas disease is caused by the parasite Trypanosoma cruzi, which is transmitted to humans by blood-sucking triatomine insects. This disease is endemic throughout Mexico and Central and South America, but only a few autochthonous cases have been reported in the United States, despite the fact that infected insects readily invade houses and feed on humans. Competent vectors defecate during or shortly after feeding so that infective feces contact the host. We thus studied the feeding and defecation behaviors of the prevalent species in southern Arizona, Triatoma rubida. We found that whereas defecation during feeding was frequent in females (93%), it was very rare in immature stages (3%), and absent in males. Furthermore, more than half of the immature insects that exhibited multiple feeding bouts (62%) defecated during interruptions of feeding, i.e., while likely on or near the host. These results indicate that T. rubida potentially could transmit T. cruzi to humans.

Local interneurons (LNs) play important roles in shaping and modulating the activity of output neurons in primary olfactory centers. Here, we studied the morphological characteristics, odor responses, and neurotransmitter content of LNs in the antennal lobe (AL, the insect primary olfactory center) of the moth Manduca sexta. We found that most LNs are broadly tuned, with all LNs responding to at least one odorant. 70% of the odorants evoked a response, and 22% of the neurons responded to all the odorants tested. Some LNs showed excitatory (35%) or inhibitory (33%) responses only, while 33% of the neurons showed both excitatory and inhibitory responses, depending on the odorant. LNs that only showed inhibitory responses were the most responsive, with 78% of the odorants evoking a response. Neurons were morphologically diverse, with most LNs innervating almost all glomeruli and others innervating restricted portions of the AL. 61 and 39% of LNs were identified as GABA-immunoreactive (GABA-ir) and non-GABA-ir, respectively. We found no correlations between odor responses and GABA-ir, neither between morphology and GABA-ir. These results show that, as observed in other insects, LNs are diverse, which likely determines the complexity of the inhibitory network that regulates AL output.

Bursting as well as tonic firing patterns have been described in various sensory systems. In the olfactory system, spontaneous bursts have been observed in neurons distributed across several synaptic levels, from the periphery, to the olfactory bulb (OB) and to the olfactory cortex. Several in vitro studies indicate that spontaneous firing patterns may be viewed as "fingerprints" of different types of neurons that exhibit distinct functions in the OB. It is still not known, however, if and how neuronal burstiness is correlated with the coding of natural olfactory stimuli. We thus conducted an in vivo study to probe this question in the OB equivalent structure of insects, the antennal lobe (AL) of the tobacco hornworm Manduca sexta. We found that in the moth's AL, both projection (output) neurons (PNs) and local interneurons (LNs) are spontaneously active, but PNs tend to produce spike bursts while LNs fire more regularly. In addition, we found that the burstiness of PNs is correlated with the strength of their responses to odor stimulation--the more bursting the stronger their responses to odors. Moreover, the burstiness of PNs was also positively correlated with the spontaneous firing rate of these neurons, and pharmacological reduction of bursting resulted in a decrease of the neurons' responsiveness. These results suggest that neuronal burstiness reflects a physiological state of these neurons that is directly linked to their response characteristics.

The survival of an animal often depends on an innate response to a particular sensory stimulus. For an adult male moth, two categories of odors are innately attractive: pheromone released by conspecific females, and the floral scents of certain, often co-evolved, plants. These odors consist of multiple volatiles in characteristic mixtures. Here, we review evidence that both categories of odors are processed as sensory objects, and we suggest a mechanism in the primary olfactory center, the antennal lobe (AL), that encodes the configuration of these mixtures and may underlie recognition of innately attractive odors. In the pheromone system, mixtures of two or three volatiles elicit upwind flight. Peripheral changes are associated with behavioral changes in speciation, and suggest the existence of a pattern recognition mechanism for pheromone mixtures in the AL. Moths are similarly innately attracted to certain floral scents. Though floral scents consist of multiple volatiles that activate a broad array of receptor neurons, only a smaller subset, numerically comparable to pheromone mixtures, is necessary and sufficient to elicit behavior. Both pheromone and floral scent mixtures that produce attraction to the odor source elicit synchronous action potentials in particular populations of output (projection) neurons (PNs) in the AL. We propose a model in which the synchronous output of a population of PNs encodes the configuration of an innately attractive mixture, and thus comprises an innate mechanism for releasing odor-tracking behavior. The particular example of olfaction in moths may inform the general question of how sensory objects trigger innate responses.

In southwestern USA, the jimsonweed Datura wrightii and the nocturnal moth Manduca sexta form a pollinator-plant and herbivore-plant association. Because the floral scent is probably important in mediating this interaction, we investigated the floral volatiles that might attract M. sexta for feeding and oviposition. We found that flower volatiles increase oviposition and include small amounts of both enantiomers of linalool, a common component of the scent of hawkmoth-pollinated flowers. Because (+)-linalool is processed in a female-specific glomerulus in the primary olfactory centre of M. sexta, we hypothesized that the enantiomers of linalool differentially modulate feeding and oviposition. Using a synthetic mixture that mimics the D. wrightii floral scent, we found that the presence of linalool was not necessary to evoke feeding and that mixtures containing (+)- and/or (-)-linalool were equally effective in mediating this behaviour. By contrast, females oviposited more on plants emitting (+)-linalool (alone or in mixtures) over control plants, while plants emitting (-)-linalool (alone or in mixtures) were less preferred than control plants. Together with our previous investigations, these results show that linalool has differential effects in feeding and oviposition through two neural pathways: one that is sexually isomorphic and non-enantioselective, and another that is female-specific and enantioselective.

We describe a flexible multisite microelectrode for insect flight biasing using neural stimulation. The electrode is made of two layers of polyimide (PI) with gold sandwiched in between in a split-ring geometry. The split-ring design in conjunction with the flexibility of the PI allows for a simple insertion process and provides good attachment between the electrode and ventral nerve cord of the insect. Stimulation sites are located at the ends of protruding tips that are circularly distributed inside the split-ring structure. These protruding tips penetrate into the connective tissue surrounding the nerve cord. We have been able to insert the electrode into pupae of the giant sphinx moth Manduca sexta as early as seven days before the adult moth emerges, and we are able to use the multisite electrode to deliver electrical stimuli that evoke multidirectional, graded abdominal motions in both pupae and adult moths. Finally, in loosely tethered flight, we have used stimulation through the flexible microelectrodes to alter the abdominal angle, thus causing the flying moth to deviate to the left or right of its intended path.

The primary olfactory centres of the sphinx moth Manduca sexta, the antennal lobes, contain a small number of sexually dimorphic glomeruli: the male-specific macroglomerular complex and the large female glomeruli. These glomeruli play important roles in sex-specific behaviours, such as the location of conspecific females and the selection of appropriate host plants for oviposition. The development of sexually dimorphic glomeruli depends strictly on the ingrowth of sex-specific olfactory receptor cell afferents. In the present study we tested the role of female-specific olfactory receptor cells (ORCs) in mediating female-specific host plant approach behaviour and in determining the response of downstream antennal lobe neurons. We generated male gynandromorphs by excising one imaginal disc from a male larva and replacing it with the antennal imaginal disc from a female donor. Most male gynandromorphs had an apparently normal female antenna and a feminised antennal lobe. These gynandromorphs were tested for flight responses in a wind tunnel towards tomato plants, a preferred host plant for oviposition in M. sexta. Male gynandromorphs landed on host plants as often as normal females, demonstrating that the presence of the induced female-specific glomeruli was necessary and sufficient to produce female-like, odour-oriented behaviour, i.e. orientation towards host plants. We also characterised the physiological and morphological properties of antennal lobe neurons of male gynandromorphs. We found that projection neurons with arborisations in the induced female-specific glomeruli showed physiological responses akin to those of female-specific projection neurons in normal females. These results therefore indicate that ORCs confer specific odour tuning to their glomerular targets and, furthermore, instruct odour-specific behaviour.

Triatomine insects (Hemiptera: Reduviidae), commonly known as kissing bugs, are a potential health problem in the southwestern United States as possible vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Although this disease has been traditionally restricted to Latin America, a small number of vector-transmitted autochthonous US cases have been reported. Because triatomine bugs and infected mammalian reservoirs are plentiful in southern Arizona, we collected triatomines inside or around human houses in Tucson and analyzed the insects using molecular techniques to determine whether they were infected with T. cruzi. We found that 41.5% of collected bugs (n = 164) were infected with T. cruzi, and that 63% of the collection sites (n = 22) yielded >or=1 infected specimens. Although many factors may contribute to the lack of reported cases in Arizona, these results indicate that the risk for infection in this region may be higher than previously thought.

With Manduca sexta as a model system, we analyzed how natural odor mixtures that are most effective in eliciting flight and foraging behaviors are encoded in the primary olfactory center in the brain, the antennal lobe. We used gas chromatography coupled with multiunit neural-ensemble recording to identify key odorants from flowers of two important nectar resources, the desert plants Datura wrightii and Agave palmeri, that elicited responses from individual antennal-lobe neurons. Neural-ensemble responses to the A. palmeri floral scent, comprising >60 odorants, could be reproduced by stimulation with a mixture of six of its constituents that had behavioral effectiveness equivalent to that of the complete scent. Likewise, a mixture of three floral volatiles from D. wrightii elicited normal flight and feeding behaviors. By recording responses of neural ensembles to mixtures of varying behavioral effectiveness, we analyzed the coding of behaviorally "meaningful" odors. We considered four possible ensemble-coding mechanisms--mean firing rate, mean instantaneous firing rate, pattern of synchronous ensemble firing, and total net synchrony of firing--and found that mean firing rate and the pattern of ensemble synchrony were best correlated with behavior (R = 41% and 43%, respectively). Stepwise regression analysis showed that net synchrony and mean instantaneous firing rate contributed little to the variation in the behavioral results. We conclude that a combination of mean-rate coding and synchrony of firing of antennal-lobe neurons underlies generalization among related, behaviorally effective floral mixtures while maintaining sufficient contrast for discrimination of distinct scents.

Olfactory cues play decisive roles in the lives of most insect species, providing information about biologically relevant resources, such as food, mates, and oviposition sites. The nocturnal moth Manduca sexta feeds on floral nectar from a variety of plants (and thus serves as a pollinator), but females oviposit almost exclusively on solanaceous plants, which they recognize on the basis of olfactory cues. Plants, however, respond to herbivory by releasing blends of volatiles that attract natural enemies of herbivores. Thus, oviposition behavior probably results from the sensory evaluation not only of attractive host plant volatiles but also of repellent volatiles that indicate the acceptability or inappropriateness, respectively, of host plants for the females' offspring. Here we describe results from chemical-ecological, neurophysiological, and behavioral experiments aimed at understanding the neural mechanisms that control oviposition behavior in M. sexta.

For animals to execute odor-driven behaviors, the olfactory system must process complex odor signals and maintain stimulus identity in the face of constantly changing odor intensities [1-5]. Surprisingly, how the olfactory system maintains identity of complex odors is unclear [6-10]. We took advantage of the plant-pollinator relationship between the Sacred Datura (Datura wrightii) and the moth Manduca sexta[11, 12] to determine how olfactory networks in this insect's brain represent odor mixtures. We combined gas chromatography and neural-ensemble recording in the moth's antennal lobe to examine population codes for the floral mixture and its fractionated components. Although the floral scent of D. wrightii comprises at least 60 compounds, only nine of those elicited robust neural responses. Behavioral experiments confirmed that these nine odorants mediate flower-foraging behaviors, but only as a mixture. Moreover, the mixture evoked equivalent foraging behaviors over a 1000-fold range in dilution, suggesting a singular percept across this concentration range. Furthermore, neural-ensemble recordings in the moth's antennal lobe revealed that reliable encoding of the floral mixture is organized through synchronized activity distributed across a population of glomerular coding units, and this timing mechanism may bind the features of a complex stimulus into a coherent odor percept.

Background: An animal navigating to an unseen odor source must accurately resolve the spatiotemporal distribution of that stimulus in order to express appropriate upwind flight behavior. Intermittency of natural odor plumes, caused by air turbulence, is critically important for many insects, including the hawkmoth, Manduca sexta, for odor-modulated search behavior to an odor source. When a moth's antennae receive intermittent odor stimulation, the projection neurons (PNs) in the primary olfactory centers (the antennal lobes), which are analogous to the olfactory bulbs of vertebrates, generate discrete bursts of action potentials separated by periods of inhibition, suggesting that the PNs may use the binary burst/non-burst neural patterns to resolve and enhance the intermittency of the stimulus encountered in the odor plume., Results: We tested this hypothesis first by establishing that bicuculline methiodide reliably and reversibly disrupted the ability of PNs to produce bursting response patterns. Behavioral studies, in turn, demonstrated that after injecting this drug into the antennal lobe at the effective concentration used in the physiological experiments animals could no longer efficiently locate the odor source, even though they had detected the odor signal., Conclusions: Our results establish a direct link between the bursting response pattern of PNs and the odor-tracking behavior of the moth, demonstrating the behavioral significance of resolving the dynamics of a natural odor stimulus in antennal lobe circuits.

Inhibitory interactions shape the activity of output neurons in primary olfactory centers and promote contrast enhancement of odor representations. Patterns of interglomerular connectivity, however, are largely unknown. To test whether the proximity of glomeruli to one another is correlated with interglomerular inhibitory interactions, we used intracellular recording and staining methods to record the responses of projection (output) neurons (PNs) associated with glomeruli of known olfactory tuning in the primary olfactory center of the moth Manduca sexta. We focused on Toroid I, a glomerulus in the male-specific macroglomerular complex (MGC) specialized to one of the two key components of the conspecific females' sex pheromone, and the adjacent, sexually isomorphic glomerulus 35, which is highly sensitive to Z-3-hexenyl acetate (Z3-6:OAc). We used the two odorants to activate these reference glomeruli and tested the effects of olfactory activation in other glomeruli. We found that Toroid-I PNs were not inhibited by input to G35, whereas G35 PNs were inhibited by input to Toroid-I PNs. We also recorded the responses of PNs arborizing in other sexually isomorphic glomeruli to stimulation with the sex pheromone and Z3-6:OAc. We found that inhibitory responses were not related to proximity to the MGC and G35: both distant and adjacent PNs were inhibited by stimulation with the sex pheromone, some others were affected by only one odorant, and yet others by neither. Similar results were obtained in female PNs recorded in proximity to female-specific glomeruli. Our findings indicate that inhibitory interactions among glomeruli are widespread and independent of their spatial proximity.

Odor-mediated insect navigation in airborne chemical plumes is vital to many ecological interactions, including mate finding, flower nectaring, and host locating (where disease transmission or herbivory may begin). After emission, volatile chemicals become rapidly mixed and diluted through physical processes that create a dynamic olfactory environment. This review examines those physical processes and some of the analytical technologies available to characterize those behavior-inducing chemical signals at temporal scales equivalent to the olfactory processing in insects. In particular, we focus on two areas of research that together may further our understanding of olfactory signal dynamics and its processing and perception by insects. First, measurement of physical atmospheric processes in the field can provide insight into the spatiotemporal dynamics of the odor signal available to insects. Field measurements in turn permit aspects of the physical environment to be simulated in the laboratory, thereby allowing careful investigation into the links between odor signal dynamics and insect behavior. Second, emerging analytical technologies with high recording frequencies and field-friendly inlet systems may offer new opportunities to characterize natural odors at spatiotemporal scales relevant to insect perception and behavior. Characterization of the chemical signal environment allows the determination of when and where olfactory-mediated behaviors may control ecological interactions. Finally, we argue that coupling of these two research areas will foster increased understanding of the physicochemical environment and enable researchers to determine how olfactory environments shape insect behaviors and sensory systems.

The nervous system copes with variability in the external and internal environment by using neuromodulators to adjust the efficacy of neural circuits. The role of serotonin (5HT) as a neuromodulator of olfactory information processing in the antennal lobe (AL) of Manduca sexta was examined using multichannel extracellular electrodes to record the responses of ensembles of AL neurons to olfactory stimuli. In one experiment, the effects of 5HT on the concentration-response functions for two essential plant oils across a range of stimulus intensities were examined. In a second experiment, the effect of 5HT on the ability of ensembles to discriminate odorants from different chemical classes was examined. Bath application of 5HT enhanced AL unit responses by increasing response duration and firing rate, which in turn increased the amount of spike time cross-correlation and -covariance between pairs of units. 5HT had the greatest effect on overall ensemble activation at higher odorant concentrations, resulting in an increase in the gain of the dose-response function of individual units. Additionally, response thresholds shifted to lower odorant concentrations for some units, suggesting that 5HT increased their sensitivity. Serotonin enhanced ensemble discrimination of different concentrations of individual odorants as well as discrimination of structurally dissimilar odors at the same concentration. Given the known circadian fluctuations of 5HT in the AL of this species, these findings support the hypothesis that 5HT periodically enhances sensitivity and responsiveness in the AL of Manduca to maximize efficiency when the requirement for olfactory acuity is the greatest.

Spatiotemporal variability in floral resources can have ecological and evolutionary consequences for both plants and the pollinators on which they depend. Seldom, however, can patterns of flower abundance and visitation in the field be linked with the behavioral mechanisms that allow floral visitors to persist when a preferred resource is scarce. To explore these mechanisms better, we examined factors controlling floral preference in the hawkmoth Manduca sexta in the semiarid grassland of Arizona. Here, hawkmoths forage primarily on flowers of the bat-adapted agave, Agave palmeri, but shift to the moth-adapted flowers of their larval host plant, Datura wrightii, when these become abundant. Both plants emit similar concentrations of floral odor, but scent composition, nectar, and flower reflectance are distinct between the two species, and A. palmeri flowers provide six times as much chemical energy as flowers of D. wrightii. Behavioral experiments with both naïve and experienced moths revealed that hawkmoths learn to feed from agave flowers through olfactory conditioning but readily switch to D. wrightii flowers, for which they are the primary pollinator, based on an innate odor preference. Behavioral flexibility and the olfactory contrast between flowers permit the hawkmoths to persist within a dynamic environment, while at the same time to function as the major pollinator of one plant species.

Carbon dioxide (CO(2)) is a ubiquitous sensory cue that plays multiple roles in insect behavior. In recent years understanding of the well-known role of CO(2) in foraging by hematophagous insects (e.g., mosquitoes) has grown, and research on the roles of CO(2) cues in the foraging and oviposition behavior of phytophagous insects and in behavior of social insects has stimulated interest in this area of insect sensory biology. This review considers those advances, as well as some of the mechanistic bases of the modulation of behavior by CO(2) and important progress in our understanding of the detection and CNS processing of CO(2) information in insects. Finally, this review briefly addresses how the ongoing increase in atmospheric CO(2) levels may affect insect life.

Serotonin (5-hydroxytryptamine; 5HT) functions in insects as a neurotransmitter, neuromodulator, and neurohormone. In the sphinx moth Manduca sexta, each of the paired antennal lobes (ALs; the primary olfactory centers in the insect brain) has one 5HT-immunoreactive (5HT-ir) neuron that projects into the protocerebrum, crosses the posterior midline, and innervates the contralateral AL; this is referred to as the contralaterally projecting, serotonin-immunoreactive deutocerebral (CSD) neuron. These neurons are thought to function as centrifugal modulators of olfactory sensitivity. To examine the phylogenetic distribution of 5HT-ir neurons apparently homologous to the CSD neuron, we imaged 5HT-like immunoreactivity in the brains of 40 species of insects belonging to 38 families in nine orders. CSD neurons were found in other Lepidoptera, Trichoptera, Diptera, Coleoptera, and Neuroptera but not in the Hymenoptera. In the paraneopteran and polyneopteran species (insects that undergo incomplete metamorphosis) examined, AL 5HT neurons innervate the ispsilateral AL and project to the protocerebrum. Our findings suggest that the characteristic morphology of the CSD neurons originated in the holometabolous insects (those that undergo complete metamorphosis) and were lost in the Hymenoptera. In a subset of the Diptera, the CSD neurons branch within the contralateral AL and project back to the ipsilateral AL via the antennal commissure. The evolution of AL 5HT neurons is discussed in the context of the physiological actions of 5HT observed in the lepidopteran AL.

Knowledge of the neuroanatomy of the sucking pump of Manduca sexta (Sphingidae) is valuable for studies of olfactory learning, pattern generators, and postembryonic modification of motor circuitry. The pump comprises a cibarial valve, a buccal pump, and an esophageal sphincter valve. Cibarial opener and closer muscles control the cibarial valve. Six pairs of dilator muscles and a compressor muscle operate the buccal pump. The cibarial opener and one pair of buccal dilator muscles are innervated by paired neurons in the tritocerebrum, and the cibarial opener has double, bilateral innervation. Their tritocerebral innervation indicates that these muscles evolved from labro-clypeal muscles. The remaining paired buccal dilator muscles each are innervated by an unpaired motor neuron in the frontal ganglion. These motor neurons project bilaterally through the frontal connectives to dendritic arborizations in the tritocerebrum. These projections also have a series of dendritic-like arborizations in the connectives. The cibarial closer and buccal compressor muscles are also innervated by motor neurons in the frontal ganglion, but only the closer muscle neuron projects bilaterally to the tritocerebrum. The innervation of the pump muscles indicates that they are associated with the stomodaeum, and, therefore, the buccal pump evolved from the anterior stomodaeum rather than from the cibarium.

The antennal lobe (AL) of insects, like the olfactory bulb of vertebrates, is characterized by discrete modules of synaptic neuropil called glomeruli. In some insects (e.g., moths and cockroaches), a few glomeruli are sexually dimorphic and function in labeled lines for processing of sensory information about sex pheromones. Controversy still exists, however, about whether projection (output) neurons (PNs) of glomeruli in the main AL are also narrowly tuned. We examined this critical issue in the AL of the moth Manduca sexta. We used intracellular recording and staining techniques to investigate the chemosensory tuning of PNs innervating an identifiable, sexually isomorphic glomerulus, G35, in the main AL. We found that the morphological features and chemosensory tuning of G35-PNs were nearly identical in females and males. G35-PNs responded to low concentrations of the plant-derived volatile compound cis-3-hexenyl acetate (c3HA), but the sensitivity threshold of female PNs was lower than that of male PNs. The propionate and butyrate homologs of c3HA could evoke excitatory responses but only at moderate-to-high concentrations. Other plant volatiles did not evoke responses from G35-PNs. Moreover, PNs innervating glomeruli near G35 (in females) showed little or no response to c3HA. Female G35-PNs were hyperpolarized by (+/-)linalool, a compound that excites PNs in an adjacent glomerulus, thus providing evidence for lateral-inhibitory interactions between glomeruli. Our results show that PNs arborizing in an identified glomerulus in the main olfactory pathway are morphologically and physiologically equivalent in both sexes and have characteristic, limited molecular receptive ranges that are highly conserved across individuals.

Octopamine is a neuroactive monoamine that functions as a neurohormone, a neuromodulator, and a neurotransmitter in many invertebrate nervous systems, but little is known about the distribution of octopamine in the brain. We therefore used a monoclonal antibody to study the distribution of octopamine-like immunoreactivity in the brain of the hawkmoth Manduca sexta. Immunoreactive processes were observed in many regions of the brain, with the distinct exception of the upper division of the central body. We focused our analysis on nine ventral unpaired median (VUM) neurons with cell bodies in the labial neuromere of the subesophageal ganglion. Seven of these neurons projected caudally through the ventral nerve cord. Two neurons projected rostrally into the brain (supraesophageal ganglion), and one of these was a bilateral neuron that sent projections to the gamma-lobe of the mushroom body and the lateral protocerebrum. Octopamine-immunoreactive processes from one or more cells originating in the subesophageal ganglion also form direct connections between the antennal lobes and the calyces of the mushroom bodies., ((c) 2005 Wiley-Liss, Inc.)

Chemotherapy-induced peripheral neuropathy (CIPN) is a common phenomenon, often resulting in serious limitations in daily functioning and compromised quality of life. Currently available toxicity grading systems typically use a combination of clinical and paraclinical parameters and relies on the judgment of clinicians and/or nurses. However, because many of the symptoms of CIPN are subjective in nature, it is only logical that an assessment of CIPN be based, at least in part, on patient self-report data. We report on the development of a patient self-report questionnaire, the CIPN20, intended to supplement the core quality of life questionnaire of the European Organization for Research and Treatment of Cancer (EORTC). Following EORTC guidelines, relevant CIPN-related issues were identified from a literature survey and interviews with health professionals (n=15) and patients (n=112). The resulting 20-item questionnaire was pre-tested in three languages and four countries and is currently being examined in a large, international clinical trial. The EORTC CIPN20 should provide valuable information on CIPN-related symptoms and functional limitations of patients exposed to potentially neurotoxic chemotherapeutic and/or neuroprotective agents.

Using whole cell recordings from antennal-lobe (AL) neurons in vitro and in situ, in semi-intact brain preparations, we examined membrane properties that contribute to electrical activity exhibited by developing neurons in primary olfactory centers of the brain of the sphinx moth, Manduca sexta. This activity is characterized by prolonged periods of membrane depolarization that resemble plateau potentials. The presence of plateau potential-generating mechanisms was confirmed using a series of tests established earlier. Brief depolarizing current pulses could be used to trigger a plateau state. Once triggered, plateau potentials could be terminated by brief pulses of hyperpolarizing current. Both triggering and terminating of firing states were threshold phenomena, and both conditions resulted in all-or-none responses. Rebound excitation from prolonged hyperpolarizing pulses could also be used to generate plateau potentials in some cells. These neurons were found to express a hyperpolarization-activated inward current. Neither the generation nor the maintenance of plateau potentials was affected by removal of Na+ ions from the extracellular medium or by blockade of Na+ currents with TTX. However, blocking of Ca2+ currents with Cd2+ (5 x 10(-4) M) inhibited the generation of plateau potentials, indicating that, in Manduca AL neurons, plateau potentials depend on Ca2+. Examining Ca2+ currents in isolation revealed that activation of these currents occurs in the absence of experimentally applied depolarizing stimuli. Our results suggest that this activity underlies the generation of plateau potentials and characteristic bursts of electrical activity in developing AL neurons of M. sexta.

In the insect antennal lobe, odor discrimination depends on the ability of the brain to read neural activity patterns across arrays of uniquely identifiable olfactory glomeruli. Less is understood about the complex temporal dynamics and interglomerular interactions that underlie these spatial patterns. Using neural-ensemble recording, we show that the evoked firing patterns within and between groups of glomeruli are odor dependent and organized in both space and time. Simultaneous recordings from up to 15 units per ensemble were obtained from four zones of glomerular neuropil in response to four classes of odorants: pheromones, monoterpenoids, aromatics, and aliphatics. Each odor class evoked a different pattern of excitation and inhibition across recording zones. The excitatory response field for each class was spatially defined, but inhibitory activity was spread across the antennal lobe, reflecting a center-surround organization. Some chemically related odorants were not easily distinguished by their spatial patterns, but each odorant evoked transient synchronous firing across a uniquely different subset of ensemble units. Examination of 535 cell pairs revealed a strong relationship between their recording positions, temporal correlations, and similarity of odor response profiles. These findings provide the first definitive support for a nested architecture in the insect olfactory system that uses both spatial and temporal coordination of firing to encode chemosensory signals. The spatial extent of the representation is defined by a stereotyped focus of glomerular activity for each odorant class, whereas the transient temporal correlations embedded within the ensemble provide a second coding dimension that can facilitate discrimination between chemically similar volatiles.

An outstanding challenge in olfactory neurobiology is to explain how glomerular networks encode information about stimulus mixtures, which are typical of natural olfactory stimuli. In the moth Manduca sexta, a species-specific blend of two sex-pheromone components is required for reproductive signaling. Each component stimulates a different population of olfactory receptor cells that in turn target two identified glomeruli in the macroglomerular complex of the male's antennal lobe. Using intracellular recording and staining, we examined how responses of projection neurons innervating these glomeruli are modulated by changes in the level and ratio of the two essential components in stimulus blends. Compared to projection neurons specific for one component, projection neurons that integrated information about the blend (received excitatory input from one component and inhibitory input from the other) showed enhanced ability to track a train of stimulus pulses. The precision of stimulus-pulse tracking was furthermore optimized at a synthetic blend ratio that mimics the physiological response to an extract of the female's pheromone gland. Optimal responsiveness of a projection neuron to repetitive stimulus pulses therefore appears to depend not only on stimulus intensity but also on the relative strength of the two opposing synaptic inputs that are integrated by macroglomerular complex projection neurons.

Insects use information about CO2 to perform vital tasks such as locating food sources. In certain moths, CO2 is involved in oviposition behavior. The labial palps of adult moths that feed as adults have a pit organ containing sensory receptor cells that project into the antennal lobes, the sites of primary processing of olfactory information in the brain. In the moth Manduca sexta and certain other species of Lepidoptera, these receptor cells in the labial-palp pit organ have been shown to be tuned to CO2, and their axons project to a single, identified glomerulus in the antennal lobe, the labial-palp pit organ glomerulus. At present, however, nothing is known about the function of this glomerulus or how CO2 information is processed centrally. We used intracellular recording and staining to reveal projection (output) neurons in the antennal lobes that respond to CO2 and innervate the labial-palp pit organ glomerulus. Our results demonstrate that this glomerulus is the site of first-order processing of sensory information about ambient CO2. We found three functional types of CO2-responsive neurons (with their cell bodies in the antennal lobe or the protocerebrum) that provide output from the antennal lobe to higher centers in the brain. Some physiological characteristics of those neurons are described.

In the moth Manduca sexta, development of the adult olfactory system depends on complex interactions between olfactory receptor neurons in the antenna, antennal-lobe neurons in the brain, and several classes of glial cells. As one approach to characterizing molecules that may play roles in these interactions, we used lectins to screen antennae and antennal lobes at different stages of adult development. We find that each of the major neural cell types has a distinct pattern of labeling by lectins. Effects of enzymatic and other treatments on lectin labeling lead us to conclude that the predominant lectin ligands are: glycosphingolipids and an O-linked, fucose-containing glycoprotein on axons of olfactory receptor neurons, O-linked glycoproteins on antennal-lobe neurons, and N-linked glycoproteins on all classes of glial cells in the primary olfactory pathway. Wheat germ agglutinin labels all olfactory axons uniformly during much of development, but labeling becomes restricted to the pheromone-responsive olfactory receptor neurons in the adult male. Succinylated WGA reveals differences in these axon classes earlier, as glomerului develop from protoglomeruli. The adult female displays a less pronounced difference in labeling of axons targeting ordinary and sexually dimorphic glomeruli. Differences in labeling of receptor axons targeted to ordinary and sexually dimorphic glomeruli may be correlated with differences in function or connectivity in different regions of the antennal lobe., (Copyright 2004 Wiley-Liss, Inc.)

Recent evidence suggests that odor-driven responses in the insect antennal lobe (AL) can be modified by associative and nonassociative processes, as has been shown in the vertebrate olfactory bulb. However, the specific network changes that occur in response to olfactory learning remain unknown. To characterize changes in AL network activity during learning, we developed an in vivo protocol in Manduca sexta that allows continuous monitoring of neural ensembles and feeding behavior over the course of olfactory conditioning. Here, we show that Pavlovian conditioning produced a net recruitment of responsive neural units across the AL that persisted after conditioning. Recruitment only occurred when odor reliably predicted food. Conversely, when odor did not predict food, a net loss of responsive units occurred. Simultaneous measures of feeding responses indicated that the treatment-specific patterns of neural recruitment were positively correlated with changes in the insect's behavioral response to odor. In addition to recruitment, conditioning also produced consistent and profound shifts in the temporal responses of 16% of recorded units. These results show that odor representations in the AL are dynamic and related to olfactory memory consolidation. We furthermore provide evidence that the basis of the learning-dependent changes in the AL is not simply an increase in activity in the neural network representing an odorant. Rather, learning produces a restructuring of spatial and temporal components of network responses to odor in the AL.