Evolution of herbivory in Drosophilidae linked to loss of behaviors, antennal responses, odorant receptors, and ancestral diet.

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.

Odorant receptors and antennal lobe morphology offer a new approach to understanding olfaction in the Asian longhorned beetle.

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.

Responses of protocerebral neurons in Manduca sexta to sex-pheromone mixtures.

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.

Synchronous firing of antennal-lobe projection neurons encodes the behaviorally effective ratio of sex-pheromone components in male Manduca sexta.

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.

The distribution and abundance of triatomine insects, potential vectors of Chagas Disease, in a metropolitan area in southern Arizona, United States.

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.

Neural correlates of behavior in the moth Manduca sexta in response to complex odors.

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.

Local interneuron diversity in the primary olfactory center of the moth Manduca sexta.

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.

Behavioral consequences of innate preferences and olfactory learning in hawkmoth-flower interactions.

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.

Infection of kissing bugs with Trypanosoma cruzi, Tucson, Arizona, USA.

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.

Antagonistic effects of floral scent in an insect-plant interaction.

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.

Local inhibition modulates odor-evoked synchronization of glomerulus-specific output neurons.

At the first stage of olfactory processing in the brain, synchronous firing across glomeruli may help to temporally bind multiple and spatially distributed input streams activated by a given odor. This hypothesis, however, has never been tested in an organism in which the odor-tuning properties of several spatially identifiable glomeruli are known. Using the sphinx moth, an insect that meets these specific criteria, we recorded odor-evoked responses simultaneously from pairs of projection neurons (PNs) innervating the same or different glomeruli in the macroglomerular complex (MGC), which is involved in processing pheromonal information. PNs that branched in the same glomerulus and were activated by the same pheromone component also showed the strongest coincident responses to each odor pulse. Glomerulus-specific PN pairs were also inhibited by the pheromone component that selectively activated PNs in the neighboring glomerulus, and about 70% of all intraglomerular pairs showed increased synchronization when stimulated with a mixture of the two odorants. Thus, when two adjacent glomeruli receive their inputs simultaneously, the temporal tuning of output from each glomerulus is enhanced by reciprocal and inhibitory interglomerular interactions.

Chemosensory selectivity of output neurons innervating an identified, sexually isomorphic olfactory glomerulus.

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.

Phylogeny of a serotonin-immunoreactive neuron in the primary olfactory center of the insect brain.

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.

Pheromonal and host-odor processing in the insect antennal lobe: how different?

In the olfactory bulb of vertebrates and the antennal lobe of insects, precise connections between sensory receptor cells and olfactory glomeruli form the basis of a highly organized chemotopic map at the first stage of central processing in the brain. Beyond this basic level of organization, the olfactory system is typically separated into two subsystems: a 'main' olfactory pathway that detects and processes information about most environmental odorants, and an 'accessory' olfactory pathway that is devoted to information about social signals such as sex pheromones. A growing number of studies show, however, that it is not always possible to draw clear functional distinctions between the two subsystems. These findings have led some to speculate that the organizational principles by which olfactory stimuli are represented across glomeruli may be more similar in these two olfactory subsystems than previously thought.

Spatial and temporal organization of ensemble representations for different odor classes in the moth antennal lobe.

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.

Enantioselectivity of projection neurons innervating identified olfactory glomeruli.

Projection neurons (PNs) with arborizations in the sexually dimorphic "lateral large female glomerulus" (latLFG) in the antennal lobe (AL) of the moth Manduca sexta previously were shown to respond preferentially to antennal stimulation with (+/-)linalool, a volatile compound commonly emitted by plants. In the present study, using intracellular recording and staining techniques, we examined the responsiveness of latLFG-PNs to the enantiomers, (+)linalool and (-)linalool and found that (1) latLFG-PNs are more responsive to antennal stimulation with (+)linalool than with (-)linalool, (2) PNs with arborizations in a glomerulus adjacent to the latLFG are preferentially responsive to (-)linalool, and (3) PNs with arborizations confined to other glomeruli near the latLFG are equally responsive to both enantiomers of linalool. Structure-activity studies showed that the hydroxyl group in this tertiary terpene alcohol is the key feature of the molecule determining the response of enantioselective PNs to linalool. In contrast, the responses of non-enantioselective PNs are less dependent on the alcoholic functionality of linalool. Our findings show that PNs innervating a uniquely identifiable glomerulus respond preferentially to a particular enantiomer of an odor substance. Moreover, PNs with arborizations in a glomerulus adjacent to the latLFG, although less sensitive than latLFG-PNs to linalool, respond preferentially to the opposite enantiomer, demonstrating that information about stimulus-absolute configuration can be encoded in different olfactory glomeruli.

Octopamine-immunoreactive neurons in the brain and subesophageal ganglion of the hawkmoth Manduca sexta.

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.

Role of neurosecretory cells in the photoperiodic induction of pupal diapause of the tobacco hornworm Manduca sexta.

In the tobacco hornworm, Manduca sexta, pupal diapause can be induced by exposure of fifth-instar larvae to a short-day photoperiod. We studied the effect of surgical ablation of tissues containing the neurosecretory cells of the brain of fifth-instar larvae on the photoperiodic induction of pupal diapause. At the end of the experiments, we immunostained the neurosecretory cells to determine the success of the ablations. Under long-day conditions (LD 16:8 at 22 degrees C), all intact larvae, most of the sham-operated larvae, and control-operated larvae developed into nondiapausing pupae. Under short-day conditions (LD 10:14 at 22 degrees C), most intact, sham-operated, and control-operated larvae developed into diapausing pupae. Removal of type-II cells did not interfere with the photoperiodic response. Under long-day conditions, elimination of type-Ia(1) cells did not affect the incidence of nondiapausing pupae. When type-Ia(1) cells were removed under short-day conditions, however, the incidence of nondiapausing pupae was higher (51%, n = 41) than that of the intact (16%, n = 75), sham-operated (24%, n = 88), control-operated larvae (5%, n = 40), and larvae with type-II cells removed (11%, n = 27). Thus, removal of type-Ia(1) cells can impede induction of diapause. These results indicate that the type-Ia(1) neurosecretory cells have an important role in the induction of pupal diapause.