Jeffrey A. Riffell

Characterization and Coding of Behaviorally Significant Odor Mixtures

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 insects brain represent odor mixtures. We combined gas chromatography and neural-ensemble recording in the moths 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 moths 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.

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.

The neurobiology of insect olfaction: sensory processing in a comparative context.

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 animals life history. We propose that this perspective is beneficial for insect olfactory neurobiology in particular and sensory neurobiology in general.

Odorant receptors and olfactory-like signaling mechanisms in mammalian sperm

Since their discovery in 1991, members of the odorant receptor (OR) family have been found in various ectopic tissues, including testis and sperm. It took, however, more than a decade for the first mammalian testicular ORs to be functionally characterized and implicated in a reproductively relevant scenario. Activation of hOR17-4 and mOR23 in human and mouse sperm, respectively, mediates distinct flagellar motion patterns and chemotactic behavior in various bioassays. For hOR17-4, receptor function and downstream signal transduction events are shown to be subject to pharmacological manipulation. Further insight into the basic principles that govern sperm OR operation as well as into the molecular logic that underlies OR-mediated signaling could set the stage for pioneering future applications in procreation and/or contraception.

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.

Physical Processes and Real-Time Chemical Measurement of the Insect Olfactory Environment

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.

Flower discrimination by pollinators in a dynamic chemical environment

How hawkmoths sniff out a flower Pollinators such as butterflies and bees are the true targets of the flower odors we love so much. Though we might imagine insects “following their noses,” the wealth of odors in the real world can drown out the smell of a flower, making it hard to find. Riffel et al. found that hawkmoths find angels trumpets by creating a neuronal picture within their antennal lobe, the part of the moth brain that receives olfactory signals from the antennae (see the Perspective by Szyszka). The picture represents both the flower and the background odors. Finding a flower involves a complex reading of both background and target odors, and changes in the background odors—including human pollutants—can hinder the process. Science, this issue p. 1515; see also p. 1454 Moths find flowers by building an odor map in their antennal lobes. [Also see Perspective by Szyszka] Pollinators use their sense of smell to locate flowers from long distances, but little is known about how they are able to discriminate their target odor from a mélange of other natural and anthropogenic odors. Here, we measured the plume from Datura wrightii flowers, a nectar resource for Manduca sexta moths, and show that the scent was dynamic and rapidly embedded among background odors. The moth’s ability to track the odor was dependent on the background and odor frequency. By influencing the balance of excitation and inhibition in the antennal lobe, background odors altered the neuronal representation of the target odor and the ability of the moth to track the plume. These results show that the mix of odors present in the environment influences the pollinator’s olfactory ability.

Neural Basis of a Pollinator’s Buffet: Olfactory Specialization and Learning in Manduca sexta

A Varied Bouquet Pollinators display innate attractions to odor, but can also learn to associate odor with a nectar reward. Riffell et al. (p. 200, published online 6 December; see the Perspective by Knaden and Hansson) characterized the odor profile for flowers to which hawkmoths are innately attracted and found that the majority contain a distinct chemical profile, which is uniquely represented on their olfactory lobe. The moths could also be trained to associate nonattractive odors with a reward and thus learn novel odor attractions. Though learning altered neurons within the antennal lobe, the innate preferences were not changed. Hawkmoths supplement their innate repertoire of attractive flower odors by learning new ones via an octopamine pathway. [Also see Perspective by Knaden and Hansson] 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.

Sex and flow: the consequences of fluid shear for sperm-egg interactions

SUMMARY Fertilization is a complex interaction among biological traits of gametes and physical properties of the fluid environment. At the scale of fertilization (0.01–1 mm), sperm encounter eggs while being transported within a laminar (or viscous) shear flow. Varying laminar-shear in a Taylor-Couette flow tank, our experiments simulated important aspects of small-scale turbulence within the natural habitats of red abalone (Haliotis rufescens), a large marine mollusk and external fertilizer. Behavioral interactions between individual cells, sperm–egg encounter rates, and fertilization success were quantified, simultaneously, using a custom-built infrared laser and computer-assisted video imaging system. Relative to still water, sperm swam faster and moved towards an egg surface, but only in comparatively slow flows. Encounter rate, swim speed and orientation, and fertilization success each peaked at the lowest shear tested (0.1 s–1), and then decayed as shear increased beyond 1.0 s–1. The decay did not result, however, from damage to either sperm or eggs. Analytical and numerical models were used to estimate the propulsive force generated by sperm swimming (Fswim) and the shear force produced by fluid motion within the vicinity of a rotating egg (Fshear). To first order, male gametes were modeled as prolate spheroids. The ratio Fswim/Fshear was useful in explaining sperm–egg interactions. At low shears where Fswim/Fshear>1, sperm swam towards eggs, encounter rates were pronounced, and fertilization success was very high; behavior overpowered fluid motion. In contrast, sperm swimming, encounter rate and fertilization success all decayed rapidly when Fswim/Fshear<1; fluid motion dominated behavior. The shears maximizing fertilization success in the lab typically characterized natural flow microenvironments of spawning red abalone. Gamete behavior thus emerges as a critical determinant of sexual reproduction in the turbulent sea.

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.