Context and the functional use of information in insect sensory ecology.

Context-specific behaviors emerge from the interaction between an animal's internal state and its external environment. Although the importance of context is acknowledged in the field of insect sensory ecology, there is a lack of synthesis on this topic stemming from challenges in conceptualizing 'context'. We address this challenge by gleaning over the recent findings on the sensory ecology of mosquitoes and other insect pollinators. We discuss internal states and their temporal dynamics, from those lasting minutes to hours (host-seeking) to those lasting days to weeks (diapause, migration). Of the many patterns reviewed, at least three were common to all taxa studied. First, different sensory cues gain prominence depending on the insect's internal state. Second, similar sensory circuits between related species can result in different behavioral outcomes. And third, ambient conditions can dramatically alter internal states and behaviors.

Eat, Drink, Live: Foraging behavior of a nectarivore when relative humidity varies but nectar resources do not.

To meet energetic and osmotic demands, animals make dynamic foraging decisions about food quality and quantity. In the wild, foraging animals may be forced to consume a less preferred or sub-optimal food source for long periods of time. Few choice feeding assays in laboratory settings approximate such contingencies. In this study the foraging behaviors of the hawkmoth Manduca sexta were measured when adult moths were placed within different relative humidity (RH) environments (20%, 40%, 60% and 80% RH) and provided with only one of the following experimental nectars: 0% (water), 12% or 24 % w/V sucrose solutions. Overall, ambient humidity influenced survivorship and foraging behaviors. Moth survivorship increased at higher ambient humidity regardless of experimental nectar. Moths that had access to experimental nectar imbibed large volumes of fluid regardless of what nectar was offered when placed at the lowest humidity (20% RH). However, when placed at the highest humidity (80% RH), moths imbibed higher volumes of fluid when given access to experimental nectar with sucrose in comparison with water. RH also influenced daily foraging behaviors: peak nectar consumption occurred earlier at lower RH levels. Consistent with previous studies in which moths could choose among nectar solutions, total energy intake was not affected by ambient RH under no-choice conditions. However, the proportion of time spent foraging and total energy consumption were significantly reduced across all RH levels in no-choice assays, when compared with previous studies of choice assays under the same conditions. Our results show that even when M. sexta moths are presented with limited options, they can alter their foraging behavior in response to environmental changes, enabling them to meet osmotic and/or energetic demands.

Innate colour preferences of a hawkmoth depend on visual context.

Solitary insects that feed on floral nectar must use innate knowledge to find their first flower. While innate preferences for flower colours are often described as fixed, species-specific traits, the nature and persistence of these preferences have been debated, particularly in relation to ontogenetic processes such as learning. Here we present evidence for a strong context-dependence of innate colour preferences in the crepuscular hawkmoth Manduca sexta. Contrary to expectations, our results show that innate colour biases shift with changes in the visual environment, namely illuminance and background. This finding reveals that innate responses might emerge from a contextual integration of sensory inputs involved in object class recognition rather than from the deterministic matching of such inputs with a fixed internal representation.

Context dependency of in-flight responses by Manduca sexta moths to ambient differences in relative humidity.

The use of sensory information to control behavior usually involves the integration of sensory input from different modalities. This integration is affected by behavioral states and experience, and it is also sensitive to the spatiotemporal patterns of stimulation and other general contextual cues. Following the finding that hawkmoths can use relative humidity (RH) as a proxy for nectar content during close-range foraging, we evaluate here whether RH could be used during locomotive flight under two simulated contexts in a wind tunnel: (1) dispersion and (2) search phase of the foraging behavior. Flying moths showed a bias towards air with a higher RH in a context devoid of foraging stimuli, but the addition of visual and olfactory floral stimuli elicited foraging responses that overrode the behavioral effects of RH. We discuss the results in relation to the putative adaptive value of the context-dependent use of sensory information.

Influence of Ambient Illumination on the Use of Olfactory and Visual Signals by a Nocturnal Hawkmoth During Close-Range Foraging.

As a goal-directed behavior, foraging for nectar functions on the basis of a sequence of innate stereotyped movements mainly regulated by sensory input. The operation of this inherited program is shaped by selective pressures acting on its efficiency, which is largely dependent upon the way the system handles sensory information. Flowers offer a wealth of signals, from odors acting as distant attractants, to colors eliciting approximation and feeding responses, to textures guiding feeding responses toward a reservoir of nectar. Thus, animals use different signals in the regulation of particular motor outputs. Nevertheless, the use of these sensory signals can be user-specific (e.g. species, motivation, experience, learning) as well as context-dependent (e.g. spatiotemporal patterns of stimulation, availability of signals, multimodal integration). The crepuscular/nocturnal hawkmoths Manduca sexta experience a wide range of illuminations during their foraging activity, which raises the question of how these environmental changes might affect the use of two important floral signals, odor and visual display. In a flight cage, we explored the use of these signals under different illuminances. Under conditions of starlight and crescent moonlight, moths showed very low levels of responsiveness to unscented feeders (artificial flowers). However, responsiveness was recovered either by increasing illumination, or by offering olfactory signals. Additionally, we recorded a bias toward white over blue feeders under dim conditions, which disappeared with increasing illumination. We discuss how this kind of experimental manipulation may provide insights to the study of how innate behavioral programs, and their underlying neural substrates, overcome selective forces imposed by the uncertainty of natural, ever-changing environments.

Out of the blue: the spectral sensitivity of hummingbird hawkmoths.

The European hummingbird hawkmoth Macroglossum stellatarum is a diurnal nectar forager like the honeybee, and we expect similarities in their sensory ecology. Using behavioural tests and electroretinograms (ERGs), we studied the spectral sensitivity of M. stellatarum. By measuring ERGs in the dark-adapted eye and after adaptation to green light, we determined that M. stellatarum has ultraviolet (UV), blue and green receptors maximally sensitive at 349, 440 and 521 nm, and confirmed that green receptors are most frequent in the retina. To determine the behavioural spectral sensitivity (action spectrum) of foraging moths, we trained animals to associate a disk illuminated with spectral light, with a food reward, and a dark disk with no reward. While the spectral positions of sensitivity maxima found in behavioural tests agree with model predictions based on the ERG data, the sensitivity to blue light was 30 times higher than expected. This is different from the honeybee but similar to earlier findings in the crepuscular hawkmoth Manduca sexta. It may indicate that the action spectrum of foraging hawkmoths does not represent their general sensory capacity. We suggest that the elevated sensitivity to blue light is related to the innate preference of hawkmoths for blue flowers.

The effect of ambient humidity on the foraging behavior of the hawkmoth Manduca sexta.

The foraging decisions of flower-visiting animals are contingent upon the need of an individual to meet both energetic and osmotic demands. Insects can alter their food preferences to prioritize one need over the other, depending on environmental conditions. In this study, preferences in nectar sugar concentrations (0, 12, 24 %) were tested in the hawkmoth Manduca sexta, in response to different levels of ambient humidity (20, 40, 60, and 80 % RH). Moths altered their foraging behavior when placed in low humidity environments by increasing the volume of nectar imbibed and by consuming more dilute nectar. When placed in high humidity environments the total volume imbibed decreased, because moths consumed less from dilute nectars (water and 12 % sucrose). Survivorship was higher with higher humidity. Daily foraging patterns changed with relative humidity (RH): moths maximized their nectar consumption earlier, at lower humidities. Although ambient humidity had an impact on foraging activity, activity levels and nectar preferences, total energy intake was not affected. These results show that foraging decisions made by M. sexta kept under different ambient RH levels allow individuals to meet their osmotic demands while maintaining a constant energy input.

Floral humidity as a reliable sensory cue for profitability assessment by nectar-foraging hawkmoths.

Most research on plant-pollinator communication has focused on sensory and behavioral responses to relatively static cues. Floral rewards such as nectar, however, are dynamic, and foraging animals will increase their energetic profit if they can make use of floral cues that more accurately indicate nectar availability. Here we document such a cue--transient humidity gradients--using the night blooming flowers of Oenothera cespitosa (Onagraceae). The headspace of newly opened flowers reaches levels of about 4% above ambient relative humidity due to additive evapotranspirational water loss through petals and water-saturated air from the nectar tube. Floral humidity plumes differ from ambient levels only during the first 30 min after anthesis (before nectar is depleted in wild populations), whereas other floral traits (scent, shape, and color) persist for 12-24 h. Manipulative experiments indicated that floral humidity gradients are mechanistically linked to nectar volume and therefore contain information about energy rewards to floral visitors. Behavioral assays with Hyles lineata (Sphingidae) and artificial flowers with appropriate humidity gradients suggest that these hawkmoth pollinators distinguish between subtle differences in relative humidity when other floral cues are held constant. Moths consistently approached and probed flowers with elevated humidity over those with ambient humidity levels. Because floral humidity gradients are largely produced by the evaporation of nectar itself, they represent condition-informative cues that facilitate remote sensing of floral profitability by discriminating foragers. In a xeric environment, this level of honest communication should be adaptive when plant reproductive success is pollinator limited, due to intense competition for the attention of a specialized pollinator.

Chromatic signals control proboscis movements during hovering flight in the hummingbird hawkmoth Macroglossum stellatarum.

Most visual systems are more sensitive to luminance than to colour signals. Animals resolve finer spatial detail and temporal changes through achromatic signals than through chromatic ones. Probably, this explains that detection of small, distant, or moving objects is typically mediated through achromatic signals. Macroglossum stellatarum are fast flying nectarivorous hawkmoths that inspect flowers with their long proboscis while hovering. They can visually control this behaviour using floral markings known as nectar guides. Here, we investigate whether this is mediated by chromatic or achromatic cues. We evaluated proboscis placement, foraging efficiency, and inspection learning of naïve moths foraging on flower models with coloured markings that offered either chromatic, achromatic or both contrasts. Hummingbird hawkmoths could use either achromatic or chromatic signals to inspect models while hovering. We identified three, apparently independent, components controlling proboscis placement: After initial contact, 1) moths directed their probing towards the yellow colour irrespectively of luminance signals, suggesting a dominant role of chromatic signals; and 2) moths tended to probe mainly on the brighter areas of models that offered only achromatic signals. 3) During the establishment of the first contact, naïve moths showed a tendency to direct their proboscis towards the small floral marks independent of their colour or luminance. Moths learned to find nectar faster, but their foraging efficiency depended on the flower model they foraged on. Our results imply that M. stellatarum can perceive small patterns through colour vision. We discuss how the different informational contents of chromatic and luminance signals can be significant for the control of flower inspection, and visually guided behaviours in general.

Look and touch: multimodal sensory control of flower inspection movements in the nocturnal hawkmoth Manduca sexta.

A crucial stage in the interaction between pollinators and plants is the moment of physical contact between them, known as flower inspection, or handling. Floral guides - conspicuous colour markings, or structural features of flower corollas - have been shown to be important in the inspecting behaviour of many insects, particularly in diurnal species. For the nocturnal hawkmoth Manduca sexta tactile input has an important role in flower inspection, but there is no knowledge about the use of visual floral guides in this behaviour. I carried out a series of experiments to first, evaluate the putative role of floral guides during flower inspection and second, to explore how simultaneous tactile and visual guides could influence this behaviour. Results show that visual floral guides affect flower inspection by M. sexta. Moths confine proboscis placement to areas of higher light reflectance regardless of their chromaticity, but do not appear to show movements in any particular direction within these areas. I also recorded inspection times, finding that moths can learn to inspect flowers more efficiently when visual floral guides are available. Additionally, I found that some visual floral guides can affect the body orientation that moths adopt while hovering in front of horizontal models. Finally, when presented with flower models offering both visual and tactile guides, the former influenced proboscis placement, whereas the latter controlled proboscis movements. Results show that innate inspection behaviour is under multimodal sensory control, consistent with other components of the foraging task. Fine scale inspection movements (elicited by diverse floral traits) and the tight adjustment between the morphology of pollinators and flowers appear to be adaptively integrated, facilitating reward assessment and effective pollen transfer.

Flexible responses to visual and olfactory stimuli by foraging Manduca sexta: larval nutrition affects adult behaviour.

Here, we show that the consequences of deficient micronutrient (beta-carotene) intake during larval stages of Manduca sexta are carried across metamorphosis, affecting adult behaviour. Our manipulation of larval diet allowed us to examine how developmental plasticity impacts the interplay between visual and olfactory inputs on adult foraging behaviour. Larvae of M. sexta were reared on natural (Nicotiana tabacum) and artificial laboratory diets containing different concentrations of beta-carotene (standard diet, low beta-carotene, high beta-carotene and cornmeal). This vitamin-A precursor has been shown to be crucial for photoreception sensitivity in the retina of M. sexta. After completing development, post-metamorphosis, starved adults were presented with artificial feeders that could be either scented or unscented. Regardless of their larval diet, adult moths fed with relatively high probabilities on scented feeders. When feeders were unscented, moths reared on tobacco were more responsive than moths reared on beta-carotene-deficient artificial diets. Strikingly, moths reared on artificial diets supplemented with increasing amounts of beta-carotene (low beta and high beta) showed increasing probabilities of response to scentless feeders. We discuss these results in relationship to the use of complex, multi-modal sensory information by foraging animals.

Why do Manduca sexta feed from white flowers? Innate and learnt colour preferences in a hawkmoth.

Flower colour is an important signal used by flowering plants to attract pollinators. Many anthophilous insects have an innate colour preference that is displayed during their first foraging bouts and which could help them locate their first nectar reward. Nevertheless, learning capabilities allow insects to switch their colour preferences with experience and thus, to track variation in floral nectar availability. Manduca sexta, a crepuscular hawkmoth widely studied as a model system for sensory physiology and behaviour, visits mostly white, night-blooming flowers lacking UV reflectance throughout its range in the Americas. Nevertheless, the spectral sensitivity of the feeding behaviour of naïve moths shows a narrow peak around 450 nm wavelengths, suggesting an innate preference for the colour blue. Under more natural conditions (i.e. broader wavelength reflectance) than in previous studies, we used dual choice experiments with blue- and white-coloured feeders to investigate the innate preference of naïve moths and trained different groups to each colour to evaluate their learning capabilities. We confirmed the innate preference of M. sexta for blue and found that these moths were able to switch colour preferences after training experience. These results unequivocally demonstrate that M. sexta moths innately prefer blue when presented against white flower models and offer novel experimental evidence supporting the hypothesis that learning capabilities could be involved in their foraging preferences, including their widely observed attraction to white flowers in nature.

Context- and scale-dependent effects of floral CO2 on nectar foraging by Manduca sexta.

Typically, animal pollinators are attracted to flowers by sensory stimuli in the form of pigments, volatiles, and cuticular substances (hairs, waxes) derived from plant secondary metabolism. Few studies have addressed the extent to which primary plant metabolites, such as respiratory carbon dioxide (CO(2)), may function as pollinator attractants. Night-blooming flowers of Datura wrightii show transient emissions of up to 200 ppm above-ambient CO(2) at anthesis, when nectar rewards are richest. Their main hawkmoth pollinator, Manduca sexta, can perceive minute variation (0.5 ppm) in CO(2) concentration through labial pit organs whose receptor neurons project afferents to the antennal lobe. We explored the behavioral responses of M. sexta to artificial flowers with different combinations of CO(2), visual, and olfactory stimuli using a laminar flow wind tunnel. Responses in no-choice assays were scale-dependent; CO(2) functioned as an olfactory distance-attractant redundant to floral scent, as each stimulus elicited upwind tracking flights. However, CO(2) played no role in probing behavior at the flower. Male moths showed significant bias in first-approach and probing choice of scented flowers with above-ambient CO(2) over those with ambient CO(2), whereas females showed similar bias only in the presence of host plant (tomato) leaf volatiles. Nevertheless, all males and females probed both flowers regardless of their first choice. While floral CO(2) unequivocally affects male appetitive responses, the context-dependence of female responses suggests that they may use floral CO(2) as a distance indicator of host plant quality during mixed feeding-oviposition bouts on Datura and Nicotiana plants.

The breath of a flower: CO(2) adds another channel-and then some-to plant-pollinator interactions.

In this article I comment on our findings that floral carbon dioxide (CO(2)) can be used by Manduca sexta hawkmoths in a scale- and context-dependent fashion. We firstly found, in wind tunnel assays, that diffusing floral CO(2) is used as long-distance cue (e.g., meters). Moths track CO(2) plumes up-wind in the same manner they track floral odors. Nevertheless, CO(2) did not appear to function as a local stimulus for flower probing, evidencing a scale-dependent role in nectar foraging. These results were further enriched by a second finding. In dual choice assays, where moths were offered two scented artificial flowers of which only one emitted above-ambient CO(2)-levels, female Manduca sexta chose to feed on the CO(2) emitting flower only when host-plant volatiles were added to the background. We discuss this apparent measurement of oviposition obligations during foraging in the context of the life histories of both insect and plant species. These findings seem to pinpoint the usually artificial nature of compartmentalizing herbivory and pollination as different, isolated aspects of insect-plant interactions. Insects do not seem to have a defined response to a certain stimulus; instead, motor programs appear to be in response to composite arrangements of external stimuli and inner states. If animal-plant interactions have evolved under these premises, I believe it may prove beneficial to include a non-linear, integrative view of plant multi-signaling and life history aspects into the study of pollination biology.

The effect of decoupling olfactory and visual stimuli on the foraging behavior of Manduca sexta.

Within an appetitive context, Manduca sexta, a nectivorous nocturnal hawkmoth, can be attracted by a range of stimuli including floral volatiles and visual display, carbon dioxide and water vapor. Several studies on this and other flower-visiting insects have shown how olfactory and visual stimulation play (or do not play) a role in attraction and feeding. Nevertheless, these studies have consistently manipulated stimuli in a 'presence-absence' manner. Here, we experimentally decoupled the presentation of both stimuli spatially and temporally in a wind tunnel, rather than entirely eliminating either one, and found that the decision-making process based on these stimuli is more flexible and complex than previously asserted. Manduca sexta was most responsive when both cues were present and emanated from the same source. When stimuli were spatially separated, responsiveness levels were comparable to those elicited by a single stimulus. However, transient olfactory stimulation either before or after visually guided approach (temporal decoupling) enhanced responsiveness to an odorless visual target. Additionally, searching times were increased by either a transient olfactory stimulation before take-off or by having the flower model spatially separated from the odor source tracked by the moths. Finally, in a dual-choice experiment, moths showed a strong bias for the visual display over the odor plume, suggesting the former to be the ultimate indicator of a nectar source. Our manipulation of floral cues shows that the feeding behavior of M. sexta, and probably of other nectivorous insects, is based not only on the sensory stimulation per se but also on the temporal and spatial pattern in which these stimuli are perceived.

The role of mechanosensory input in flower handling efficiency and learning by Manduca sexta.

Nectar-foraging animals are known to utilize nectar guides-- patterns of visual contrast in flowers-- to find hidden nectar. However, few studies have explored the potential for mechanosensory cues to function as nectar guides, particularly for nocturnal pollinators such as the tobacco hornworm moth, Manduca sexta. We used arrays of artificial flowers to investigate the flower handling behavior (the ability to locate and drink from floral nectaries) of naïve moths, looking specifically at: (1) how the shape and size of flat (two-dimensional) artificial corollas affect nectar discovery and (2) whether three-dimensional features of the corolla can be used to facilitate nectar discovery. In these experiments, we decoupled visual from tactile flower features to explore the role of mechanosensory input, putatively attained via the extended proboscides of hovering moths. In addition, we examined changes in nectar discovery times within single foraging bouts to test whether moths can learn to handle different kinds of artificial flowers. We found that corolla surface area negatively affects flower handling efficiency, and that reliable mechanosensory input is crucial for the moths' performance. We also found that three-dimensional features of the corolla, such as grooves, can significantly affect the foraging behavior, both positively (when grooves converge to the nectary) and negatively (when grooves are unnaturally oriented). Lastly, we observed that moths can decrease nectar discovery time during a single foraging bout. This apparent learning ability seems to be possible only when reliable mechanosensory input is available.

Non-random nectar unloading interactions between foragers and their receivers in the honeybee hive.

Nectar acquisition in the honeybee Apis mellifera is a partitioned task in which foragers gather nectar and bring it to the hive, where nest mates unload via trophallaxis (i.e. mouth-to-mouth transfer) the collected food for further storage. Because forager mates exploit different feeding places simultaneously, this study addresses the question of whether nectar unloading interactions between foragers and hive-bees are established randomly, as it is commonly assumed. Two groups of foragers were trained to exploit a different scented food source for 5 days. We recorded their trophallaxes with hive-mates, marking the latter ones according to the forager group they were unloading. We found non-random probabilities for the occurrence of trophallaxes between experimental foragers and hive-bees, instead, we found that trophallactic interactions were more likely to involve groups of individuals which had formerly interacted orally. We propose that olfactory cues present in the transferred nectar promoted the observed bias, and we discuss this bias in the context of the organization of nectar acquisition: a partitioned task carried out in a decentralized insect society.