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.

SUMMARY: 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.

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.

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.