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

Abstract

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.

Fig. 1.

Visualization of predicted outcomes for alternative hypotheses on the behavioral function of floral CO₂ in nectar foraging behavior by M. sexta, as tested in experiment 1. The vertical axis represents a unit-free relative measure of moth response (e.g., tracking and probing). The horizontal axis contrasts the predicted outcomes when moths are exposed to artificial flowers with different subsets of floral stimuli, including visual target (V) with floral odor (V+O), carbon dioxide (V+CO₂), or the combination thereof (V+O+CO₂). Hypothesis names reflect how CO₂ would interact with floral odor. Here, synergism is broadly defined as a nonadditive (+ or −) interaction with odor, as indicated by the two-headed arrow within the bar. The potential for strict synergism, in which only the combination of odor and CO₂ elicits the appropriate behavior, has already been falsified for tracking, floral approach, and probing in previous experiments (7, 43, 44).

Fig. 2.

Behavioral responses of adult M. sexta in a wind tunnel with a single surrogate flower. (A) Responsiveness, expressed in percentage, of the recorded behaviors. Categories in the abscissa represent the different sets of stimuli offered by the surrogate flower: Different type of letters (uppercase, lowercase, and Greek) are used for comparisons between treatments for each behavior. Different letters indicate significant differences with an α-level of 0.05 (see Results). (B) Latencies to approach (i.e., time from takeoff to flower approach, in seconds) of male and female moths. Data points and error bars represent means ± SEM. Number of replicates for each treatment in parenthesis.

Fig. 3.

Proportion of first flower choices by adult M. sexta in dual-choice assays between two scented surrogate flowers, of which only one emitted above-ambient CO₂ levels (V+O+CO₂). Assays were performed in a wind tunnel either without (experiment 2; A) or with (experiment 3; B) four tomato plants at the upstream end. * and **, significant departure from random probabilities with α-levels of 0.05 and 0.005, respectively.