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Chemical Ecology Special Feature
CHEMICAL ECOLOGY SPECIAL FEATURE / BIOLOGICAL SCIENCES / RESEARCH ARTICLES / ECOLOGY
Context- and scale-dependent effects of floral CO2 on nectar foraging by Manduca sexta
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*Department of Biological Sciences, University of South Carolina, Columbia, SC 29208; and Oberlin College, Oberlin, OH 44074
Edited by Thomas Eisner, Cornell University, Ithaca, NY, and approved November 30, 2007 (received for review September 11, 2007)
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Abstract |
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fragrance | odor | olfaction | pollination | Sphingidae
Our knowledge of how insects use CO2 as a sensory cue is derived primarily from studies of insects that vector diseases or attack crop plants. For example, many haematophagous insects use CO2 to locate their animal hosts from a distance (8). Discontinuous CO2 plumes modulate host-seeking behavior by mosquitoes (9, 10), suggesting that CO2 acts as a long-range orientation stimulus (11). Tsetse flies and biting midges also use CO2 as a long-range attractant that can synergize the attractiveness of other host odors (12, 13). However, CO2 also functions as a close-range feeding stimulus (on the host's skin) for mosquitoes (8). CO2 synergizes the attractiveness of some human skin odors to female yellow fever mosquitoes (14) and has been suggested to synergize triatomine bug responses to L(+)-lactic acid (15).
Some herbivorous insects show similar responses to CO2, although typically at closer range to its source than haematophagous insects. CO2 alone is sufficient to guide Diabrotica virgifera beetle larvae toward corn roots (16), and larvae of noctuid (Helicoverpa armigera) (17) and pyralid (Elasmopalpus lignosellus) (18) moths orient to above-ambient CO2 sources. Females of the pyralid moth Cactoblastis cactorum search for CO2 sinks on photosynthetic stem surfaces of Opuntia stricta, as indicators of high carbon fixation activity (19). In this case, CO2 gradients are used as close-range stimuli, as is true for tephritid flies that oviposit on fruit wounds, which provide a localized source of CO2 and other olfactory oviposition stimulants (20). Finally, responses to CO2 may be context-dependent. Tephritid flies respond to CO2 when presented with a fruit-like visual stimulus (20). Within a certain concentration range, Drosophila melanogaster adults and larvae are repelled by CO2, and it has been suggested that these responses depend on the olfactory context (21).
It is clear from the studies reviewed above that CO2 may function alone or in concert with host odors, at a distance or at close range, via several behavioral mechanisms. Recent studies suggest that CO2 may also contribute to the interactions between flowers and insect pollinators. Floral CO2 is primarily associated with elevated respiratory activity in thermogenic flowers, including deceptive flowers that mimic carrion and other decaying brood sites (22, 23). Although CO2 emission is a by-product of elevated floral temperature, the compound stimulus of CO2, heat, and foul odors most accurately mimics microbial activity in rotting meat (24, 25). Less well studied are the patterns of CO2 production in nondeceptive flowers that proffer nectar as an energetic reward. In principle, if newly opened, unvisited flowers emit elevated CO2, nectivorous insects could use such information to find the most profitable flowers or patches thereof (26). If floral CO2 levels were ephemeral, insects might use them as more "honest" indicators of nectar availability in real time than floral color or scent (2), in much the same way that tarsal secretions are used as flower-marking cues by foraging bees (27, 28). Given that CO2 is a component of ambient air in plant communities, flowers would need to emit amounts that could be detected by the insect with a sufficiently high signal-to-noise ratio.
These conditions are met in a night-blooming plant, Datura wrightii [Solanaceae (29, 30)], and its primary pollinator, the crepuscular hawkmoth, Manduca sexta (Sphingidae), in the Sonoran Desert. Hawkmoth-pollinated flowers often undergo dramatic bud elongation, nectar secretion, scent biosynthesis, and emission during the 6–12 h before opening (31, 32). Flowers of D. wrightii open explosively at dusk, releasing concentrations of CO2 up to 200 ppm above ambient levels (29). Adult M. sexta moths can detect such differences with their CO2-sensing organ [labial-palp pit organ (LPO)], whose threshold may be as low as 0.5 ppm (33, 34). In the aforementioned C. cactorum, males have vestigial LPOs, whereas females use their well developed LPOs to detect gradients of CO2 concentration for oviposition on Opuntia cactus. In contrast, the LPOs of M. sexta are well developed in both sexes (35), suggesting that they contribute to adult nectar foraging rather than, or in addition to, oviposition (36). Thom and collaborators (26) performed dual-choice assays with naïve male M. sexta moths in a flight cage, in which the moths preferred to feed from an artificial flower emitting higher than ambient CO2 concentrations (765 ppm) over one emitting ambient levels of CO2 (438 ppm). These findings provide direct evidence that floral CO2 affects the foraging behavior of adult Lepidoptera.
In this study, we examined the behavioral mechanisms of CO2-mediated nectar foraging in M. sexta by addressing three scale- or context-related questions. (i) Do M. sexta moths use floral CO2 as an orientation cue or a local feeding stimulus? (ii) How does CO2 interact with visual and olfactory cues during foraging? (iii) Do the behavioral responses of moths to floral CO2 change in the presence of host-plant volatiles? We explored these questions by presenting naïve adult moths with subsets of sensory stimuli in a laminar flow wind tunnel, scoring tracking, approaching, and probing behaviors to distinguish between alternative hypotheses on the behavioral role(s) of floral CO2 (Table 1, Table 2, and Fig. 1). Additionally, we performed a choice experiment similar to that published by Thom et al. (26), but in a laminar flow wind tunnel, in which we tested the foraging behavior of unmated females in addition to males. Because male moths already had been shown to choose scented paper flowers with above-ambient CO2 (19), we simply expected female moths to behave similarly. When they did not, we repeated this experiment with the addition of tomato plant volatiles to test whether female responses to floral CO2 depend on contextual information provided by host-plant odors.
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Experiment 2.
Approximately 74% of the males and 82% of the females tested flew and probed at the experimental flowers, showing a significant preference of first approaches to the V+O+CO2 flower instead of the V+O flower [
2 test; P = 0.04, n = 60 (Fig. 3A)]. However, when analyzing gender differences, we found this result to be due exclusively to male behavior (2 test for males: P = 0.002, n = 28; 2 test for females: P = 1.00, n = 32). Females took 64 ± 15 sec from take-off to probing on the V+O+CO2 flowers and 61 ± 18 sec on V+O flowers. For males, latency times were 48 ± 7 sec and 28 ± 7 sec, respectively, on these treatments. However, latencies did not differ significantly between gender under ANOVA [F(1, 58) = 1.91, P = 0.17]. Analysis also showed that neither females nor males differed significantly in latency times when responding to the different flower models [females: F(1, 30) = 0.01, P = 0.91; males: F(1, 26) = 2.00, P = 0.17], but females tended to probe the V+O+CO2 flower for a longer time [7 ± 1 sec vs. 4 ± 1 sec; F(1, 30) = 3.87, P = 0.06], whereas males did not show any probing time difference between the two flower models [7 ± 2 sec probing on the V+O+CO2 flower and 9 ± 4 sec on the V+O flower; F(1, 26) = 0.58, P = 0.45].
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2 test, P = 0.002). The addition of background tomato plant volatiles did not change first choice preferences in males (Fig. 3), which first chose the V+O+CO2 flower (2 test, P = 0.025, n = 20) as in experiment 2. Nevertheless, tomato volatiles appeared to have a strong effect on female moths, which departed from their random choice (experiment 2) to show a significant bias toward the V+O+CO2 flower over the V+O flower (2 test, P = 0.018, n = 17) (Fig. 3). |
Discussion |
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Nectar foraging by naïve M. sexta follows a sequence of behavioral events in response to sensory stimuli at different distances from the flower. For example, upwind flight is released by a floral odor plume (42, 43) and floral approach is elicited by olfactory and/or visual stimuli (43) that are most effective when combined (44), after which probing is guided by visual and tactile inputs (7). Upon closer inspection, these behavioral "rules" are somewhat flexible, depending on the spatial and temporal scale (44) and the context in which specific stimuli are perceived [e.g., after appetitive conditioning (45, 46)]. With this framework in mind, we explored how CO2 might interact with other floral stimuli in behavioral assays with M. sexta.
Scale Dependence of Responses to CO2 in No-Choice Assays. Our first approach (experiment 1) was to decouple floral traits to examine how CO2 affects nectar foraging by M. sexta at artificial flowers with different subsets of sensory stimuli. As outlined in Tables 1 and 2, our goal was to determine the scale (if any) at which CO2 affects appetitive responses, using no-choice behavioral assays. The tracking and probing behaviors observed (Fig. 2A) allow us to reject the hypotheses stating that CO2 has no behavioral effect (HA0) or acts as a local feeding stimulus (HA2). Instead, our results support the hypothesis that CO2 acts as an orientation stimulus (Table 1, HA1). The CO2 plume evoked the typical zigzag casting flight pattern indicative of odor-guided behavior in M. sexta (30, 47, 48) and was slightly more likely to elicit upwind casting flight than was floral odor. These results suggest that floral CO2 is perceived by M. sexta as an odor, a hypothesis that is consistent with the fact that CO2-receptor cells in the LPO project their axons through the suboesophageal ganglion into both antennal lobes (34), the primary centers for the processing of olfactory inputs from the antennae.
Even though CO2 alone (V+CO2) had an effect on tracking behavior, approaches to the flower did not differ significantly from the negative control (i.e., Fig. 2A, V). Probing behavior was unaffected by the presence of CO2, with and without floral odor, which allows us to reject the hypotheses of redundant (HB1), additive (HB2), and synergistic (HB3) effects in favor of the null hypothesis (Table 2 and Fig. 1, HB0). Thus, in no-choice assays, CO2 affects the foraging behavior of naïve M. sexta as an orientation stimulus. Additionally, CO2 appears to have no effect or interaction with floral odor as a local feeding stimulus, either in terms of probing frequency or duration. Latency times did not show any treatment effect, although females tended to spend longer times than males in the wind tunnel before approaching the artificial flower when it emitted CO2 (Fig. 2B). Also, females were less responsive than males when no CO2 was present (see Results). We suspect a conflict or overlap in task specificity between female foraging and reproduction that will be discussed below in the light of experiments 2 and 3.
Context Dependence of Female Responses in Binary-Choice Assays. Having found that CO2 effectively elicits orientation toward flowers with no apparent effect on the probing response at close range, we designed experiment 2 to evaluate whether the tracking response elicited by CO2 might impact individual flower choice. Strikingly, we found that females showed no bias in their first probing choice between scented flowers (V+O) and scented flowers emitting above-ambient CO2 (V+O+CO2) but that males significantly preferred the latter (Fig. 3A). When host-plant (tomato vegetation) odors were added upstream of the flowers (experiment 3), males maintained the CO2 bias observed in experiment 2, but females now chose to probe the flower with above-ambient CO2 in the same proportion as did males (Fig. 3B). Our assays, like those of Thom et al. (26), presented moths with a binary choice between single flowers with markedly different local CO2 concentrations. Our protocols differed (besides the fact that we flew females in addition to males) in that we presented two flowers in the wind tunnel with distinct odor plumes that did not overlap near their sources, whereas Thom et al. (26) mounted artificial flowers on D. wrightii plants in a small flight cage with no air flow. These observations suggest that M. sexta can perceive differences in CO2 on the scale of a single plant, amidst background contributions from its vegetation (e.g., ref. 36). It remains to be tested whether moths can distinguish between closely spaced individual flowers with different CO2 concentrations on the same plant.
Stimuli, Context, and Task Specificity in Female M. sexta. Male M. sexta show identical first-flower preferences for scented artificial flowers with CO2, regardless of whether host plant odors are present (Fig. 3), suggesting that their responses to floral CO2 are not influenced by the presence of host-plant volatiles. In contrast, female M. sexta showed clear first-choice biases only in the presence of tomato plant volatiles (Fig. 3). Mechaber et al. (48) demonstrated that virgin female M. sexta show host-plant tracking behavior in response to tomato leaf volatiles, and abdominal curling typical of oviposition when in contact with the plant, 3 days after eclosion. Thus, our unmated females—all of them 3 or more days posteclosion—were expected to show some response to host-plant volatiles. However, in experiment 3, females could not physically contact tomato plants, nor did they show reduced interest in the flowers due to the presence of host-plant odors; their approaches ended in floral probing while hovering rather than landing and abdominal curling. Host-plant volatiles had a dramatic influence on female responses, which strongly suggests that their feeding behavior is influenced by stimuli related to oviposition.
Female M. sexta often mix nectar foraging and oviposition bouts on host plants with nectar-rich flowers [e.g., Datura (48)] and derive a 10-fold fitness benefit in number of eggs matured when they consume nectar as adults (49). The observation that female M. sexta lay more eggs on plants with experimentally enhanced nectar volumes (50) prompted Kessler and Baldwin (51) to suggest that females use nectar as a proxy measure of host-plant quality. In experiment 2, CO2 had no bearing on the first flower probed by females when host-plant odors were absent, whereas the addition of tomato odors in experiment 3 resulted in a first-choice preference for the above-ambient CO2 flower. However, only first choices appeared to be affected because moths continued to forage; and in all cases, scented flower models with and without CO2 were probed. Following Kessler and Baldwin (51) and considering the positive correlation between nectar production and CO2 emission found by Guerenstein and collaborators (29), we suggest that floral respiratory CO2, when presented in the context of host-plant odors, is used by female moths as a long-distance indicator of host-plant quality, releasing upwind orientation flight as shown here. Both floral nectar and CO2 could function as redundant plant-quality indicators, each affecting behavioral responses at different scales. A moth could be attracted from a distance to a plant by floral CO2; then, floral nectar could act as a local indicator of host-plant quality as proposed by Kessler and Baldwin (51).
Conditionality and Opportunism in the Use of Floral Stimuli. We have documented two dimensions of sensory conditionality in the use of floral CO2 by M. sexta during nectar foraging. The first dimension was scale dependence, such that CO2 functioned as a redundant stimulus (with floral odor) for distance orientation and floral approach but was superfluous as a local feeding stimulus (Fig. 2A). This pattern calls to mind the phenomenon of postpollination color change, in which older flowers turn color but remain turgid and are retained on the plant [reviewed by Weiss (52)]. Such flowers, though unrewarding, enhance pollinator attraction from a distance but are ignored once the insects arrive at the inflorescence because of learned color preferences (53). The second dimension of conditionality was context dependence, such that male first-visit preference for flowers with above-ambient CO2 was constant irrespective of host-plant odor, whereas female preference for such flowers was observed only when they were presented in the context of oviposition cues (Fig. 3). For these hawkmoths, responses to floral CO2 clearly depend on the sex of the moth and the conditions under which it perceives this stimulus; non-host-plant floral odors attract both sexes of M. sexta in the absence of CO2 (44). The context-dependent responses of female moths to the combined stimulus of host-plant odor and CO2 are conflated with nonappetitive behaviors likely to be associated with oviposition, as discussed above.
Given the multitude of scales, contexts and mechanisms by which CO2 functions in plant–insect interactions, the conditionality of its significance in nectar foraging by M. sexta is not surprising. The perception of CO2 as a floral odor by M. sexta adds to the small but growing list of "secondary functions" (e.g., pollinator attraction and herbivore deterrence) attributable to primary plant metabolites among plant–insect interactions (54, 55). Floral CO2 is a stimulus whose relevance to moth foraging behavior depends on the specific task at hand (e.g., oviposition) and the extent to which other floral information is available. In night-blooming plants such as D. wrightii, floral CO2 might provide the most honest indication of nectar profitability to foraging moths because it decreases markedly within the first hour after anthesis (29), when nectar feeding by M. sexta is most intense (56, 57), whereas floral odor and visual display remain unchanged after nectar has been drained from individual flowers (26, 29). Future experiments should test the extent to which moths' foraging behavior in a natural setting reflects local differences in floral CO2, and whether females are capable of using such information at non-host nectar sources such as Oenothera caespitosa (Onagraceae) and Mirabilis longiflora (Nyctaginaceae). We have found increased CO2 levels at the corolla openings of these night-blooming plants [1,912 ± 195 ppm (n = 8) and 1,281 ± 173 ppm (n = 9), respectively], which also are visited by M. sexta and related hawkmoths (58, 59). At the same time, our results call for more detailed investigations of the role of CO2 in mixed nectar foraging–oviposition bouts shown by female M. sexta in nature.
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Animals. We used M. sexta adults reared from eggs provided by Lynn Riddiford (University of Washington, Seattle). Larvae were fed ad libitum on an artificial diet (60) and were kept as pupae under a 16:8 light/dark, 24/21°C cycle. Moths were separated by sex as pupae and housed in different incubators (Precision 818) under the same ambient regime and emerged within 45 x 45 x 45-cm screen cages (BioQuip). Adults were starved for 3–4 days after eclosion to increase their appetitive motivation.
Sensory Stimuli. Artificial flower. The visual stimulus consisted of a white conical paper cup (7 cm in diameter and 10 cm in depth; no reward present) attached perpendicularly to a black vertical ring stand at a height of 50 cm against a black background. Spectrophotometer readings of flowers (data not shown) revealed that the paper absorbed UV wavelengths but reflected light nearly uniformly from 400 to 700 nm. Volatile analysis (data not shown) using solid-phase microextraction combined with gas chromatography–mass spectrometry revealed that the artificial flower did not emit volatile compounds.
Olfactory stimulus. A cotton swab was soaked with 25 µl of bergamot essential oil (Body Shop) and was refreshed every 30 min to standardize odor intensity. This odor source is a reliable feeding stimulant for M. sexta (7) and, like many night-blooming flowers visited by this species, is dominated by linalool and related monoterpenoid odors (61). The wooden stem of the swab (2.5 cm) was affixed to a 1-cm3 piece of dark gray modeling clay, 2 cm inside the flower. When odor was not present, we arranged flowers in the same way but with a dry cotton swab.
Carbon dioxide (CO2). Carbon dioxide was delivered from a CO2 tank (National Welders) through Teflon tubing directly into a blue aquarium "air stone" positioned at the bottom of the artificial flower. Treatments with no CO2 emission were set up in the same way but with the CO2 tank in the closed position. Ambient and "flower-emitted" CO2 levels were measured with a LI-COR 840 CO2/H2O detector previously calibrated with three different CO2 concentrations (392, 798, and 1,032 ppm ± 2%). We obtained the following calibration curve: R = (A – 8.1199)/0.0432, where A is the actual CO2 concentration and R is the one read by the LI-COR detector. This linear regression was statistically significant (P < 0.05) with an R2 of 0.99. CO2 emission from the artificial flower was set at a flow of 10 ml/min, achieving a CO2 concentration at the flower model opening of 787 ± 33 ppm (mean ± SEM), based on concentrations measured by Thom and collaborators (26).
General Procedure in the Wind Tunnel and Recorded Variables. At the beginning of scotophase, naïve, starved adult moths were placed individually at the downwind end of a 3 x 1.5 x 1.5-m wind tunnel with a laminar flow rate of 1m/sec. Each moth was allowed to fly freely inside the wind tunnel for 5 min, during which its behavior was recorded.
Experiment 1 (no-choice assay). To investigate moth responsiveness to different combinations of stimuli, we designed an experiment with four treatments in which a single artificial flower was presented at the upwind end of the wind tunnel. The first treatment encompassed the artificial flower alone (only V). The following two treatments consisted of the addition of either odor (V+O) or CO2 (V+CO2). Finally, the fourth treatment consisted of the addition of both odor and CO2 (V+O+CO2). In all cases, the surrogate flower was identically attached to a ring-stand, with an air-stone diffuser. In treatments lacking CO2, the diffuser emitted ambient air at the same flow rate (10 ml/min).
We recorded "responsiveness," the percentage (%) of moths flown in each treatment that (i) showed the stereotypic anemotactic "zigzag" odor plume tracking behavior (62), (ii) approached the artificial flower (i.e., hovered in front of it), and (iii) probed at the artificial flower at least once with their extended proboscides. We also recorded the amount of time (latency, in seconds) during which moths flew inside the tunnel before probing the artificial flower, and the time spent probing (probing time, in seconds).
Experiment 2 (binary-choice assay). The second experiment was a dual-choice assay in which we presented individual moths with two artificial flowers that were 40 cm apart and equidistant to the release site of the moths at the upwind end of the wind tunnel. This distance effectively decouples visual and olfactory stimuli under our test conditions (44), reducing the potential for ambiguous binary choices by moths. The physical characterization of CO2 plumes is beyond the scope of this article, but we confirmed that TiCl smoke plumes do not overlap for the first meter under our experimental conditions. We recorded the proportion of moths that probed the artificial flower emanating odor (no CO2 emission) vs. the one emanating odor plus above ambient CO2. As in experiment 1, we also recorded latency and probing time at each flower model.
Experiment 3 (binary-choice assay with host plant). Given the gender asymmetries found in experiment 2 (see Results), the possible role that CO2 may play in oviposition behavior (36), and the observation that female M. sexta may show mixed bouts of nectar feeding and oviposition (50), we decided to investigate responses to floral CO2 in the presence of stimuli related to oviposition (host-plant volatiles). Thus, in this experiment, we replicated the design of experiment 2 but now positioned four tomato plants (Solanum lycopersicum cv. "Better boy"; South Carolina Farmers Market, Columbia) 1 m upstream from the flowers. This provided the odor of undamaged host-plant vegetation in addition to bergamot oil and CO2. Tomato plants were physically and visually separated from the flight arena of the wind tunnel by means of a fine, dark aluminum screen that spanned its entire transverse section.
Statistical Analysis.
The effects of treatment on the display of tracking, approach, and probing behaviors were analyzed by means of G-tests. Latency and probing times were subjected to two-way ANOVA, accounting for treatment, gender, and their interaction. For this analysis, data from experiment 1 were log-transformed. 2 tests of independence were performed to determine any deviation from a random choice pattern in experiments 2 and 3.
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To whom correspondence should be addressed. E-mail: jg549{at}cornell.edu
Present address: Department of Neurobiology and Behavior, Cornell University, Seeley G. Mudd Hall, Ithaca, NY 14853. 
Author contributions: J.G., P.M.M., and R.A.R. designed research; J.G. and P.M.M. performed research; J.G. analyzed data; and J.G. and R.A.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
© 2008 by The National Academy of Sciences of the USA
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  J. Meinwald and T. Eisner Chemical Ecology Special Feature: Chemical ecology in retrospect and prospect PNAS, March 25, 2008; 105(12): 4539 - 4540. [Full Text] [PDF]
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-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.