QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online April 18, 2006
Journal of Experimental Biology 209, 1585-1593 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02169

This Article Summary Figures Only Full Text (PDF) A corrigendum has been published Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JEB Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Goyret, J. Articles by Raguso, R. A. Search for Related Content PubMed PubMed Citation Articles by Goyret, J. Articles by Raguso, R. A.

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

Joaquín Goyret^* and Robert A. Raguso

Department of Biological Sciences, Coker Life Sciences Building, 700 Sumter Street, University of South Carolina, Columbia, SC 29208, USA

^* Author for correspondence (e-mail: jgoyret{at}biol.sc.edu)

Accepted 13 February 2006

	Summary

TOP Summary Introduction Materials and methods Results Discussion References

 
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.

Key words: pollination, Lepidoptera, sensory, multimodal, Sphingidae

	Introduction

TOP Summary Introduction Materials and methods Results Discussion References

 
One of the `mysteries of nature' revealed by Sprengel's landmark (Sprengel, 1793) publication was the concept of nectar guides – visually contrasting markings or aspects of flower morphology – that indicate the location of nectar to animal pollinators. The ubiquity of such markings, particularly those perceived in ultraviolet (UV) wavelengths, is one of the primary arguments made for the importance of contrasting flower colors to the visual perception and foraging behavior of insect pollinators (Menzel and Schmida, 1993; Chittka et al., 1994; Lunau et al., 1996). For example, honeybees show an innate proboscis extension reflex (PER) to UV marks at the center of Helianthus rigidus sunflowers, and probe at the periphery of the flower when the orientation of the ray florets is reversed (Daumer, 1958). However, it is unlikely that vision is the only sensory modality used by animals to find the nectar within flowers. Kevan and Lane (Kevan and Lane, 1985) showed that honeybees can detect differences in petal surface cell texture, and can learn such differences in conjunction with nectar rewards. Thus, tactile floral cues also could function as nectar guides (Leppik, 1956; Glover and Martin, 1998), especially for animals with poor vision, or those that forage under low light conditions, such as crepuscular or nocturnal hawkmoths (Lepidoptera: Sphingidae).

Hawkmoths are abundant in tropical and warm-temperate habitats worldwide, where they constitute an important class of pollinators (Grant, 1983; Nilsson et al., 1987; Haber and Frankie, 1989). Olfactory and visual floral stimuli are known to attract several species within an appetitive context (Knoll, 1926; Kugler, 1971; Haber, 1984; Kelber, 1997). The European Deilephila elpenor and Macroglossum stellatarum utilize true color vision even under starlit conditions (Kelber and Hénique, 1999; Kelber et al., 2002), and modify their innate odor and color preferences through associative learning (Kelber, 1996; Balkenius and Kelber, 2004). Manduca sexta, a large nocturnal hawkmoth native to the Americas, also can learn particular odors associated with nectar rewards (Daly and Smith, 2000; Daly et al., 2001a). The blue photoreceptors have been identified as the major visual mediators of feeding behavior in M. sexta (Cutler et al., 1995), whereas ultraviolet wavelengths were found to inhibit its feeding response (White et al., 1994). Floral odors attract M. sexta from a distance (3 m) in wind tunnel assays (Raguso and Willis, 2003; Raguso et al., 2005), and synergize with visual cues to activate feeding behavior (i.e. proboscis extension while hovering) in both naïve and wild moths (Raguso and Willis, 2002; Raguso and Willis, 2005).

However, successful approach to floral nectar sources and release of feeding behavior must be followed by reliable nectar assessment of individual flowers. Locating the nectary within a flower (evaluating the energy resource) is as critical as searching efficiently in order to find that flower. The hovering flight of M. sexta is an energetically expensive activity (Heinrich, 1971; Ziegler and Schulz, 1986), thus, the efficiency with which these moths handle flowers should be subject to selective pressures. Manduca sexta has a broad geographical distribution with several generations per year and it visits a wide variety of flower types across its range (Fleming, 1970; Raguso et al., 2003; Nattero et al., 2003). These observations led us to ask whether M. sexta can handle some flower morphologies more easily than others, and whether they can learn to handle flowers more efficiently with time. Such abilities would be consistent with their generalist foraging behavior and would allow these moths to efficiently assess flower profitability, as do other generalist flower visiting insects, such as bumblebees (Laverty and Plowright, 1988; Chittka and Thomson, 1997) and Pieris butterflies (Lewis, 1986).

The question remains as to which sensory modalities adult M. sexta might utilize for such a task. The diurnal hawkmoth Macroglossum stellatarum utilizes contrasting marks on the surface of flower corollas by preferentially placing its proboscis on such visual nectar guides (Knoll, 1922). Thus, M. stellatarum uses visual cues not only while searching (in flight) for nectar sources (Kelber, 1997), but also while hovering a relatively short distance (proboscis length: 2.5 cm) in front of individual flowers. Owing to its long (8–10 cm) tongue, M. sexta also hovers at a distance from flowers while feeding, such that in most cases, its only physical contact with flowers is through the proboscis. Here we ask whether mechanosensory input to the proboscis is redundant or complementary to the visual stimuli used by M. sexta when freely foraging on artificial flowers. In the first experiment, we decoupled visual from tactile stimuli by placing flat square transparency film sheets over the corolla portion of plain-white artificial flowers to test whether these moths use mechanosensory stimuli to find nectar within individual flowers. If visual stimuli are sufficient, hawkmoths should show comparable handling efficiencies on the same flower models, whether or not they are covered with transparency film. We repeated this comparison among five different artificial flower morphologies, systematically varying corolla shape and surface area.

In the second experiment, we tested whether groove-like folds, usually found in the corollas of flowers visited by hawkmoths, affect flower handling by M. sexta. We also evaluated flower handling performance in relation to different artificial flower morphologies by comparing total, successful and unsuccessful visits of individual moths foraging on arrays of 12 flowers. Finally, we examined whether moths can learn to handle different flower morphs more efficiently within a single foraging bout by examining the time they took to find nectaries as foraging bouts progressed.

	Materials and methods

TOP Summary Introduction Materials and methods Results Discussion References

 
Animal care
This study was carried out from September to December 2004 at the University of South Carolina, Columbia, SC, USA. We used 3- to 5-day-old M. sexta L. adults reared from eggs provided by Dr Lynn Riddiford, University of Washington, Seattle, WA, USA. Larvae were fed ad libitum on an artificial diet (Bell and Joachim, 1976) and were kept, as pupae, under a 16 h:8 h light:dark cycle (24:21°C), in a humidified atmosphere. Male and female pupae were kept in separate incubators (Precision 818, Winchester, VA, USA) under the same ambient regime and emerged within 45x45x45 cm screen cages (BioQuip, Inc., Rancho Dominguez, CA, USA). Adults were starved for 3–4 days before being used in experiments.

Experimental arena and flight assays
At the beginning of scotophase (15:00 h, temperature range: 22–25°C), naïve moths were placed individually within a closed Tedlar mesh flight enclosure (Bioquip; 2 mx2 mx2 m). The flight cage included an experimental floral array (20 cmx30 cmx45 cm) placed over a dark, odor-permeable box constructed by covering a matte-black-painted aluminum grid with black cheesecloth. To provide appropriate olfactory cues and humidity, we placed the cheesecloth-covered grid over two 200 ml glass beakers filled with water, each of which contained a cotton-tipped applicator swab impregnated with two drops of undiluted bergamot oil (Body Shop, Columbia, SC, USA). Thus, odor and water vapor passed through the cheese cloth and permeated the flight chamber. Bergamot oil is chemically similar to the odors of many hawkmoth-pollinated flowers (Kaiser, 1993; Knudsen and Tollsten, 1993; Mondello et al., 1998), and pilot experiments revealed it to be a potent releaser of feeding behavior in M. sexta. Visual floral stimuli were provided by a 3x4 array of artificial flowers (see below), in which each flower was separated from its neighbor by 10 cm. Artificial flowers were bathed in odor and water vapor that diffused freely through the cheesecloth. The flight enclosure was lit with a dim red light [wavelengths >600 nm (see Raguso and Willis, 2002)]. Each trial involved only one moth, which was allowed to fly freely. If the moth did not find, approach or probe the flowers within 5 min, it was captured and discarded. If it found the flowers, it was allowed to forage for a maximum of 10 min after the first floral approach. Foraging bouts were recorded with a video camera (Sony Digital 8–TRV120 Best Buy, Columbia, SC, USA) in `night-shot' mode placed outside the flight enclosure.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Five different two-dimensional flower morphs tested in Experiment 1. (A) `No transparency' paper flowers, whose surfaces are covered with acetate film cut to their exact shape, to control for fine texture. (B) `Transparency' flowers covered with a square (9 cmx9 cm) sheet of acetate film. Arrows and brackets indicate a priori comparisons: (I) `half lobes vs medium disks' compares flowers that have different surface area but the same edge-to-center distances. (II) `Half lobes vs small disks' compares flowers with similar surface area but different edge-to-center distances. Note: all flowers have accessible nectaries at their centers.

Variables recorded
We recorded the foraging efficiency (number of successfully exploited flowers over 10 min) after each trial. The number and duration of total, successful and unsuccessful flower visits were recorded from video-tape playback and timed with a Mistral chronometer (Buenos Aires, Argentina) to a resolution of 1 s.

Each flower visit began at the moment the proboscis made contact with the flower. Unsuccessful visits ended when the proboscis lost contact with the artificial flower without having reached the nectary. Successful visits were recorded until the proboscis was inserted into the nectary; when drinking time was recorded as the time elapsed until the proboscis was removed. The ratio of successful to total visits (successful visits/total visits; where total visits = unsuccessful visits + successful visits) was established as an indicator of the animals' efficiency when foraging on the different flower morphs.

Given that we had recorded the time moths took visiting each flower, we tested whether moths could learn to handle the different flower morphs during a single foraging bout. Discovery time was defined by the time elapsed between the initiation of flower probing and the entry of the proboscis into the nectary. This does not include the time flying from one flower to another or drinking, but only the time spent probing at the flower's threshold. We measured discovery time for the first eight successful visits, as did Lewis (Lewis, 1986).

Experiment 2
A second experiment was carried out to evaluate whether M. sexta can use morphological features of flowers involving a third dimension (i.e. depth) to improve its foraging efficiency. Many night-blooming flowers (e.g. Datura, Mirabilis) have conspicuously grooved petals, which could in theory be used as tactile guides for the moths' proboscides (Fig. 6). Thus, three treatments were designed. The first was `medium disks', the same flat flowers used in Experiment 1. The second and third were paper disks of the same diameter as medium disks, with two groove-like folds (see Fig. 2). In the second treatment, the folds were oriented parallel to each other (`chord grooves') and were placed 1.5 cm apart from the origin (nectary) of the disk (Fig. 2). In the third treatment, the folds were placed as two orthogonal diameters of the disk (`radial grooves'), intersecting at the nectary (Fig. 2).

View larger version (110K): [in this window] [in a new window]   Fig. 6. Features of flower handling are illustrated in this photo of Manduca sexta feeding from a flower of Mirabilis multiflora (Nyctaginaceae). Note the extended proboscis (grey arrow), the distance of the moth's body from the flower, and the radial grooves in the flower's perianth (white arrows). Scale bar, 1 cm. Photo© Robert A. Raguso.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Three-dimensional flower morphs tested in Experiment 2. Medium disk: same disk as in Fig. 1. Radial folds: medium disk with two groove-like folds along two perpendicular diameters of the disk. Chord folds: medium disk with two groove-like folds along two parallel chords, each 1.5 cm apart from the origin of the disk.

Because the variables measuring moths' foraging success on model flowers (emptied flowers and ratio of successful to total visits) showed equivalent results in Experiment 1 (see Results), we only analyzed emptied flowers data in Experiment 2. Two a priori comparisons were planned (control group, i.e. medium disks vs radial grooves, and control vs chord grooves). Our evaluation of emptied flowers and the appropriate contrasts were performed using one-way analysis of variance (ANOVA) and t-tests, respectively, because the assumptions of the model (normality and homogeneity of variances) were met.

	Results

TOP Summary Introduction Materials and methods Results Discussion References

 
Experiment 1
Inside the flight cage, 71.4% of the experimental animals (N=172) approached and probed the artificial flowers, with no significant gender differences observed (females: 66.4%; males: 76.2%; G_h=1.81; P=0.6).

There were no differences in the overall proportions of responses to the different flower morphs, either with or without square transparency film (G_h=5.85; P=0.56; Table 1). Variation in flower shape and size did not account for any difference in initial feeding responses (i.e. approaches and probes). The presence of the square transparency film had a significant effect on the number of artificial flowers that moths successfully exploited (`emptied flowers') during each foraging bout (Kruskal–Wallis test; transparency vs no transparency: ²_(1,0.005)=18.43; P<0.0001; Table 1, Fig. 3). In addition, variation in emptied flowers was significantly affected by flower morphology among the no transparency treatments (Kruskal–Wallis test; within no transparency: ²_(4,0.005)=44.64; P<0.0001). This effect was not observed among transparency treatments; in this case differences were not as pronounced, only accounting for a trend (Kruskal–Wallis median test; within transparency: ²_(4,0.005)=10.18; P=0.04).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Number of emptied flowers (foraging efficiency; mean ± s.e.m.) after a 10 min foraging bout by individual Manduca sexta inside the flight chamber. In each treatment (abscissa) an array of 12 artificial flowers of the same morph was present. Black bars represent responses to artificial flowers without square transparency film; gray bars represent responses to the same artificial flowers covered with a square transparency film. Different letters denote statistically significant differences with a corrected -level for significance of 0.008 (see text).

Among the no transparency treatments, moths clearly were more successful when handling full lobe and small disk morphs (see Fig. 3). As flower surface area increased from small disk (12.6 cm²) to large disk (63.6 cm²), moth performance declined (see regression analysis below). this same effect was observed when comparing full lobe (21.4 cm²), half lobe (33.7 cm²) and large disk (Kruskal–Wallis test; full lobe vs half lobe: ²_(1,0.005)=6.07; P=0.014; large disk vs medium disk: ²_(1,0.005)=16.63; P<0.0001). Because surface area is not the only flower feature that varied among treatments, we tested whether the minimum distance from the edge to the nectary (center) of the flower could affect moths' performance independently. Half lobe and medium disk flowers have similar surface areas (33.7 cm² and 33.2 cm², respectively) but different edge-to-center distances (2 cm and 3.25 cm, respectively). Similarly, half lobe and small disk flowers have the same minimum edge-to-center distance (2 cm) but different surface areas (33.7 cm² and 12.6 cm², respectively). Surface area appeared to be a more important flower feature than edge-to-center distance, as feeding effectiveness did not differ significantly between half lobe and medium disk flowers (Kruskal–Wallis test; ²_(1,0.005)=0.005; P=0.945), but differed significantly between half lobe and small disk flowers (Kruskal–Wallis test; ²₍₁₎=18.34; P<0.0001; -level: 0.005). Moreover, the number of emptied flowers was significantly correlated with flower surface area (a+ßx=y; a=14.33; ß=–0.75; R²=0.56; F_(1,0.005,84)=105.5; P<0.0001).

Analysis of the ratio of successful/total visits yielded the same results as obtained from the analysis of emptied flowers (Kruskal–Wallis tests; transparency vs no transparency: ²₍₁₎=30.48; P<0.0001; within transparency: ²₍₄₎=59.29; P<0.0001; within no transparency: ²_(4,0.005)=7.39; P=0.12). Contrasts between flower morphs on this variable show the same significance levels as those on the emptied flowers variable.

Discovery times generally decreased when moths foraged on the artificial flowers (no transparency; exponential decline fit: R²=84.35; P=0.001) as illustrated by Fig. 4A (full lobe with no transparency; exponential decline function fit: R²=84.87; P=0.001). Nevertheless, this was not the case for the large disk treatment (Fig. 4C; exponential decline function fit: R²=37.99; P=0.10). When flowers were covered by a transparency film, discovery times did not conform to a typical learning curve (transparency treatment; exponential decline function fit: R²=56.58; P=0.031; corrected -level of significance: 0.005), as shown in Fig. 4B,C (full lobe with transparency: R²=8.89; P=0.47; large disk with transparency: R²=0.0; P=1.0).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Discovery time (probing time between feeding attempts) for four different treatments. (A) Full lobe, (B) full lobe with transparency film, (C) large disk and (D) large disk with transparency film. Data points are medians, whiskers represent first and third quartiles. Statistical values refer to goodness-of-fit to an exponential decline function (one factor), a classical `learning curve'. Moths exploiting full lobe flowers with no transparency film show exponentially decreasing discovery times. When exploiting large disks, or either shape with transparency film, moths show larger variances in their responses, which do not fit an exponential decline function.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Three-dimensional corolla features affect foraging efficiency by Manduca sexta. The vertical bars represent number of flowers (mean ± s.e.m.) emptied after a 10 min foraging bout by individual moths inside the flight chamber. In each treatment (abscissa) an array of 12 artificial flowers of the same morph was present. Different letters denote statistically significant differences (see text).

	Discussion

TOP Summary Introduction Materials and methods Results Discussion References

In our experiments, moths were effectively and equally attracted to the different artificial paper flowers, regardless of the fact that they differed in shape and size (which in turn, greatly affected performance), when paper flower arrays were presented with Bergamot oil as an olfactory stimulus (see Results). This result indicates that probing responses (i.e. emptied flowers) to different treatments were not confounded by innate differences in attractiveness, and that no biases in moth preference or attraction were associated with the transparency films used to de-couple visual and mechanical stimuli.

Vision and mechanoreception during flower probing
What is the innate probing strategy of M. sexta? Is the proboscis guided visually or are there other sensory systems involved? The use of different artificial flowers affected the efficiency with which M. sexta foraged on them. The `lobes' series and the `disks' series (both of which include the large disk treatment) showed improvements in moth performance correlated with decreased surface area. As surface area increases, edge-to-center distance also increases, but the a priori comparisons (Figs 1, 3) strongly suggest that for the set of artificial flowers used in this study, surface area was the main corolla feature affecting performance. Furthermore, this hypothesis is supported by the significant linear regression between surface area and performance (i.e. emptied flowers). On flat disk flowers with no surface features, probing by naïve M. sexta is ineffectual on larger disks, as the moths probe across the disk's surface and rarely find the centrally located nectary. Similarly, Knoll (Knoll, 1926) showed that when Hyles lineata livornica hawkmoths forage on artificial flowers, they probe the entire surface of the paper models. Our findings suggest that the innate strategy of M. sexta is to perform a `random walk' of probing across the flower's surface.

The disruption of reliable tactile information clearly interferes with flower handling by M. sexta, showing that mechanoreception, in addition to vision and olfaction, is involved in nectar feeding by these moths (Fig. 3). Tactile cues constitute an important component of many flower–pollinator systems (Kevan and Lane, 1985; Borg-Karlson, 1990), but are rarely investigated from a behavioral standpoint. Interestingly, in the treatments with transparency film, we observed an overall reduced performance to the point where variation in flower shape had no significant effect on handling efficiency. Moths performed equally poorly on the different flower models without reliable tactile information, despite the fact that visual differences were preserved. Further investigation of the influence of mechanoreception on probing behavior led to Experiment 2, in which we found that corolla grooves positively affect the handling performance when they converge at the nectary and negatively affect it when they are incorrectly oriented (Fig. 5). This suggests that three-dimensional features have a hierarchical precedence on nectar-searching behavior at the flower handling scale as proposed by Brantjes and Bos (Brantjes and Bos, 1980). At this level, the spatial resolution of M. sexta's eyes does not allow for accurate feedback about proboscis position (A. Kelber, personal communication). The low signal-to-noise ratio of the visual modality at this scale could have imposed selective pressures for M. sexta to efficiently assess floral nectar content by other means. Such means include mechanoreception, as suggested by Leppik (Leppik, 1956) for some butterflies and as showed in this study, and probably gustation, given the responses of chemoreceptive sensilla positioned along the tip of the lepidopteran proboscis (Krenn, 1998; Kelber, 2003).

Sprengel (Sprengel, 1793) introduced the concept of nectar guides as floral features that could be used by pollinators to visually locate the nectar. Subsequent experiments revealed that the diurnal hawkmoth Macroglossum stellatarum (Knoll, 1922), bumblebees (Manning, 1956; Kugler, 1966), honeybees (Daumer, 1958; Free, 1979) and bee-flies (Johnson and Dafni, 1998), among other insects, successfully utilize visual nectar guides. Here we show that the utility of Spengel's idea extends beyond the visual system, as the tactile sensitivity of the proboscis of M. sexta allows these moths to exploit the physical features of flowers in order to find nectar (Figs 3, 5, 6). Our experiments, unlike those of Knoll (Knoll, 1922; Knoll, 1926), varied the contours of artificial flowers, rather than testing moth responses to natural flowers. Further experiments will be required to test whether visual nectar guides of color contrast can be used by M. sexta.

Context dependence of the floral visual display
This study indicates that once moths approach a flower patch, they extend their proboscides towards a visual target and then appear to rely on mechanosensory input. At this point, when probing is relatively random, any irregularity on the corolla surface could guide moths' searching behavior, such that the proboscis `rides' along the length of petal grooves, nectary openings or the margins of highly divided corollas.

The funnel-shaped flowers of Datura wrightii, a favored nectar source of M. sexta in the Sonoran Desert (Raguso et al., 2003) are comparable in diameter to the large disk models in Experiment 1, but previous experiments indicate that Datura flowers are learned very quickly by naïve M. sexta (Desai and Raguso, 2001), which is not the case when foraging on our large disks (Fig. 3). It appears that the decrement in flower handling by M. sexta on flowers with high surface area is offset by floral depth. However, attraction from a distance is enhanced by the increased visual display provided by flowers with larger diameters (Knoll, 1922). Tubular flowers appear to offer a compromise solution to this hypothetical trade-off, while simultaneously providing for high nectar volumes and appropriate physical contact between the body of the moth and the sexual organs of the flower (Nilsson, 1988). It is tempting to consider how differences in handling efficiency associated with corolla form might impact competition between night blooming flowers for hawkmoths as pollinators (see Haber and Frankie, 1989), however, most flowers in nature are likely to be visited by experienced moths. Additional experiments will be needed to determine whether the handling differences identified in this study have an impact on subsequent foraging decisions.

Flower handling improves with experience
We analyzed whether M. sexta could learn to improve its handling abilities (i.e. reduce the time to find nectar) during a single foraging bout. Indeed, M. sexta adults improve their handling of artificial flowers within an extended feeding bout (Fig. 4). We analyzed improvement in flower handling overall (with and without transparency film) and within two specific treatments – full lobe and large disk – as examples of flowers that elicited high and low performances, respectively. Flower handling did not improve on model flowers in which the nectary was difficult to find (large circles), nor when square transparency films prevented the acquisition of reliable mechanosensory information (see Fig. 4B,D). This suggests that reliable tactile information is needed not only to forage efficiently (Fig. 3), but also to learn to forage more efficiently (slope of learning curves, see Results and Fig. 4).

Learned improvement in flower handling has been shown in a variety of nectivorous insects, including other lepidopterans (Lewis, 1986; Hartlieb, 1996; Cunningham et al., 1998) and hymenopterans (Harder, 1983; Laverty and Plowright, 1988; Chittka and Thomson, 1997). This ability gives animals the opportunity to decrease the time they spend on individual flowers and thus, directly increase their foraging efficiency and caloric intake (Pyke et al., 1977; Hughes and Seed, 1981). Learned flower handling (and its attendant constraints, i.e. the inability to learn more than one or a few floral species) has been hypothesized to account for facultative flower specialization through the advantage that generalist pollinators gain by learning to handle a particular floral species (Darwin, 1895; Lewis, 1986). This is supported by Lewis' (Lewis, 1986) observation that Pieris rapae butterflies trained to one flower type find it more difficult (than do naïve butterflies) to learn a second flower type. Moreover, bumblebees can associate the morphology of artificial flowers with their color (Chittka and Thomson, 1997). On natural flowers, preference for flowers that are more easily handled is shown by the specialist bumble bee Bombus consobrinus (Laverty and Plowright, 1988), and by two species of hummingbirds and bumblebees for blue-colored (over albino) flowers of Delphinium nelsonii (Waser and Price, 1983).

We have shown that naïve Manduca sexta hawkmoths are equally likely to feed from several different homogeneous arrays of artificial flowers with different morphologies. However, we did not explicitly test whether the moths have innate preferences for flower morphology in a dual choice setting, nor whether they develop preferences for different flower models after learning to handle them. Future studies should test whether naïve M. sexta prefer specific flower morphologies when faced with a mixed array, and if so, whether such preferences can be modified through experience.

	Acknowledgments

 
We would like to thank Glenn Svensson and members of John Hildebrand's laboratory for fruitful discussions, David Wethey for statistical advice, and Richard Vogt and Addie Williams for help with moth culture. We also thank Michael Hickman and Anna Claire Vaughn for help with the flower models, initial data recording and analysis. Thanks to Valentin Bãrca, Sheetal Desai and Melissa Jurkiewicz for the insights gained through their student projects. This study was funded by NSF grant IOB-0444163 to R.A.R.

	References

TOP Summary Introduction Materials and methods Results Discussion References

 

Balkenius, A. and Kelber, A. (2004). Colour constancy in diurnal and nocturnal hawkmoths. J. Exp. Biol. 207,3307 -3316.[Abstract/Free Full Text]

Bell, R. A. and Joachim, F. G. (1976). Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms. Ann. Entomol. Soc. Am. 69,365 -373.

Borg-Karlson, A. K. (1990). Chemical and ethological studies of pollination in the genus Ophrys (Orchidaceae). Phytochemistry 29,1359 -1387.[CrossRef]

Brantjes, N. B. M. (1978). Sensory responses to flowers in night-flying moths. In The Pollination of Flowers by Insects (ed. A. J. Richards), pp. 13-19. London: Academic Press.

Brantjes, N. B. M. and Bos, J. J. (1980). Hawkmoth behaviour and flower adaptation reducing self-pollination in two Liliiflorae. New Phytol. 84,139 -143.[CrossRef]

Chittka, L. and Thomson, J. D. (1997). Sensori-motor learning and its relevance for task specialization in bumble bees. Behav. Ecol. Sociobiol. 41,385 -398.[CrossRef]

Chittka, L., Shmida, A., Troje, N. and Menzel, R. (1994). Ultraviolet as a component of flower reflections, and the colour perception of Hymenoptera. Vision Res. 34,1489 -1508.[CrossRef][Medline]

Cunningham, J. P., West, S. A. and Wright, D. J. (1998). Learning in the nectar foraging behaviour of Helicoverpa armigera. Ecol. Entomol. 23,363 -369.

Cutler, D. E., Bennett, R. R., Stevenson, R. D. and White, R. H. (1995). Feeding behavior in the nocturnal moth Manduca sexta is mediated mainly by blue receptors, but where are they located in the retina? J. Exp. Biol. 198,1909 -1917.[Medline]

Daly, K. C. and Smith, B. H. (2000). Associative olfactory learning in the moth Manduca sexta. J. Exp. Biol. 203,2025 -2038.[Abstract]

Daly, K. C., Chandra, S., Durtschi, M. L. and Smith, B. H. (2001). The generalization of an olfactory-based conditioned response reveals unique but overlapping odour representations in the moth Manduca sexta. J. Exp. Biol. 204,3085 -3095.[Abstract/Free Full Text]

Darwin, C. (1895). On the Effects of Cross- and Self-fertilization in the Vegetable Kingdom. New York: Appleton.

Daumer, K. (1958). Blumenfarben, wie sie die Bienen sehen. Z. Vergl. Physiol. 41, 49-110.

Desai, S. and Raguso, R. A. (2001). Flower morphology, learning ability and nectar discovery time by Manduca sexta.Am. Zool. 41,1426 .

Fleming, R. C. (1970). Food plants of some adult sphinx moths (Lepidoptera: Sphingidae). Mich. Entomol. 3,17 -23.

Free, J. B. (1979). Effect of flower shape and nectar guides on the behaviour of foraging honeybees. Behavior 37,269 -281.

Glover, B. J. and Martin, C. (1998). The role of petal cell shape and pigmentation in pollination success in Antirrhinum majus. Heredity 80,778 -784.[CrossRef]

Grant, V. (1983). The systematic and geographical distribution of hawkmoth flowers in the temperate North American flora. Bot. Gaz. 144,439 -449.[CrossRef]

Haber, W. A. (1984). Pollination by deceit in a mass-flowering tropical tree Plumeria rubra L. (Apocynaceae). Biotropica 16,269 -275.[CrossRef]

Haber, W. A. and Frankie, G. W. (1989). A tropical hawkmoth community: Costa Rican dry forest Sphingidae. Biotropica 21,155 -172.[CrossRef]

Harder, L. D. (1983). Flower handling efficiency of bumble bees: morphological aspects of probing time. Oecologia 57,274 -280.[CrossRef]

Hartlieb, E. (1996). Olfactory conditioning in the moth Heliothis virescens. Naturwissenschaften 83,87 -88.

Heinrich, B. (1971). Temperature regulation of the sphinx moth, Manduca sexta. I. Flight energetics and body temperature during free and tethered flight. J. Exp. Biol. 54,141 -152.[Abstract/Free Full Text]

Hilgard, E. R. and Bower, G. H. (1966). Theories of Learning. New York: Appleton-Century-Crofts.

Hughes, R. N. and Seed, R. (1981). Optimal diets under the energy maximization premise: the effects of recognition and learning. Am. Nat. 113,209 -221.[CrossRef]

Johnson, S. D. and Dafni, A. (1998). Response of bee flies to the shape and pattern of model flowers: implications for floral evolution in Mediterranean herbs. Funct. Ecol. 12,289 -297.[CrossRef]

Kaiser, R. (1993). The Scent of Orchids – Olfactory and Chemical Investigations. Amsterdam: Elsevier.

Kelber, A. (1996). Colour learning in the hawkmoth Macroglossum stellatarum. J. Exp. Biol. 199,1227 -1231.

Kelber, A. (1997). Innate preferences for flower features in the hawkmoth Macroglossum stellatarum. J. Exp. Biol. 200,827 -836.[Abstract]

Kelber, A. (2003). Sugar preferences and feeding strategies in the hawkmoth Macroglossum stellatarum. J. Comp. Physiol. 189A,661 -666.

Kelber, A. and Hénique, U. (1999). Trichromatic colour vision in the hummingbird hawkmoth, Macroglossum stellatarum L. J. Comp. Physiol. 184A,535 -541.[CrossRef]

Kelber, A., Balkenius, A. and Warrant, E. J. (2002). Scotopic colour vision in nocturnal hawkmoths. Nature 419,922 -925.[CrossRef][Medline]

Kevan, P. G. and Lane, M. A. (1985). Flower petal microtexture is a tactile cue for bees. Proc. Natl. Acad. Sci. USA 82,4750 -4752.[Abstract/Free Full Text]

Knoll, F. (1922). Lichtsinn und blumenbesuch des falters Macroglossum stellatarum. Abh. Zool. Bot. Ges. Wien 12,125 -377.

Knoll, F. (1926). Insekten und blumen. Abh. Zool. Bot. Ges. Wien 12, 1-645.

Knudsen, J. and Tollsten, L. (1993). Trends in floral scent chemistry in pollination syndromes: floral scent composition in moth pollinated taxa. Bot. J. Linn. Soc. 113,263 -284.[CrossRef]

Krenn, H. W. (1998). Proboscis sensilla in Vanessa cardui (Nymphalidae, Lepidoptera): functional morphology and significance in flower-probing. Zoomorphology 118, 23-30.[CrossRef]

Kugler, H. (1966). UV-male auf blüten. Ber. Dtsch. Bot. Ges. 79, 57-70.

Kugler, H. (1971). Zur bestäubung grossblumiger Datura arten. Flora 160,511 -517.

Laverty, T. M. and Plowright, R. C. (1988). Flower handling by bumblebees: a comparison of specialists and generalists. Anim. Behav. 36,733 -740.[CrossRef]

Leppik, E. E. (1956). The form and function of numerical patterns in flowers. Am. J. Bot. 43,445 -455.[CrossRef]

Lewis, A. C. (1986). Memory constraints and flower choice in Pieris rapae. Science 232,863 -865.[Abstract/Free Full Text]

Lunau, K., Wacht, S. and Chittka, L. (1996). Colour choices of naïve bumble bees and their implications for colour perception. J. Comp. Physiol. A 178,477 -489.

Manning, A. (1956). The effect of honey-guides. Behavior 9,114 -139.

Menzel, R. and Shmida, A. (1993). The ecology of flower colours and the natural colour vision of insect pollinators: the Israeli flora as a case study. Biol. Rev. 68, 81-120.

Mondello, L., Verzera, A., Previti, P., Crispo, F. and Dugo, G. (1998). Multidimensional capillary GC-GC for the analysis of complex samples. 5. Enantiomeric distribution of monoterpene hydrocarbons, monoterpene alcohols and linalyl acetate of bergamot (Citrus bergamia Risso et Poiteau) oils. J. Agric. Food Chem. 46,4275 -4282.[CrossRef]

Nattero, J., Moré, M., Sérsic, A. N. and Cocucci, A. A. (2003). Possible tobacco progenitors share long-tongued hawkmoths as pollen vectors. Plant Sys. Evol. 241, 47-54.[CrossRef]

Nilsson, L. A. (1988). The evolution of flowers with deep corolla tubes. Nature 334,147 -149.[CrossRef]

Nilsson, L. A., Jonsson, L., Ralison, L. and Randrianjohany, E. (1987). Angraecoid orchids and hawkmoths in central Madagascar: specialized pollination systems and generalist foragers. Biotropica 19,310 -318.[CrossRef]

Pyke, G. H., Pulliam, H. R. and Charnov, E. L. (1977). Optimal foraging: a selective review of theory and tests. Q. Rev. Biol. 52,137 -154.[CrossRef]

Raguso, R. A. and Willis, M. A. (2002). Synergy between visual and olfactory cues in nectar feeding by naive hawkmoths. Anim. Behav. 64,685 -695.[CrossRef]

Raguso, R. A. and Willis, M. A. (2003). Hawkmoth pollination in Arizona's Sonoran Desert: behavioral responses to floral traits. In Evolution and Ecology Taking Flight: Butterflies as Model Systems, Rocky Mountain Biological Lab Symposium Series (ed. C. L. Boggs, W. B. Watt and P. R. Ehrlich), pp.43 -65. Chicago: University of Chicago Press.

Raguso, R. A. and Willis, M. A. (2005). Synergy between visual and olfactory cues in nectar feeding by wild hawkmoths Manduca sexta. Anim. Behav. 69,407 -418.[CrossRef]

Raguso, R. A., Henzel, C., Buchmann, S. L. and Nabhan, G. P. (2003). Trumpet flowers of the Sonoran Desert: floral biology of Peniocereus cacti and Sacred Datura. Int. J. Plant Biol. 164,877 -892.

Raguso, R. A., LeClere, A. R. and Schlumpberger, B. O. (2005). Sensory flexibility in hawkmoth foraging behavior: lessons from Manduca sexta and other species. Chem. Senses 30,i295 -i296.[Free Full Text]

Sprengel, F. C. (1793). Das entdeckte Geheimnis der Natur im Bau und in der Befruchtung der Blumen. Leipzig: Englemann.

Waser, N. M and Price, M. V. (1983). Pollinator behaviour and natural selection for flower colour in Delphinium nelsonii.Nature 302,422 -424.[CrossRef]

White, R. H., Stevenson, R. D., Bennett, R. R., Cutler, D. E. and Haber, W. A. (1994). Wavelength discrimination and the role of ultraviolet vision in the feeding behavior of hawkmoths. Biotropica 26,427 -435.

Ziegler, R. and Schultz, M. (1986). Regulation of lipid metabolism during flight in Manduca sexta. J. Insect Physiol. 32,903 -908.[CrossRef]

Related articles in JEB:

PROBING PROBOSCIS LEADS MOTH TO NECTAR

Kathryn Phillips
JEB 2006 209: 0. [Full Text]  

This article has been cited by other articles:

				J. Goyret, P. M. Markwell, and R. A. Raguso Chemical Ecology Special Feature: Context- and scale-dependent effects of floral CO2 on nectar foraging by Manduca sexta PNAS, March 25, 2008; 105(12): 4565 - 4570. [Abstract] [Full Text] [PDF]

				J. Goyret, P. M. Markwell, and R. A. Raguso The effect of decoupling olfactory and visual stimuli on the foraging behavior of Manduca sexta J. Exp. Biol., April 15, 2007; 210(8): 1398 - 1405. [Abstract] [Full Text] [PDF]

				D. J. Martins and S. D. Johnson Hawkmoth pollination of aerangoid orchids in Kenya, with special reference to nectar sugar concentration gradients in the floral spurs Am. J. Botany, April 1, 2007; 94(4): 650 - 659. [Abstract] [Full Text] [PDF]

				J. D. H. Sprayberry and T. L. Daniel Flower tracking in hawkmoths: behavior and energetics J. Exp. Biol., January 1, 2007; 210(1): 37 - 45. [Abstract] [Full Text] [PDF]

				T. Seidl and R. Wehner Visual and tactile learning of ground structures in desert ants J. Exp. Biol., September 1, 2006; 209(17): 3336 - 3344. [Abstract] [Full Text] [PDF]

				K. Phillips PROBING PROBOSCIS LEADS MOTH TO NECTAR J. Exp. Biol., May 1, 2006; 209(9): ii - ii. [Full Text] [PDF]

This Article Summary Figures Only Full Text (PDF) A corrigendum has been published Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JEB Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Goyret, J. Articles by Raguso, R. A. Search for Related Content PubMed PubMed Citation Articles by Goyret, J. Articles by Raguso, R. A.