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

The role of mechanosensory input in flower handling efficiency and learning by Manduca sexta The Journal of Experimental Biology 209, 2238 Published by The Company of Biologists 2006 doi:10.1242/jeb.02291 Corrigendum Goyret, J. and Raguso, R. A. (2006). The role of mechanosensory input in flower handling efficiency and learning by Manduca sexta. J. Exp. Biol. 209, 1585-1593. The legend to Fig. 1 was published incorrectly in both the print and on-line versions of the article. The correct legend should read: 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·cm9·cm) sheet of acetate film. Arrows and brackets indicate a priori comparisons: (I) ‘Half lobes vs medium disks’ compares flowers with similar surface area but different edge-to-center distances. (II) ‘Half lobes vs small disks’ – compares flowers that have different surface area but the same edge- to-center distances. Note: all flowers have accessible nectaries at their centers. The authors apologise for this error and any inconvenience caused. 1585 The Journal of Experimental Biology 209, 1585-1593 Published by The Company of Biologists 2006 doi:10.1242/jeb.02169 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: [email protected]) Accepted 13 February 2006 Summary Nectar-foraging animals are known to utilize nectar foraging bouts to test whether moths can learn to handle guides – patterns of visual contrast in flowers – to find different kinds of artificial flowers. We found that corolla hidden nectar. However, few studies have explored the surface area negatively affects flower handling efficiency, potential for mechanosensory cues to function as nectar and that reliable mechanosensory input is crucial for the guides, particularly for nocturnal pollinators such as the moths’ performance. We also found that three- tobacco hornworm moth, Manduca sexta. We used arrays dimensional features of the corolla, such as grooves, can of artificial flowers to investigate the flower handling significantly affect the foraging behavior, both positively behavior (the ability to locate and drink from floral (when grooves converge to the nectary) and negatively nectaries) of naïve moths, looking specifically at: (1) how (when grooves are unnaturally oriented). Lastly, we the shape and size of flat (two-dimensional) artificial observed that moths can decrease nectar discovery time corollas affect nectar discovery and (2) whether three- during a single foraging bout. This apparent learning dimensional features of the corolla can be used to facilitate ability seems to be possible only when reliable nectar discovery. In these experiments, we decoupled mechanosensory input is available. visual from tactile flower features to explore the role of mechanosensory input, putatively attained via the extended proboscides of hovering moths. In addition, we Key words: pollination, Lepidoptera, sensory, multimodal, examined changes in nectar discovery times within single Sphingidae. Introduction nectar guides (Leppik, 1956; Glover and Martin, 1998), especially for animals with poor vision, or those that forage One of the ‘mysteries of nature’ revealed by Sprengel’s under low light conditions, such as crepuscular or nocturnal landmark (Sprengel, 1793) publication was the concept of hawkmoths (Lepidoptera: Sphingidae). nectar guides – visually contrasting markings or aspects of Hawkmoths are abundant in tropical and warm-temperate flower morphology – that indicate the location of nectar to habitats worldwide, where they constitute an important class animal pollinators. The ubiquity of such markings, particularly of pollinators (Grant, 1983; Nilsson et al., 1987; Haber and those perceived in ultraviolet (UV) wavelengths, is one of the Frankie, 1989). Olfactory and visual floral stimuli are known primary arguments made for the importance of contrasting to attract several species within an appetitive context (Knoll, flower colors to the visual perception and foraging behavior of 1926; Kugler, 1971; Haber, 1984; Kelber, 1997). The insect pollinators (Menzel and Schmida, 1993; Chittka et al., European Deilephila elpenor and Macroglossum stellatarum 1994; Lunau et al., 1996). For example, honeybees show an utilize true color vision even under starlit conditions (Kelber innate proboscis extension reflex (PER) to UV marks at the and Hénique, 1999; Kelber et al., 2002), and modify their center of Helianthus rigidus sunflowers, and probe at the innate odor and color preferences through associative learning periphery of the flower when the orientation of the ray florets is reversed (Daumer, 1958). However, it is unlikely that vision (Kelber, 1996; Balkenius and Kelber, 2004). Manduca sexta, is the only sensory modality used by animals to find the nectar a large nocturnal hawkmoth native to the Americas, also can learn particular odors associated with nectar rewards (Daly and within flowers. Kevan and Lane (Kevan and Lane, 1985) showed that honeybees can detect differences in petal surface Smith, 2000; Daly et al., 2001a). The blue photoreceptors have cell texture, and can learn such differences in conjunction with been identified as the major visual mediators of feeding nectar rewards. Thus, tactile floral cues also could function as behavior in M. sexta (Cutler et al., 1995), whereas ultraviolet THE JOURNAL OF EXPERIMENTAL BIOLOGY 1586 J. Goyret and R. A. Raguso wavelengths were found to inhibit its feeding response (White artificial flower morphologies by comparing total, successful et al., 1994). Floral odors attract M. sexta from a distance (3·m) and unsuccessful visits of individual moths foraging on arrays in wind tunnel assays (Raguso and Willis, 2003; Raguso et al., of 12 flowers. Finally, we examined whether moths can learn 2005), and synergize with visual cues to activate feeding to handle different flower morphs more efficiently within a behavior (i.e. proboscis extension while hovering) in both single foraging bout by examining the time they took to find naïve and wild moths (Raguso and Willis, 2002; Raguso and nectaries as foraging bouts progressed. Willis, 2005). However, successful approach to floral nectar sources and Materials and methods release of feeding behavior must be followed by reliable nectar Animal care assessment of individual flowers. Locating the nectary within a flower (evaluating the energy resource) is as critical as This study was carried out from September to December searching efficiently in order to find that flower. The hovering 2004 at the University of South Carolina, Columbia, SC, USA. flight of M. sexta is an energetically expensive activity We used 3- to 5-day-old M. sexta L. adults reared from eggs (Heinrich, 1971; Ziegler and Schulz, 1986), thus, the efficiency provided by Dr Lynn Riddiford, University of Washington, with which these moths handle flowers should be subject to Seattle, WA, USA. Larvae were fed ad libitum on an artificial selective pressures. Manduca sexta has a broad geographical diet (Bell and Joachim, 1976) and were kept, as pupae, under distribution with several generations per year and it visits a a 16·h:8·h light:dark cycle (24:21°C), in a humidified wide variety of flower types across its range (Fleming, 1970; atmosphere. Male and female pupae were kept in separate Raguso et al., 2003; Nattero et al., 2003). These observations incubators (Precision 818, Winchester, VA, USA) under the led us to ask whether M. sexta can handle some flower same ambient regime and emerged within 454545·cm morphologies more easily than others, and whether they can screen cages (BioQuip, Inc., Rancho Dominguez, CA, USA). learn to handle flowers more efficiently with time. Such Adults were starved for 3–4·days before being used in abilities would be consistent with their generalist foraging experiments. behavior and would allow these moths to efficiently assess Experimental arena and flight assays flower profitability, as do other generalist flower visiting insects, such as bumblebees (Laverty and Plowright, 1988; At the beginning of scotophase (15:00·h, temperature Chittka and Thomson, 1997) and Pieris butterflies (Lewis, range: 22–25°C), naïve moths were placed individually 1986). within a closed Tedlar mesh flight enclosure (Bioquip; The question remains as to which sensory modalities adult 2·m2·m2·m). The flight cage included an experimental M. sexta might utilize for such a task. The diurnal hawkmoth floral array (20·cm30·cm45·cm) placed over a dark, odor- Macroglossum stellatarum utilizes contrasting marks on the permeable box constructed by covering a matte-black-painted surface of flower corollas by preferentially placing its aluminum grid with black cheesecloth. To provide proboscis on such visual nectar guides (Knoll, 1922). Thus, M. appropriate olfactory cues and humidity, we placed the stellatarum uses visual cues not only while searching (in flight) cheesecloth-covered grid over two 200·ml glass beakers filled for nectar sources (Kelber, 1997), but also while hovering a with water, each of which contained a cotton-tipped relatively short distance (proboscis length: 2.5·cm) in front of applicator swab impregnated with two drops of undiluted individual flowers. Owing to its long (8–10·cm) tongue, M. bergamot oil (Body Shop, Columbia, SC, USA). Thus, odor sexta also hovers at a distance from flowers while feeding, such and water vapor passed through the cheese cloth and that in most cases, its only physical contact with flowers is permeated the flight chamber. Bergamot oil is chemically through the proboscis. Here we ask whether mechanosensory similar to the odors of many hawkmoth-pollinated flowers input to the proboscis is redundant or complementary to the (Kaiser, 1993; Knudsen and Tollsten, 1993; Mondello et al., visual stimuli used by M. sexta when freely foraging on 1998), and pilot experiments revealed it to be a potent artificial flowers. In the first experiment, we decoupled visual releaser of feeding behavior in M. sexta. Visual floral stimuli from tactile stimuli by placing flat square transparency film were provided by a 34 array of artificial flowers (see sheets over the corolla portion of plain-white artificial flowers below), in which each flower was separated from its neighbor to test whether these moths use mechanosensory stimuli to find by 10·cm. Artificial flowers were bathed in odor and water nectar within individual flowers. If visual stimuli are sufficient, vapor that diffused freely through the cheesecloth. The flight hawkmoths should show comparable handling efficiencies on enclosure was lit with a dim red light [wavelengths >600·nm the same flower models, whether or not they are covered with (see Raguso and Willis, 2002)]. Each trial involved only one transparency film. We repeated this comparison among five moth, which was allowed to fly freely. If the moth did not different artificial flower morphologies, systematically varying find, approach or probe the flowers within 5·min, it was corolla shape and surface area. captured and discarded. If it found the flowers, it was allowed In the second experiment, we tested whether groove-like to forage for a maximum of 10·min after the first floral folds, usually found in the corollas of flowers visited by approach. Foraging bouts were recorded with a video camera hawkmoths, affect flower handling by M. sexta. We also (Sony Digital 8 –TRV120 Best Buy, Columbia, SC, USA) in evaluated flower handling performance in relation to different ‘night-shot’ mode placed outside the flight enclosure. THE JOURNAL OF EXPERIMENTAL BIOLOGY Flower handling by Manduca sexta 1587 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 II (9·cm9·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 Full lobes Half lobes Large disk Medium disk Small disk their centers. Experiment 1 were recorded from video-tape playback and timed with a In each trial, individual moths were offered different Mistral chronometer (Buenos Aires, Argentina) to a resolution homogeneous arrays (12 flowers of the same morph) displayed of 1·s. as described above. We used light-grey paper with low UV Each flower visit began at the moment the proboscis made reflectance (Kinkos ‘Grey fleck’; wavelength reflectance 80% contact with the flower. Unsuccessful visits ended when the of a barium sulfate ‘white’ standard above 420·nm, <50% proboscis lost contact with the artificial flower without having below 400·nm) to construct five different flower morphs reached the nectary. Successful visits were recorded until the (Fig.·1A), as follows. Full lobes: four elliptical lobes or petals proboscis was inserted into the nectary; when drinking time with a semi-major axis of 2.2·cm and a semi-minor axis of was recorded as the time elapsed until the proboscis was 0.8·cm each. Total area: 21.4·cm . Flower span: 9·cm. Half removed. The ratio of successful to total visits (successful lobes: four elliptical lobes with a semi-major axis of 3.2·cm visits/total visits; where total visits = unsuccessful visits + and a semi-minor axis of 1.3·cm each. Every lobe overlaps with successful visits) was established as an indicator of the the adjacent ones leaving a squared center with sides of 2.7·cm. animals’ efficiency when foraging on the different flower Half of each ellipse appears as a petal. Total area, 33.7·cm ; morphs. flower span, 9·cm. Large disk: a disk with a diameter of 9·cm Given that we had recorded the time moths took visiting and an area of 63.6·cm . Medium disk: a disk with a diameter each flower, we tested whether moths could learn to handle the of 6.5·cm and an area of 33.2·cm . Small disk: a disk with a different flower morphs during a single foraging bout. diameter of 4·cm and an area of 12.57·cm . Additionally, the Discovery time was defined by the time elapsed between the corolla portion of each of the five flower morphs was covered initiation of flower probing and the entry of the proboscis into with a square transparency film sheet (henceforth called the nectary. This does not include the time flying from one transparency treatments; Fig.·1B), accounting for a total of 10 flower to another or drinking, but only the time spent probing treatments. In this way, we could evaluate the foraging at the flower’s threshold. We measured discovery time for the behavior under circumstances where no reliable tactile stimuli first eight successful visits, as did Lewis (Lewis, 1986). were available to the animals, but visual stimuli could be Experiment 2 preserved. To control for any surface texture effect, flowers in the treatments lacking a transparency square above them A second experiment was carried out to evaluate whether M. (henceforth called no transparency treatments) were covered sexta can use morphological features of flowers involving a with transparency film that had been cut to match the exact third dimension (i.e. depth) to improve its foraging efficiency. shape of the underlying paper flower. Each flower from every Many night-blooming flowers (e.g. Datura, Mirabilis) have treatment offered 20·l of a 20% (w/w) sucrose solution in a conspicuously grooved petals, which could in theory be used nectary (5·cm long by 0.5·cm opening diameter pipette tip) as tactile guides for the moths’ proboscides (Fig.·6). Thus, placed at its center; nectaries were accessible to moths through three treatments were designed. The first was ‘medium disks’, a 0.5·cm opening cut into the transparency film in both the same flat flowers used in Experiment 1. The second and treatments. Each nectary was attached to the flower such that third were paper disks of the same diameter as medium disks, it did not protrude above the flower surface. with two groove-like folds (see Fig.·2). In the second treatment, the folds were oriented parallel to each other (‘chord Variables recorded grooves’) and were placed 1.5·cm apart from the origin We recorded the foraging efficiency (number of successfully (nectary) of the disk (Fig.·2). In the third treatment, the folds exploited flowers over 10·min) after each trial. The number and were placed as two orthogonal diameters of the disk (‘radial duration of total, successful and unsuccessful flower visits grooves’), intersecting at the nectary (Fig.·2). THE JOURNAL OF EXPERIMENTAL BIOLOGY 1588 J. Goyret and R. A. Raguso Statistical analysis Response levels of male and female M. sexta to different flower morphs in Experiment 1 were tested by means of log- likelihood tests (G-tests using the Gh test statistic). Foraging efficiency, measured with the variables, emptied flowers and ratio of successful/total visits, was tested with the Kruskal–Wallis non-parametric test using a corrected -level of significance of 0.005. Thus, we performed ten statistical tests using the same set of data: six for emptied flowers, three for ratio of successful/total visits and one linear regression). Discovery time as a function of the sequence of feeding attempts was tested to fit the classic exponential decline learning curve described by Hilgard and Bower (Hilgard and Bower, 1966). A corrected -level of significance of 0.008 was used in these tests (six regression analyses). 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 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 =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 =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:  =18.43; P<0.0001; Table·1, Fig.·3). In (1,0.005) Medium disk Radial folds Chord folds 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. THE JOURNAL OF EXPERIMENTAL BIOLOGY Table·1. Variables recorded in relation to flower handling by Manduca sexta on different flower morphs with and without a square transparency film No transparency film Transparency film Number of visits (mean) FL(16) HL(18) LD(17) MD(16) SD(16) FL(17) HL(19) LD(17) MD(16) SD(16) Total 85.11±8.7 73.20±15.4 48.25±10.3 81.69±10.5 77.88±11.1 65.53±16.4 74.88±9.2 54.44±10 39.20±6.9 85.11±11.81 Successful 50.39±5.9 21.4±4.4 5.81±2.2 18.38±3.7 51.18±6 8.63±2.7 13.47±3.6 6.89±2.8 4.13±1.4 22.69±5.6 Failed 34.72±5.6 51.8±7.9 42.44±9.5 63.31±6.5 26.71±4.2 60.94±13.9 61.41±8.8 47.56±14.1 35.07±8.8 41.63±8.6 Ratio of successful/ 0.6±0.04 0.24±0.04 0.08±0.02 0.21±0.02 0.65±0.05 0.08±0.02 0.15±0.03 0.09±0.03 0.07±0.02 0.26±0.07 total All values are means ± s.e.m. Numbers in parentheses are the number of replicates for each treatment. FL, full lobe; HL, half lobe; LD, large disc; MD, medium disc; SD, small disk. Flower handling by Manduca sexta 1589 (Kruskal–Wallis test;  =18.34; P<0.0001; -level: 0.005). (1) Moreover, the number of emptied flowers was significantly correlated with flower surface area (a+x=y; a=14.33; =–0.75; R =0.56; F =105.5; P<0.0001). (1,0.005,84) 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 2 =30.48; P<0.0001; within transparency:  =59.29; (1) (4) 4 P<0.0001; within no transparency:  =7.39; P=0.12). a (4,0.005) 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: R =84.35; P=0.001) as illustrated by Fig.·4A (full lobe with no Fig.·3. Number of emptied flowers (foraging efficiency; mean ± transparency; exponential decline function fit: R =84.87; s.e.m.) after a 10·min foraging bout by individual Manduca sexta P=0.001). Nevertheless, this was not the case for the large disk inside the flight chamber. In each treatment (abscissa) an array of 12 treatment (Fig.·4C; exponential decline function fit: R =37.99; artificial flowers of the same morph was present. Black bars represent P=0.10). When flowers were covered by a transparency film, responses to artificial flowers without square transparency film; gray discovery times did not conform to a typical learning curve bars represent responses to the same artificial flowers covered with a (transparency treatment; exponential decline function fit: square transparency film. Different letters denote statistically R =56.58; P=0.031; corrected -level of significance: 0.005), significant differences with a corrected -level for significance of as shown in Fig.·4B,C (full lobe with transparency: R =8.89; 0.008 (see text). P=0.47; large disk with transparency: R =0.0; P=1.0). Experiment 2 addition, variation in emptied flowers was significantly affected by flower morphology among the no transparency Flower morphology was significantly associated with the treatments (Kruskal–Wallis test; within no transparency: number of emptied flowers when moths foraged on different =44.64; P<0.0001). This effect was not observed flower arrays (ANOVA: F =21.11; P<0.0001). Both kinds (4,0.005) (2,57) among transparency treatments; in this case differences were of grooved artificial flowers affected the performance of not as pronounced, only accounting for a trend foraging moths, but in opposite ways (Fig.·5). Moths (Kruskal–Wallis median test; within transparency: performed worse on flowers with chord grooves than on flat =10.18; P=0.04). control flowers (medium disk vs chord grooves: F =78.9; (4,0.005) (1,39) Among the no transparency treatments, moths clearly were P<0.0001), whereas moth performance on flowers with radial more successful when handling full lobe and small disk morphs grooves was significantly better than on flat control flowers (see Fig.·3). As flower surface area increased from small disk (medium disk vs radial grooves: F =328.11; P<0.0001). (1,37) 2 2 (12.6·cm ) to large disk (63.6·cm ), moth performance declined (see regression analysis below). this same effect was 2 Discussion observed when comparing full lobe (21.4·cm ), half lobe Behavioral sequence of flower foraging and its distinct (33.7·cm ) and large disk (Kruskal–Wallis test; full lobe vs half sensory modalities lobe:  =6.07; P=0.014; large disk vs medium disk: (1,0.005) =16.63; P<0.0001). Because surface area is not the The foraging behavior of Manduca sexta appears to follow (1,0.005) only flower feature that varied among treatments, we tested a sequential pattern involving different sensory modalities at whether the minimum distance from the edge to the nectary each stage (Raguso and Willis, 2003). Thus, a moth under (center) of the flower could affect moths’ performance appetitive motivation will first fly upwind when encountering independently. Half lobe and medium disk flowers have similar an appropriate fragrance (Brantjes, 1978). At closer range, 2 2 surface areas (33.7·cm and 33.2·cm , respectively) but flower approach by M. sexta is guided by either olfactory or different edge-to-center distances (2·cm and 3.25·cm, visual stimuli, whereas proboscis extension requires the respectively). Similarly, half lobe and small disk flowers have combination of visual and olfactory cues (Raguso and Willis, the same minimum edge-to-center distance (2·cm) but different 2002; Raguso and Willis, 2005). Here we show that 2 2 surface areas (33.7·cm and 12.6·cm , respectively). Surface mechanoreception is an additional sensory modality that area appeared to be a more important flower feature than edge- contributes to the final stage of the feeding sequence, once the to-center distance, as feeding effectiveness did not differ proboscis is extended and moths must locate and drink from significantly between half lobe and medium disk flowers floral nectaries, a process frequently referred to as ‘flower (Kruskal–Wallis test;  =0.005; P=0.945), but differed handling’. (1,0.005) significantly between half lobe and small disk flowers In our experiments, moths were effectively and equally THE JOURNAL OF EXPERIMENTAL BIOLOGY Number of emptied flowers 1590 J. Goyret and R. A. Raguso R =8.89 P=0.47 20 R =84.87 Fig.·4. Discovery time (probing P=0.0012 time between feeding attempts) for four different treatments. 10 10 (A) Full lobe, (B) full lobe with transparency film, (C) large disk 5 5 and (D) large disk with transparency film. Data points are 0 0 medians, whiskers represent first and third quartiles. Statistical values refer to goodness-of-fit to 2 40 R =0 R =37.99 an exponential decline function P=1.0 P=0.10 (one factor), a classical ‘learning curve’. Moths exploiting full lobe flowers with no transparency film show exponentially decreasing discovery times. When exploiting 10 10 large disks, or either shape with transparency film, moths show larger variances in their 0 02468 02468 responses, which do not fit an exponential decline function. Number of attempts attracted to the different artificial paper flowers, regardless of transparency films used to de-couple visual and mechanical the fact that they differed in shape and size (which in turn, stimuli. greatly affected performance), when paper flower arrays were Vision and mechanoreception during flower probing presented with Bergamot oil as an olfactory stimulus (see Results). This result indicates that probing responses (i.e. What is the innate probing strategy of M. sexta? Is the emptied flowers) to different treatments were not confounded proboscis guided visually or are there other sensory systems by innate differences in attractiveness, and that no biases in involved? The use of different artificial flowers affected the moth preference or attraction were associated with 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 B 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 8 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 0 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 N=20 N=19 N=21 ‘random walk’ of probing across the flower’s surface. The disruption of reliable tactile information clearly Fig.·5. Three-dimensional corolla features affect foraging efficiency interferes with flower handling by M. sexta, showing that by Manduca sexta. The vertical bars represent number of flowers mechanoreception, in addition to vision and olfaction, is (mean ± s.e.m.) emptied after a 10·min foraging bout by individual involved in nectar feeding by these moths (Fig.·3). Tactile cues moths inside the flight chamber. In each treatment (abscissa) an array constitute an important component of many flower–pollinator of 12 artificial flowers of the same morph was present. Different letters denote statistically significant differences (see text). systems (Kevan and Lane, 1985; Borg-Karlson, 1990), but are THE JOURNAL OF EXPERIMENTAL BIOLOGY Number of emptied flowers Discovery time (s) Flower handling by Manduca sexta 1591 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 Fig.·6. Features of flower handling are illustrated in this photo of of M. sexta’s eyes does not allow for accurate feedback about Manduca sexta feeding from a flower of Mirabilis multiflora proboscis position (A. Kelber, personal communication). The (Nyctaginaceae). Note the extended proboscis (grey arrow), the low signal-to-noise ratio of the visual modality at this scale distance of the moth’s body from the flower, and the radial grooves could have imposed selective pressures for M. sexta to © in the flower’s perianth (white arrows). Scale bar, 1·cm. Photo efficiently assess floral nectar content by other means. Such Robert A. Raguso. 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 disks (Fig.·3). It appears that the decrement in flower handling sensilla positioned along the tip of the lepidopteran proboscis by M. sexta on flowers with high surface area is offset by floral (Krenn, 1998; Kelber, 2003). depth. However, attraction from a distance is enhanced by the Sprengel (Sprengel, 1793) introduced the concept of nectar increased visual display provided by flowers with larger guides as floral features that could be used by pollinators to diameters (Knoll, 1922). Tubular flowers appear to offer a visually locate the nectar. Subsequent experiments revealed compromise solution to this hypothetical trade-off, while that the diurnal hawkmoth Macroglossum stellatarum (Knoll, simultaneously providing for high nectar volumes and 1922), bumblebees (Manning, 1956; Kugler, 1966), honeybees appropriate physical contact between the body of the moth and (Daumer, 1958; Free, 1979) and bee-flies (Johnson and Dafni, the sexual organs of the flower (Nilsson, 1988). It is tempting to 1998), among other insects, successfully utilize visual nectar consider how differences in handling efficiency associated with guides. Here we show that the utility of Spengel’s idea extends corolla form might impact competition between night blooming beyond the visual system, as the tactile sensitivity of the flowers for hawkmoths as pollinators (see Haber and Frankie, proboscis of M. sexta allows these moths to exploit the physical 1989), however, most flowers in nature are likely to be visited features of flowers in order to find nectar (Figs·3, 5, 6). Our by experienced moths. Additional experiments will be needed to experiments, unlike those of Knoll (Knoll, 1922; Knoll, 1926), determine whether the handling differences identified in this varied the contours of artificial flowers, rather than testing study have an impact on subsequent foraging decisions. moth responses to natural flowers. Further experiments will be Flower handling improves with experience required to test whether visual nectar guides of color contrast can be used by M. sexta. We analyzed whether M. sexta could learn to improve its handling abilities (i.e. reduce the time to find nectar) during a Context dependence of the floral visual display single foraging bout. Indeed, M. sexta adults improve their This study indicates that once moths approach a flower handling of artificial flowers within an extended feeding bout patch, they extend their proboscides towards a visual target and (Fig.·4). We analyzed improvement in flower handling overall then appear to rely on mechanosensory input. At this point, (with and without transparency film) and within two specific when probing is relatively random, any irregularity on the treatments – full lobe and large disk – as examples of flowers corolla surface could guide moths’ searching behavior, such that elicited high and low performances, respectively. Flower that the proboscis ‘rides’ along the length of petal grooves, handling did not improve on model flowers in which the nectary openings or the margins of highly divided corollas. nectary was difficult to find (large circles), nor when square The funnel-shaped flowers of Datura wrightii, a favored transparency films prevented the acquisition of reliable nectar source of M. sexta in the Sonoran Desert (Raguso et al., mechanosensory information (see Fig.·4B,D). This suggests 2003) are comparable in diameter to the large disk models in that reliable tactile information is needed not only to forage Experiment 1, but previous experiments indicate that Datura efficiently (Fig.·3), but also to learn to forage more efficiently flowers are learned very quickly by naïve M. sexta (Desai and (slope of learning curves, see Results and Fig.·4). Raguso, 2001), which is not the case when foraging on our large Learned improvement in flower handling has been shown in THE JOURNAL OF EXPERIMENTAL BIOLOGY 1592 J. Goyret and R. A. Raguso a variety of nectivorous insects, including other lepidopterans Chittka, L. and Thomson, J. D. (1997). Sensori-motor learning and its relevance for task specialization in bumble bees. Behav. Ecol. Sociobiol. 41, (Lewis, 1986; Hartlieb, 1996; Cunningham et al., 1998) and 385-398. hymenopterans (Harder, 1983; Laverty and Plowright, 1988; Chittka, L., Shmida, A., Troje, N. and Menzel, R. (1994). Ultraviolet as a Chittka and Thomson, 1997). This ability gives animals the component of flower reflections, and the colour perception of Hymenoptera. Vision Res. 34, 1489-1508. opportunity to decrease the time they spend on individual Cunningham, J. P., West, S. A. and Wright, D. J. (1998). 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(1987). flight in Manduca sexta. J. Insect Physiol. 32, 903-908. THE JOURNAL OF EXPERIMENTAL BIOLOGY http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Biology The Company of Biologists

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

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The Journal of Experimental Biology 209, 2238 Published by The Company of Biologists 2006 doi:10.1242/jeb.02291 Corrigendum Goyret, J. and Raguso, R. A. (2006). The role of mechanosensory input in flower handling efficiency and learning by Manduca sexta. J. Exp. Biol. 209, 1585-1593. The legend to Fig. 1 was published incorrectly in both the print and on-line versions of the article. The correct legend should read: 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·cm9·cm) sheet of acetate film. Arrows and brackets indicate a priori comparisons: (I) ‘Half lobes vs medium disks’ compares flowers with similar surface area but different edge-to-center distances. (II) ‘Half lobes vs small disks’ – compares flowers that have different surface area but the same edge- to-center distances. Note: all flowers have accessible nectaries at their centers. The authors apologise for this error and any inconvenience caused. 1585 The Journal of Experimental Biology 209, 1585-1593 Published by The Company of Biologists 2006 doi:10.1242/jeb.02169 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: [email protected]) Accepted 13 February 2006 Summary Nectar-foraging animals are known to utilize nectar foraging bouts to test whether moths can learn to handle guides – patterns of visual contrast in flowers – to find different kinds of artificial flowers. We found that corolla hidden nectar. However, few studies have explored the surface area negatively affects flower handling efficiency, potential for mechanosensory cues to function as nectar and that reliable mechanosensory input is crucial for the guides, particularly for nocturnal pollinators such as the moths’ performance. We also found that three- tobacco hornworm moth, Manduca sexta. We used arrays dimensional features of the corolla, such as grooves, can of artificial flowers to investigate the flower handling significantly affect the foraging behavior, both positively behavior (the ability to locate and drink from floral (when grooves converge to the nectary) and negatively nectaries) of naïve moths, looking specifically at: (1) how (when grooves are unnaturally oriented). Lastly, we the shape and size of flat (two-dimensional) artificial observed that moths can decrease nectar discovery time corollas affect nectar discovery and (2) whether three- during a single foraging bout. This apparent learning dimensional features of the corolla can be used to facilitate ability seems to be possible only when reliable nectar discovery. In these experiments, we decoupled mechanosensory input is available. visual from tactile flower features to explore the role of mechanosensory input, putatively attained via the extended proboscides of hovering moths. In addition, we Key words: pollination, Lepidoptera, sensory, multimodal, examined changes in nectar discovery times within single Sphingidae. Introduction nectar guides (Leppik, 1956; Glover and Martin, 1998), especially for animals with poor vision, or those that forage One of the ‘mysteries of nature’ revealed by Sprengel’s under low light conditions, such as crepuscular or nocturnal landmark (Sprengel, 1793) publication was the concept of hawkmoths (Lepidoptera: Sphingidae). nectar guides – visually contrasting markings or aspects of Hawkmoths are abundant in tropical and warm-temperate flower morphology – that indicate the location of nectar to habitats worldwide, where they constitute an important class animal pollinators. The ubiquity of such markings, particularly of pollinators (Grant, 1983; Nilsson et al., 1987; Haber and those perceived in ultraviolet (UV) wavelengths, is one of the Frankie, 1989). Olfactory and visual floral stimuli are known primary arguments made for the importance of contrasting to attract several species within an appetitive context (Knoll, flower colors to the visual perception and foraging behavior of 1926; Kugler, 1971; Haber, 1984; Kelber, 1997). The insect pollinators (Menzel and Schmida, 1993; Chittka et al., European Deilephila elpenor and Macroglossum stellatarum 1994; Lunau et al., 1996). For example, honeybees show an utilize true color vision even under starlit conditions (Kelber innate proboscis extension reflex (PER) to UV marks at the and Hénique, 1999; Kelber et al., 2002), and modify their center of Helianthus rigidus sunflowers, and probe at the innate odor and color preferences through associative learning periphery of the flower when the orientation of the ray florets is reversed (Daumer, 1958). However, it is unlikely that vision (Kelber, 1996; Balkenius and Kelber, 2004). Manduca sexta, is the only sensory modality used by animals to find the nectar a large nocturnal hawkmoth native to the Americas, also can learn particular odors associated with nectar rewards (Daly and within flowers. Kevan and Lane (Kevan and Lane, 1985) showed that honeybees can detect differences in petal surface Smith, 2000; Daly et al., 2001a). The blue photoreceptors have cell texture, and can learn such differences in conjunction with been identified as the major visual mediators of feeding nectar rewards. Thus, tactile floral cues also could function as behavior in M. sexta (Cutler et al., 1995), whereas ultraviolet THE JOURNAL OF EXPERIMENTAL BIOLOGY 1586 J. Goyret and R. A. Raguso wavelengths were found to inhibit its feeding response (White artificial flower morphologies by comparing total, successful et al., 1994). Floral odors attract M. sexta from a distance (3·m) and unsuccessful visits of individual moths foraging on arrays in wind tunnel assays (Raguso and Willis, 2003; Raguso et al., of 12 flowers. Finally, we examined whether moths can learn 2005), and synergize with visual cues to activate feeding to handle different flower morphs more efficiently within a behavior (i.e. proboscis extension while hovering) in both single foraging bout by examining the time they took to find naïve and wild moths (Raguso and Willis, 2002; Raguso and nectaries as foraging bouts progressed. Willis, 2005). However, successful approach to floral nectar sources and Materials and methods release of feeding behavior must be followed by reliable nectar Animal care assessment of individual flowers. Locating the nectary within a flower (evaluating the energy resource) is as critical as This study was carried out from September to December searching efficiently in order to find that flower. The hovering 2004 at the University of South Carolina, Columbia, SC, USA. flight of M. sexta is an energetically expensive activity We used 3- to 5-day-old M. sexta L. adults reared from eggs (Heinrich, 1971; Ziegler and Schulz, 1986), thus, the efficiency provided by Dr Lynn Riddiford, University of Washington, with which these moths handle flowers should be subject to Seattle, WA, USA. Larvae were fed ad libitum on an artificial selective pressures. Manduca sexta has a broad geographical diet (Bell and Joachim, 1976) and were kept, as pupae, under distribution with several generations per year and it visits a a 16·h:8·h light:dark cycle (24:21°C), in a humidified wide variety of flower types across its range (Fleming, 1970; atmosphere. Male and female pupae were kept in separate Raguso et al., 2003; Nattero et al., 2003). These observations incubators (Precision 818, Winchester, VA, USA) under the led us to ask whether M. sexta can handle some flower same ambient regime and emerged within 454545·cm morphologies more easily than others, and whether they can screen cages (BioQuip, Inc., Rancho Dominguez, CA, USA). learn to handle flowers more efficiently with time. Such Adults were starved for 3–4·days before being used in abilities would be consistent with their generalist foraging experiments. behavior and would allow these moths to efficiently assess Experimental arena and flight assays flower profitability, as do other generalist flower visiting insects, such as bumblebees (Laverty and Plowright, 1988; At the beginning of scotophase (15:00·h, temperature Chittka and Thomson, 1997) and Pieris butterflies (Lewis, range: 22–25°C), naïve moths were placed individually 1986). within a closed Tedlar mesh flight enclosure (Bioquip; The question remains as to which sensory modalities adult 2·m2·m2·m). The flight cage included an experimental M. sexta might utilize for such a task. The diurnal hawkmoth floral array (20·cm30·cm45·cm) placed over a dark, odor- Macroglossum stellatarum utilizes contrasting marks on the permeable box constructed by covering a matte-black-painted surface of flower corollas by preferentially placing its aluminum grid with black cheesecloth. To provide proboscis on such visual nectar guides (Knoll, 1922). Thus, M. appropriate olfactory cues and humidity, we placed the stellatarum uses visual cues not only while searching (in flight) cheesecloth-covered grid over two 200·ml glass beakers filled for nectar sources (Kelber, 1997), but also while hovering a with water, each of which contained a cotton-tipped relatively short distance (proboscis length: 2.5·cm) in front of applicator swab impregnated with two drops of undiluted individual flowers. Owing to its long (8–10·cm) tongue, M. bergamot oil (Body Shop, Columbia, SC, USA). Thus, odor sexta also hovers at a distance from flowers while feeding, such and water vapor passed through the cheese cloth and that in most cases, its only physical contact with flowers is permeated the flight chamber. Bergamot oil is chemically through the proboscis. Here we ask whether mechanosensory similar to the odors of many hawkmoth-pollinated flowers input to the proboscis is redundant or complementary to the (Kaiser, 1993; Knudsen and Tollsten, 1993; Mondello et al., visual stimuli used by M. sexta when freely foraging on 1998), and pilot experiments revealed it to be a potent artificial flowers. In the first experiment, we decoupled visual releaser of feeding behavior in M. sexta. Visual floral stimuli from tactile stimuli by placing flat square transparency film were provided by a 34 array of artificial flowers (see sheets over the corolla portion of plain-white artificial flowers below), in which each flower was separated from its neighbor to test whether these moths use mechanosensory stimuli to find by 10·cm. Artificial flowers were bathed in odor and water nectar within individual flowers. If visual stimuli are sufficient, vapor that diffused freely through the cheesecloth. The flight hawkmoths should show comparable handling efficiencies on enclosure was lit with a dim red light [wavelengths >600·nm the same flower models, whether or not they are covered with (see Raguso and Willis, 2002)]. Each trial involved only one transparency film. We repeated this comparison among five moth, which was allowed to fly freely. If the moth did not different artificial flower morphologies, systematically varying find, approach or probe the flowers within 5·min, it was corolla shape and surface area. captured and discarded. If it found the flowers, it was allowed In the second experiment, we tested whether groove-like to forage for a maximum of 10·min after the first floral folds, usually found in the corollas of flowers visited by approach. Foraging bouts were recorded with a video camera hawkmoths, affect flower handling by M. sexta. We also (Sony Digital 8 –TRV120 Best Buy, Columbia, SC, USA) in evaluated flower handling performance in relation to different ‘night-shot’ mode placed outside the flight enclosure. THE JOURNAL OF EXPERIMENTAL BIOLOGY Flower handling by Manduca sexta 1587 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 II (9·cm9·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 Full lobes Half lobes Large disk Medium disk Small disk their centers. Experiment 1 were recorded from video-tape playback and timed with a In each trial, individual moths were offered different Mistral chronometer (Buenos Aires, Argentina) to a resolution homogeneous arrays (12 flowers of the same morph) displayed of 1·s. as described above. We used light-grey paper with low UV Each flower visit began at the moment the proboscis made reflectance (Kinkos ‘Grey fleck’; wavelength reflectance 80% contact with the flower. Unsuccessful visits ended when the of a barium sulfate ‘white’ standard above 420·nm, <50% proboscis lost contact with the artificial flower without having below 400·nm) to construct five different flower morphs reached the nectary. Successful visits were recorded until the (Fig.·1A), as follows. Full lobes: four elliptical lobes or petals proboscis was inserted into the nectary; when drinking time with a semi-major axis of 2.2·cm and a semi-minor axis of was recorded as the time elapsed until the proboscis was 0.8·cm each. Total area: 21.4·cm . Flower span: 9·cm. Half removed. The ratio of successful to total visits (successful lobes: four elliptical lobes with a semi-major axis of 3.2·cm visits/total visits; where total visits = unsuccessful visits + and a semi-minor axis of 1.3·cm each. Every lobe overlaps with successful visits) was established as an indicator of the the adjacent ones leaving a squared center with sides of 2.7·cm. animals’ efficiency when foraging on the different flower Half of each ellipse appears as a petal. Total area, 33.7·cm ; morphs. flower span, 9·cm. Large disk: a disk with a diameter of 9·cm Given that we had recorded the time moths took visiting and an area of 63.6·cm . Medium disk: a disk with a diameter each flower, we tested whether moths could learn to handle the of 6.5·cm and an area of 33.2·cm . Small disk: a disk with a different flower morphs during a single foraging bout. diameter of 4·cm and an area of 12.57·cm . Additionally, the Discovery time was defined by the time elapsed between the corolla portion of each of the five flower morphs was covered initiation of flower probing and the entry of the proboscis into with a square transparency film sheet (henceforth called the nectary. This does not include the time flying from one transparency treatments; Fig.·1B), accounting for a total of 10 flower to another or drinking, but only the time spent probing treatments. In this way, we could evaluate the foraging at the flower’s threshold. We measured discovery time for the behavior under circumstances where no reliable tactile stimuli first eight successful visits, as did Lewis (Lewis, 1986). were available to the animals, but visual stimuli could be Experiment 2 preserved. To control for any surface texture effect, flowers in the treatments lacking a transparency square above them A second experiment was carried out to evaluate whether M. (henceforth called no transparency treatments) were covered sexta can use morphological features of flowers involving a with transparency film that had been cut to match the exact third dimension (i.e. depth) to improve its foraging efficiency. shape of the underlying paper flower. Each flower from every Many night-blooming flowers (e.g. Datura, Mirabilis) have treatment offered 20·l of a 20% (w/w) sucrose solution in a conspicuously grooved petals, which could in theory be used nectary (5·cm long by 0.5·cm opening diameter pipette tip) as tactile guides for the moths’ proboscides (Fig.·6). Thus, placed at its center; nectaries were accessible to moths through three treatments were designed. The first was ‘medium disks’, a 0.5·cm opening cut into the transparency film in both the same flat flowers used in Experiment 1. The second and treatments. Each nectary was attached to the flower such that third were paper disks of the same diameter as medium disks, it did not protrude above the flower surface. with two groove-like folds (see Fig.·2). In the second treatment, the folds were oriented parallel to each other (‘chord Variables recorded grooves’) and were placed 1.5·cm apart from the origin We recorded the foraging efficiency (number of successfully (nectary) of the disk (Fig.·2). In the third treatment, the folds exploited flowers over 10·min) after each trial. The number and were placed as two orthogonal diameters of the disk (‘radial duration of total, successful and unsuccessful flower visits grooves’), intersecting at the nectary (Fig.·2). THE JOURNAL OF EXPERIMENTAL BIOLOGY 1588 J. Goyret and R. A. Raguso Statistical analysis Response levels of male and female M. sexta to different flower morphs in Experiment 1 were tested by means of log- likelihood tests (G-tests using the Gh test statistic). Foraging efficiency, measured with the variables, emptied flowers and ratio of successful/total visits, was tested with the Kruskal–Wallis non-parametric test using a corrected -level of significance of 0.005. Thus, we performed ten statistical tests using the same set of data: six for emptied flowers, three for ratio of successful/total visits and one linear regression). Discovery time as a function of the sequence of feeding attempts was tested to fit the classic exponential decline learning curve described by Hilgard and Bower (Hilgard and Bower, 1966). A corrected -level of significance of 0.008 was used in these tests (six regression analyses). 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 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 =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 =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:  =18.43; P<0.0001; Table·1, Fig.·3). In (1,0.005) Medium disk Radial folds Chord folds 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. THE JOURNAL OF EXPERIMENTAL BIOLOGY Table·1. Variables recorded in relation to flower handling by Manduca sexta on different flower morphs with and without a square transparency film No transparency film Transparency film Number of visits (mean) FL(16) HL(18) LD(17) MD(16) SD(16) FL(17) HL(19) LD(17) MD(16) SD(16) Total 85.11±8.7 73.20±15.4 48.25±10.3 81.69±10.5 77.88±11.1 65.53±16.4 74.88±9.2 54.44±10 39.20±6.9 85.11±11.81 Successful 50.39±5.9 21.4±4.4 5.81±2.2 18.38±3.7 51.18±6 8.63±2.7 13.47±3.6 6.89±2.8 4.13±1.4 22.69±5.6 Failed 34.72±5.6 51.8±7.9 42.44±9.5 63.31±6.5 26.71±4.2 60.94±13.9 61.41±8.8 47.56±14.1 35.07±8.8 41.63±8.6 Ratio of successful/ 0.6±0.04 0.24±0.04 0.08±0.02 0.21±0.02 0.65±0.05 0.08±0.02 0.15±0.03 0.09±0.03 0.07±0.02 0.26±0.07 total All values are means ± s.e.m. Numbers in parentheses are the number of replicates for each treatment. FL, full lobe; HL, half lobe; LD, large disc; MD, medium disc; SD, small disk. Flower handling by Manduca sexta 1589 (Kruskal–Wallis test;  =18.34; P<0.0001; -level: 0.005). (1) Moreover, the number of emptied flowers was significantly correlated with flower surface area (a+x=y; a=14.33; =–0.75; R =0.56; F =105.5; P<0.0001). (1,0.005,84) 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 2 =30.48; P<0.0001; within transparency:  =59.29; (1) (4) 4 P<0.0001; within no transparency:  =7.39; P=0.12). a (4,0.005) 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: R =84.35; P=0.001) as illustrated by Fig.·4A (full lobe with no Fig.·3. Number of emptied flowers (foraging efficiency; mean ± transparency; exponential decline function fit: R =84.87; s.e.m.) after a 10·min foraging bout by individual Manduca sexta P=0.001). Nevertheless, this was not the case for the large disk inside the flight chamber. In each treatment (abscissa) an array of 12 treatment (Fig.·4C; exponential decline function fit: R =37.99; artificial flowers of the same morph was present. Black bars represent P=0.10). When flowers were covered by a transparency film, responses to artificial flowers without square transparency film; gray discovery times did not conform to a typical learning curve bars represent responses to the same artificial flowers covered with a (transparency treatment; exponential decline function fit: square transparency film. Different letters denote statistically R =56.58; P=0.031; corrected -level of significance: 0.005), significant differences with a corrected -level for significance of as shown in Fig.·4B,C (full lobe with transparency: R =8.89; 0.008 (see text). P=0.47; large disk with transparency: R =0.0; P=1.0). Experiment 2 addition, variation in emptied flowers was significantly affected by flower morphology among the no transparency Flower morphology was significantly associated with the treatments (Kruskal–Wallis test; within no transparency: number of emptied flowers when moths foraged on different =44.64; P<0.0001). This effect was not observed flower arrays (ANOVA: F =21.11; P<0.0001). Both kinds (4,0.005) (2,57) among transparency treatments; in this case differences were of grooved artificial flowers affected the performance of not as pronounced, only accounting for a trend foraging moths, but in opposite ways (Fig.·5). Moths (Kruskal–Wallis median test; within transparency: performed worse on flowers with chord grooves than on flat =10.18; P=0.04). control flowers (medium disk vs chord grooves: F =78.9; (4,0.005) (1,39) Among the no transparency treatments, moths clearly were P<0.0001), whereas moth performance on flowers with radial more successful when handling full lobe and small disk morphs grooves was significantly better than on flat control flowers (see Fig.·3). As flower surface area increased from small disk (medium disk vs radial grooves: F =328.11; P<0.0001). (1,37) 2 2 (12.6·cm ) to large disk (63.6·cm ), moth performance declined (see regression analysis below). this same effect was 2 Discussion observed when comparing full lobe (21.4·cm ), half lobe Behavioral sequence of flower foraging and its distinct (33.7·cm ) and large disk (Kruskal–Wallis test; full lobe vs half sensory modalities lobe:  =6.07; P=0.014; large disk vs medium disk: (1,0.005) =16.63; P<0.0001). Because surface area is not the The foraging behavior of Manduca sexta appears to follow (1,0.005) only flower feature that varied among treatments, we tested a sequential pattern involving different sensory modalities at whether the minimum distance from the edge to the nectary each stage (Raguso and Willis, 2003). Thus, a moth under (center) of the flower could affect moths’ performance appetitive motivation will first fly upwind when encountering independently. Half lobe and medium disk flowers have similar an appropriate fragrance (Brantjes, 1978). At closer range, 2 2 surface areas (33.7·cm and 33.2·cm , respectively) but flower approach by M. sexta is guided by either olfactory or different edge-to-center distances (2·cm and 3.25·cm, visual stimuli, whereas proboscis extension requires the respectively). Similarly, half lobe and small disk flowers have combination of visual and olfactory cues (Raguso and Willis, the same minimum edge-to-center distance (2·cm) but different 2002; Raguso and Willis, 2005). Here we show that 2 2 surface areas (33.7·cm and 12.6·cm , respectively). Surface mechanoreception is an additional sensory modality that area appeared to be a more important flower feature than edge- contributes to the final stage of the feeding sequence, once the to-center distance, as feeding effectiveness did not differ proboscis is extended and moths must locate and drink from significantly between half lobe and medium disk flowers floral nectaries, a process frequently referred to as ‘flower (Kruskal–Wallis test;  =0.005; P=0.945), but differed handling’. (1,0.005) significantly between half lobe and small disk flowers In our experiments, moths were effectively and equally THE JOURNAL OF EXPERIMENTAL BIOLOGY Number of emptied flowers 1590 J. Goyret and R. A. Raguso R =8.89 P=0.47 20 R =84.87 Fig.·4. Discovery time (probing P=0.0012 time between feeding attempts) for four different treatments. 10 10 (A) Full lobe, (B) full lobe with transparency film, (C) large disk 5 5 and (D) large disk with transparency film. Data points are 0 0 medians, whiskers represent first and third quartiles. Statistical values refer to goodness-of-fit to 2 40 R =0 R =37.99 an exponential decline function P=1.0 P=0.10 (one factor), a classical ‘learning curve’. Moths exploiting full lobe flowers with no transparency film show exponentially decreasing discovery times. When exploiting 10 10 large disks, or either shape with transparency film, moths show larger variances in their 0 02468 02468 responses, which do not fit an exponential decline function. Number of attempts attracted to the different artificial paper flowers, regardless of transparency films used to de-couple visual and mechanical the fact that they differed in shape and size (which in turn, stimuli. greatly affected performance), when paper flower arrays were Vision and mechanoreception during flower probing presented with Bergamot oil as an olfactory stimulus (see Results). This result indicates that probing responses (i.e. What is the innate probing strategy of M. sexta? Is the emptied flowers) to different treatments were not confounded proboscis guided visually or are there other sensory systems by innate differences in attractiveness, and that no biases in involved? The use of different artificial flowers affected the moth preference or attraction were associated with 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 B 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 8 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 0 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 N=20 N=19 N=21 ‘random walk’ of probing across the flower’s surface. The disruption of reliable tactile information clearly Fig.·5. Three-dimensional corolla features affect foraging efficiency interferes with flower handling by M. sexta, showing that by Manduca sexta. The vertical bars represent number of flowers mechanoreception, in addition to vision and olfaction, is (mean ± s.e.m.) emptied after a 10·min foraging bout by individual involved in nectar feeding by these moths (Fig.·3). Tactile cues moths inside the flight chamber. In each treatment (abscissa) an array constitute an important component of many flower–pollinator of 12 artificial flowers of the same morph was present. Different letters denote statistically significant differences (see text). systems (Kevan and Lane, 1985; Borg-Karlson, 1990), but are THE JOURNAL OF EXPERIMENTAL BIOLOGY Number of emptied flowers Discovery time (s) Flower handling by Manduca sexta 1591 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 Fig.·6. Features of flower handling are illustrated in this photo of of M. sexta’s eyes does not allow for accurate feedback about Manduca sexta feeding from a flower of Mirabilis multiflora proboscis position (A. Kelber, personal communication). The (Nyctaginaceae). Note the extended proboscis (grey arrow), the low signal-to-noise ratio of the visual modality at this scale distance of the moth’s body from the flower, and the radial grooves could have imposed selective pressures for M. sexta to © in the flower’s perianth (white arrows). Scale bar, 1·cm. Photo efficiently assess floral nectar content by other means. Such Robert A. Raguso. 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 disks (Fig.·3). It appears that the decrement in flower handling sensilla positioned along the tip of the lepidopteran proboscis by M. sexta on flowers with high surface area is offset by floral (Krenn, 1998; Kelber, 2003). depth. However, attraction from a distance is enhanced by the Sprengel (Sprengel, 1793) introduced the concept of nectar increased visual display provided by flowers with larger guides as floral features that could be used by pollinators to diameters (Knoll, 1922). Tubular flowers appear to offer a visually locate the nectar. Subsequent experiments revealed compromise solution to this hypothetical trade-off, while that the diurnal hawkmoth Macroglossum stellatarum (Knoll, simultaneously providing for high nectar volumes and 1922), bumblebees (Manning, 1956; Kugler, 1966), honeybees appropriate physical contact between the body of the moth and (Daumer, 1958; Free, 1979) and bee-flies (Johnson and Dafni, the sexual organs of the flower (Nilsson, 1988). It is tempting to 1998), among other insects, successfully utilize visual nectar consider how differences in handling efficiency associated with guides. Here we show that the utility of Spengel’s idea extends corolla form might impact competition between night blooming beyond the visual system, as the tactile sensitivity of the flowers for hawkmoths as pollinators (see Haber and Frankie, proboscis of M. sexta allows these moths to exploit the physical 1989), however, most flowers in nature are likely to be visited features of flowers in order to find nectar (Figs·3, 5, 6). Our by experienced moths. Additional experiments will be needed to experiments, unlike those of Knoll (Knoll, 1922; Knoll, 1926), determine whether the handling differences identified in this varied the contours of artificial flowers, rather than testing study have an impact on subsequent foraging decisions. moth responses to natural flowers. Further experiments will be Flower handling improves with experience required to test whether visual nectar guides of color contrast can be used by M. sexta. We analyzed whether M. sexta could learn to improve its handling abilities (i.e. reduce the time to find nectar) during a Context dependence of the floral visual display single foraging bout. Indeed, M. sexta adults improve their This study indicates that once moths approach a flower handling of artificial flowers within an extended feeding bout patch, they extend their proboscides towards a visual target and (Fig.·4). We analyzed improvement in flower handling overall then appear to rely on mechanosensory input. At this point, (with and without transparency film) and within two specific when probing is relatively random, any irregularity on the treatments – full lobe and large disk – as examples of flowers corolla surface could guide moths’ searching behavior, such that elicited high and low performances, respectively. Flower that the proboscis ‘rides’ along the length of petal grooves, handling did not improve on model flowers in which the nectary openings or the margins of highly divided corollas. nectary was difficult to find (large circles), nor when square The funnel-shaped flowers of Datura wrightii, a favored transparency films prevented the acquisition of reliable nectar source of M. sexta in the Sonoran Desert (Raguso et al., mechanosensory information (see Fig.·4B,D). This suggests 2003) are comparable in diameter to the large disk models in that reliable tactile information is needed not only to forage Experiment 1, but previous experiments indicate that Datura efficiently (Fig.·3), but also to learn to forage more efficiently flowers are learned very quickly by naïve M. sexta (Desai and (slope of learning curves, see Results and Fig.·4). Raguso, 2001), which is not the case when foraging on our large Learned improvement in flower handling has been shown in THE JOURNAL OF EXPERIMENTAL BIOLOGY 1592 J. Goyret and R. A. Raguso a variety of nectivorous insects, including other lepidopterans Chittka, L. and Thomson, J. D. (1997). Sensori-motor learning and its relevance for task specialization in bumble bees. Behav. Ecol. Sociobiol. 41, (Lewis, 1986; Hartlieb, 1996; Cunningham et al., 1998) and 385-398. hymenopterans (Harder, 1983; Laverty and Plowright, 1988; Chittka, L., Shmida, A., Troje, N. and Menzel, R. (1994). Ultraviolet as a Chittka and Thomson, 1997). This ability gives animals the component of flower reflections, and the colour perception of Hymenoptera. Vision Res. 34, 1489-1508. opportunity to decrease the time they spend on individual Cunningham, J. P., West, S. A. and Wright, D. J. (1998). 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Journal of Experimental Biology – The Company of Biologists

Published: May 1, 2006

References