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ANIMAL BEHAVIOUR, 2005, 69, 407–418doi:10.1016/j.anbehav.2004.04.015

Synergy between visual and olfactory cues in nectar feeding

by wild hawkmoths, Manduca sexta

ROBERT A. RAGUSO*†‡ & MARK A. WILLIS*

*Center for Insect Science and ARL Division of Neurobiology, University of Arizona, Tucson

yDepartment of Ecology and Evolutionary Biology, University of Arizona, Tucson

zArizona-Sonora Desert Museum, Tucson

(Received 31 October 2003; initial acceptance 31 December 2003;

final acceptance 7 April 2004; published online 15 December 2004; MS. number: A9741)

We performed field experiments to measure the relative importance of olfactory and visual cues in nectarforaging by wild tobacco hornworm moths, Manduca sexta (Lepidoptera, Sphingidae) in the SonoranDesert of Arizona, U.S.A. We manipulated flowers of sacred Datura (Datura wrightii; Solanaceae) toexperimentally decouple floral scent from visual display and presented these cues to free-flying moths inmixed and homogeneous arrays. Moths did not feed from cloth-bagged fragrant flowers lacking strongvisual contrast, nor did they feed from paper model flowers lacking plant odours. Unexpectedly, moths fedfrom paper model flowers that were associated solely with vegetative odours, albeit at lower levels thanwhen floral scent was present. Subsequent experiments revealed that the combination of floral andvegetative odours did not incrementally increase nectar feeding and that floral scent without vegetationwas sufficient to elicit feeding when paper flowers were present. Thus, wild M. sexta in our study did notshow generalized feeding responses to natural or artificial flowers with single sensory stimuli; like naıvelaboratory-reared moths, they required a combination of visual and olfactory cues. However, given theprior foraging experience of our study population on Datura flowers, their lack of generalized feedingresponses may reflect a learned preference for the full complement of floral cues, rather than thepersistence of innate sensory constraints. Future efforts to distinguish between these hypotheses shouldfocus on whether M. sexta can be conditioned to associate nectar with visual cues alone, and whethermoths that feed ad libitum from nectar-rich plants learn floral attributes as a search image or generalize tosingle-modality stimuli.

� 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

The dynamic interplay between an organism’s innateresponses to sensory stimuli and the modification of suchresponses with experience remains a central theme inethology, particularly in plant–animal communication(Papaj & Lewis 1993; Hansson 1999). Recent studies haveexplored how innate pollinator responses to visual, olfac-tory and gustatory floral cues change during an animal’slifetime (Chittka & Thomson 2001). For example, al-though both laboratory-reared and wild Glossophaga batsprefer flowers with sulphurous odours, these and otherbats learn to visit odourless plastic feeders in captivity andin the wild (von Helversen et al. 2000; Winter & von

Correspondence and present address: R. A. Raguso, Department ofBiological Sciences, University of South Carolina, Coker Life SciencesBuilding, Columbia, SC 29208, U.S.A. (email: raguso@biol.sc.edu).M. A. Willis is now at the Department of Biology, Case Western ReserveUniversity, 10900 Euclid Avenue, Cleveland, OH 44106-7080, U.S.A.(email: maw27@po.cwru.edu).

407003–3472/04/$30.00/0 � 2004 The Association for the St

Helversen 2001), and can use echolocation to find scent-less flowers in the dark (von Helversen & von Helversen2003). Such flexibility is important in food-based plant–pollinator communication, in which generalized flower-feeding animals such as honeybees must learn (and forget)a broad spectrum of multimodal sensory signals (Smith &Getz 1994; Menzel 2001). The degree to which flower-visiting animals’ innate sensory preferences are modifiedwith experience is likely to vary within and betweendifferent kinds of pollinators (Weiss 2001), in ways thatdirectly impact the evolution of floral colour (e.g. Menzel& Schmida 1993; Chittka 1996) and odour as signals thatadvertise the presence of food rewards.One distinctive group of insect pollinators, hawkmoths

(Sphingidae; Lepidoptera), meets the energetic demandsof hovering flight, dispersal and migration by foragingfrom a broad spectrum of nectar-rich flowers (Haber &Frankie 1989; Raguso & Willis 2003). In a previous study(Raguso & Willis 2002) we showed that floral odours and

udy of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

ANIMAL BEHAVIOUR, 69, 2408

bright visual displays, when presented independently, areinnately attractive to naıve, laboratory-reared adult malesof a nocturnally active species, the tobacco hornworm(Manduca sexta L.), whereas only the combination ofolfactory and visual cues elicits spontaneous proboscisextension and nectar feeding. Baerends (1950), Tinbergen(1958) and Brantjes (1973) initially described the upwind‘searching flight’ and visually guided probing by noctur-nal hawkmoths such as M. sexta as ‘fixed action patterns’released by the sign stimulus of fragrance. Subsequentexperiments have revealed that these moths’ innatenectar-feeding behaviours are modified by at least threeforms of olfactory or visual learning. Daly & Smith (2000)demonstrated rapid associative (appetitive) learning byM. sexta when single odours were forward-paired withsugar rewards in a Pavlovian context, and symmetricaldiscrimination learning using pairs of rewarded and un-rewarded odours as conditioned stimuli in proboscisextension-cibarial pump reflex (PER) assays (Daly et al.2001a). Kelber et al. (2002) showed that free-flyinghawkmoths of another species, Deilephila elpenor (L.), learnto discriminate between rewarding and unrewardingpaper flowers using true colour vision under dimly lit,nocturnal conditions. Similar experiments with diurnalMacroglossum stellatarum (L.) hawkmoths indicate thatinnate visual preferences for floral colour and patterncan be reversed through operant conditioning (Kelber1996, 1997, 2002; Kelber & Henique 1999).The available evidence thus indicates that M. sexta and

related hawkmoths can learn to associate specific floralcues with nectar rewards. Does foraging experience modifythe responses of wild, free-flying M. sexta to such stimuli?We designed a series of experiments to examine howM. sexta use visual and olfactory cues to find and feedfrom flowers in natural populations. One goal was to testwhether wildM. sextawill feed when floral cues are limitedto a single sensory modality, something that naıve malemoths will not do under our experimental conditions(Raguso & Willis 2002). Sensory generalization, the degreeto which a novel stimulus elicits feeding based on itssimilarity to stimuli used to condition the feeding response(Daly et al. 2001b), is widespread among flower-visitinganimals, as is suggested by the historical effectiveness ofartificial flowers in behavioural assays (Clements & Long1923; Stiles 1976; Real 1981; Smithson 2001). GivenM. sexta’s broad geographical range and catholic feedinghabits (Fleming 1970; Hodges 1971), we expected wildmoths to show generalized responses to modified orartificial flowers with subsets of sensory cues. Another goalwas to test whether the moths’ behavioural responseschange at different spatial scales. For instance, a plant’sodour plume might provide context for visual cuespresented within it, but may be irrelevant when physicallydecoupled from visual targets. To address these goals, wemanipulated flowers of Datura wrightii (Regel; Solanaceae)to present either visual or olfactory floral cues, but notboth, and asked whether M. sexta could distinguishbetween these and control flowers in mixed arrays. Then,we performed a similar experiment at a larger spatial scalewith homogeneous arrays of scentless or visually con-cealed flowers, to test whether moths would visit patches

of flowers lacking scent or visual targets. Finally, we usedmovable stations and artificial plants to control for theinfluence of landmark learning and vegetative odours,respectively, on the moths’ foraging decisions.

MATERIALS AND METHODS

Experimental Setting and Study Organisms

Experiments were performed at the Arizona-SonoraDesert Museum (ASDM), in thorn scrub habitat in theTucson Mountains, Pima Country, Arizona, U.S.A.(800 m), where large native populations of M. sexta occur.Foraging behaviour was monitored at D. wrightii flowersduring the monsoon season (July–September) in 1996–1998. Field observations occurred on 13 nights over 2years and were limited by thunderstorms or fluctuationsin moth or flower abundance. Manduca sexta was distin-guished from similar species by size (it is larger than Hyleslineata Fab. and smaller than M. rustica Fab.) and byabundance (light trapping showed that M. quinquemacu-lata (Haworth) and Agrius cingulatus (Fab.) were rare orabsent at ASDM during the period of study). Daturawrightii flowers are large, trumpet-shaped and white tothe human eye and emit a chemically complex fragrance(Raguso et al. 2003a). The flowers open at dusk and arevisited for nectar by M. sexta and other hawkmothsthroughout southwestern North America (Baker 1961;Grant & Grant 1983). Datura wrightii plants also are larvalhosts forM. sexta in the Sonoran Desert (Bernays & Woods2000; Mechaber & Hildebrand 2000), and female M. sextaoften feed from Datura flowers during oviposition(Mechaber et al. 2002).

In each experiment, moths foraged freely at open(control) or manipulated flowers. Observations extended40–60 min from sunset to dusk, until moths could not beobserved unaided. (Nectar feeding by M. sexta sometimesextended for 1–2 h, but never at the levels observed duringthe first hour; Gregory 1963). This protocol, combinedwith the absence of any significant ANOVA terms (seeTable 2), confirms that the data were not biased byobservation time. Nearly all nectar feeding by M. sextaoccurred at D. wrightii flowers, although other plants (e.g.Oenothera caespitosa (Onagraceae), Hymenocallis sonorensis(Amaryllidaceae)) growing at ASDM were visited whenDatura flowers were not abundant or their nectar had beendepleted.

We observed individual moths’ foraging bouts (sequen-ces of consecutive flower visits) and scored two behav-ioural events: (1) approaches, in which moths deceleratedand hovered within 10 cm of a flower, and (2) visits, inwhich approaching moths extended the proboscis andprobed for nectar. We did not mark or capture mothsduring any of these experiments, and some individualsmay have performed multiple bouts during observationalperiods. However, tens to hundreds of M. sexta visitedD. wrightii flowers on nonexperimental plants during mostevenings. We tracked moths that moved from one treat-ment to the next on an extended bout and routinelyobserved simultaneous bouts by several moths at different

RAGUSO & WILLIS: WILD HAWKMOTH FEEDING BEHAVIOUR 409

treatments or stations. Thus, it is unlikely that only a fewmoths were responsible for a disproportionate percentageof our data.Datura wrightii flowers were manipulated to modify

floral scent and visual display. First, we covered flowerswith transparent oven bags (Reynolds, Inc., Richmond,Virginia, U.S.A.), which lack a strong scent of their own(Pellmyr et al. 1990), to prevent fragrance emission andmoth access to nectar. Second, we concealed naturalflowers beneath brown-green dyed cheesecloth bags (Rit,Inc., Indianapolis, Indiana, U.S.A.), permitting fragranceemission while eliminating the corolla’s visual display(Fig. 1). Third, we used white paper to construct conicalmodel flowers (Fig. 2a) terminating in 2-ml plastic micro-centrifuge tubes filled with 50 ml of 25% sucrose solution.Model flowers provided an unscented visual stimulus witha nectar reward and were comparable to natural D. wrightiiflowers in nectar volume, sugar concentration and corollalength (Raguso et al. 2003a). However, both model flowersand plastic-bagged flowers differed from controls in shape,profile and ultraviolet (UV) reflectance (Figs 1, 2). The baseof each model flower was attached to a D. wrightii stemusing a binder clip. Equal sample sizes of floral treatmentsand controls were used in each experiment. Superfluousbuds or wilted flowers were removed from plants 30 minbefore sunset to standardize replication and spread thepotential for wounding artefacts between all treatments.

Spectral Reflectance

The spectral properties of experimental stimuli weremeasured using a Spectral Instruments, Inc. (Tucson,Arizona) SI-440 CCD array UV–VIS spectrophotometerwith a tungsten light source, a 400-mm fibre optic probeand a Labsphere, Inc. (North Sutton, New Hampshire,U.S.A.) 9-cm ID integration sphere. We collected reflec-tance data from 350 nm (UV) to 700 nm (infrared, IR)wavelengths by placing an integration sphere fitted with

0

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350 400 450 500 550 600 650 700Wavelength (nm)

Ref

lect

ance

(%

of

stan

dar

d) Paper flower

D. wrightii leaf

Flower in cloth bag

D. wrightii flower

Flower in plastic bag

Figure 1. Spectral reflectance of D. wrightii flowers and leavesrelative to white reference standard, within the visual spectrum of

M. sexta. Plastic bags increased UV reflectance by 30%, but did not

alter floral spectral properties above 400 nm. Floral reflectance when

camouflaged by dyed cheesecloth bags was comparable to that offoliage. The paper model flower is a supernormal visual stimulus and

was 50% more reflective than D. wrightii flowers in all wavelengths.

a 1.5-cm diameter sampling port over paper flowers,D. wrightii flowers, leaves or bags laid flat over flowers,with a black felt cloth as background. This methodallowed us to capture transmitted light scattered bytextured surfaces. Data were collected as percentage trans-mission (1/absorbance) in comparison to a white pigment(Duraflect; Labsphere Inc., North Sutton, New Hampshire,U.S.A) standard. This standard reflected evenly in all non-UV wavelengths, with a 5% drop-off from 350–400 nm(Labsphere, Inc. product literature). The reflectance of theblack cloth background was negligible (0.5–1% of stan-dard) for all wavelengths tested.

Experiment 1: Control for Bags Usedto Modify Flowers

We tested for potential bias in moth responses to dyedcheesecloth and clear plastic bags by marking 30 openingbuds on two large, adjacent D. wrightii plants andwrapping dyed cheesecloth or plastic bagging beneaththe flared corollas of 20 of them, leaving 10 controlflowers without bags. Flowers were evenly spaced andtreatments were haphazardly assigned. (Paper was nottested for bias becauseM. sexta had previously been shownnot to differentiate between them and natural flowers ofOenothera neomexicana ((Small) Munz; Onagraceae) flowers(Raguso & Willis 2002), whose reflectance spectrumclosely matches that of D. wrightii flowers; Fig. 1.)The sequence of floral approaches and visits by each

moth to different treatments was scored during two 15-min periods. These data were analysed for homogeneity ofpreference using the binomial test (Waser 1986; Jones1997), testing whether visit bouts by different animals inan array-like setting could be combined for statisticalanalysis (Waser 1986; Jones 1997). First, we comparedthe observed visits (by all moths) to treatments andcontrol flowers with expected values using the chi-squaretest of independence, testing the null hypothesis ofrandom visits to each treatment in proportion to eachtreatment’s relative abundance. Second, we tested forpreference (assortative visits) by comparing the propor-tion of visits to control flowers versus the relativefrequency of those flowers (0.33) for each moth usingsingle sample t tests on arcsine-transformed data (Jones1997). Significant overvisiting of control flowers wouldindicate that the presence of bags reduced floral attrac-tiveness. We also recorded which treatment received thefirst visit in each bout, testing the null hypothesis of equalattraction using the chi-square test of independence.Finally, we tested for constancy by asking whethertransitions between different treatments during foragingbouts departed significantly from random expectations.The expected values for transitions were calculated fromthe observed proportion of visits to each treatment, asdescribed by Jones (1997). The observed number of ‘like’and ‘unlike’ transitions were compared with expectedvalues using the chi-square test of independence, suchthat significant deviations from random visits wouldindicate bias associated with cloth or plastic bags.

ANIMAL BEHAVIOUR, 69, 2410

Figure 2. (a) Manduca sexta ‘warming up’ after feeding from a paper flower for 40 s. Most hawkmoths hover while feeding, but Datura flowers

and paper models were deep enough to require landing. (b) Layout of control and manipulated D. wrightii flowers in experiment 2. Whitearrows indicate flowers covered with dyed cheesecloth bags; only a subset of one mixed plot is shown.

Experiment 2: Decoupling Visual andOlfactory Stimuli in Mixed Plots

We created twomixed arrays of control andmanipulatedD. wrightii flowers and paper flower models to test whetherthe selective removal of odour or visual cues reducesthe attractiveness of individual flowers. Six flowers ofeach treatment (cloth-bagged, plastic-bagged and paperflowers C sucrose solution), plus six open control flowerswere spaced roughly 20 cm apart and assigned haphazardlybetween each of two D. wrightii plants growing 10 m apart(Fig. 2b), totalling 12 flowers for each treatment. We useddark plastic garbage bags to conceal additional flowering

plants, such as Mentzelia multiflora ((Nuttall) Gray; Loasa-ceae) and Glandularia goodingii ((Briq.) Solbrig; Verbena-ceae), effectively removing superfluous floral cues andalternative nectar sources from the experimental arena.

We observed individual moths as they approachedexperimental plants and scored the number of approachesand visits to flowers of each treatment and the sequence ofmovement between treatments for each moth. Behaviou-ral data were collected during two consecutive 20-minperiods on three successive nights, with two observersexchanging places between periods. In this and subse-quent experiments, data were analysed for overall trendsand individual bouts in three way as follows.

RAGUSO & WILLIS: WILD HAWKMOTH FEEDING BEHAVIOUR 411

(1) We converted raw data into proportions of totalapproaches (those that did and did not result in a visit) pertime replicate. We performed ANOVA on arcsine-trans-formed proportion data, with treatment as a fixed effectand time, day and their interaction with treatment asrandom effects (see Roy & Raguso 1997). This was doneto determine whether there were consistent differencesin attraction to different treatments overall, regardlessof natural, well-documented fluctuations in hawkmothabundance (Miller 1981; Willmott & Burquez 1996). In-teraction termswere not significant andwere dropped fromANOVAmodels in subsequent experiments (Table 2).Whena significant term was associated with treatment, we testedwhether the open control flowers elicited a significantlygreater proportion of total approaches than each single-modality treatment (one-tailed tests) using Dunnett’s test(Zar 1996). Such data are presented in Table 2 and Fig. 4.(2) We analysed data from individual bouts by testing

for homogeneity of preference, then combining bouts andtesting for preference and constancy in total approaches,as described for experiment 1. Again, we tested for first-visit preference using the chi-square test of independence.The same approach was used to test whether the patternof last flowers visited or approached in each bout departedfrom null expectations.(3) We used cumulative behavioural data to determine

the relative effectiveness of different combinations offloral cues as feeding stimuli. We defined a ‘feeding index’by calculating the ratio of visits to total approaches foreach treatment (see Table 1), in which values approachingunity indicate the most attractive flowers. We comparedthese values using Fisher’s exact test.

Experiment 3: Decoupling Floral Stimuliin Homogeneous Plots

Floral scent and visual cues were manipulated at thewhole-plant level to remove the effect of diffuse floralscent plumes on treatments lacking fragrance. We pre-sented three homogeneous floral arrays, each with a dif-ferent treatment, using three large D. wrightii plants thatformed a triangular arena 10 m on a side. In the first array,eight evenly spaced flowers were covered with dyedcheesecloth bags, eliminating visual floral cues whilepermitting scent emission. In the second array, all flowersand mature buds were removed and eight paper flowers(with sucrose solution) were attached to the foliage toproduce a visual display lacking floral scent. In the thirdarray, eight paper flowers were attached directly aboveeight cloth-bagged flowers, reconstituting visual andolfactory floral cues with a sugar reward. The spacing oftreatments and removal of additional floral attractantsfrom the experimental arena was as described for exper-iment 2. Three observers counted floral approaches andvisits for each moth at each treatment during three 15-min periods (45 min total), and treatments were rotatedclockwise among plants each evening for 5 days. Becauseno visits were observed at cloth-bagged flowers, we testedwhether a greater proportion of total approaches wasmade to paper flowers with versus without floral scent

using one-tailed, unpaired t tests after ANOVA. As inexperiment 2, we compared the feeding index for thesetwo treatments using Fisher’s exact test.

Experiment 4: Subsets of Floral Stimuliwith Fixed versus Movable Positions

The design of experiment 3 was modified to control forDatura vegetative odour and proximity of individualplants to physical landmarks. We established a pentagonalexperimental arena bounded by two D. wrightii plants(15 m apart) growing in soil near a pavement cul-de-sacand three mobile positions 15 m apart within the cul-de-sac (Fig. 3). The first treatment was a control plant with 12evenly spaced open flowers each evening. Visits to theseflowers by M. sexta during discrete time replicates allowedus to interpret the absence of moths at other treatments asreduced attractiveness, rather than scarcity of moths. Thesecond treatment was a plant growing 5 m from a largegreenhouse, with 12 paper flowers clipped above fragrant,cloth-bagged flowers, as in the third treatment of exper-iment 3. The third treatment was identical to the second,but comprised four potted, movable D. wrightii plants,which could be rotated among three stations within thecul-de-sac (Fig. 3). The comparison of these two treat-ments allowed us to determine whether ‘landmark’ learn-ing contributed to moth attraction to experimental plants.The remaining two treatments were designed to test theimportance of visual cues with or without Datura floralscent, in the absence of Datura vegetation. We constructedartificial plants using dead branches of paloverde (Cerci-dium microphyllum (Torrey) Rose & I. M. Johnston; Faba-ceae) placed in 4-litre plastic pots filled with fine gravel. Inthe fourth treatment, 12 excised D. wrightii flowers werecovered with cheesecloth bags, placed into plastic test-tubes and clipped to the branches. Twelve paper modelflowers were clipped directly above cloth-bagged flowers,combining visual cues and sugar water with floral scent,without any D. wrightii vegetation. In the fifth treatment,12 paper flowers with sugar water were attached to nakedbranches, presenting visual cues and nectar rewardswithout any D. wrightii plant odours (Fig. 3).Five observers scored visits and approaches at each

station during two 20-min periods each evening. Thethree mobile treatments were rotated clockwise within thecul-de-sac each evening for 6 days. ANOVA was performedas before on arcsine-transformed data, excluding thosefrom the open control plant. When a significant ANOVAterm was associated with treatment, we performed one-tailed, unpaired t tests (aZ 0.017) to address the follow-ing three questions.(1) Did the presence of a large visual landmark signif-

icantly increase the proportion of total approaches whenall plant cues were present?(2) Were paper flowers with a combination of floral and

vegetative odours significantly more attractive than paperflowers with floral scent alone? (We also contrastedfeeding index for these two treatments using Fisher’s exacttest, as above.)

ANIMAL BEHAVIOUR, 69, 2412

Figure 3. Schematic of D. wrightii floral manipulations for experiment 4, with five homogeneous treatments spaced 15 m apart. Upright conesrepresent paper flowers, hatched ellipses with a curled line denote fragrant flowers concealed within dyed cheesecloth bags, and the presence

of foliage is indicated with leaves. Treatments are figured with only four floral units for clarity of presentation; actual plants had 12 floral units

each. Mobile treatments are drawn in pots; stationary treatments are drawn with roots, and the building at lower left depicts the large

greenhouse. Clockwise arrows indicate the rotation of mobile treatments each night of the experiment. Grey club-tipped bars indicatetreatments to be compared using a priori contrasts.

(3) Did the addition of floral scent to paper flowerswithout Datura vegetation significantly increase the pro-portion of total approaches by M. sexta?

RESULTS

Experiment 1

We observed 26 feeding bouts, 172 approaches and 166visits by M. sexta to flowers of D. wrightii. More than 95%of all approached flowers were visited, regardless oftreatment (Table 1). The moths showed homogeneity ofpreference (binomial test: S25 Z 19.7, P Z 0.77), enablingus to combine separate foraging bouts for further analysis.The number of total visits to control flowers (62), thosesubtended by plastic (47) or cloth bags (57) did not departsignificantly from expected values given the null hypoth-esis of random visits (chi-square test: c2

2 Z 2.1, P Z 0.348).There was no significant preference for control flowers(t25 Z 1.4, P Z 0.16), nor were there significant differ-ences between the distribution of first visits for each bout(c2

2 Z 4.9, PZ 0.09). Finally, constancy was not observed,as the frequency of transitions within and between classes

did not depart significantly from expected values gener-ated under the null hypothesis of random transitions(c1

2 Z 1.7, P Z 0.2).

Experiment 2

We observed 40 foraging bouts, 273 approaches and 169floral visits by M. sexta during a 3-day period (Table 1). Allfloral treatments were approached, but 95% of all ap-proaches and visits occurred at open control or paperflowers. Moths probed only five flowers within plasticbags, and did not probe at flowers concealed by cheese-cloth. Variance in the proportion of total approaches wassignificantly associated with treatment but not with time,day, treatment ! time or treatment! day (ANOVA, Table2). The proportion of total approaches to control flowerswas significantly greater than for any other treatment(Fig. 4a).

More than half of all approaches and visits occurred atcontrol flowers in 26 of 40 foraging bouts and at paperflowers in six bouts, with attraction split evenly in fivebouts. The hypothesis of homogeneity of preference couldnot be rejected (binomial test: S39 Z 32.3, P Z 0.78), and

RAGUSO & WILLIS: WILD HAWKMOTH FEEDING BEHAVIOUR 413

Table 1. Manduca sexta combined behavioural responses to Datura wrightii flower manipulations

Experiment Treatment Visits Total approaches Feeding index Total bouts

Experiment 1 Open control 62 65 0.95 26OpenCplastic bag 47 48 0.98OpenCcloth bag 57 59 0.97

Experiment 2 Open control 128 166 0.77 40Paper flowers 36 92 0.39Plastic-bagged 5 12 0.42Cloth-bagged 0 3 0.0

Experiment 3 Cloth-bagged 0 6 (C48)* 0.0 50Paper flowers 11 26 0.42Paper flowersCscent 37 131 0.28

Experiment 4 Open control 144 163 0.88 172PaperCscent, veg.; rooted 31 77 0.40PaperCscent, veg.; mobile 49 75 0.65PaperCfloral scent 30 48 0.63Paper flowers only 0 3 0.0

*The value in parentheses indicates the total number of times that M. sexta moths hovered or passed closely (!1 m) above flowers in thistreatment, but did not approach individual flowers. This behaviour was not observed in response to other experimental treatments, and iscomparable to tracking an odour plume in a wind tunnel (Raguso & Willis 2003).

individual bouts were combined for further analyses.Moths showed significant preference for control flowers(single sample t test: t39 Z 8.41, P! 0.001) but not forpaper flowers (t39 Z 1.13, PZ 0.13), given the null expect-ations of equal attraction (25%) to each of four treat-ments. When alternative expected values (0.47 each) weregenerated to reflect the observed dominance (95% of totalattraction) of control and paper flowers, controls re-mained significantly more attractive than expected(t39 Z 3.64, P! 0.001), whereas paper flowers were sig-nificantly less attractive than expected (t39 Z�3.79,P! 0.001).Floral constancy was not observed when ‘like’ (control–

control, paper–paper) and ‘unlike’ transitions (control–paper, paper–control) were combined (chi-square test:c12 Z 0.23, P Z 0.64). However, moths visiting control

flowers were nearly twice as likely to visit another controlflower (c1

2 Z 5.88, PZ 0.015) than a paper flower, whereasthose at paper flowers were twice as likely to switchto control flowers (c1

2 Z 4.5, PZ 0.034). Thirty-one of

40 observed foraging bouts began at control flowers, withonly nine first visits to paper flowers (c3

2 Z 13.56,P! 0.001) and no first visits to flowers concealed withinplastic or cloth bags. Similarly, 27 of 40 bouts ended atcontrol flowers, whereas only 12 ended at paper flowers(c3

2 Z 29.30, P! 0.001). However, when expected valueswere adjusted to reflect the relative attractiveness ofcontrol (0.61) and paper flowers (0.34), first visits(c1

2 Z 1.55, PZ 0.21) and last visits (c12 Z 0.11,

PZ 0.74) did not depart significantly from the overallbias in attraction to control flowers. The feeding index forcontrol flowers (0.77) was significantly greater than thatfor paper flowers (0.39; Fisher’s exact test: P ! 0.0001,NZ 258 events), as approaches to the former were twiceas likely as the latter to end in a floral visit.

Experiment 3

We scored 50 foraging bouts, 163 approaches and48 floral visits by M. sexta during 5 days of observation

Table 2. ANOVA results for behavioural experiments

Factors and effects SS df MS F P

Experiment 2 Treatment 2.01 3 0.67 62.11 0.02Time 0.002 1 0.002 0.15 0.72Day 0.001 2 !0.001 0.04 0.96Treatment!time 0.03 3 0.01 1.43 0.30Treatment!day 0.04 6 0.007 0.93 0.52

Experiment 3 Treatment 1.53 2 0.76 3.61 0.04Time 0.72 2 0.36 1.69 0.20Day 0.52 4 0.13 0.61 0.66

Experiment 4 Treatment 1.44 3 0.48 4.15 0.01Time 0.001 1 0.001 0.005 0.94Day 0.11 5 0.02 0.20 0.96

In each case, the dependent variable is the arcsine-transformed proportion of total approaches per time replicate. Values in bold weresignificant at an alpha level of 0.05.

ANIMAL BEHAVIOUR, 69, 2414

(Table 1). Moths were attracted to each experimentaltreatment, but visits only occurred when visual stimuliwere present. In addition, moths displayed a novel be-haviour, decelerating and hovering within a metre of

Cloth bag

Plastic bag

Paper flowers

Open flowers

q' = 13.63, P< 0.001

q' = 13.15, P < 0.001

q' = 7.65, P < 0.001

(a)

(b)

(c)

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Cloth bag

Paper flowers

Paper flowers +scent

t28= -2.22, P = 0.017

0 0.1 0.2 0.3 0.4 0.5 0.6

Paper flowers, noscent

Paper flowers +floral scent, – veg

Paper flowers +scent, mobile

Paper flowers +scent, ‘landmark’

t22= 1.21, P = 0.12

t22= 2.91, P = 0.004

t22 = 0.43,P = 0.34

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Mean + SE proportion of total approachesper time replicate

Figure 4. Relative attractiveness of floral manipulations. (a)

Experiment 2: brackets indicate a priori contrasts between open

control and each manipulated treatment (Dunnett’s test). (b)

Experiment 3: the hatched stacked bar for cloth-bagged treatmentindicates close hovering events, which were not observed for other

treatments or experiments. Brackets show comparison between

paper flowers with versus without floral scent in the absence of

Datura vegetation (unpaired t test). (c) Experiment 4: bracketsindicate a priori contrasts between manipulated treatments (un-

paired t tests). Results for open control plant are not shown.

cheesecloth-concealed flowers a total of 48 times, butnever attempting to feed. This behaviour was not ob-served in response to other treatments. The proportion oftotal approaches was significantly associated with treat-ment but not with time or day (ANOVA, Table 2). Therewas a significantly greater proportion of total approachesper unit time to paper flowers when floral scent waspresent (Fig. 4b), reflecting a five-fold increase in ap-proaches and more than a three-fold increase in visits tothe latter treatment (Table 1). However, the feeding indexdid not differ significantly between paper flowers lackingfloral scent (0.42) and those with floral scent (0.28;Fisher’s exact test: two-tailed PZ 0.17, NZ 157 events).

Experiment 4

We observed 172 foraging bouts, 366 approaches and254 visits byM. sexta over 6 days (Table 1). Moths fed fromcontrol flowers during all observational periods, thus theirabsence at other treatments was attributed to lack of floralattraction. The mean G SE proportion of total approachesto control flowers (0.61 G 0.13) exceeded that of allmanipulated treatments combined (0.39 G 0.13) by a fac-tor of 1.5 (Fig. 4c), as in experiment 2. Variance in theproportion of total approaches per replicate was signifi-cantly associated with treatment but not with time or day(ANOVA, Table 2). When paper flowers, floral scent andvegetation were present, the treatment located near thegreenhouse was not significantly more attractive than itsmobile counterpart (Fig. 4c). To the contrary, the feedingindex of the mobile treatment (0.65) was significantlygreater than that of the fixed ‘landmark control’ (0.40,Fisher’s exact test: two-tailed PZ 0.002, N Z 152 events).Moth attraction to scented paper flowers did not increasesignificantly when Datura vegetation also was present(Fig. 4c), and the feeding index was nearly identicalbetween treatments with and without foliage (0.65 and0.63, respectively; Fisher’s exact test: two-tailed P Z 0.85,N Z 123 events). However, paper flowers with floral scentalone attracted a significantly greater proportion of mothsthan did scentless paper flowers (Fig. 4c), the latter ofwhich were not visited once (Table 1).

DISCUSSION

Visual Cues are Necessary but Insufficientfor Nectar Feeding

Visual or olfactory cues alone attracted wild M. sextamoths to floral nectar resources, but the combinationthereof was required to elicit proboscis extension andfeeding. When cheesecloth bags masked the visual cues ofDatura flowers in mixed (experiment 2) and homogeneous(experiment 3) arrays, moths consistently approached butnever probed at the fragrant, concealed flowers. Theabsence of foraging preference or constancy associatedwith cheesecloth bags in experiment 1 suggests that theywere neither overly attractive nor repellent to M. sexta,and could be utilized with confidence. Cheeseclothbags effectively camouflaged the flowers among D. wrightii

RAGUSO & WILLIS: WILD HAWKMOTH FEEDING BEHAVIOUR 415

vegetation (Fig. 1), creating the only experimental manip-ulation devoid of visual contrast. Our results are consistentwith our previous findings for naıve, laboratory-reared,male M. sexta, which track floral odours to their sources ingreenhouse (Raguso & Willis 2002) and wind tunnelbioassays (Raguso & Willis 2003) but do not feed withoutvisual cues. Similarly, Clements & Long (1923) concealedOenothera caespitosa macroglottis (Rydb.) Wagner, Stock-house & Klein (Onagraceae) flowers beneath green crepepaper and found that odour alone did not elicit probing bywild M. quinquemaculata or H. lineata moths. In contrast,several species of noctuid and geometrid moths probethrough cloth bags at hidden, fragrant flowers underlaboratory and field conditions (Brantjes 1976; Nilsson1978; Nilsson et al. 1990; Plepys et al. 2002), and may notrequire visual contrast as a feeding cue.

Plant Odours Synergize Visually GuidedFeeding from Model Flowers

Our attempts to experimentally remove floral scentrevealed that wild M. sexta use plant odours as synergisticor contextual information during nectar feeding. Paperflowers were visited most frequently in experiments 2–4when plant odours were present, whereas the truly scent-less flowers in experiment 4 were at least 15 m distantfrom any source of plant odour and were ignored. Theseresults mirror our laboratory studies, in which naıve maleM. sexta hovered above or passed over arrays of paperflowers with sugar water, but fed only when plant odourswere added to the arrays (Raguso & Willis 2002). In theformer Yugoslavia, Kugler (1971) found that Agrius con-volvuli L. only probed at paper disks and flower modelswhen they were placed among scented Datura inoxia(Mill.) flowers. Similarly, Clements & Long (1923) ob-served approaches by M. quinquemaculata and H. lineatato white crepe paper strewn among scented Oenotheracaespitosa flowers in Colorado, U.S.A., and Haber (1984)induced several species of Costa Rican hawkmoths tovisit plastic flowers in captivity when strongly fragrantPlumeria rubra (L.; Apocynaceae) flowers were present.Thus, the presence of a ‘scent cloud’ above a patch offlowering plants, as described by Nilsson et al. (1987) inMadagascar and Eisikowitch & Rotem (1987) in Israel,elicits visually guided approaches, proboscis extensionand feeding in nocturnal hawkmoth species worldwide.

Can Host Plant Vegetative Odours Functionas Surrogate Floral Attractants?

Unexpectedly, M. sexta approached and visited paperflowers when D. wrightii vegetation provided the soleodour stimulus in experiment 3 (Fig. 4b). However, theaddition of vegetative odours to floral scent in experiment4 did not increase moth attraction, suggesting that thefloral and vegetative odours of D. wrightii are redundantsignals. This interpretation is supported by the experi-ments of White et al. (1994) and Cutler et al. (1995), inwhich laboratory-raised M. sexta visited plastic artificial

flowers in the presence of tobacco vegetation alone.Datura wrightii foliage has a rancid peanut odour tohuman perception, in strong contrast to its perfume-likefloral scent, which is dominated by geraniol, benzylalcohol and ocimene-derived compounds (Raguso et al.2003a). However, Fraser et al. (2003) recently showed thatmale and female M. sexta display the strongest olfactory(electroantennogram) responses to minor components ofD. wrightii foliar odour, some of which (e.g. methylsalicylate, benzyl alcohol) are abundant floral scent com-ponents in D. wrightii (Raguso et al. 2003a). Recentanalyses by Levin et al. (2001) and Raguso et al. (2003b)suggest that scented vegetation is more widespread innight-blooming (and perhaps all) plants than has beenappreciated, and may contribute generally to hawkmothattraction and feeding (Baker 1961; Cruden 1970).The alternative explanation that moths in our study

learned landmarks to return to the same plant each nightwas not supported by the results of experiment 4 (Fig. 4c),in which moths did not distinguish between scentedpaper flowers in landmark versus mobile treatments.However, further experiments will be needed to fullyexplore the spatial learning capabilities of M. sexta.Landmark learning may be critical to long-lived flower-visiting animals that use trap-lining strategies duringforaging (Janzen 1971; Ackerman et al. 1982; Murawski& Gilbert 1986), as suggested by Linhart & Mendenhall(1977) but not yet demonstrated for nocturnal hawk-moths. Another potential explanation is that most or allof the moths that visited paper flowers with D. wrightiivegetation in experiment 3 were female M. sexta searchingfor host plants on which to lay their eggs. At thistreatment, feeding bouts were significantly shorter(meanG SE durationZ 1.9 G 0.4 total approaches perbout, NZ 14) than those observed at paper flowers withfloral scent (3.6G 0.6, NZ 33; t45 Z �1.81, P Z 0.04).The few obvious oviposition bouts observed duringexperiment 3 were omitted from statistical analysis, butwe did not capture or sex all individuals observed in thisstudy. Female M. sexta regularly feed from D. wrightiiflowers before or after oviposition bouts and are twice aslikely as males to feed in laboratory settings (Ziegler 1991;White et al. 1994). Future experiments should addresswhether mated or unmated M. sexta females use differentsensory cues than males when feeding from flowers, andwhether their foraging bouts are shorter, on average, thanthose of males.

Sensory Discrimination and Flower Models:Why are Paper Flowers Less Attractive?

Moths approached and visited paper flowers when plantodours were present, but were significantly more likely toapproach or visit control Datura flowers during first (andsubsequent) visits of foraging bouts. Feeding index wasvariable but always lower in scented paper flowers (28–65%) than in open control flowers (77–87%). Visualdifferences between paper flowers and controls may havediscouraged initial visits to the former by M. sexta. Our

ANIMAL BEHAVIOUR, 69, 2416

model flowers were just as rewarding as control D. wrightiiflowers, but differed in shape and reflectance in 300–400 nm (UV) wavelengths. We also used plastic bags toexperimentally remove fragrance as a stimulus, andalthough Datura flowers subtended by bags were notavoided in experiment 1, flowers placed within bags werenearly ignored in experiment 2, and plastic bags wereabandoned as manipulative tools in subsequent experi-ments. These bags’ increased UV reflectance and modifi-cation of Datura flower shape are unlikely to explain theirunattractiveness, given the allure of paper flowers witheven greater UV reflectance and conical profile (Figs 1, 2).Instead, mirror reflections from the bag’s surface (Fig. 2),although not repellent per se in experiment 1, may havemodified the appearance of Datura flowers beyond themoths’ threshold of recognition.The apparent discrimination by M. sexta against arti-

ficial or bagged natural flowers with subtle visual dif-ferences is plausible given that the related diurnal(Macroglossum stellatarum) and nocturnal (Deilephila elpe-nor) hawkmoths can learn to distinguish rewarding fromunrewarding flower models that differ only in UV re-flectance (Kelber & Henique 1999; Kelber et al. 2002).White et al. (1994) found that UV reflecting flower modelsinhibited feeding by M. sexta in flight cage assays. Thismay stem from low visibility, as Kevan et al. (1996) arguethat ‘insect-white’ flowers (reflecting in all insect-visiblewavelengths) provide poor colour contrast with vegeta-tion, and Kelber et al. (2002) report relatively low photoncapture by UV photoreceptors in the compound eyes ofD. elpenor. Alternatively, UV wavelengths have beenassociated with ‘escape’ or dispersal flight towards openspace in Pieris brassicae L. (Pieridae) and other Lepidoptera(Scherer and Kolb 1987), and may trigger similar behav-iour in M. sexta (Ramaswamy 1988).The lack of a generalized feeding response to single-

modality floral stimuli in this study suggests (1) thatnectar feeding in M. sexta is constrained by an innatesensory requirement for multimodal floral cues, or, morelikely, (2) that the moths in our study learned visual andolfactory cues from D. wrightii flowers as a compoundstimulus or search image (e.g. Pietrewicz & Kamil 1979)and discriminated against floral manipulations lackingappropriate signal components (Gegear & Laverty 2001).This observation highlights the difficulty of distinguish-ing innate from learned behaviours in the field, and theneed for further experiments using free-flying moths withknown histories in captive settings (Cunningham et al.2004). Cross-paradigm learning experiments, such asthose performed by Gerber et al. (1996) with honeybees,in which operant-conditioned animals are tested for thetransfer of visual or olfactory memories to a classicalconditioning context, would be one way to test the‘compound stimulus’ hypothesis for M. sexta.Fragrance and visual display are not the only sensory

cues proffered by flowers; moths may use additionalsensory information to discriminate between natural andartificial flowers. Our paper flower models contained 25%sucrose solution, whereas D. wrightii nectar also containshexose sugars (Raguso et al. 2003a), amino acids andtropane alkaloids (Grant & Grant 1983), and has a sharp,

unpleasant odour that is chemically distinct from that ofthe corolla (Raguso 2004). Similarly, D. wrightii flowersrespire actively and emit significantly more carbon dioxidethan do leaves or other plant parts (Guerenstein et al.2004). The labial pit organs of M. sexta respond sensitivelyto carbon dioxide, which appears to be processed as anolfactory signal (Kent et al. 1986; P. G. Guerenstein &J. G. Hildebrand, unpublished data). On a larger scale, theodour plume structure of control flowers would differ fromthat of cheesecloth-bagged flowers, which could affect theinformation content or perception of floral scent by moths(Vickers 2000). Unexpected cues such as floral movementin wind (e.g. Lehrer 1988) are highly attractive to M. sexta(Moreno et al. 2000; Sprayberry 2002) and are likely todiffer markedly between control and paper flowers. Alter-natively, hawkmoth experiences during visits to paperflowers may have reduced the probability of further visits.Such experiences could stem from differences in floraltexture, nonsucrose nectar constituents and gustatorylearning, perceived vulnerability to predation or othercontextual factors (Dobson 1994; Dukas 2001).

Conclusions

Wild, presumably experienced M. sexta moths requiredboth visual and olfactory floral stimuli in order to feed,and did not generalize to single-modality cues in any ofour experiments. This unexpected result indicates thatM. sexta are capable of fine sensory discriminationbetween our floral models and authentic D. wrightiiblossoms, and suggests that the learning paradigms offree-flying and captive moths may differ. The observedfunctional redundancy between floral and vegetativeodours of D. wrightii as contextual cues for nectar feedingby M. sexta suggests that the sensory requirement forodour is not chemically strict. Future experiments shouldexplore this apparent flexibility by comparing the effec-tiveness of chemically diverse fragrances as contextualfeeding cues for M. sexta. We have shown that dual-modality floral signals with visual and olfactory compo-nents play a highly predictable role in the behaviours thatdefine nectar feeding by M. sexta (Raguso & Willis 2003).However, it is likely that feeding behaviour is impacted bya more complex array of sensory stimuli and experiences,several of which are currently being studied in laboratoriesaround the world, including our own.

Acknowledgments

We thank M. Dimmitt and G. Nabhan for their enthusi-astic support and permission to work at the Arizona-Sonora Desert Museum. J. Hildebrand, R. Schneider,M. Wicklein and the late R. Chapman contributed toexperimental design, and members of the Willis labo-ratory provided valuable editorial assistance. Specialthanks to E. Pichersky for suggesting the odour-freetreatment in experiment 4, and to L.A. Nilsson andA. Kelber for comments that greatly improved the man-uscript. We thank R. Papke, O. Pellmyr and T. Tullyfor help in the field, K. Jones and C. Martinez del Rıofor statistical advice and M. Clauss and K. Selchow for

RAGUSO & WILLIS: WILD HAWKMOTH FEEDING BEHAVIOUR 417

translating and discussing Kugler’s work. Special thanksare due to our colleagues too numerous to mention whovolunteered to watch moths. K. Copeland and G. Simms(Spectral Instruments, Inc.) provided spectral data fornatural and paper flowers, and N. Armstrong and P. Lee(University of Arizona, Department of Chemistry) dis-cussed the properties of moonlit flowers. R. A. R. wassupported by a National Institutes of Health TrainingGrant T32 AI07475 through the Center for Insect Science,a National Science Foundation (NSF) grant BIR-9602246 tothe Research Training Group in Biological Diversification,University of Arizona, Department of Ecology and Evolu-tionary Biology, an NSF grant DEB-9806840 and a Univer-sity of Arizona Foundation Small Grant. M. A. W. wassupported by NSF grant IBN-9511742.

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