Reversal Learning and Risk-Averse Foraging Behavior in the Monarch Butterfly, Danaus plexippus...

RE SEAR CH PAP ER

Reversal Learning and Risk-Averse Foraging Behavior in theMonarch Butterfly, Danaus plexippus (Lepidoptera:Nymphalidae)Daniela Rodrigues*, Brad W. Goodner� & Martha R. Weiss*

* Department of Biology, Georgetown University, Washington, DC, USA

� Department of Biology, Hiram College, Hiram, OH, USA

Introduction

Learning allows insects to respond to variable or

unpredictable environments (Stephens 1993;

reviewed in Dukas 2008). Many studies have dem-

onstrated that bees, wasps, flies, butterflies, and

grasshoppers, among other insects, can develop a

positive association between visual and ⁄ or olfactory

cues and resources such as nectar or oviposition sites

(e.g., Shafir 1996; Dukas 1999; Dukas & Bernays

2000; Weiss & Papaj 2003; Cnaani et al. 2005). Addi-

tionally, insects in a range of taxa can learn to

associate visual and olfactory cues with negative or

aversive stimuli such as salt, shock, toxins, poor

quality food or distractors (e.g., Berenbaum & Mili-

czky 1984; Tully & Quinn 1985; Lee & Bernays

1990; Bernays 1993; Bowdish & Bultman 1993;

Dukas & Bernays 2000; Matsumoto & Mizunami

2002; Chittka et al. 2003; Dyer & Chittka 2004;

Davis 2005; Vergoz et al. 2007; Blackiston et al.

2008). Learning to associate biologically relevant

cues with either positive or negative stimuli can

increase an insect’s fitness (e.g., Dukas & Bernays

2000).

Correspondence

Daniela Rodrigues, Departamento de Biologia

Animal, IB, UNICAMP, Campinas, SP, Brazil.

E-mail: [email protected]

Received: September 22, 2009

Initial acceptance: November 16, 2009

Final acceptance: December 12, 2009

(D. Zeh)

doi: 10.1111/j.1439-0310.2009.01737.x

Abstract

Learning ability allows insects to respond to a variable environment,

and to adjust their behaviors in response to positive or negative experi-

ences. Pollinating insects readily learn to associate floral characteristics,

such as color, shape, or pattern, with appetitive stimuli, such as the

presence of a nectar reward. However, in nature pollinators may also

encounter flowers that contain distasteful or toxic nectar, or offer highly

variable nectar volumes, providing opportunities for aversive learning or

risk-averse foraging behavior. Whereas some bees learn to avoid flowers

with unpalatable or unreliable nectar rewards, little is known about

how Lepidoptera respond to such stimuli. We used a reversal learning

paradigm to establish that monarch butterflies learn to discriminate

against colored artificial flowers that contain salt solution, decreasing

both number of probes and probing time on flowers of a preferred color

and altogether avoiding artificial flowers of a non-preferred color. In

addition, when we offered butterflies artificial flowers of two different

colors, both of which contained the same mean nectar volume but

which differed in variance, the monarchs exhibited risk-averse foraging:

they probed the constant flowers significantly more than the variable

ones, regardless of flower color or butterfly sex. Our results add to our

understanding of butterfly foraging behavior, as they demonstrate that

monarchs can respond to aversive as well as appetitive stimuli, and can

also adjust their foraging behavior to avoid floral resources with high

variance rewards.

Ethology

270 Ethology 116 (2010) 270–280 ª 2010 Blackwell Verlag GmbH

ethology international journal of behavioural biology

Flowers often provide their visitors with opportu-

nities for positive (appetitive) learning, and insects

are able to associate nectar characteristics with floral

cues such as color, odor, or pattern (Laverty 1994;

Weiss 1995, 1997; Kelber 1996, 2002; Gumbert

2000; Andersson & Dobson 2003). Flowers, however,

can also provide opportunities for negative (aversive)

learning, as nectars that contain alkaloids or pheno-

lics, and are thus toxic or repellent to some floral vis-

itors, are produced by plants in over 20 families

(Adler 2000). The ecological significance of toxic nec-

tar, if any, is not well understood, although various

adaptive explanations, including deterrence of nectar

robbers, encouragement of specialist pollinators, pre-

vention of microbial growth, and alteration of polli-

nator behavior leading to reduction of geitonogamy,

have been proposed to explain the phenomenon

(Johnson et al. 2006; Gegear et al. 2007).

Floral nectar volume may also vary markedly

within and between plants of a given species. Such

variation may be a result of pollinator foraging activ-

ity (Zimmerman & Pleasants 1982; Biernaskie et al.

2002; Biernaskie & Cartar 2004) or the variability

itself may be a heritable phenotypic trait (McDade &

Weeks 2004). In either case, some groups of flower

visitors respond to the variable rewards; ‘risk-averse’

foragers preferentially select flowers that offer low

variance in nectar volume, even if other flowers

with an equivalent mean nectar volume are avail-

able (Real 1981; Real et al. 1982; Dukas & Real

1993; Cnaani et al. 2005).

Among pollinators, investigations of negative

conditioning and risk-averse foraging have focused

largely on bees. Bumblebees have been shown to

avoid both real and artificial flowers that contain

alkaloids in their nectar (Adler & Irwin 2005;

Gegear et al. 2007), and several bee species are

risk-averse foragers (Real 1981; Real et al. 1982;

Dukas & Real 1993; Cnaani et al. 2005). Whereas

butterflies’ responses to positive floral rewards have

been well studied (e.g. Goulson & Cory 1993;

Weiss 1995, 1997; Kelber 1996, 2002; Kinoshita

et al. 1999), little is known about their responses

to aversive stimuli, or to variable rewards. For

example, Agraulis vanillae adults were deterred by

monocrotaline offered in combination with sucrose

solution in one case (Masters 1991), but not

another (Landolt & Lenczewski 1993). The contrast-

ing results may be explained by differences in the

corresponding methods, as butterflies were allowed

to learn in the first case but not in the second. No

studies are available on risk-averse foraging behav-

ior in lepidopterans.

The monarch butterfly (Danaus plexippus Linnaeus)

(Nymphalidae), a wide-ranging, long-lived nympha-

lid (Scott 1986; Hay-Roe et al. 2007), is perhaps the

most widely recognized and popular insect in North

America. Its intimate relationship with the milkweed

plant (e.g. Malcolm 1994; Zalucki et al. 2001;

Helmus & Dussourd 2005), its role as a Batesian

model (Brower 1988; Ritland & Brower 1991), and

its remarkable long-distance migration (Calvert &

Brower 1986; Mouritsen & Frost 2002) have been

the subject of considerable attention. However, sur-

prisingly little is known about the monarch’s color

vision and color learning abilities. Recent work has

demonstrated that monarchs have true color vision,

and that they can learn to associate colors with

nectar rewards following as little as 1 or 2 min of

training (Blackiston 2007; Blackiston & Weiss,

unpublished data; see Briscoe & Chittka 2001). Mon-

archs can also learn to associate floral patterns with

a nectar reward (Weiss, Wadlington & Rodrigues,

unpublished data). Monarchs are generalist forag-

ers (http://www.monarchwatch.com) and present

innate preferences for orange flower models fol-

lowed by yellow (Blackiston 2007). In this study,

we examine the responses of monarch butterflies to

artificial flowers (1) containing aversive salt solution,

and (2) offering constant means, but variable vol-

umes of sucrose solution.

Methods

General Methods

Butterflies

Monarchs were obtained as pupae from several com-

mercial sources (Sassyfrass Butterfly Ranch, Minne-

sota; Live Monarch Foundation, Florida; and Great

House Butterfly Farm, FL, USA). Following emer-

gence, adults were separated by sex, individually

marked, and placed in green 0.3 m3 mesh cages,

with a maximum of five butterflies per cage. Cages

were kept in the laboratory under controlled condi-

tions (25 � 2�C; 14 L:10 D). Approximately 24 h

after emergence, butterflies were allowed to feed for

1 min on a 20% sucrose solution contained in the

central well of a black model flower. The butterfly’s

proboscis was gently unrolled into the nectar well

under a 75 W soft white incandescent lamp.

Artificial flowers

Artificial flowers were constructed of a 10-ll plastic

pipette tip (0.5 cm in diameter at the top) inserted

D. Rodrigues, B. W. Goodner & M. R. Weiss Aversive Learning in Monarch Butterflies

Ethology 116 (2010) 270–280 ª 2010 Blackwell Verlag GmbH 271

into the center of a 4 cm circle of matte-finish, satu-

rated Color-Aid� colored paper (Hudson Falls, NY,

USA). The paper disc was creased along two perpen-

dicular diameters to provide a tactile nectar guide for

the insects.

General training and testing protocol

Trials commenced 2 d after emergence, and were

conducted under controlled conditions as described

above. Butterflies were trained and tested individu-

ally in a 0.6 m3 green mesh cage illuminated by two

250 W non-UV halogen lamps, one suspended above

each side of the cage. Artificial flowers were inserted

into a base of white styrofoam (56 cm2 · 2 cm high)

on the bottom of the cage. Butterflies were allowed to

feed from an artificial black flower for 5 s to stimulate

foraging prior to the start of a given training or testing

session. After training or testing was completed, each

butterfly was again fed at the black flower for 55 s.

During training and testing, we recorded the number

of times each butterfly probed the center of an artifi-

cial flower, as well as the color of the artificial flower

probed. In addition, we also recorded duration of

probes, commencing when the butterfly accessed the

center of the flower with its proboscis.

The ‘nectar’ used in both sets of experiments was

a 20% sucrose solution, with volumes varying

according to each experimental protocol, whereas a

20% sodium chloride solution (10 ll per artificial

flower) was used as the aversive stimulus in the

reversal learning experiment.

Experiments

Reversal Learning Involving Appetitive and Aversive

Stimuli

Flowers used in these set of experiments were con-

structed of yellow (Yw-HUE) and blue (B-HUE)

papers (see Fig. 1 for reflectance). Previous work has

shown that monarchs have an innate preference for

yellow over blue (Blackiston 2007; Rodrigues &

Weiss, unpublished data; this study) when offered a

choice between those two colors, and we were inter-

ested in learning whether it was possible to condi-

tion a butterfly against an innately preferred color.

Twelve artificial flowers of each color were arranged

alternately in a 4 · 6 grid (9.2 cm apart center to

center; total n = 24 artificial flowers). In a series of

pilot experiments, we found that after encountering

salt water in a model flower, butterflies tended to

cease visiting any flowers in the array, and instead

remained on the side of the cage. Therefore, to

assess the response of butterflies to an aversive stim-

ulus, we chose to use a reversal learning paradigm

in which we tested the relative visitation of mon-

archs to flowers containing either saline or sucrose

solution (see Matsumoto & Mizunami 2002).

The training and testing regime consisted of one

control and two treatments, each lasting 3 d. For a

given control ⁄ treatment, each butterfly was allowed

to forage individually for 2 d (15 min ⁄ d) concomi-

tantly on flowers containing either sucrose or salt

solution (10 ll ⁄ artificial flower), and tested on the

third day (10 min) on empty artificial flowers. The

same butterflies participated in control and all treat-

ments (n = 20; 10 males and 10 females).

Training began with a control (C), in which naıve

monarchs were offered yellow and blue flowers,

both of which contained sucrose. Following 2 d of

training, butterflies were tested on day 3 on empty

yellow and blue flowers. Treatment one (T1) took

place on days 4 and 5, when butterflies were pro-

vided with yellow flowers (the preferred color) that

contained salt solution and blue flowers (the non-

preferred color) that contained salt solution. The

butterflies were again tested on empty flowers on

day 6. In treatment two (T2), on days 7 and 8, the

pattern was reversed, and salt solution was offered

in the blue flowers while sucrose was offered in the

yellow. The final testing session, once again with

empty flowers, took place on day 9. For each butter-

fly, we calculated the total number of flowers of

each color probed, as well as the proportion of time

spent probing each color.

Risk-Averse Foraging

To investigate whether butterflies exhibit risk-averse

foraging, we constructed artificial flowers out of

Fig. 1: Reflectance spectra of Color-Aid� papers used for construct-

ing artificial flowers.

Aversive Learning in Monarch Butterflies D. Rodrigues, B. W. Goodner & M. R. Weiss


purple (V-HUE) and green (Gw-HUE) papers

(Fig. 1). Choice tests with naıve butterflies demon-

strated that these are two of the monarchs’ least pre-

ferred colors when offered an array of yellow, red,

orange, blue, green, or purple artificial flowers

(Blackiston 2007). Additionally, we performed a bin-

ary choice test using empty purple vs. green artificial

flowers prior to the risk-aversion experiment (trial

duration = 20 min), and found no preference for

either color (purple: 1.3 � 0.50 visits; green:

1.3 � 0.60 visits [mean � SE]; paired t-test, t = 0;

p = 1; n = 10 butterflies).

We ran two experiments, each with a cohort of

10 butterflies (five males and five females; total

n = 20 butterflies). In each experiment, one color

offered a constant reward and the other a variable

reward for 4 d (phase one); the color–reward associ-

ation was switched for a second 4 d (phase two). For

phase one of experiment one, purple flowers offered

constant nectar volumes and green flowers offered

variable nectar volumes; the colors were switched in

phase two. For phase one of experiment two, green

flowers offered constant nectar volumes and purple

flowers offered variable nectar volumes; again, colors

were switched in phase two. All butterflies were 2

and 6 d post-emergence at the beginning of phases

one and two, respectively. The green and purple

flowers were arranged alternated in six rows and six

columns, and were 9.2 cm apart center to center (18

flowers of each color; total n = 36 artificial flowers).

All of the constant flowers (C) contained 2-ll nec-

tar (mean = 2.0, variance = 0), whereas half of the

variable flowers (V) contained 4-ll nectar, and the

other half contained no nectar at all (mean 2.0, vari-

ance = 4.23). Volumes were chosen based on Real

(1981) and Goodner & Sgambat (unpublished data).

Butterflies were allowed to forage individually for

15 min ⁄ d for the 4 d of a phase, for a total foraging

time of 1 h ⁄ phase. Flowers were refilled with nectar

after each 15-min training ⁄ testing session.

Unlike the reversal learning trials, we did not sep-

arate training and testing sessions in the risk-averse

experiments; instead, we recorded the number of

artificial flowers visited during each 15-min trial

over the entire 8 d of the experiment (4 d for each

phase). We recorded total number rather than dura-

tion of probes, as probing time could be positively

correlated with available nectar volume. To be

included in the analyses, butterflies had to have vis-

ited at least one flower, regardless of its status, by

the second day of training ⁄ testing within each phase.

We summed each individual’s visits to green and

purple over the 4 d of each phase, determined the

percentage of visits made to each color, and calcu-

lated means for both number and percentage of

probes across all butterflies within each experiment.

Statistical Analysis

Reversal learning involving appetitive and aversive stimuli

Data were not normally distributed (Shapiro–Wilk

normality test), and were then arcsine transformed.

Within the control and each treatment, we used

paired t-tests to compare the proportions of probes

and time spent on yellow and blue artificial flow-

ers. In addition, we used a repeated measures

anova followed by Tukey’s post hoc tests to compare

proportions of probes and time spent on each color

across treatments, to assess changes in monarch

learned responses to a given color in relation to the

innate preferences (i.e. naıve level) as well as

between treatments (Sokal & Rohlf 1995). Only

monarchs that probed at least one of the colors in

each of the three testing sections were included in

the analyses.

Risk-averse foraging

Effects of flower color and butterfly sex on monarch

choice were analyzed for each phase of each experi-

ment through a two-way anova. Because these

effects were not significant (data not shown), we

grouped the data from both experiments by reward

category (constant vs. variable) rather than by

flower color. Both percentage and number of probes

of constant and variable flowers within and across

phases showed a normal distribution (Shapiro–Wilk

normality test). We used a paired t-test to compare

percentage and number of visits to constant vs. vari-

able artificial flowers, available simultaneously

within each phase (C1 vs. V1 and C2 vs. V2), irre-

spective of flower color (Sokal & Rohlf 1995).

Within each phase, to assess how monarch

responses to reward variability change over time, we

used paired t-tests to compare percentage of probes

to each flower category in the first half of the phase

(days 1 and 2) with those during the second half of

the phase (days 3 and 4). We also used paired t-tests

to evaluate how both percentage and number of

probes to green and purple flowers changed when

the reward status of the colors switched between the

first and second phases (i.e., C1 vs. V2 and V1 vs.

C2). Finally, we compared the overall mean number

of probes in phase one vs. phase two, again using a

paired t-test. Results were analyzed with Vassar Stats

(http://faculty.vassar.edu/lowry/VassarStats.html)



and GraphPad Software (La Jolla, CA, USA) (Motul-

sky 1999).

Results

Reversal Learning Involving Appetitive and Aversive

Stimuli

Of the original 20 butterflies, 16 made choices in the

testing session following the control (eight females

and eight males), 11 in testing following treatment

one (four females and seven males) and 13 in testing

following treatment two (six females and seven

males) (Table 1). When nectar was offered in both

blue and yellow flowers (control), the butterflies in

the subsequent testing session spent 10 times longer

probing yellow than blue empty flowers (paired

t-test, t15 = 3.16, p < 0.01), and made seven times

more visits to the yellow flowers (paired t-test,

t15 = 2.83, p < 0.01) (Table 1; Fig. 2a). In treatment

one, when yellow flowers offered salt solution and

blue flowers offered nectar in the training, neither

time spent probing nor number of probes to empty

yellow and blue flowers differed significantly during

the testing session (paired t-test, time: t10 = 0.74,

p = 0.47; probes: t10 = 0.17; p = 0.86) (Table 1;

Fig. 2b). When salt solution was present in the blue

flowers and nectar in the yellow ones (treatment

two), none of the 13 participating butterflies probed

the empty blue flowers at all during the test phase,

so that empty yellow flowers received all of the visits

(paired t-test, t12 = 8.06, p < 0.01 for both time

spent and probes) (Table 1; Fig. 2c).

Within colors, responses of monarchs changed sig-

nificantly over time (repeated measures anova, time

spent – yellow: F2,15 = 11.42, p < 0.01; blue:

F2,15 = 6.92, p < 0.01; probes – yellow: F2,15 = 8.57

p < 0.01; blue: F2,15 = 6.38, p < 0.01). Both propor-

tion of probes and time spent on yellow flowers by

monarchs declined significantly between the test ses-

sions following the control and treatment one, when

the contents of the yellow flowers changed from

nectar to salt solution (Table 2; Fig. 2a,b). A signifi-

cant recovery to the naıve level occurred between

the test sessions following treatments one and two,

when the contents of the yellow flowers changed in

the opposite direction, from salt to sugar (Table 2;

Fig. 2a–c). With respect to blue flowers, proportion

of probes and time spent by monarchs did not

change significantly between the test sessions follow-

ing the control and treatment one, as blue flowers in

both of the training sessions contained sugar

Table 1: Reversal learning: response of monarch butterflies to positive and negative stimuli in association with flower color (mean � SE)

Parameter

Control Treatment one Treatment two

Blue (sucrose) Yellow (sucrose) Blue (sucrose) Yellow (salt) Blue (salt) Yellow (sucrose)

Time (s) 6.06 � 2.72 60.56 � 10.87 13.36 � 4.28 17.27 � 5.54 0 � 0 47.46 � 8.31

Time (%) 20.12 � 8.51 79.88 � 8.51 59 � 10.74 41 � 10.74 0 � 0 100 � 0

Probes (n) 1.19 � 0.39 8.44 � 1.41 2 � 0.51 3.27 � 1.03 0 � 0 7.85 � 1.07

Probes (%) 22.68 � 9.0 77.32 � 9.0 51.24 � 10.73 48.76 � 10.73 0 � 0 100 � 0

Monarchs visiting each color (n) 9 14 9 7 0 13

(a) (b) (c)

Fig. 2: Relative frequency of the amount of time spent by monarchs probing blue (white bars) or yellow (black bars) artificial flowers (mean � SE)

following learning on: (a) sucrose solution in both yellow and blue artificial flowers (control; n = 16); (b) salt solution in the yellow flowers and

sucrose solution in blue (treatment one; n = 11) and (c) salt solution in blue flowers and sucrose solution in yellow (treatment two; n = 13). Aster-

isks indicate significant differences in probing time within treatments (paired t-tests, a = 0.05).



(Table 2; Fig. 2a,b). In contrast, numbers of probes

and time spent on blue flowers by monarchs

decreased significantly between the training sessions

following treatments one and two, that is, when

nectar was replaced by salt in the corresponding

training (Tables 2; Fig. 2b,c).

Risk-Averse Foraging

In phase one, the butterflies probed a significantly

higher percentage of constant than of variable flow-

ers (paired t-test, t19 = 3.10; p < 0.01; Fig. 3a),

regardless of color. In phase two, the difference

between constant and variable flowers was no longer

significant (paired t-test, t19 = 1.05, p > 0.05;

Fig. 3a). During phase one, the butterflies’ foraging

preferences changed with experience. After the first

2 d of foraging on constant and variable flowers,

mean number of probes to the two categories did

not differ significantly; however, after 4 d, the but-

terflies visited constant flowers significantly more

often than they did variable flowers (Table 3). In

phase 2, the lack of difference between visitation to

constant and variable flowers persisted throughout

all 4 d (Table 3).

The butterflies’ behavior towards a given color

changed significantly when its reward status chan-

ged across experimental phases. On an individual

level, for 17 of 20 butterflies the percentage of visits

to the color that had been rewarding in phase one

decreased when that color became variable in phase

two. Furthermore, on a population level, the per-

centage of visits made by all butterflies to artificial

flowers that was initially variable but became

constant increased significantly (V1 vs. C2, paired

t-test, t19 = 4.66, p < 0.01; Fig. 3a); conversely, the

Table 2: Reversal learning: Tukey’s post hoc

tests of the relative time spent and number of

probes by monarchs on each color across

treatments (a = 0.05)

Parameter Comparison

Mean

difference q p

Time (%) Yellow C (sucrose) vs. Yellow T1 (salt) 0.76 5.56 <0.05*

Yellow C (sucrose) vs. Yellow T2 (sucrose) )0.08 0.56 >0.05

Yellow T1 (salt) vs. Yellow T2 (sucrose) )0.83 6.11 <0.05*

Blue C (sucrose) vs. Blue T1 (sucrose) )0.27 2.19 >0.05

Blue C (sucrose) vs. Blue T2 (salt) 0.37 3.05 >0.05

Blue T1 (sucrose) vs. Blue T2 (salt) 0.64 5.24 <0.05*

Probes (%) Yellow C (sucrose) vs. Yellow T1 (salt) 0.65 4.65 <0.05*

Yellow C (sucrose) vs. Yellow T2 (sucrose) )0.11 0.76 >0.05

Yellow T1 (salt) vs. Yellow T2 (sucrose) )0.76 5.41 <0.05*

Blue C (sucrose) vs. Blue T1 (sucrose) )0.16 1.42 >0.05

Blue C (sucrose) vs. Blue T2 (salt) 0.40 3.49 <0.05*

Blue T1 (sucrose) vs. Blue T2 (salt) 0.56 4.91 <0.05*

Asterisks denote significant differences.

(a)

(b)

Fig. 3: Relative (a) and absolute (b) number of monarch probes

(mean � SE) in relation to artificial flowers offering constant and vari-

able sucrose rewards. Flowers that offer variable reward in phase one

offer constant reward in phase two, and vice versa. Different capital

letters indicate significant differences in probes within phase one; non-

capital letters, significant differences within phase two. Asterisks indi-

cate significant differences in probes across phases (paired t-tests,

a = 0.05). Numbers inside bars represent total number of probes on

each flower category ⁄ phase.



percentage of visits from C1 to V2 significantly

declined.

With respect to number, rather than percentage of

probes, when green and purple flowers changed

from constant to variable across phases, number of

probes to the artificial flowers did not differ signifi-

cantly (C1 vs. V2, paired t-test: t19 = 1.18, p = 0.25).

In contrast, probe number increased significantly fol-

lowing the change from variable to constant (V1 vs.

C2, paired test, t19 = 4.20, p < 0.01). Overall, the

mean number of visits to artificial flowers of both

colors was significantly higher in phase two (paired

test, t39 = 3.05, p < 0.01) (Fig. 3b).

Discussion

To date, research on learning in Lepidoptera in the

context of nectar foraging has focused on appetitive

associative learning. Butterflies and moths are able

to associate colors, patterns, or odors with rewarding

stimuli, and can rapidly change their visitation pat-

terns in response to a change in reward status (e.g.,

Andersson & Dobson 2003; Weiss 1995, 1997; Kan-

dori & Ohsaki 1996; Kelber 1996; Blackiston 2007).

Our results clearly demonstrate that monarch butter-

flies also learn to respond to aversive stimuli, and

thus they avoid high variance in nectar volume.

Using a reversal-learning paradigm, we found that

the butterflies’ preference for a color significantly

decreases when that color is paired with salt, an

aversive stimulus. These butterflies are also ‘risk-

averse’ foragers and prefer to forage on flowers with

constant rather than variable nectar volumes. Con-

sidering that monarchs are generalist foragers, such

behavioral plasticity in the context of feeding is eco-

logically relevant for this wide-ranged and long-lived

butterfly species. Nectarivorous insects such as

honey bees and bumble bees have been shown to

associate aversive stimuli with color or odor cues

(e.g., Chittka et al. 2003) and are also risk-averse

foragers with respect to nectar volume (Real 1981;

Real et al. 1982), and nectar concentration (Cnaani

et al. 2005). To our knowledge, this is the first dem-

onstration that butterflies can, like bees, adjust their

foraging behavior in response to both unpalatable

stimuli and to variance in reward volume.

In the reversal-learning experiment, flower color

choice and the number of butterflies that partici-

pated in the tests seem to reflect an interaction

between innate preference and floral reward status.

In the control, when both artificial flowers provided

a nectar reward, butterflies preferentially visited yel-

low over blue, corroborating the monarchs’ innate

preference for yellow (Blackiston 2007). In the sec-

ond test period, following training in which reward

countered innate preference (yellow flowers offered

salt solution and blue flowers offered nectar), butter-

flies behaved similarly towards yellow and blue

flowers, with respect to both time spent and number

of visits. Here, in comparison with the control, half

as many butterflies chose to visit yellow flowers,

perhaps reflecting a compromise between negative

reward and innately preferred color, whereas the

number that visited blue did not change. It is not

surprising that visits to blue flowers did not change

from control to treatment one, as the reward status

of these flowers did not change. In the test period

following treatment two, when the rewards offered

by the flowers supported innate preferences (yellow

offered nectar, blue offered salt solution), the butter-

flies visited only yellow flowers, and stopped visiting

blue altogether. Here the number of butterflies visit-

ing yellow flowers returned almost to the level of

the control, reflecting reinforcement of interest in

the preferred color, whereas visitors to blue dropped

to zero, presumably dissuaded by the learned

association of a non-preferred color and a negative

stimulus.

The ecological significance of toxic or distasteful

compounds in nectar, if any, is not clear (see Adler

2000; Gardener & Gillman 2002). One adaptive

hypothesis is that the toxic compounds can be toler-

ated by specialist but not generalist pollinators

through detoxification or sequestration mechanisms

(Baker & Baker 1975; Rhoades & Bergdahl 1981). In

a study of nectar constituents other than sugars,

Baker & Baker (1975) found that the plants that

tested positive for presence of alkaloids in nectar

Table 3: Risk-averse foraging: Comparison of

the relative number of monarch probes on

constant vs. variable flowers at the beginning

and end of each phase (mean � SE)

Phase Time Constant Variable t p

One Days 1–2 (beginning) 44.62 � 7.06 37.88 � 6.84 0.54 0.59

Days 3–4 (end) 58.84 � 4.01 41.16 � 4.01 2.21 <0.01*

Two Days 1–2 (beginning) 52.76 � 3.79 47.24 � 3.79 0.73 0.47

Days 3–4 (end) 50.83 � 4.27 44.17 � 4.16 0.87 0.39

Asterisk denotes significant difference (paired t-tests, a = 0.05).



were generally pollinated by bees, whereas of the 46

species of flowers known to be pollinated by Lepi-

doptera, none contained alkaloids in their nectar.

This observation led the Bakers to speculate that the

presence of alkaloids in nectar would encourage pol-

lination by specialist bees and discourage less effec-

tive butterfly visitors. It is not clear, however, that

the presence of alkaloids in nectar is in fact deterrent

to pollinators, be they generalists or specialists. The

alkaloid monocrotaline, when added to sucrose in

artificial flowers, deters visits by the generalist but-

terfly visitor A. vanillae (Masters 1991), but the same

does not occur for other generalist butterfly and

moth species (Landolt & Lenczewski 1993). In addi-

tion, high levels of alkaloids in the nectar of Gelsemi-

num sempervirens do not strongly deter visits of bee

pollinators or nectar robbers in the field (Adler &

Irwin 2005) but deters naıve bees in the lab (Gegear

et al. 2007). Regarding other compounds, Stephen-

son (1982) found that iridoid glycosides in the nectar

of Catalpa speciosa deterred potential nectar thieves

(ants and a skipper butterfly) but regular bee pollin-

ators did not seem to be affected. The bitter pheno-

lics present in the nectar of Aloe vryheidensis are

unpalatable for opportunistic visitors as sunbirds and

honeybees, but do not affect other nectar-feeding

birds, which are potential pollinators (Johnson et al.

2006). Thus the role of toxic or distasteful com-

pounds as filters to opportunistic visitors may

depend on the system and on the ecological context

of the situation (Gegear et al. 2007), and the extent

of avoidance will depend on several factors as flower

color, abundance, scent, and other floral traits.

Whether flowers commonly visited by monarchs

contain secondary compounds in their nectar, how

monarchs might respond to such compounds, and

how effective these butterflies are as pollinators, all

remain unknown.

In nature, floral nectar volume can vary widely

between plants as well as between flowers on the

same plant either because of prior insect visitation or

as a phenotypic trait (Zimmerman & Pleasants 1982;

McDade & Weeks 2004). Therefore, pollinators are

likely to encounter variable reward levels. Our data

show that monarchs are clearly ‘risk-averse’ foragers,

in that they reliably choose constant over variable

artificial flowers, regardless of color, when they are

first offered a simultaneous choice between the two.

Furthermore, the change in monarch’s response to

flower categories as phase one progressed clearly

shows learning. The butterflies’ foraging behaviors

track the switch of each color’s reward status in the

second phase of the experiments: relative visits to

the newly constant colors increase, and to the newly

variable colors decrease, such that the differences in

visitation to the two groups are no longer significant.

An examination of the number, rather than percent-

age of probes to constant and variable artificial flow-

ers reveals the cause of the difference in preference

between phases one and two. In phase two, the but-

terflies continue to visit the flowers that had offered

a constant reward in phase one, but also increase

their level of visitation to the flowers that had been

variable in phase one, so that overall, the total num-

ber of visits to all flowers in phase two is signifi-

cantly greater than that in phase one. Thus the

butterflies seem to remember the positive association

between color and constant reward for days, a pat-

tern that is supported by a preliminary memory

study (Rodrigues & Weiss, in preparation). Other

studies have shown a similar duration of associative

memory (on the order of days) in butterflies (e.g.

Goulson & Cory 1993; Kandori & Ohsaki 1996). Fur-

thermore, earlier work has shown that butterflies

continue to visit colored flowers or flowers that had

once been rewarding long after the flowers had

ceased offering nectar (Goulson & Cory 1993; Weiss

1997).

In an earlier pilot study investigating risk-averse

foraging, we found that monarchs preferentially visit

constant blue flowers (mean = 2 ll) even if the vari-

able yellow flowers offer a higher mean reward

(mean = 3 ll), and are of a preferred color. This

result corroborates our findings in the reversal learn-

ing experiment, where monarchs were successfully

trained against the preferred color when it contained

salt solution. Similarly, a pilot study of painted lady

butterflies showed that these insects preferentially

visited constant over variable artificial flowers when

both offered the same mean reward level, but the

constant color offered 4 ll of nectar, and the variable

color offered no nectar in two-thirds of the flowers,

and 12 ll in the other third (Goodner & Sgambat,

unpublished data).

Several studies have demonstrated that bumble-

bees are risk-averse with respect to nectar volume

(e.g., Real 1981; Real et al. 1982; Cartar 1991) and

may or may not be risk-sensitive in relation to nec-

tar concentration, depending on the situation (see

Banschbach & Waddington 1994; Waddington 1995;

Cnaani et al. 2005). Further, Drezner-Levy & Shafir

(2007) demonstrated that honeybees are risk-averse

foragers only if the variable treatment consists of

some flowers that offer a zero nectar volume. Mod-

els proposed to explain risk-averse behavior in bees

have related constant rewards with rate of energy



uptake (Harder & Real 1987), or energy state with

fitness (see Possigham et al. 1990; Smallwood 1996),

among other factors. From the plant perspective,

risk-averse foraging might be adaptive for reducing

geitonogamy (Biernaskie et al. 2002). In addition,

some studies have demonstrated that bees may

change their sensitivity and become risk-prone for-

agers, depending on variables such as nectar avail-

ability in the colonies (Cartar 1991), nectar volume

of constant and variable flowers, and innate color

preference (Real et al. 1982). However, because for-

aging dynamics of bees and butterflies differ in sev-

eral ways (e.g. flower location and handling time,

need to provision for colony energy stores, or for

individual use, etc.), further studies are necessary to

clarify the factors that influence foraging style in

butterflies, and to determine whether butterflies are

risk-averse foragers under field conditions.

In the early days of behavioral ecology, entomolo-

gists and behaviorists assumed that well-developed

learning abilities would be found primarily in the

social insects (especially bees), and that the behav-

iors of solitary insects were more or less instinct-dri-

ven. However, a robust and growing body of

evidence now demonstrates that butterflies, moths,

flies, crickets, grasshoppers, and other solitary insects

are indeed capable learners in a variety of behavioral

contexts. The current study, demonstrating that but-

terflies, like bees, can learn aversive stimuli and are

risk-averse foragers, further underscores the behav-

ioral flexibility and learning ability of solitary insects

(see Weiss 2001).

Acknowledgements

We thank Jessamyn S. Manson, Douglas J. Blackis-

ton, David W. Zeh, and two anonymous reviewers

for helpful comments on the manuscript. Douglas J.

Blackiston also assisted us on methodological proce-

dures and statistical analysis. We are also grateful to

Shannon Murphy, Gina M. Wimp, Peter Armbruster,

Divya B. Uma, Heather S. Mallory, Aaron Howard

and Jose R. Trigo for their help with statistical analy-

sis. D. Rodrigues was supported by CAPES Founda-

tion ⁄ Brazil (Proc. BEX 0018 ⁄ 06-6) and Georgetown

University.

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