Naturwissenschaften (2012) 99:705–713 DOI 10.1007/s00114-012-0952-y

ORIGINAL PAPER

Reward and non-reward learning of flower colours in the Byasa alcinous (: Papilionidae)

Ikuo Kandori & Takafumi Yamaki

Received: 17 January 2012 /Revised: 13 July 2012 /Accepted: 14 July 2012 /Published online: 1 August 2012 # Springer-Verlag 2012

Abstract Learning plays an important role in food acquisition social interaction and sexual behaviour (Dukas 2008). Learning for a wide range of . To increase their foraging efficien- is a fundamental mechanism for adjusting behaviour to envi- cy, flower-visiting insects may learn to associate floral cues ronmental change (Stephens 1993; Dunlap and Stephens 2009). with the presence (so-called reward learning) or the absence Although some aspects of learning are costly (e.g. Mery and (so-called non-reward learning) of a reward. Reward learning Kawecki 2003, 2004;Burgeretal.2008), learning generally whilst foraging for flowers has been demonstrated in many increasesfitness(DukasandBernays2000; Dukas and Duan taxa, whilst non-reward learning in flower-visiting 2000; Raine and Chittka 2008). Many studies have shown that insects has been demonstrated only in honeybees, bumblebees insects can develop a positive association between visual and/or and hawkmoths. This study examined both reward and non- olfactory cues and resources such as nectar or oviposition sites reward learning abilities in the butterfly Byasa alcinous whilst (e.g. Shafir 1996;Cnaanietal.2006;Dukas1999;Dukasand foraging among artificial flowers of different colours. This Bernays 2000; Weiss and Papaj 2003). This is often called butterfly showed both types of learning, although ‘positive (associative) learning’ (e.g. Papaj et al. 1994;Costa of both sexes learned faster via reward learning. In addition, et al. 2010; Rodrigues et al. 2010), ‘reward learning’ (e.g. females learned via reward learning faster than males. To the Menzel 1999;Thumetal.2007)or‘appetitive learning’ (e.g. best of our knowledge, these are the first empirical data on the Vergoz et al. 2007; Rodrigues et al. 2010). In addition, insects in learning speed of both reward and non-reward learning in a range of taxa can learn to associate visual and olfactory cues insects. We discuss the adaptive significance of a lower learn- with negative or aversive stimuli such as salt, shock, and toxins ing speed for non-reward learning when foraging on flowers. and to avoid or to escape from those cues (e.g. Berenbaum and Miliczky 1984; Tully and Quinn 1985; Lee and Bernays 1990; Keywords Positive associative learning . Appetitive Matsumoto and Mizunami 2002). This is often called ‘aversion learning . Negative associative learning . Aversive learning . learning’ (e.g. Bernays 1993; Bowdish and Bultman 1993; Aversion learning . Habituation Blackiston et al. 2008)or‘aversive learning’ (e.g. Vergoz et al. 2007; Rodrigues et al. 2010). Learning to associate biolog- ically relevant cues with either positive or negative stimuli can increase an insect’s fitness (e.g. Dukas and Bernays 2000). Introduction Some insects can also learn to associate the cues with the absence of rewards or unrewarding stimuli and to avoid those Avariety of insects rely extensively on learning for all major life cues (e.g. Papaj et al. 1994). This type of learning is sometimes activities, including feeding, predator avoidance, aggregation, called ‘non-reward learning’ (e.g. Dicke et al. 2011). Whilst in aversive learning insects learn to avoid cues associated with Communicated by: Sven Thatje negative or minus stimuli, in non-reward learning, they learn to I. Kandori (*) : T. Yamaki avoid cues associated with absent or zero stimuli. Non-reward Laboratory of Entomology, Faculty of Agriculture, learning can also increase an insect’s fitness because unsuccess- Kinki University, ful foraging may be costly in terms of energy and time expen- Naka-machi, Nara 631-8505, Japan diture. However, very few studies have demonstrated non- e-mail: [email protected] reward learning in insects. 706 Naturwissenschaften (2012) 99:705–713

Among pollinator insects, positive or reward learning Materials and methods whilst foraging on flowers has been investigated in many taxa, including bees (e.g. Menzel 1985, 1993; Dukas and Real Experimental preparation 1991), wasps (e.g. Shafir 1996;SatoandTakasu2000; Takasu et al. 2007), blowflies (Fukushi 1989), butterflies (e.g. Swihart Larvae and pupae of B. alcinous were obtained in Kyota- and Swihart 1970; Lewis 1986; Kinoshita et al. 1999;Kandori nabe, Kyoto, Japan, from June to July 2003 and from et al. 2009) and moths (e.g. Kelber 1996, 2002; Cunningham September to October 2005. Approximately 10 larvae were et al. 1998, 2004). Meanwhile, few studies have explored reared together on fresh debilis Sieb. et Zucc. aversive or non-reward learning, although many studies in- leaves in 450-ml transparent plastic cups at 25 °C and a cluded aversive or non-reward conditioning procedures in the light/dark (L/D) cycle of 14 h:10 h in a growth chamber in experiments. For example, when insects are trained to two the laboratory. After eclosion, each adult was numbered on types of cue—each associated with rewarding and unreward- the hind wings with an oil-soluble marker and kept without ing or deterrent stimuli—during the conditioning procedure food at 15 °C and 12-h:12-h L/D until the experiments were (so-called differential conditioning), the choice of the reward- started. All butterflies were of similar age (1–3 days) at the ing cue often increases, which inevitably decreases the choice start of the experiments. of the unrewarding or deterrent cue (apparent avoidance; e.g. Masters 1991;Gigordetal.2002; Chittka et al. 2003;Dyer Experimental location and Chittka 2004;AdlerandIrwin2005;Gegearetal.2007; Internicola et al. 2007, 2008; Rodrigues et al. 2010). However, We examined the foraging behaviour of butterflies in an this phenomenon alone does not demonstrate aversive or non- indoor incubation room (2.7×1.8×2.0 m) on the Nara cam- reward learning, and it can be explained by positive learning pus of Kinki University (see Kandori et al. 2009 for details). alone. To our knowledge, non-reward learning or aversive In the incubation room, light was provided from the ceiling learning of pollinator insects has been demonstrated only in by 10 fluorescent tubes (Truelite EX-VS, 40 W; ELC, Phil- honeybees (Srinivasan et al. 1994;Horridge2007; Vergoz et adelphia, PA, USA). The walls inside the room were cov- al. 2007), bumblebees (Simonds and Plowright 2004) and ered with a black nylon net, except for the ceiling, and room hawkmoths (Kelber 1996; Blackiston et al. 2008). It is not temperature was controlled at 25 °C. known whether butterflies possess non-reward or aversive learning abilities. In nature, pollinators may experience unre- Artificial flowers warding and rewarding flowers more frequently than flowers with aversive stimuli, such as shock, salt or toxins. Conse- Artificial flowers made from disks of coloured paper (5 cm quently, they may have a greater chance to learn via non- in diameter) were used as the stimuli (Daiei Training colour reward learning than through aversive learning. 200, Tokyo, Japan). Rewarding flowers were made from a Dukas and Real (1993) suggested that under certain exper- disk of coloured paper that had a 5-mm-wide hole at the imental conditions, bumblebees learn and remember only the centre with an attached Eppendorf tube (1.5 ml) containing a rewarding flowers they visit, even if they experience both 10 % sucrose solution. Unrewarding flowers consisted only rewarding and unrewarding flowers (Dukas and Real 1993). of a disk of coloured paper with a hole at the centre. All This suggests that because of their limited abilities to process flowers were set on a green plastic circular frame (30 cm in and store information (Waser 1986), insects preferentially diameter). The number of flowers and their colours differed remember more useful information about rewarding flowers according to the experiment (see below). The plastic frame than less useful information about unrewarding flowers, mak- was elevated 60–70 cm above the floor from the centre of ing it more difficult and slower to learn via non-reward learn- the experimental room and exposed to the butterflies. ing than reward learning. However, there has been no experimental comparison of the learning speeds of these two General experimental rules types of learning within pollinator species. In this study, we examined both the reward and non- We defined a ‘visit’ as a positive response when a butterfly reward learning abilities of the butterfly Byasa alcinous landed and extended its proboscis towards the coloured (Klug) whilst foraging for artificial flowers of different paper. In all behavioural experiments, 10–40 butterflies colours. We specifically addressed two questions: Does this were released at the same time and allowed to visit the butterfly possess both reward and non-reward learning abil- artificial flowers. When a butterfly visited any flower for a ities? If so, by which type of learning do the butterflies learn set number of times (i.e. five times, except during the faster? To the best of our knowledge, this is the first empir- training session for the second part of experiment 2, in ical comparison of reward and non-reward learning speeds which each butterfly was allowed to visit only once; see in insects. below), we temporarily removed that butterfly until the end Naturwissenschaften (2012) 99:705–713 707 of the experiment, part or session. Recent studies have presented eight flowers (four flowers of each colour) that reported that bumblebees can copy the flower choice of we set alternatively on the frame without rewards, and each experienced foragers (Leadbeater and Chittka 2005; Worden individual butterfly was allowed to visit the flowers five and Papaj 2005). However, in butterflies, such behaviour times. The frame was rotated 45° every 20 min so that has not been reported. A preliminary experiment revealed flower position would not be a factor. All individuals were that naive Peris rapae butterflies chose an artificial flower then separated into two groups: those that visited red more with and without another individual on it nearly equally (I. often and those that visited orange more often. In the second Kandori, unpublished data). This means that the presence of part of the experiment, we examined flower colour learning another conspecific on a flower does not act as either an through a reward training session, followed by a test ses- attractant or a deterrent stimulus for P. rapae. We assume sion. In the reward training session, butterflies from each of that this is also the case for B. alcinous and that multiple the two groups were trained to feed on the colour less visited individuals in the same place would not have a significant during the first part; that is, four flowers of the less visited effect on their flower choices. colour were set with rewards, and each individual was allowed to visit only one of the four flowers, drink the Experiment 1: Innate colour choices among 12 colours reward and spontaneously leave the flower. Butterflies that did not visit a flower spontaneously were placed on the To determine which colours should be used in the colour- flower manually. These individuals usually learned to feed learning experiment, innate colour choices were investigat- spontaneously on the flower within two training sessions. ed by allowing naive butterflies to visit 12 artificial flowers The test session was similar to the first part of this experi- coloured red, red-purple, purple, blue, green, yellow-green, ment in that we set eight flowers (four of each of the two yellow, orange, brown, light blue, white and pink. The colours) without rewards, and the colour choice of each preference of each butterfly was recorded over five visits. individual was recorded over five visits. The two sessions The experiment was conducted in July 2003. During the were conducted alternately every day and repeated five experiment, the order of 12 artificial flowers on the frame times (days) each. Therefore, the second part of this exper- was changed randomly every 20 min so that flower position iment was conducted for 10 days. Each training or test would not become a factor. session was conducted daily between 1000 and 1400 hours; that is, the interval between training and testing for each individual was approximately 1 day (20–28 h), and the inter- Experiment 2: Reward learning training interval was approximately 2 days. We rejected individual butterflies that did not finish the task within This experiment examined reward colour learning and its 4 h. Approximately half of the individuals completed all of learning speed. Based on the results of experiment 1, red the tasks (six tests, including the innate colour choice test, and orange were used as the two artificial flower colours with five training sessions between each test). Only these “ ” (see Results ). Colour traits, which were measured using a were used in the statistical analysis (8 and 12 females and reflectance spectrophotometer (High Sensitivity Spectro- 8 and 7 males that were trained to red and orange, respec- photometer USB2000, Ocean Optics Inc., Dunedin, FL, tively). The experiment was conducted from July to August USA), are shown in Fig. 1. The experiment consisted of 2003. two parts (Fig. 2). In the first part, we examined the innate colour choice for two colours in naive butterflies. We Experiment 3: Non-reward learning 100 90 This experiment examined whether a butterfly utilises non- 80 reward colour learning. Assuming that non-reward learning is 70 “ ” 60 more difficult (see Introduction ), we designed an experi- red 50 ment to directly detect any effects of non-reward learning. 40 orange The experiment consisted of two parts (Fig. 2). In the first

Reflectance (%) 30 20 part, we examined the innate colour choice between red and 10 orange in naive butterflies using the same method described 0 in the first part of experiment 2. Subsequently, we chose 300 400 500 600 700 Wavelength (nm) only individuals that visited red three times or more out of five visits to effectively detect possible non-reward learning Fig. 1 Spectral reflectance of the artificial flowers of different colour for red. In the second part, we trained these butterflies to used in the reward and non-reward learning experiments (experiments 2, 3 and 4). Relative spectral reflectance is normalised to a white unrewarding red and then retested their choice between red standard (magnesium oxide) and orange. This part was conducted on the day after 708 Naturwissenschaften (2012) 99:705–713

Fig. 2 Schematic diagram 1st part Innately 2nd part Group Repetition of showing the sequence of testing more visited Innate choice testassignment Training Test the 2nd part and training in experiments 2, 3 colour Red: 10% sucrose and 4. In the second part of Orange Red-training experiments 3 and 4, we used Experiment 2: 1 visit 5 times Reward learning Orange RedOrange- Orange: 10% sucrose Orange Red Total 10 days Red only individuals that innately training 1 visit chose red more often, and we Red: no rewards allowed them to visit non- Experiment 3:Red Red-training Free visits: numbers reward red flowers freely for Non-reward learningNo rewards not counted No rewards 5 visits 5 visits Once 2 h in the training session Red Control None (5 choices) (5 choices) Total 1 day Experiment 4: Red: no rewards Speed ofRed Red-training Free visits: numbers non-reward learning counted individually completion of the first part. It consisted of two sessions: a five choices without rewards in the test session equals five non-reward training session in the morning (1000– non-reward trainings, which masks the effect of one non- 1200 hours) followed by a test session in the afternoon reward training in the training session. Therefore, we used (1200–1600 hours). Therefore, the inter-training interval the same experimental procedure as that used for the group for each individual was between a few seconds and 2 h, with unrewarded experience in experiment 3, except that we and the interval between the last training and testing was counted the number of visits (trainings) to unrewarding red between a few minutes and 6 h. In the non-reward training flowers by each individual butterfly during the non-reward session, four red flowers were set without rewards and training session in the second part of the experiment (Fig. 2). butterflies were allowed to visit them freely. The test session Then, the count data were used to investigate whether the was similar to the first part of this experiment in that we set percentage choice for red in the test session decreases when eight flowers (four red and four orange) without rewards, the individual butterfly visits unrewarding red more times and the colour choice of each individual was recorded over during the training session. If so, the rate of decrease (i.e. the five visits. Then, we compared the percentage choice of the speed of non-reward learning) was compared with that of butterflies for red between before and after non-reward reward learning. The experiment was conducted only for training to red. In addition to the group with unrewarded females in November 2005. Different individuals were used experience on red, we examined a control group that had no in each of experiments 1–4. unrewarded experience on red (Fig. 2). The experimental procedure for the control group was the same as the group Statistical analysis with unrewarded experience, except that the control group was not exposed to any flower colours in the non-reward In experiment 2, to compare the reward learning speeds training session in the second part of the experiment. Only between the two sexes or colours, we used a general non- those that finished all tasks were used in the statistical linear learning model: analysis, i.e. 27 females and 24 males for the group with aN unrewarded experience and 26 females and 25 males for the P ¼ 1 ðÞ1 P0 e ; control group. The experiment was conducted in October 2005. where P is the proportion of butterfly visits to a rewarding flower colour, a is the learning speed, and N is the number

Experiment 4: Speed of non-reward learning of trainings on a rewarding flower colour. P0 refers to P at N00, or the innate colour preference. This model illustrates We examined whether the avoidance of the unrewarding the general form of the learning curve: The rate of change in colour increased with the amount of non-reward training to learning is initially higher and diminishes gradually as the that colour and estimated the speed of increase (i.e. non- individual is trained on more flowers. The proportion of reward learning speed). To compare the speeds of reward visits to a rewarding flower colour approaches 1 asymptot- and non-reward learning, the experimental procedure for the ically. This model describes the butterfly learning seen in two learning experiments (i.e. experiments 2 and 4) must be our previous experiments (Kandori et al. 2009). This learn- identical, except whether they were trained to flowers with ing curve was fitted to six data points, representing six tests, or without reward. If we were to apply the protocol of for each individual butterfly to estimate P0 and a. Then, we experiment 2 to experiment 4, the design of the latter would used a fixed effects analysis of variance [ANOVA; general be a repetition of one non-reward training in the training linear model (GLM) with type III sums of squares] to test session followed by five choices without rewards in the test for effects on learning speed (log+1-transformed), with sex, session. However, it would not allow us to detect the precise colour and their interactions as independent factors. In ex- effect of non-reward learning and its speed. This is because periment 3, we used the Wilcoxon signed-rank test to Naturwissenschaften (2012) 99:705–713 709 compare the proportion of visits to red before and after the 1.0 non-reward training session for the group with unrewarded experience and the control group. To compare the learning speeds between sexes or groups, we used the same nonlinear 0.5 learning model described above. Here, all parameters are the

same as above, except that P is the proportion of butterfly trained colour visits to an opposite colour (i.e. orange) than the non-reward

Proportion of visits to the 0.0 training colour (i.e. red) and N consists of only 0 and 1, 012345 which represent the tests before and after the non-reward Number of trainings to a rewarding colour training session, respectively. This learning curve was fitted to two data points for each individual butterfly to estimate Fig. 3 Effect of training to a rewarding colour for females (closed circles) and males (open circles)inB. alcinous. Because flower colour P0 and a. Then, we used a fixed effect ANOVA (GLM with did not affect learning speed (Table 1), the two groups with different type III sums of squares) to test for effects on learning speed training colours (red and orange) were combined within each sex. (log+1-transformed), with sex, group (whether butterflies There were 20 females and 15 males. Values represent the means± had unrewarded experience or not) and their interactions as standard deviation independent factors. In experiment 4, linear regression anal- ysis was performed to determine whether the percentage suggests that females learn a rewarding colour faster than choice for red decreased with increasing unrewarded expe- males and that the learning speeds are not different between rience on red flowers. To analyse the difference in learned the two training colours for each sex. performance between reward and non-reward learning, the preference rate (the percentage choice) of the rewarding Non-reward learning colour in females in experiment 2 and the avoidance rate (the percentage choice of the opposite colour) of the unre- Butterflies in the group with unrewarded experience on red warding colour in females in experiment 4 were compared significantly reduced their proportion of visits to red in both – for the same number of trainings using the Mann Whitney sexes (26–30 % reduction; Table 2). In contrast, butterflies in U test. IBM SPSS statistics 20 (IBM SPSS 2011) was used the control group that had no unrewarded experience on red did for all statistical analyses. not significantly reduce their proportion of visits to red (Ta- ble 2). The ANOVA for learning speed showed that there was a significant effect between groups (Table 3). These results sug- Results gest that the butterflies learned to avoid the unrewarding colour. However, contrary to reward learning, there was not a signifi- Innate colour choices cant effect of sex on the speed of non-reward learning (Table 3).

From the 12 possible colours, naive females chose red (23 %), followed by purple (21 %), orange (20 %) and blue (9 %) in a Comparison of learning speed between reward total of 160 visits. Naive males chose purple (36 %), followed and non-reward learning by red (22 %), orange (14 %) and blue (11 %) in a total of 110 visits. Since the percentage choice of the two colours was The more times that female butterflies experienced unre- similar within each sex, we selected red and orange as the warding red, the less frequently they visited red (regression: 0− 20 0 0 two flower colours used in subsequent experiments. y 0.087x+0.700, r 0.715, n 34, F 80.39, P<0.0001; Fig. 4). This demonstrates non-reward learning in B. alcinous. Reward learning Although the starting points of the two types of learning curve

Table 1 ANOVA of reward learning speed When they were trained to rewarding colours, both females and males of B. alcinous exhibited typical learning curves; Source df MS FP that is, as the number of times an individual was rewarded on a particular flower colour increased, the rate of selection of that Sex 1 0.109 4.850 0.035 colour increased (Fig. 3). When the two training colour groups Colour 1 0.008 0.366 0.550 were combined, the mean proportion of visits to a trained Sex×colour 1 0.007 0.324 0.573 colour was always higher for females compared to males after Error 31 0.023 one to five trainings (Fig. 3). The ANOVA for learning speed Sex refers to male and female effects. Colour refers to the effect of the indicated that there was a significant effect of sex and a non- two flower colours (red and orange) that were used in reward training significant effect of colour and their interaction (Table 1). This for the butterfly 710 Naturwissenschaften (2012) 99:705–713

Table 2 Proportion of visits to red before and after the non-reward 1 training session in which butterflies had an unrewarded experience on red and butterflies had no unrewarded experience 17 1 n % visits to red (mean ± SD) Pa 2 0.5 Before After 4 Butterflies with unrewarded experience on red 3 4

Proportion of visits to red 3 Females 27 69.6±12.9 40.0±17.5 <0.0001 0 Males 24 67.5±11.5 41.7±17.6 <0.0001 01234567 Butterflies with no unrewarded experience Number of trainings to unrewarded red Females 26 69.2±12.9 62.3±15.3 NS Fig. 4 Effect of training to unrewarding red artificial flowers on visits Males 25 64.8±15.6 56.8±19.7 NS to that colour in B. alcinous females. The numbers in the figure refer to the number of individuals. Values represent the means±standard NS not significant deviation a Wilcoxon signed-rank test more efficiently, which has selected for enhanced learning were similar, there was a relatively rapid increase in the ability (Kandori et al. 2009). In contrast, the speed of non- preference rate via reward learning and a more gradual in- reward learning did not differ by sex in the present study crease in the avoidance rate via non-reward learning (Fig. 5). (Tables 2 and 3). However, further investigations should be Performance was significantly higher for reward learning than carried out to confirm this result. Experiment 3 was designed non-reward learning after three to five training sessions only to determine whether non-reward colour learning oc- (Fig. 5). This indicates that B. alcinous learns via reward curred, not to investigate the speed of non-reward learning. learning faster than it does via non-reward learning. Many studies have shown that learning speed differs by flower colour (e.g. Fukushi 1989; Weiss 1997; Kinoshita et al. 1999). This may occur partly because the learning speed Discussion on a certain colour positively correlates with an innate preference for that colour (Dukas and Real 1991;Weiss We found that the butterfly B. alcinous learns to associate 1997). Our studies failed to detect a difference in the learn- flower colours not only with the presence of nectar but also ing speed between flower colours, perhaps because the with the absence of nectar; that is, B. alcinous uses both butterflies innately preferred both colours used in the dual- reward and non-reward colour learning whilst foraging on choice test (red and orange in this study). flowers. The speed of reward learning differed by sex, but not by flower colour (Table 1 and Fig. 3), consistent with our previous study (Kandori et al. 2009). 1 To explain the faster reward learning in female than in male ** *** butterflies, Kroutov et al. (1999) hypothesised that the com- ** plexity of female behaviour, such as locating oviposition sites and determining host-plant suitability, may enhance their 0.5 learning behaviour. Alternatively, we hypothesised that fe- male butterflies require more nectar than their male counter- parts to produce and oviposit eggs; thus, they must forage Preference/avoidance rate Preference/avoidance 0 012345 Table 3 ANOVA of non-reward learning speed Number of trainings

Source df MS FPFig. 5 Performance comparison of reward learning (circles) and non- reward learning (squares)inB. alcinous females. Mean values are Sex 1 0.002 0.128 0.722 taken from Figs. 2 and 3. The y-axis shows the preference rate for a Group 1 0.595 34.522 0.000 rewarding colour and the avoidance rate of an unrewarding colour for reward and non-reward learning, respectively. Asterisks in the figure Sex×group 1 0.005 0.277 0.600 indicate that the proportion of avoidance in non-reward learning dif- Error 98 0.017 fered significantly from the proportion of preference in reward learning for the same number of trainings (Mann–Whitney U test: **P<0.01; Sex refers to male and female effects. Group refers to the effect of the ***P<0.001). Note that the inter-training interval for reward learning two groups (butterflies with and without an unrewarded experience on was approximately 2 days, whilst that of non-reward learning was red) between a few seconds and 2 h Naturwissenschaften (2012) 99:705–713 711

Asymmetric confusion may also affect the learning speed the difference in the speed of the learning types even larger. of new colours. Blackiston et al. (2011) found that monarch Alternatively, we may not have detected non-reward learn- butterflies (Danaus plexippus) trained to red also visited ing in butterflies. This is because, in addition to the diffi- orange, but not vice versa (Blackiston et al. 2011). They culty in establishing non-reward learning, butterflies may explained this phenomenon from the viewpoint of stimulus have a shorter memory for non-reward learning than for brightness; that is, butterflies trained to the dimmer colour reward learning. According to Matsumoto and Mizunami always made mistakes on the brighter colour, whereas the (2002), in the cricket Gryllus bimaculatus, appetitive con- reverse was not true. However, their explanation may not be ditioning leads to long-lasting memory retention with no the case for B. alcinous (this study) and Idea leuconoe significant decay 4 days after training, whereas retention (Kandori et al. 2009), in which both butterflies showed after aversive conditioning disappears 1 day after training neither different learning speeds nor asymmetric confusion (Matsumoto and Mizunami 2002). Camponotus fellah ants between red and orange. We think that asymmetric confu- may also have difficulty in establishing non-reward or aver- sion occurs between two colours of different innate prefer- sive learning or in retaining their memories because they did ence rather than different stimulus brightness. From this not show repellence to an odour paired with quinine, but perspective, monarch butterflies showed asymmetric confu- only showed preference to an odour paired with sugar, after sion because they innately prefer orange to red, whilst B. differential conditioning in a Y-maze (Josens et al. 2009). alcinous and I. leuconoe did not show asymmetric confu- At this time, it is unclear whether non-reward learning sion because they innately prefer both red and orange. reflects negative associative learning or a specific type of We also found that B. alcinous learned faster via reward habituation to an initially preferred colour. To our knowl- learning than via non-reward learning. Our results suggest edge, only four studies have demonstrated the non-reward that one rewarded experience on a certain flower colour may learning of flower-visiting insects (a decrease in preference have a more positive learning effect on a subsequent flower for the unrewarded flower colour or pattern). However, none choice than the negative learning effect of even four unre- of these studies clarified whether this was due to negative warded experiences on the same colour: In experiment 2, associative learning or habituation. It has been proposed to most butterflies ultimately approached 100 % preference for be negative associative learning (Srinivasan et al. 1994; the rewarding colour, i.e. they chose the rewarding colour Kelber 1996; Horridge 2007) as well as habituation five times in the test session, which means that following (Simonds and Plowright 2004). It is very difficult to design one rewarded experience, they continued to choose the same experiments to address this issue. In our opinion, habitua- flower colour despite four unrewarded experiences. The tion to floral visual and olfactory cues may be rare because it differences in speed between the two types of learning can would interfere with positive associative learning that be considered adaptive for two reasons. First, even if a enhances constant visits to the rewarding flower type. certain plant species in the field produces nectar that butter- Therefore, the observed non-reward learning likely reflected flies can utilise, a plant may have more unrewarding flowers negative associative learning. than rewarding flowers due to nectar consumption by other We conclude that naive pollinators in nature can find flower visitors. In this case, to achieve a learned preference rewarding flower species more efficiently via both reward for that plant species, the increase in preference with one and non-reward learning; that is, insects may initially visit a rewarded experience on a flower should be larger than the certain flower by innate preference. If this flower is reward- decrease in preference with one unrewarded experience on ing, they quickly increase their constancy to that flower the same plant species. Second, because of limited abilities species through reward learning. If the flower species is to process and store information (Waser 1986), insects may unrewarding and common, frequent visits to that flower remember more useful information about rewarding flowers species enhance non-reward learning to avoid that flower in preference to less useful information about unrewarding species. They can identify new rewarding flower species flowers. Thus, non-reward learning is more difficult and with a reduced loss of energy and time if they avoid foraging takes longer than reward learning. on such abundant, innately attractive but unrewarding flow- We could not accurately compare the learning speed er species. However, if the flower species is unrewarding between the two types of learning because the experimental and uncommon, the speed of non-reward learning is so slow designs differed somewhat between experiments 2 and 4, that they may locate a new rewarding flower species before e.g. the interval between trainings was 2 days in experiment they learn to avoid the unrewarding flower species. 2, but <2 h in experiment 4. However, since learned associ- ation may weaken over time (e.g. Fukushi 1989; Kelber Acknowledgments We sincerely thank Drs. T. Sugimoto, Y. 1996), if we had trained butterflies successively in experi- Sakuratani, E. Yano and D. R. Papaj for their valuable advice. We also thank H. Nakai, H. Narita and Y. Kinoshita for assistance ment 4 using the same interval as in experiment 2, the non- with the preliminary experiments. All experiments complied with reward learning speed might have been even lower, making the current laws of Japan. 712 Naturwissenschaften (2012) 99:705–713

References Gegear RJ, Manson JS, Thomson JD (2007) Ecological context influ- ences pollinator deterrence by alkaloids in floral nectar. Ecol Lett 10:375–382. doi:10.1111/j.1461-0248.2007.01027.x Adler LS, Irwin RE (2005) Ecological costs and benefits of defenses in Gigord LDB, Macnair MR, Stritesky M, Smithson A (2002) The nectar. Ecology 86:2968–2978 potential for floral mimicry in rewardless orchids: an experimental Berenbaum MR, Miliczky E (1984) Mantids and milkweed bugs: study. Proc R Soc B 269:1389–1395. doi:10.1098/rspb.2002.2018 efficacy of aposematic coloration against invertebrate predators. Horridge A (2007) The preferences of the honeybee (Apis mellifera) Am Midl Nat 111:64–68 for different visual cues during the learning process. J Insect Bernays EA (1993) Aversion learning and feeding. In: Papaj DR, Physiol 53:877–889. doi:10.1016/j.jinsphys.2006.12.002 Lewis AC (eds) Insect learning: ecological and evolutionary IBM SPSS (2011) IBM SPSS statistics 20. IBM Corp., New York perspectives. Chapman & Hall, New York, pp 1–17 Internicola AI, Page PA, Bernasconi G, Gigord LDB (2007) Competi- Blackiston DJ, Casey ES, Weiss MR (2008) Retention of memory through tion for pollinator visitation between deceptive and rewarding metamorphosis: can a moth remember what it learned as a caterpillar? artificial inflorescences: an experimental test of the effects of PLoS One 3:e1736. doi:e173610.1371/journal.pone.0001736 floral colour similarity and spatial mingling. Funct Ecol 21:864– Blackiston D, Briscoe AD, Weiss MR (2011) Color vision and 872. doi:10.1111/j.1365-2435.2007.01303.x learning in the monarch butterfly, Danaus plexippus Internicola AI, Bernasconi G, Gigord LDB (2008) Should food- (Nymphalidae). J Exp Biol 214:509–520. doi:10.1242/ deceptive species flower before or after rewarding species? An jeb.048728 experimental test of pollinator visitation behaviour under contrast- Bowdish TI, Bultman TL (1993) Visual cues used by mantids in ing phenologies. J Evol Biol 21:1358–1365. doi:10.1111/j.1420- learning aversion to aposematically colored prey. Am Midl Nat 9101.2008.01565.x 129:215–222 Josens R, Eschbach C, Giurfa M (2009) Differential conditioning and Burger JMS, Kolss M, Pont J, Kawecki TJ (2008) Learning long-term olfactory memory in individual Camponotus fellah ability and longevity: a symmetrical evolutionary trade-off ants. J Exp Biol 212:1904–1911. doi:10.1242/jeb.030080 in Drosophila. Evolution 62:1294–1304. doi:10.1111/j.1558- Kandori I, Yamaki T, Okuyama S, Sakamoto N, Yokoi T (2009) 5646.2008.00376.x Interspecific and intersexual learning rate differences in four Chittka L, Dyer AG, Bock F, Dornhaus A (2003) Bees trade off butterfly species. J Exp Biol 212:3810–3816. doi:10.1242/ foraging speed for accuracy. Nature 424:388. doi:10.1038/ jeb.032870 424388a Kelber A (1996) Colour learning in the hawkmoth Macroglossum Cnaani J, Thomson JD, Papaj DR (2006) Flower choice and learning in stellatarum. J Exp Biol 199:1127–1131 foraging bumblebees: effects of variation in nectar volume and Kelber A (2002) Pattern discrimination in a hawkmoth: innate prefer- concentration. Ethology 112:278–285 ences, learning performance and ecology. Proc R Soc B Costa A, Ricard I, Davison AC, Turlings TCJ (2010) Effects of 269:2573–2577. doi:10.1098/rspb.2002.2201 rewarding and unrewarding experiences on the response to host- Kinoshita M, Shimada N, Arikawa K (1999) Colour vision of the induced plant odors of the generalist parasitoid Cotesia margin- foraging Papilio xuthus.JExpBiol iventris (Hymenoptera: Braconidae). J Insect Behav 23:303–318. 202:95–102 doi:10.1007/s10905-010-9215-y Kroutov V, Mayer MS, Emmel TC (1999) Olfactory conditioning of Cunningham JP, West SA, Wright DJ (1998) Learning in the nectar the butterfly Agraulis vanillae (L.) (Lepidoptera, Nymphalidae) to foraging behaviour of Helicoverpa armigera. Ecol Entomol floral but not host-plant odors. J Insect Behav 12:833–843 23:363–369 Leadbeater E, Chittka L (2005) A new mode of information transfer in Cunningham JP, Moore CJ, Zalucki MP, West SA (2004) Learning, foraging bumblebees? Curr Biol 15:R447–R448 odour preference and flower foraging in moths. J Exp Biol Lee JC, Bernays EA (1990) Food tastes and toxic effects: associative 207:87–94. doi:10.1242/Jeb.00733 learning by the polyphagous grasshopper Schistocerca americana Dicke U, Heidorn A, Roth G (2011) Aversive and non-reward learning (Drury) (Orthoptera: Acrididae). Anim Behav 39:163–173 in the fire-bellied toad using familiar and unfamiliar prey stimuli. Lewis AC (1986) Memory constraints and flower choice in Pieris Current Zoology 57:709–716 rapae. Science 232:863–865 Dukas R (1999) Ecological relevance of associative learning in fruit fly Masters AR (1991) Dual role of pyrrolizidine alkaloids in nectar. J larvae. Behav Ecol Sociobiol 45:195–200 Chem Ecol 17:195–205 Dukas R (2008) Evolutionary biology of insect learning. Annu Rev Matsumoto Y, Mizunami M (2002) Temporal determinants of long- Entomol 53:145–160. doi:10.1146/annurev.ento.53.103106.093343 term retention of olfactory memory in the cricket Gryllus bima- Dukas R, Bernays EA (2000) Learning improves growth rate in grass- culatus. J Exp Biol 205:1429–1437 hoppers. Proc Natl Acad Sci USA 97:2637–2640 Menzel R (1985) Learning in honeybees in an ecological and behav- Dukas R, Duan JJ (2000) Potential fitness consequences of asso- ioral context. In: Hölldobler B, Lindauer M (eds) Experimental ciative learning in a parasitoid wasp. Behav Ecol 11:536– behavioral ecology. Fischer, Stuttgart, pp 55–74 543. doi:10.1093/beheco/11.5.536 Menzel R (1993) Associative learning in honey bees. Apidologie Dukas R, Real LA (1991) Learning foraging tasks by bees: a compar- 24:157–168 ison between social and solitary species. Anim Behav 42:269–276 Menzel R (1999) Memory dynamics in the honeybee. J Comp Physiol, Dukas R, Real LA (1993) Learning constraints and floral choice A 185:323–340. doi:10.1007/s003590050392 behavior in bumble bees. Anim Behav 46:637–644 Mery F, Kawecki TJ (2003) A fitness cost of learning ability in Dunlap AS, Stephens DW (2009) Components of change in the evo- Drosophila melanogaster. Proc R Soc B 270:2465–2469. lution of learning and unlearned preference. Proc R Soc B doi:10.1098/rspb.2003.2548 276:3201–3208. doi:10.1098/rspb.2009.0602 Mery F, Kawecki TJ (2004) An operating cost of learning in Dyer AG, Chittka L (2004) Fine colour discrimination requires differ- Drosophila melanogaster. Anim Behav 68:589–598 ential conditioning in bumblebees. Naturwissenschaften 91:224– Papaj DR, Snellen H, Swaans K, Vet LEM (1994) Unrewarding 227. doi:10.1007/s00114-004-0508-x experiences and their effect on foraging in the parasitic wasp Fukushi T (1989) Learning and discrimination of colored papers in the Leptopilina heterotoma (Hymenoptera: Eucoilidae). J Insect walking blowfly, Lucilia cuprina. J Comp Physiol, A 166:57–64 Behav 7:465–481 Naturwissenschaften (2012) 99:705–713 713

Raine NE, Chittka L (2008) The correlation of learning speed and Takasu K, Rains GC, Lewis WJ (2007) Comparison of detection ability natural foraging success in bumble bees. Proc R Soc B 275:803– of learned odors between males and females in the larval parasit- 808. doi:10.1098/rspb.2007.1652 oid Microplitis croceipes. Entomol Exp Appl 122:247–251. Rodrigues D, Goodner BW, Weiss MR (2010) Reversal learning and doi:10.1111/j.1570-7458.2006.00511.x risk-averse foraging behavior in the monarch butterfly, Danaus Thum AS, Jenett A, Ito K, Heisenberg M, Tanimoto H (2007) plexippus (Lepidoptera: Nymphalidae). Ethology 116:270–280. Multiple memory traces for olfactory reward learning in Dro- doi:10.1111/j.1439-0310.2009.01737.x sophila. J Neurosci 27:11132–11138. doi:10.1523/jneurosci. Sato M, Takasu K (2000) Food odor learning by both sexes of the 2712-07.2007 pupal parasitoid Pimpla alboannulatus Uchida (Hymenoptera: Tully T, Quinn WG (1985) Classical conditioning and retention in Ichneumonidae). J Insect Behav 13:263–272 normal and mutant Drosophila melanogaster. J Comp Physiol, Shafir S (1996) Color discrimination conditioning of a wasp, Polybia A 157:263–277 occidentalis (Hymenoptera: Vespidae). Biotropica 28:243–251 Vergoz V, Roussel E, Sandoz JC, Giurfa M (2007) Aversive Simonds V, Plowright CMS (2004) How do bumblebees first find learning in honeybees revealed by the olfactory conditioning flowers? Unlearned approach responses and habituation. Anim of the sting extension reflex. PLoS One 2:e288. doi: Behav 67:379–386. doi:10.1016/j.anbehav.2003.03.020 e28810.1371/journal.pone.0000288 Srinivasan MV, Zhang SW, Witney K (1994) Visual discrimination of Waser NM (1986) Flower constancy - definition, cause, and measure- pattern orientation in honeybees: performance and implication for ment. Am Nat 127:593–603 cortical processing. Philos Trans R Soc B 343:199–210 Weiss MR (1997) Innate colour preferences and flexible colour Stephens DW (1993) Learning and behavioral ecology: incomplete learning in the pipevine swallowtail. Anim Behav 53:1043– information and environmental predictability. In: Papaj DR, Lewis 1052 AC (eds) Insect learning: ecological and evolutionary perspectives. Weiss MR, Papaj DR (2003) Colour learning in two behavioural Chapman & Hall, New York, pp 195–218 contexts: how much can a butterfly keep in mind? Anim Behav Swihart CA, Swihart SL (1970) Color selection and learned feeding 65:425–434. doi:10.1006/anbe.2003.2084 preferences in the butterfly, Heliconius charitonius Linn. Anim Worden BD, Papaj DR (2005) Flower choice copying in bumblebees. Behav 18:60–64 Biol Lett 1:504–507. doi:10.1098/rsbl.2005.0368