Different roles for innate and learnt behavioral responses to odors in insect host location

Transcription

1 Behavioral Ecology doi: /beheco/ars172 Advance Access publication 17 October 2012 Origianl Article Different roles for innate and learnt behavioral responses to odors in insect host location Ben Webster, a Erika Qvarfordt, a Ulf Olsson, b and Robert Glinwood a a Department of Ecology and b Department of Economics, Swedish University of Agricultural Sciences, Uppsala, Sweden Volatile chemical cues are used by herbivorous insects to locate and identify their host plants. Many species show a preference for volatiles experienced during development in the natal habitat. The reliability of this learnt information, however, may be limited. Many insects develop in restricted habitats, often on a single plant. Large between-plant variability in volatile emission, due to genetic differences and different exposure to biotic and abiotic factors, means that the volatile profile of a single plant may not be representative of the entire species. Insects must, therefore, balance the benefits of learning with the risks associated with its reliability. This is especially important for insects for which habitat exploration is costly. We hypothesize that information gained in the natal habitat is most likely to be utilized in situations where the cost of habitat exploration is lowest. To test this hypothesis, the black bean aphid, Aphis fabae, was reared on artificial diet while exposed to volatiles from its host, broad bean, and an unsuitable host, mustard. When offered the choice between bean and mustard leaves as adults, aphids showed a preference for the leaves whose odor they had experienced during development. When only exposed to volatiles from the two plants, in the absence of cues to indicate proximity or accessibility of the odor source, aphids preferred bean volatiles, regardless of experience. This suggests that information acquired from the natal habitat is only utilized when the perceived cost of habitat assessment is low, with innate preferences dominating otherwise. Key words: aphid, host location, learning, olfaction, volatiles. [Behav Ecol] Introduction Learning is the acquisition and retention of neuronal representations of new information (Dukas 2008). It is a widespread trait among insects and provides numerous selective advantages when foraging in an unpredictable environment. Many insects learn from experience acquired in the natal habitat and this can provide several adaptive advantages (Davis and Stamps 2004; Stamps and Davis 2006). One advantage of learning from natal experience is that it can improve flexibility in host range and facilitate in the exploitation of novel host plants. Many insects show strong preferences for their preferred hosts but will attempt to settle on novel, unfamiliar plants should they be unable to locate anything more suitable (Stamps and Davis 2006). If a phytophagous insect successfully colonizes a novel host plant and its offspring survive to maturity, its offspring will be able to continue to take advantage of the new host species by learning to recognize cues associated with it. This would facilitate the expansion of host range and could be especially adaptive in a changing environment. Some insect species can develop physiological adaptations to novel host plants, and learning allows insects to best take advantage of such adaptations by allowing subsequent generations to continue exploiting the host they become adapted to. For example, Gorur et al. (2005, 2007) demonstrated that different clones of the black bean aphid, Aphis fabae, may be more adapted to either bean (Vicia faba) or nasturtium (Tropaeolum majus) showing decreased performance when transferred to the alternative host. It was found, however, that some clones were able to adapt to the Address correspondence to B. Webster. ben.webster@slu.se. Received 23 March 2012; revised 12 July 2012; accepted 27 August The Author Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. All rights reserved. For permissions, please journals.permissions@oup.com alternative host after being reared on it for a few generations of asexual reproduction, showing increased performance and survival on the new host. In some cases, such adaptations may be subject to trade-offs, reducing performance on other hosts (Tosh et al. 2004). Insects that display such adaptations are, therefore, likely to show increased performance on the host species they were reared on, which should favor learning from natal experience. The use of volatile chemical cues in host plant location is well documented among phytophagous insects, and many species show the ability to learn from such cues (Dukas 2008). Most herbivorous insects develop in restricted habitats, often on a single plant. Although information gained from the host plant may be useful, there is a risk that it may not be representative of the entire host species. This is an idea that has received little attention and could have important implications for the use of learnt information in many animal species. Plants show large variability in their volatile profiles. Infestation with herbivores can cause dramatic differences in volatile blends that can vary depending on the species of herbivore and magnitude of infestation (Hare 2011). Insects developing on an infested plant are, therefore, exposed to volatile blends that may not be emitted by new, uninfested plants they seek during host location. Plants of the same species can show considerable variability in volatile emission even when subjected to infestation with the same herbivores due to genetic differences between individuals (Degen et al. 2004). Other environmental stress factors that may vary spatially, including humidity, temperature, light, and nitrogen availability, can also result in considerable variability in volatile emission between plants (Gouinguené and Turlings 2002). Variability between plants also exists in the absence of biotic or abiotic stress. When bean plants of the same cultivar were grown in unstressed conditions in the absence of any

2 Webster et al. Innate and learnt responses to odors 367 insect herbivores, plants showed considerable variability in the volatile blends they emitted (Webster et al. 2010b). Insects are, therefore, faced with the task of identifying volatile blends that likely possess differences to the blend they experienced during development. Consistent signals may exist within highly variable blends (Webster et al. 2010b), but there is no way for insects to discern these without first sampling a range of different plants. Information acquired in the natal habitat may, therefore, not be entirely reliable. Innate behavioral responses, on the other hand, are the product of many generations of natural selection and are, therefore, more likely to be tuned in to the most reliable components of the host volatile blend. The benefits of learning from information gained in the natal habitat must be balanced with the cost associated with its lower reliability, particularly in insects for which habitat exploration and assessment is costly. Host location in insects can be divided into two stages, with olfaction playing an important role in both (Bruce et al. 2005). Prior to contact with the plant, olfaction can be used to locate and recognize potential host plants at a distance. Once visual, tactile and/or gustatory cues indicate that the insect is in close proximity to the plant, olfaction is used alongside these other cues in order to make a final decision as to whether or not to settle. Following an odor plume over a distance subjects insects to many challenges such as the risk of predation, dehydration, and starvation. Information used to locate new hosts at a distance, or when the precise location or accessibility of the odor source is unknown, must therefore be reliable. Due to its lower reliability, we hypothesize that information gained from the natal habitat is most likely to be utilized when in close proximity to the plant, when the cost of habitat exploration is low, with innate preferences playing a stronger role when the distance to the odor source is unknown and perceived costs are higher. Aphids are a model species in the study of insect behavior, showing strong behavioral responses to volatiles from their host plants (Webster 2012). Due to high rates of asexual reproduction, it is possible to generate large numbers of genetically identical aphids, allowing for the control of genetic variation among individuals used in experiments. It is not yet known whether behavioral responses of aphids to plant volatiles are innate or can be modified by experience, though evidence for learning of gustatory cues has been found (van Emden et al. 2009). Aphids are phloem feeders and develop as nymphs on a single plant. Winged morphs are produced in response to overcrowding and migrate to new hosts (Powell and Hardie 2001), but wingless aphids also play an important role in dispersal by walking to neighboring plants and forming new colonies (Hodgson 1991; Alyokhin and Sewell 2003). Aphids also show a striking ability to adapt to novel hosts (Douglas 1997; Gorur et al. 2005, 2007). This trait should favor the ability to learn from natal experience (Davis and Stamps 2004). Habitat exploration is costly for aphids. Wingless aphids play an important role in dispersal over short to medium distances, expanding to form new colonies on neighboring plants (Hodgson 1991). Wingless aphids will also readily walk over the soil to form colonies up to several meters away from the natal plant (Wiktelius 1989; Alyokhin and Sewell 2003). Walking to a new host plant, however, incurs a high mortality risk as aphids are subject to predation, starvation, and dehydration (Wiktelius 1989; Alyokhin and Sewell 2003). Following an odor plume to its source may be difficult due to meandering of plumes or physical barriers encountered en-route. This makes the task of locating a new host over any great distance difficult for walking aphids. Although learning from natal experience would provide benefits to aphids, aiding in the expansion of host range and allowing aphids to best take advantage of physiological adaptations to new hosts, we predict that learnt behavior will be restricted to when aphids are in close proximity to familiar plants, where the cost of habitat exploration is low. This would minimize the costs associated with misidentifying a plant odor while still allowing aphids to take advantage of learnt information in favorable situations. The aim of this study was to test the hypothesis that behavioral responses to plant volatiles can be modified by experience acquired during development, but this tends to manifest only when in close proximity to a potential host with innate preferences tending to dominate at a distance. The black bean aphid, A. fabae, was used as a model system to test this hypothesis. Its host selection behavior has been studied extensively, making it an ideal study species (Nottingham et al. 1991; Gorur et al. 2007; Webster et al. 2008, 2010a). Wingless aphids were used because they will readily settle on neighboring plants as well as walking longer distances to find new host plants and so will engage in both low- and high-risk habitat exploration. Aphids were reared on artificial diet and exposed to the odors of a host plant, broad bean (V. faba), and mustard (Sinapis alba), a plant on which preliminary studies showed aphids were unable to survive to maturity and so can be considered an unsuitable host. The aphids behavior was then assessed once they had reached maturity. Aphids were first offered the choice between leaves of the two plants, to determine preference when in close proximity to the potential hosts to simulate low-risk habitat exploration. To simulate higher risk habitat exploration, all other cues that might indicate close proximity to the plants were removed, with aphids being exposed only to plant volatiles in an olfactometer. Methods Plants Broad bean, V. faba (var. Sutton dwarf), and mustard, S. alba, were grown individually in 8.5-cm pots in a glasshouse (22 C, 16:8 h light:dark cycle). Plants were used in experiments when they were 2 3 weeks old, just prior to inflorescence emergence. Insects A colony of A. fabae was derived from aphids provided by Rothamsted Research, UK. A colony of summer morphs (virginoparae) were kept Harpenden, on V. faba plants in a Perspex rearing cabinet in a room maintained at 22 C with a 16:8 h light:dark cycle. Aphid rearing system The artificial diet composition and feeding system were as described previously (van Emden 2009). Briefly, the system consisted of approximately 1 ml of diet contained between two layers of Parafilm M stretched over a Perspex tube (2.5 cm tall, diameter 3 cm), into which aphids were placed. To allow air to circulate, the diet tube was attached to a Perspex ventilation tube (4 cm tall, diameter 3 cm). The ventilation tube contained 28 holes (diameter 0.3 cm) spaced equally around the tube and covered with mesh to prevent aphids escaping. Diet tubes were contained in a sealed plastic rearing chamber ( cm) connected to a Perspex odor chamber ( cm) via polytetrafluoroethylene (PTFE) tubing (inner diameter [i.d.] 0.3 cm). White pieces of paper were placed around the rearing chamber in order to remove any visual cues. Charcoal-filtered air was pumped into the

3 368 Behavioral Ecology odor chamber at a rate of 600 ml min 1 and pulled out from the rearing chamber at a rate of 300 ml min 1, creating a constant flow of air between the two. This allowed volatiles to be carried from the plant to the rearing chamber; the ventilation tubes allowing odor-laden air to reach the aphids feeding within the diet tubes. The difference in flow rate created a positive pressure that prevented volatiles entering from outside. Because the odor chamber was not completely airtight, air could escape from the system thus preventing unnatural build up of air pressure. Aphid exposure to volatiles during development Four different rearing experiences were tested: 1) Diet-reared aphids exposed to volatiles from a bean plant 2) Diet-reared aphids exposed to volatiles from a mustard plant 3) Diet-reared aphids exposed to volatiles from an empty rearing chamber (blank) 4) Aphids reared on bean plants (control). Twelve rearing/odor chambers were used simultaneously, four for each of the three diet-reared aphids. At the start of the rearing process, 10 wingless adults of A. fabae were collected from the aphid colony maintained on bean plants, added to a diet tube, and placed inside a rearing chamber. Aphids were left for 2 days to produce nymphs after which adults were removed and the nymphs gently transferred to a new diet tube using a fine paint brush where they were left to develop to maturity. Diet tubes and plants were refreshed twice a week. Aphids were used in experiments after approximately 10 days, shortly after they had reached maturity and stopped feeding, indicating that they were attempting to disperse. Only wingless adults were used in experiments. Arena bioassay Host preference was assessed using an arena bioassay. The arena was a flattened cylinder, 3 cm tall with a diameter of 15 cm, and covered with mesh to prevent aphids escaping. At opposite sides of the arena were two small gaps to accommodate the stem of a leaf. Mustard and bean leaves were inserted on opposite sides. Where possible, leaves were selected so that they had approximately equal surface area, and both were undamaged and still attached to the plant. Using intact leaves was necessary because volatiles released upon mechanical damage have previously been found to cause bean leaves to become unattractive to A. fabae (Nottingham et al. 1991). Once leaves were positioned in the arena, 10 wingless adult aphids were added to the centre and the number of aphids on each leaf was recorded every 30 min, 1 h, 2 h, 3 h, and 24 h after the experiment had started. The experiment was repeated 12 times for each rearing experience. Aphids were used only once, with new aphids being used for each new replicate. Numbers of aphids on each leaf were converted to proportions for statistical analysis and generalized linear models with a logit link were used. The data were of a repeated-measures type, so mixed generalized linear models were employed (Littell et al. 2006; Olsson 2002, 2011). The model included fixed effects of treatment (rearing experience), time, and treatment*time. Block was included as a random factor. Correlations between observations over time were modeled using an unstructured covariance matrix. Specific questions on comparisons between treatments at each time period were answered by post-hoc least squares means tests. An alpha level of P < 0.05 was used to determine if differences were statistically significant. Olfactometry An olfactometer, identical in size and dimensions to the fourarm olfactometer described previously (Webster et al. 2010a) but with two arms instead of four, was used to assess responses of aphids to plant volatiles in the absence of other plant cues. Plants used as odor sources were enclosed in polyethylene terephthalate (PET) oven bags (35 43 cm; Toppits, Klippan, Sweden) that were sealed around the pots using a rubber band. PTFE tubing (i.d. 3 mm) was inserted under the rubber bands and connected to one of the olfactometer arms. Air was drawn out of the olfactometer through a hole in the top at a rate of 200 ml min 1, drawing in air through both of the olfactometer arms. Charcoal-filtered air was pumped into the PET bags containing the plants at 250 ml min 1, creating a slight positive pressure to ensure that air from the laboratory did not enter the system. At the start of the experiment, a single aphid was placed in the olfactometer and allowed 10 min to acclimatize, after which its location (left arm or right arm) was recorded every 2 min for 20 min. Between 26 and 36 replicates were completed for each experiment, depending on availability of aphids. If an aphid remained motionless for the duration of the experiment, it was removed from the analysis. Two experiments were carried out. In the first, aphids were offered a choice between volatiles from a bean plant and volatiles from a mustard plant. In the second experiment, aphids were offered a choice between volatiles from either a bean or mustard plant and volatiles from a pot of soil used as a control. Aphids were used only once, with new aphids being used in each replicate. Number of recordings in one arm was converted to proportion of total number of recordings in both arms. The analysis used generalized linear models with a logit link (Olsson 2002), and the model studied the effects of experience on arm choice. Least significant means post-hoc analysis permitted pair-wise comparisons between treatments and tests of the hypothesis of equal probability for selecting either arm (P = 0.5) for the different experiences. An alpha level of P < 0.05 was used to determine if differences were statistically significant. Results Arena bioassay The results show that experience of volatiles during development has a significant effect on host preference in adult A. fabae when offered the choice between two leaves. Dietreared aphids with experience of mustard volatiles showed a strong tendency to settle on mustard leaves compared with diet-reared aphids exposed to bean volatiles, which showed a slight tendency to settle on bean leaves. Aphids with no prior experience of plant volatiles showed an intermediate distribution. Figure 1 shows number of aphids recorded on bean and mustard leaves at different times and from different rearing experiences. Statistical analysis revealed that there was a significant effect of rearing experience on distribution of aphids between the two leaves (F 3,15 = 4.60, P = ). Post-hoc least squares means analysis showed distribution of diet-reared aphids with experience of bean volatiles and diet-reared aphids with experience of mustard volatiles were significant at 0.5 h (t 80 = 2.41, P = ), 1 h (t 80 = 2.37, P = ), and 3 h (t 80 = 2.00, P = ), with no significant differences at 2 h (t 80 = 1.86, P = ) or at 24 h (t 80 = 1.54, P = ). Diet-reared aphids with no prior experience of plant volatiles (blank) did not show significantly different

4 Webster et al. Innate and learnt responses to odors 369 Figure 1 Responses of aphids to mustard and bean leaves in an arena bioassay. A positive value indicates more aphids were present on the bean leaf; a negative value indicates more aphids were present on the mustard leaf. Blank: diet-reared aphids reared in odorless conditions; bean: dietreared aphids exposed to bean volatiles; mustard: diet-reared aphids exposed to mustard volatiles; control: aphids reared on bean plants. Different letters in the same time period indicate significantly different distributions (P < 0.05). distributions to either of the other diet-reared aphids at any time period (P > 0.05). Plant-reared aphids (control) showed a tendency to distribute themselves on bean leaves, which increased over time. This was significantly different to the distribution of dietreared aphids exposed to mustard volatiles at all time periods (0.5 h: t 80 = 2.47, P = ; 1 h: t 80 = 2.92, P = ; 2 h: t 80 = 2.96, P = 0.004; 3 h: t 80 = 3.51, P = ; 24 h: t 80 = 3.41, P = 0.001). No significant differences were found between plant-reared aphids and diet-reared aphids exposed to bean volatiles at any time period (P > 0.05) except at 24 h (t 80 = 2.22, P = Distribution of aphids exposed to an empty rearing chamber was significantly different to those of bean-reared aphids at 3 h (t 80 = 2.17, P = ) and 24 h (t 80 = 2.43, P = ). Olfactometry The olfactometer results suggested that experience of plant odors did not have an effect on behavioral responses to plant volatiles alone. In the first experiment, aphids with different rearing experiences were offered the choice between volatiles from a mustard plant and volatiles from a bean plant (Figure 2). A significant preference for bean volatiles was displayed by diet-reared aphids with experience of bean volatiles (t 132 = 2.24, P = ), diet-reared aphids with experience of mustard volatiles (t 132 = 2.98, P = ), and aphids reared on bean plants (t 132 = 2.88, P = ). Diet-reared aphids exposed to an empty rearing chamber (blank) showed no preference for either odor source (t 132 = 1.10, P = ). Differences between rearing experiences were significant (F 3,132 = 3.61, P = ). Least squares means post-hoc analysis revealed that aphids reared in the absence of plant volatiles showed significantly different responses to each of the other three rearing experiences (blank/bean volatiles: t 132 = 2.37, P = ; blank/mustard volatiles: t 132 = 2.88, P = ; blank/bean plants: t 132 = 2.76, P = ). No significant differences were found between other pairs of treatments (P > 0.05). In the second experiment, aphids were offered the choice between volatiles from either a bean or a mustard plant and volatiles from a pot of soil control (Figure 3). Diet-reared aphids with experience of bean volatiles showed a significant preference for bean volatiles (t 115 = 2.26, P = ), but did not show any preference for mustard volatiles (t 120 = 0.51, P = ). Similar preferences were shown for diet-reared aphids exposed to an empty rearing chamber (bean/soil: t 115 = 2.48, P = ; mustard/ soil: t 120 = 0.24, P = ), and also for aphids reared on bean plants (bean/soil: t 115 = 3.83, P = ; mustard/ soil: t 120 = 1.43, P = ). Diet-reared aphids exposed to mustard volatiles showed a preference for both bean volatiles and mustard volatiles, but neither of these were significant (bean/soil: t 31 = 1.637, P = 0.11; mustard/soil: t 30 = 1.4, P = 0.172). No significant differences were found between different rearing experiences in their responses to either bean or mustard volatiles (bean/soil: F 3,115 = 1.03, P = ; mustard/soil: F 3,120 = 1.09, P = 0.357), suggesting that all aphids responded in a similar way to both test odors. Figure 2 Responses of aphids to volatiles from bean and mustard plants in an olfactometer. A positive value indicates an overall preference for bean odor; a negative value indicates an overall preference for mustard odor. Significance *P < 0.05; **P < 0.01.

5 370 Behavioral Ecology Figure 3 Responses of aphids to volatiles from bean and mustard plants and a pot of soil control in an olfactometer. A positive value indicates an overall preference for bean or mustard odor; a negative value indicates an overall preference for a pot of soil control. Significance *P < 0.05; **P < Discussion The results of the arena bioassay show that host selection behavior in A. fabae can be modified by experience of volatiles acquired during development. This is the first study to show evidence of learning of volatile chemical cues in aphids. However, innate preferences for bean volatiles appeared to dominate when not in close proximity to the plants, suggesting that learnt information is only utilized when familiar plants are close by. In the arena bioassay, diet-reared aphids with experience of mustard volatiles showed a strong preference for mustard leaves compared with aphids with previous experience of bean volatiles. It is surprising that diet-reared aphids exposed to bean volatiles did not show a strong preference for bean leaves when compared with aphids reared on bean plants (Figure 1). This result may be explained by considering the responses of diet-reared aphids reared without any experience of plant volatiles, which showed a tendency to settle on mustard leaves rather than bean leaves. This suggests that, in the absence of previous experience of plant volatiles, dietreared A. fabae preferentially settle on mustard. Therefore, experience of bean volatiles seems to have caused a shift in preference away from mustard and toward bean compared with the inexperienced diet-reared aphids. The tendency for naive diet-reared aphids to settle on mustard is surprising. One possible explanation is that the mustard leaves offered a stronger visual stimulus to the dietreared aphids because aphids are known to respond to visual cues (Doring and Chittka 2007). Although care was taken to use leaves of similar size, the smallest available mustard leaves still tended to be larger than the largest bean leaf. This means that, in the arena bioassay, the mustard leaves tended to be slightly larger and may have been more visually attractive. Aphids reared on bean plants had previous experience of visual cues associated with bean and so these may have offered a stronger visual stimulus, despite the large size of the mustard leaves. This raises the interesting possibility that aphids can also learn from visual cues. Learning of such cues, and the possible interaction with learning of olfactory cues (Siddall and Marples 2008), warrants further investigation. Despite the bias for diet-reared aphids to select mustard leaves, the effect of rearing experience on host preference is clear. Experience of either bean or mustard volatiles caused a clear shift in preference toward the plants they had experienced during development when compared with the preference of aphids with no previous experience of plant volatiles. The effect of rearing experience diminished gradually over time, with no significant differences found between diet treatments after 24 h. Olfaction plays an important role in the host settling process, but final acceptance does not occur until after sampling of the phloem, which generally does not take place until after at least 1 2 h of sustained stylet penetration (Tosh et al. 2003). The decline in effect of experience after 24 h is probably because aphids had time to sample the phloem and make a decision based on gustatory rather than olfactory cues. It is known that host selection behavior in some aphids can be altered by experience of gustatory cues (van Emden et al. 2009). Because none of the diet-reared aphids had previously sampled phloem from either bean or mustard, this could explain why aphids showed little preference for either plant toward the end of the experiment. Aphids reared on bean plants, on the other hand, had prior experience of gustatory cues associated with bean, which could explain their strong preference for bean toward the end of the experiment. In olfactometer bioassays, aphids showed a preference for bean volatiles over mustard volatiles regardless of whether they had been previously exposed to mustard or bean volatiles during development. When offered the choice between bean volatiles and volatiles from a pot of soil control, aphids spent more time in the olfactometer arm exposed to bean volatiles. For diet-reared aphids with experience of mustard volatiles, however, this preference was not significant (Figure 3). This could suggest that experience of mustard volatiles switched off the aphids responses to bean odor. Post-hoc analysis, however, failed to support this hypothesis as no significant differences in response to bean odor were found between the different aphid treatments, suggesting they all responded in a similar way. Although it is possible that experience exerted some influence, this was clearly less pronounced than when observed in the arena bioassay, where experience of mustard odor caused a strong and significant shift in host preference.

6 Webster et al. Innate and learnt responses to odors 371 There is an interesting divergence between the results of the arena bioassay and olfactometer tests, with experience shown to have a strong effect on behavior in the former, where the perceived cost of habitat assessment was low, but little effect in the latter, where the perceived cost was unknown and, therefore, higher risk. This provides insight into the different roles of innate and learnt behavioral responses to plant volatiles. Information gained from the natal plant can provide significant advantages in the expansion of host range and continued exploitation of novel host plants, but the information may be less reliable due to between-plant variability in volatile emission. Innate behavioral responses are likely to be more reliable but do not facilitate in the expansion of host range and continued exploitation of novel host plants in the same way that learnt information does. The use of learnt and innate behavioral responses is, therefore, a balance of risk and reward based on the perceived cost of habitat exploration and the reliability of learnt information. This is an idea that has received little attention to date, but it is supported by the results of this study. This may be a widespread trait among animals, particularly among those for which habitat exploration is costly, and warrants further investigation in other species. This study also highlights the fact that care should be taken when designing experiments to assess learning behavior to avoid drawing hasty conclusions. Failure to identify such behavior could be the result of failing to test responses in the proper ecological context. Conversely, too much emphasis may be placed on the importance of learning if responses are only tested in experimental designs that simulate low-cost habitat exploration. An unexpected finding was that diet-reared aphids exposed to an empty odor chamber did not show a preference for bean volatiles over mustard volatiles. This could suggest an impaired olfactory sense and could be a parallel to recent work showing a similar effect in Drosophila melanogaster (Hare 2011). Fruit flies reared on a synthetic medium relatively devoid of odors showed differences in olfactory receptor neuron firing patterns compared with flies reared on the same medium supplemented with odorants, indicating a reduction in sensitivity and acuity in the odor-deprived flies. This study may demonstrate a similar effect, showing how lack of stimulation can impair behavioral responses to odors. However, aphids reared in odorless conditions were still able to respond to bean odor when offered the choice between bean volatiles and a pot of soil control. It could be that the olfactory sense was only partly diminished so that when offered the relatively simpler choice between bean volatiles and soil, aphids were able to discriminate between the two. Volatile blends emitted by bean and mustard likely share similarities because many plant volatiles are found almost universally throughout the plant kingdom and different species often emit similar blends, with key differences being in the quantities and ratios of volatiles rather than volatiles specific to either plant (Bruce et al. 2005). The task of discriminating between the two blends could, therefore, be difficult for an aphid with an even somewhat-impaired olfactory sense. The effect of sensory deprivation on behavioral responses to odors is interesting and warrants further investigation. In conclusion, learning of olfactory cues was demonstrated for the first time in a species of aphid. It was shown that learnt information plays a prominent role when in close proximity to the plant, but innate behavior appears to dominate when cues indicating the precise location of the odor source are unavailable and so the risk of habitat exploration is higher. The ability to weigh the risks and rewards of using learnt information at different stages of the host location process could confer selective advantages, particularly for insects for which habitat exploration is costly. This trait may be widespread among other animal species and warrants further investigation. Funding This work was supported by Carl Tryggers Stiftelse and by Mistra through the PlantComMistra program. We would like to thank Prof Jan Pettersson for his helpful comments on the manuscript. We would also like to thank Prof Helmut van Emden and Ms Elizabeth Wild for their advice on the aphid diet/ rearing system. References Alyokhin A, Sewell G On-soil movement and plant colonization by walking wingless morphs of three aphid species (Homoptera:Aphididae) in greenhouse arenas. Environ Entomol. 32: Bruce TJ, Wadhams LJ, Woodcock CM Insect host location: a volatile situation. Trends Plant Sci. 10: Davis JM, Stamps JA The effect of natal experience on habitat preferences. Trends Ecol Evol. 19: Degen T, Dillmann C, Marion-Poll F, Turlings TC High genetic variability of herbivore-induced volatile emission within a broad range of maize inbred lines. Plant Physiol. 135: Doring TF, Chittka L Visual ecology of aphids-a critical review on the role of colours in host finding. Arthropod-Plant Interact. 1:3 16. Douglas AE Provenance, experience and plant utilisation by the polyphagous aphid, Aphis fabae. Entomol Exp Appl. 83: Dukas R Evolutionary biology of insect learning. Annu Rev Entomol. 53: van Emden H Artificial diet for aphids - thirty years experience. Redia. 92: van Emden H, Kilbane J, Pettersson J Changing the host selection responses of aphids through a short experience of a novel secondary plant compound. Redia. 92: Gorur G, Lomonaco C, Mackenzie A Phenotypic plasticity in host-plant specialisation in Aphis fabae. Ecol Entomol. 30: Gorur G, Lomonaco C, Mackenzie A Phenotypic plasticity in host choice behavior in the black bean aphid, Aphis fabae (Homoptera: Aphididae). Arthropod-Plant Interact. 1: Gouinguené SP, Turlings TC The effects of abiotic factors on induced volatile emissions in corn plants. Plant Physiol. 129: Hare JD Ecological role of volatiles produced by plants in response to damage by herbivorous insects. Annu Rev Entomol. 56: Hodgson C Dispersal of apterous aphids (Homoptera, Aphididae) from their host plant and its significance. B Entomol Res. 81: Littell R, Milliken G, Stroup W, Wolfinger R, Schabenberger O SAS for mixed models. 2nd ed. Cary (NC): SAS Institute Inc. Nottingham SF, Hardie J, Dawson GW, Hick AJ, Pickett JA, Wadhams LJ, Woodcock CM Behavioral and electrophysiological responses of aphids to host and nonhost plant volatiles. J Chem Ecol. 17: Olsson U Generalized linear models: an applied approach. Lund (Sweden): Studentlitteratur. Olsson U Statistics for Life Science 2. Lund (Sweden): Studentlitteratur. Powell G, Hardie J The chemical ecology of aphid host alternation: how do return migrants find the primary host plant? Appl Entomol Zool. 36: Siddall EC, Marples NM Better to be bimodal: the interaction of color and odor on learning and memory. Behav Ecol. 19: Stamps JA, Davis JM Adaptive effects of natal experience on habitat selection by dispersers. Anim Behav. 72: Tosh CR, Morgan D, Walters KFA, Douglas AE The significance of overlapping plant range to a putative adaptive trade-off in the black bean aphid Aphis fabae Scop. Ecol Entomol. 29:

7 372 Behavioral Ecology Tosh CR, Powell G, Hardie J Decision making by generalist and specialist aphids with the same genotype. J Insect Physiol. 49: Webster B The role of olfaction in aphid host location. Physiol Entomol. 37: Webster B, Bruce T, Dufour S, Birkemeyer C, Birkett M, Hardie J, Pickett J Identification of volatile compounds used in host location by the black bean aphid, Aphis fabae. J Chem Ecol. 34: Webster B, Bruce T, Pickett J, Hardie J. 2010a. Volatiles functioning as host cues in a blend become nonhost cues when presented alone to the black bean aphid. Anim Behav. 79: Webster B, Gezan S, Bruce T, Hardie J, Pickett J. 2010b. Between plant and diurnal variation in quantities and ratios of volatile compounds emitted by Vicia faba plants. Phytochemistry. 71: Wiktelius S Migration of apterous Rhopalosiphum padi. WPRS Bulletin. 12:1 6.