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The effects of novel odour and colour cues on food acceptance by the zebra finch, Taeniopygia guttata
ANIMAL BEHAVIOUR, 2004, 68, 1049–1054 doi:10.1016/j.anbehav.2004.07.001
The effects of novel odour and colour cues on food acceptance by the zebra finch, Taeniopygia guttata DAVID J . KELLY & N ICOLA M . M ARP LES
Department of Zoology, Trinity College, Dublin (Received 28 October 2003; initial acceptance 18 December 2003; final acceptance 12 March 2004; published online 15 September 2004; MS. number: 7891)
Studies on domestic chicks Gallus gallus domesticus, have shown an advantage of multimodal advertisement by prey species. An interaction of novel colour and novel odour cues creates a synergistic aversive reaction (that is, the effect of the two cues presented simultaneously is greater than predicted by their individual presentation). Such reactions have not been tested empirically with passerine species, which account for about 60% of all bird species. We investigated the reactions of a passerine model, the zebra finch to novel colour and novel odour cues within food by measuring latencies to make contact with and to eat it. Zebra finches showed a synergistic reaction towards a combination of novel colour (red) and novel odour (pyrazine) cues despite showing no reaction to novel odour alone. As domestic chicks behave similarly, our results reveal that avian predators from different families show the same behaviour and suggest it might be a general behaviour of all birds. A comparison of the contact and consumption latencies for food of novel colour, and food of novel colour and odour, found that contact latencies remained the same while consumption latencies increased. We propose this is evidence of independent regulation of the processes controlling contact with a novel food (neophobia) and consumption of that food (dietary conservatism). Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Aposematic animals advertise their unprofitability with conspicuous signals (Cott 1940; Edmunds 1974). Many of these animals are known to signal multimodally (Kirchner & Roschard 1999; Rowe 1999; de Cock & Matthysen 2001; Hatle et al. 2001) and many insects use a combination of bright colours and pyrazine odour (Rothschild & Moore 1987). Domestic chicks, Gallus gallus domesticus, show a synergistic aversive reaction to these cues, that is, their responses to the combined cues are greater than would be predicted by their responses to the individual cues (Marples & Roper 1996; Rowe & Guilford 1996). Work by Avery and collaborators (Avery & Mason 1997; Nelms & Avery 1997; Avery et al. 1998) has shown an increased aversion of passerine species to ‘aposematic’ food (multimodally advertised, defended food), but there have been no laboratory studies on the reactions of passerine species to palatable food of novel colour and novel odour. By using palatable food, it is possible to examine the protection afforded to the food by its appearance or odour alone. This avoids the considerations of aversion learning which would be necessary with aposematic prey. Correspondence: D. J. Kelly, Department of Zoology, Trinity College, Dublin 2, Ireland (email: [email protected]). 0003–3472/04/$30.00/0
A great number of dietary studies have focused on the domestic chicken (Ha¨rlin & Ha¨rlin 2003). As well as poultry being readily accessible, there are economic incentives for understanding (and potentially being able to manipulate) their dietary preferences (Leeson et al. 2001; Urdaneta-Rincon & Leeson 2002). Although such studies may prove invaluable to poultry farmers, they do not necessarily inform us about the behaviour of other species (Greenberg 1990; Exnerova´ et al. 2003) or even poultry in the wild (Schlenoff 1984). Domestic chickens have been bred for high weight gain (and probably reduced wariness of novel foods as a result) for many generations (Mench 2002). One may anticipate that such a lineage would predispose these animals to be much less neophobic towards novel foods. For this reason it is important to determine the generality of the chicks’ synergistic aversive reaction to combinations of novel colour and novel odour. In an attempt to do so, we selected a passerine model species, the zebra finch. As passerine species account for approximately 60% of all bird species (Sibley & Monroe 1990) and include many insectivores, an appreciation of passerine behaviour could add significantly to an understanding of avian predator– invertebrate prey coevolution.
1049 Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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Numerous terrestrial arthropods have been documented as aposematic (Cott 1940; Edmunds 1974). Presumably all of these species are potential prey for birds of one sort or another. Indeed, some avian predators are known to consume aposematic prey (Yosef & Whitman 1992; Davies 2000). Despite the choice of colour and odour cues here, our intention was not to test the efficacy of warning signals per se, but to test a specific multimodal combination. Marples & Kelly (1999) identified a number of stages within the incorporation of a new food into the diet of a bird. These stages were broadly grouped into neophobia (time to initial contact) and dietary conservatism (from initial contact to full incorporation into the diet). At the point of first approach to a novel food, a bird is no longer showing neophobia, as it has ceased to avoid the food (Brigham & Sibly 1999). There inevitably follows a period (albeit very short in some cases) when a bird has to decide whether to eat this new food. This period is termed dietary conservatism (Marples & Kelly 1999). Such a definition means that dietary conservatism is not interchangeable with other intuitively similar terms. Food neophobia (Roberts & Cheney 1974; Jones 1987; Pliner & Hobden 1992; Cheney & Miller 1997; Johnson 2000), feeding neophobia (Greenberg 1984; Dutoit et al. 1991; Sunnucks 1998; Webster & Lefebvre 2001), gustatory neophobia (Ayyagari et al. 1991; Pelleymounter & Cullen 1993; Roozendaal & Cools 1994; Hamm et al. 1995) and ingestional neophobia (Franchina et al. 1994) all refer to the period between first encounter and first consumption of the food. These terms effectively incorporate both neophobia and dietary conservatism, so cannot be directly compared to either. We believe that neophobia is psychologically distinct from dietary conservatism, as neophobia is demonstrated for both food (see above) and nonfood (Coleman & Mellgren 1994; Webster & Lefebvre 2001) items, whereas dietary conservatism is, by its nature, restricted to food items. The more restricted conditions under which dietary conservatism is found argue for a separate process from the more general neophobia response. We are unaware of any direct evidence to support this distinction. The separation of dietary conservatism and neophobia allows a reconsideration of predator–prey interactions. When a predator comes to the point of inspecting a novel prey (protected or not) neophobia has already been overcome, and it is dietary conservatism that determines when the predator will sample that prey. It is this aspect of protection of novel prey morphs by dietary conservatism that is rarely considered, as many authors believe that novel, conspicuous prey are under increased predation pressure compared with more cryptic familiar morphs (Yachi & Higashi 1998; Riipi et al. 2001). However, recent laboratory and field studies (Thomas et al. 2003, 2004) have demonstrated that dietary conservatism may be important in the evolution of aposematic animals. The dietary history of the predator may be a further important consideration. At any time, even a generalist predator will be able to identify a selection of familiar prey. The appearance of a novel prey (be it a new morph or a new type) in that predator’s home range can lead to a number of possible outcomes. (1) The prey may not be
recognized as prey, and may not be investigated by the predator. (2) The prey may be investigated, but the predator will learn that such prey are unprofitable. (3) The prey may be sampled and found to be palatable and incorporated into the diet. (4) The prey may be recognized as potential prey, but will not be sampled because the predator already has an adequate food supply. Point (4), the reluctance of a predator to incorporate the novel prey into its diet, is dietary conservatism. We designed experiments to discover whether zebra finches were sensitive to novel odour in combination with novel colour, as chicks had been shown to be (Marples & Roper 1996). The latencies of the zebra finches to eat the various novel treatments were considered in terms of both neophobia and dietary conservatism.
METHODS The zebra finches were housed as same-sex pairs in wooden cages (30 ! 35 cm and 60 cm high) with a vertical slot 1 cm wide in the centre of the back wall. This slot allowed the insertion of a plastic divider to separate the cage in half during the trials. The birds were maintained in a 12:12 h light:dark regime at 20 C. Water was available ad libitum. Training food was a high-nutrient food (Ce´De´ Egg Food for Canaries, Ce´De´ Vogelvoeders B.V., Tilburg, The Netherlands), which was made up into a rough paste with water (ca. 2 parts water to 1 part food). The birds were familiarized with the training food as a pair over a week. Food was presented daily, in a white plastic dish (6 cm diameter, 2.5 cm deep) and left for the birds to investigate. The training food was normally preferred to the birds’ usual food (foreign finch seed, PetStop Superstores, Dublin, Ireland). Birds were acquired from local breeders. All birds had experience of the finch seed, and many of the female birds had experience of the egg food. However, none of the birds had experience of red food or pyrazine odours. At testing time, the subjects were tested individually in divided cages. To reduce the stress of isolated birds (Sille´n-Tullberg 1985), we tested a number of boxes simultaneously. This allowed vocal communication between the individuals under test, but no observation of the choices made by those individuals. The birds were deprived of food for 90 min, and then tested with one of four treatments: control (the training food), novel odour (the training food in the presence of pyrazine odour), novel colour (the training food made up with red dye) and doubly novel (the training food made up with red dye and in the presence of pyrazine). Nine individuals were allocated to each treatment. The pyrazine was a 0.0003% solution of 2-methoxy-3-isopropylpyrazine (Pyrazine Specialties Inc., Atlanta, Georgia, U.S.A.), which is sufficiently strong to elicit a response in chicks (Marples & Roper 1996). The red dye was a 25% solution of O’Brien’s Christmas Red (4R) (O’Brien’s Ingredients, Dublin, Ireland), which is an effective concentration for avian foods (Marples & Roper 1996). All feeding cups contained a band of filter paper on their inner rim, which was soaked with water (when no odour was required) or the solution of pyrazine.
KELLY & MARPLES: DIETARY CONSERVATISM AND NEOPHOBIA
Statistical Analysis To determine whether latency to incorporate a treatment differed between treatments, we used two-tailed Mann–Whitney U tests, and corrected the results for multiple tests using step-up sequential Bonferroni correction (Hochberg 1988). RESULTS The welfare test described above (birds presented with control food before and after test food) resulted in four of
the 469 trials (i.e. !1%) being discarded. In each instance the birds had sat at the back of the cages and not moved during exposure to the test food. By ignoring these trials, we feel we more accurately reflected the choices of the birds. Some of the birds exposed to the doubly novel treatment had not incorporated the food by the end of the experimental period, so minimum possible values were calculated for them. Minimum possible values needed to be derived only for birds in the doubly novel group (five of the nine individuals). We calculated the values by adding either 1 s or one presentation to the cumulative latency scores of these birds. This gave a value of the shortest possible time any bird could have taken to incorporate a treatment, had the experiment been allowed to continue. These values were guaranteed to be conservative, as none of the birds were sampling the food regularly before we discontinued the trials. Figure 1 shows the mean latencies to peck at and incorporate the four treatments. There were no differences in latency to peck for the control and novel odour groups or for the novel colour and doubly novel groups. Analysis of the time data showed a difference in the latencies to incorporate the four treatments (Kruskal–Wallis test adjusted for ties: c23 Z 30.0, P ! 0.001). There was no main effect of odour (control versus novel odour; Table 1), but there was a main effect of colour (control versus novel colour, novel odour versus doubly novel; Table 1). There was a significant difference between the novel colour and
Latency (presentations)
40 35
(a) Contact
30
Incorporation
25 20 15 10 5 0 50000
C
NO
NC
NCO
C
NO
NC
NCO
(b)
40000 Latency (s)
During testing, birds initially received the control food, then the test treatment, and then the control food again. This protocol allowed the birds to demonstrate their habituation to the test conditions. If a bird refused to eat the control food on the initial presentation within 10 s, it was considered to be ‘uncomfortable’ in the test environment, and was not given the subsequent presentations. Birds display fear by adopting frozen postures known as tonic immobility (Ratner 1967). Birds usually responded immediately to the presentation of the control food, unless they were in this state. We recorded latencies to make contact with (peck at) and to incorporate (eat within 10 s on three successive presentations) the treatments. After testing with a treatment it was removed, and replaced with control food. This acted as a second test on the welfare of the birds. If birds failed to eat either control treatment within 10 s, then we discarded their corresponding test presentation. Birds were allowed up to 1 h with their test foods. This meant that birds were never without food for more than 2.5 h (following EC directive 86/609/EC). As birds were not guaranteed to eat a particular treatment on their first presentation, we added together the latencies from consecutive trials to derive a final total latency score. To prevent inappropriate exposure of test birds to pyrazine odours, we conducted the trials using novel odours after the completion of trials without novel odours. However, this meant that we could still test birds from the control and novel colour groups simultaneously, as well as birds from the novel odour and doubly novel groups. As birds from both the control and novel odour groups incorporated their test foods very quickly, such simultaneous testing lasted for only short periods. When birds had eaten a given treatment on three consecutive presentations in under 10 s (i.e. it was eaten as rapidly as control food), they were considered to have incorporated that food. The point of incorporation was recorded as the first of these three presentations. Some birds (N Z 7) sampled food on single occasions, and subsequently refused that same food. By imposing the criterion that food must be eaten on three successive presentations, we avoided confusing sampling with full acceptance of the food by the bird. Latencies were recorded both as the number of seconds and the number of presentations before contact and incorporation. Bird husbandry and our experimental procedures were approved by the Department of Health and Children.
30000 20000 10000 0
Figure 1. Comparative latencies of zebra finches to peck at (contact) and eat (incorporation) three novel treatments of food: control (C), novel odour (NO), novel colour (NC) and novel colour and odour (NCO). (a) Latency in terms of number of presentations; (b) latency in terms of time (s). Means are shown GSEM where they are significantly greater than zero. N Z 9 for all treatments.
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Table 1. Comparison of incorporation latencies for the four treatment groups using Mann–Whitney U tests Control Novel odour U P Novel colour U P Doubly novel U P
Novel odour
Novel colour
32.0 0.443 0.0 0.0003*
0.0 0.0003*
0.0 0.0003*
0.0 0.0003*
6.0 0.002*
U and P values are given in their uncorrected form. Significant values (following step-up sequential Bonferroni correction using f Z 0.05) are indicated with an asterisk.
doubly novel groups (Table 1), which demonstrates an interaction between novel colour and novel odour. The interaction between novel colour and novel odour can be described as synergistic, as it was greater than the combined effects of these two independent cues. The mean incorporation time for the novel odour treatment GSE was 4 G 0.7 s (N Z 9), and for novel colour 13 262 G 3772 s (N Z 9). This gives an additive incorporation time of 13 266 G 3773 s, but the actual mean incorporation time for the doubly novel treatment was 43 555 G 5711 s (N Z 9). This was an increase from about 3.7 h (novel colour) to about 12.1 h (doubly novel). Some birds were taking over 10 weeks of continuous testing before incorporating the doubly novel treatment, as only one trial was conducted on any individual per day. DISCUSSION The zebra finches showed the same trends towards novel colours and odours of food as has been shown in chicks (Marples & Roper 1996). This result is interesting for a number of reasons. Importantly, it indicates that chicks and adult zebra finches react similarly to the same colour and odour cues independently and in combination, under laboratory conditions. This suggests that results from chick feeding studies are relevant to zebra finches, and probably to a number of other related species. Equally importantly, it indicates that zebra finches possess a sense of smell, and use this sense to determine the acceptability of novel foods. Passerines have similar odour thresholds to those of galliformes (Clark et al. 1993), despite having relatively smaller olfactory bulbs (Bang & Cobb 1968). These thresholds were determined by monitoring pulse rate while the birds were presented with odours from a dilution olfactometer. However, Clark et al. (1993) highlighted an important difference between an individual’s capacity and its tendency to attend to an odour cue. That is, just because a bird has the ability to detect an odour, it does not mean that it will attend to the cue when it encounters it. Our study represents a clear demonstration of a passerine attending to such a cue. Despite the evidence that novel odour does not generate a neophobic effect on its own, the role of odour in food choice should not be underestimated. Odours have been
shown to trigger hidden, unlearned colour preferences in chicks (Rowe & Guilford 1996; Jetz et al. 2001). Pyrazine odour also produces a feeding bias against conspicuous prey (Lindstro¨m et al. 2001a). Clearly the interaction of colour and odour cues is important for these birds. In taste aversion studies with domestic chicks, odour is a preferential learning cue to colour (Roper & Marples 1997b). Roper & Marples (1997b) found that almond odour was a more salient cue than green colour for avoiding quinine-flavoured water. However, vanilla did not show this same overshadowing effect, so the effect appears to be specific to certain odours. Indeed, not all combinations of novel odours and novel colours produce synergistic aversive reactions in domestic chicks (Marples & Roper 1996). The two cues we chose are traditionally associated with aposematic animals. Red is frequently used (sometimes in combination with other colours) as a warning signal (Sille´n-Tullberg 1985; Marples et al. 1994; Exnerova´ et al. 2003; Hagen et al. 2003), and pyrazines are used by a number of aposematic insects (Rothschild & Moore 1987). Perhaps a combination of these two cues elicits an exceptional response. Only further work with other novel colour and odour combinations will answer this question. The latencies of zebra finches to sample the novel colour treatment (red; 13 262 s, N Z 9) were greater than for chicks (21 s, N Z 15; Roper & Marples 1997a), by more than two orders of magnitude. There are many differences between the two groups of animals used, including species, order (Galliformes/Passeriformes), developmental strategy (precocial/altricial), diet (omnivore/granivore), age (juvenile/sexually mature) and perhaps experience (younger/older). It is unclear which of these differences are important in determining the responses of the birds. The literal definition of neophobia is ‘fear of the new’ (Barnett 1958), but this definition is not precise enough for our experimental conditions. Brigham & Sibly (1999) defined neophobia as ‘the initial avoidance of novel objects in an otherwise familiar environment’, where ‘objects’ may include food. Our experimental feeding environment was unmodified once birds began to be tested, so the presence of test food would have been truly novel only on the first presentation. The unfamiliarity of novel objects must decrease with increased exposure to them, and hence neophobia decreases with experience (Coppinger 1969, 1970; Schlenoff 1984; Jones 1987; Mastrota & Mench 1995). It is reasonable to assume that some birds are more neophobic than others, but observations of the zebra finches gave no impression that they were avoiding the area around the novel colour or doubly novel treatments. A number of birds that were not eating the novel colour or doubly novel treatments perched on the presentation cups during their trials. Clearly neophobia (both of the experimental equipment and of the coloured food) was no longer in operation (Brigham & Sibly 1999). The term dietary conservatism (Marples et al. 1998) can be used to describe the stages of incorporation once neophobia has been overcome. Neophobia and dietary conservatism can be considered separately if latency to peck at food is considered as a measure of neophobia, and latency to eat it as a measure of dietary conservatism (Marples & Kelly 1999).
KELLY & MARPLES: DIETARY CONSERVATISM AND NEOPHOBIA
Dietary conservatism may be considered a form of wariness by the birds. Although the novel foods in these trials were not dangerous to be close to (as assessed by the relatively low contact latencies), there was no immediate information available to the birds about the costs or benefits of eating the food. As the natural world contains many aposematic animals (Cott 1940; Edmunds 1974), wariness would appear to be a profitable trait, especially as certain cryptic (or weakly signalling) invertebrates prove to be defended (Codella & Raffa 1996; Krall et al. 1999; Larsson et al. 2000). It is likely that novel foods need to be sampled for some time until they can be assessed as ‘safe’ (Kalat & Rozin 1973). Such a strategy may even lead to less-experienced predators repeatedly sampling aposematic prey before ultimately rejecting them (Lindstro¨m et al. 2001b). We deliberately chose to omit any ‘unprofitable’ aspect to the novel foods from these trials, to investigate the effect of the novel cues on their own. The zebra finches showed increased neophobia (latency to peck at food) towards the coloured treatments (Fig. 1b). However, there was no further increase in neophobia shown towards the doubly novel treatment compared with the novel colour treatment. The zebra finches also showed increased dietary conservatism (latency to eat food) towards the coloured treatments (Fig. 1b). Unlike the neophobic responses of the zebra finches, the dietary conservatism ‘effect’ was much greater for the doubly novel treatment than for the novel colour treatment. This suggests it is the dietary conservatism aspect of a predator’s reaction towards multimodally novel prey that enhances that prey’s protection, rather than the predator’s neophobic responses. Such an analysis is possible only with palatable prey. If the prey were unpalatable, an aversion learning element would also need to be considered. These data represent a clear demonstration of the neophobic and dietary conservative behaviours of an animal being independently modified when confronted with novel foods. It would seem appropriate that neophobia and dietary conservatism should be considered discrete processes, as proposed by Marples & Kelly (1999). An animal’s reaction towards new food can be more fully explained when these two processes are considered individually. If we now consider the evolutionary strategy of an aposematic prey animal, that animal will potentially exploit a predator’s dietary conservatism by advertising itself multimodally. These results suggest that multimodal advertisement would not increase the neophobic response of the predator (i.e. approach), but would increase its latency to sample the prey if it were novel. Such a situation is particularly relevant to avian predators in tropical climates, where there are many potential invertebrate prey (Owen 1977). Many aposematic insects use colour and odour to advertise their unprofitability, which capitalizes on the dietary conservatism response. Indeed, perhaps all multimodal advertisement exploits this behaviour. Acknowledgments We are grateful to the staff of the BioResources Unit in Trinity College for their care of the finches and their patience over the protracted length of the study. We thank Mark
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Owen, D. F. 1977. Latitudinal gradients in clutch size: an extension of David Lack’s theory. In: Evolutionary Ecology (Ed. by B. Stonehouse & C. Perrins), pp. 171–179. London: Macmillan. Pelleymounter, M. A. & Cullen, M. J. 1993. Effects of Idebenone on information processing in aged Long-Evans rats. Pharmacology, Biochemistry and Behavior, 46, 415–421. Pliner, P. & Hobden, K. 1992. Development of a scale to measure the trait of food neophobia in humans. Appetite, 19, 105–120. Ratner, S. 1967. Comparative aspects of hypnosis. In: Handbook of Clinical and Experimental Hypnosis (Ed. by J. Gordon), pp. 550–587. New York: MacMillan. ¨ m, L. & Mappes, J. 2001. Multiple Riipi, M., Alatalo, R. V., Lindstro benefits of gregariousness cover detectability costs in aposematic aggregations. Nature, 413, 512–514. Roberts, B. & Cheney, C. 1974. Food neophobia and taste aversion in tropical fish. Proceedings of the Utah Academy of Sciences, Arts and Letters, 51, 201. Roozendaal, B. & Cools, A. R. 1994. Influence of the noradrenergic state of the nucleus accumbens in basolateral amygdala mediated changes in neophobia of rats. Behavioral Neuroscience, 108, 1107– 1118. Roper, T. J. & Marples, N. M. 1997a. Colour preferences of domestic chicks in relation to food and water presentation. Applied Animal Behaviour Science, 54, 207–213. Roper, T. J. & Marples, N. M. 1997b. Odour and colour as cues for taste-avoidance learning in domestic chicks. Animal Behaviour, 53, 1241–1250. Rothschild, M. & Moore, B. P. 1987. Pyrazines as alerting signals in toxic plants and insects. In: Insect-Plants (Ed. by V. Labeyrie, G. Fabres & D. Lachaise), pp. 97–107. Dordrecht: W. Junk. Rowe, C. 1999. Receiver psychology and the evolution of multicomponent signals. Animal Behaviour, 58, 921–933. Rowe, C. & Guilford, T. 1996. Hidden colour aversions in domestic chicks triggered by pyrazine odours of insect warning displays. Nature, 383, 520–522. Schlenoff, D. H. 1984. Novelty: a basis for generalisation in prey selection. Animal Behaviour, 32, 919–921. Sibley, C. G. & Monroe, B. L. 1990. Distribution and Taxonomy of Birds of the World. New Haven, Connecticut: Yale University Press. Sille´n-Tullberg, B. 1985. The significance of coloration per se, independent of background, for predator avoidance of aposematic prey. Animal Behaviour, 33, 1382–1384. Sunnucks, P. 1998. Avoidance of novel objects by rabbits (Oryctolagus cuniculus L.). Wildlife Research, 25, 273–283. Thomas, R. J., Marples, N. M., Cuthill, I. C., Takahashi, M. & Gibson, E. A. 2003. Dietary conservatism may facilitate the initial evolution of aposematism. Oikos, 101, 458–466. Thomas, R. J., Bartlett, L., Marples, N. M., Kelly, D. J. & Cuthill, I. C. 2004. Prey selection by wild birds can allow novel and conspicuous colour morphs to spread in prey populations. Oikos, 106, 285–294. Urdaneta-Rincon, M. & Leeson, S. 2002. Quantitative and qualitative feed restriction on growth characteristics of male broiler chickens. Poultry Science, 81, 679–688. Webster, S. J. & Lefebvre, L. 2001. Problem solving and neophobia in a columbiform-passeriform assemblage in Barbados. Animal Behaviour, 62, 23–32. Yachi, S. & Higashi, M. 1998. The evolution of warning signals. Nature, 394, 882–884. Yosef, R. & Whitman, D. W. 1992. Predator exaptations and defensive adaptations in evolutionary balance: no defense is perfect. Evolutionary Ecology, 6, 527–536.
The effects of novel odour and colour cues on food acceptance by the zebra finch, Taeniopygia guttata DAVID J . KELLY & N ICOLA M . M ARP LES
Department of Zoology, Trinity College, Dublin (Received 28 October 2003; initial acceptance 18 December 2003; final acceptance 12 March 2004; published online 15 September 2004; MS. number: 7891)
Studies on domestic chicks Gallus gallus domesticus, have shown an advantage of multimodal advertisement by prey species. An interaction of novel colour and novel odour cues creates a synergistic aversive reaction (that is, the effect of the two cues presented simultaneously is greater than predicted by their individual presentation). Such reactions have not been tested empirically with passerine species, which account for about 60% of all bird species. We investigated the reactions of a passerine model, the zebra finch to novel colour and novel odour cues within food by measuring latencies to make contact with and to eat it. Zebra finches showed a synergistic reaction towards a combination of novel colour (red) and novel odour (pyrazine) cues despite showing no reaction to novel odour alone. As domestic chicks behave similarly, our results reveal that avian predators from different families show the same behaviour and suggest it might be a general behaviour of all birds. A comparison of the contact and consumption latencies for food of novel colour, and food of novel colour and odour, found that contact latencies remained the same while consumption latencies increased. We propose this is evidence of independent regulation of the processes controlling contact with a novel food (neophobia) and consumption of that food (dietary conservatism). Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Aposematic animals advertise their unprofitability with conspicuous signals (Cott 1940; Edmunds 1974). Many of these animals are known to signal multimodally (Kirchner & Roschard 1999; Rowe 1999; de Cock & Matthysen 2001; Hatle et al. 2001) and many insects use a combination of bright colours and pyrazine odour (Rothschild & Moore 1987). Domestic chicks, Gallus gallus domesticus, show a synergistic aversive reaction to these cues, that is, their responses to the combined cues are greater than would be predicted by their responses to the individual cues (Marples & Roper 1996; Rowe & Guilford 1996). Work by Avery and collaborators (Avery & Mason 1997; Nelms & Avery 1997; Avery et al. 1998) has shown an increased aversion of passerine species to ‘aposematic’ food (multimodally advertised, defended food), but there have been no laboratory studies on the reactions of passerine species to palatable food of novel colour and novel odour. By using palatable food, it is possible to examine the protection afforded to the food by its appearance or odour alone. This avoids the considerations of aversion learning which would be necessary with aposematic prey. Correspondence: D. J. Kelly, Department of Zoology, Trinity College, Dublin 2, Ireland (email: [email protected]). 0003–3472/04/$30.00/0
A great number of dietary studies have focused on the domestic chicken (Ha¨rlin & Ha¨rlin 2003). As well as poultry being readily accessible, there are economic incentives for understanding (and potentially being able to manipulate) their dietary preferences (Leeson et al. 2001; Urdaneta-Rincon & Leeson 2002). Although such studies may prove invaluable to poultry farmers, they do not necessarily inform us about the behaviour of other species (Greenberg 1990; Exnerova´ et al. 2003) or even poultry in the wild (Schlenoff 1984). Domestic chickens have been bred for high weight gain (and probably reduced wariness of novel foods as a result) for many generations (Mench 2002). One may anticipate that such a lineage would predispose these animals to be much less neophobic towards novel foods. For this reason it is important to determine the generality of the chicks’ synergistic aversive reaction to combinations of novel colour and novel odour. In an attempt to do so, we selected a passerine model species, the zebra finch. As passerine species account for approximately 60% of all bird species (Sibley & Monroe 1990) and include many insectivores, an appreciation of passerine behaviour could add significantly to an understanding of avian predator– invertebrate prey coevolution.
1049 Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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Numerous terrestrial arthropods have been documented as aposematic (Cott 1940; Edmunds 1974). Presumably all of these species are potential prey for birds of one sort or another. Indeed, some avian predators are known to consume aposematic prey (Yosef & Whitman 1992; Davies 2000). Despite the choice of colour and odour cues here, our intention was not to test the efficacy of warning signals per se, but to test a specific multimodal combination. Marples & Kelly (1999) identified a number of stages within the incorporation of a new food into the diet of a bird. These stages were broadly grouped into neophobia (time to initial contact) and dietary conservatism (from initial contact to full incorporation into the diet). At the point of first approach to a novel food, a bird is no longer showing neophobia, as it has ceased to avoid the food (Brigham & Sibly 1999). There inevitably follows a period (albeit very short in some cases) when a bird has to decide whether to eat this new food. This period is termed dietary conservatism (Marples & Kelly 1999). Such a definition means that dietary conservatism is not interchangeable with other intuitively similar terms. Food neophobia (Roberts & Cheney 1974; Jones 1987; Pliner & Hobden 1992; Cheney & Miller 1997; Johnson 2000), feeding neophobia (Greenberg 1984; Dutoit et al. 1991; Sunnucks 1998; Webster & Lefebvre 2001), gustatory neophobia (Ayyagari et al. 1991; Pelleymounter & Cullen 1993; Roozendaal & Cools 1994; Hamm et al. 1995) and ingestional neophobia (Franchina et al. 1994) all refer to the period between first encounter and first consumption of the food. These terms effectively incorporate both neophobia and dietary conservatism, so cannot be directly compared to either. We believe that neophobia is psychologically distinct from dietary conservatism, as neophobia is demonstrated for both food (see above) and nonfood (Coleman & Mellgren 1994; Webster & Lefebvre 2001) items, whereas dietary conservatism is, by its nature, restricted to food items. The more restricted conditions under which dietary conservatism is found argue for a separate process from the more general neophobia response. We are unaware of any direct evidence to support this distinction. The separation of dietary conservatism and neophobia allows a reconsideration of predator–prey interactions. When a predator comes to the point of inspecting a novel prey (protected or not) neophobia has already been overcome, and it is dietary conservatism that determines when the predator will sample that prey. It is this aspect of protection of novel prey morphs by dietary conservatism that is rarely considered, as many authors believe that novel, conspicuous prey are under increased predation pressure compared with more cryptic familiar morphs (Yachi & Higashi 1998; Riipi et al. 2001). However, recent laboratory and field studies (Thomas et al. 2003, 2004) have demonstrated that dietary conservatism may be important in the evolution of aposematic animals. The dietary history of the predator may be a further important consideration. At any time, even a generalist predator will be able to identify a selection of familiar prey. The appearance of a novel prey (be it a new morph or a new type) in that predator’s home range can lead to a number of possible outcomes. (1) The prey may not be
recognized as prey, and may not be investigated by the predator. (2) The prey may be investigated, but the predator will learn that such prey are unprofitable. (3) The prey may be sampled and found to be palatable and incorporated into the diet. (4) The prey may be recognized as potential prey, but will not be sampled because the predator already has an adequate food supply. Point (4), the reluctance of a predator to incorporate the novel prey into its diet, is dietary conservatism. We designed experiments to discover whether zebra finches were sensitive to novel odour in combination with novel colour, as chicks had been shown to be (Marples & Roper 1996). The latencies of the zebra finches to eat the various novel treatments were considered in terms of both neophobia and dietary conservatism.
METHODS The zebra finches were housed as same-sex pairs in wooden cages (30 ! 35 cm and 60 cm high) with a vertical slot 1 cm wide in the centre of the back wall. This slot allowed the insertion of a plastic divider to separate the cage in half during the trials. The birds were maintained in a 12:12 h light:dark regime at 20 C. Water was available ad libitum. Training food was a high-nutrient food (Ce´De´ Egg Food for Canaries, Ce´De´ Vogelvoeders B.V., Tilburg, The Netherlands), which was made up into a rough paste with water (ca. 2 parts water to 1 part food). The birds were familiarized with the training food as a pair over a week. Food was presented daily, in a white plastic dish (6 cm diameter, 2.5 cm deep) and left for the birds to investigate. The training food was normally preferred to the birds’ usual food (foreign finch seed, PetStop Superstores, Dublin, Ireland). Birds were acquired from local breeders. All birds had experience of the finch seed, and many of the female birds had experience of the egg food. However, none of the birds had experience of red food or pyrazine odours. At testing time, the subjects were tested individually in divided cages. To reduce the stress of isolated birds (Sille´n-Tullberg 1985), we tested a number of boxes simultaneously. This allowed vocal communication between the individuals under test, but no observation of the choices made by those individuals. The birds were deprived of food for 90 min, and then tested with one of four treatments: control (the training food), novel odour (the training food in the presence of pyrazine odour), novel colour (the training food made up with red dye) and doubly novel (the training food made up with red dye and in the presence of pyrazine). Nine individuals were allocated to each treatment. The pyrazine was a 0.0003% solution of 2-methoxy-3-isopropylpyrazine (Pyrazine Specialties Inc., Atlanta, Georgia, U.S.A.), which is sufficiently strong to elicit a response in chicks (Marples & Roper 1996). The red dye was a 25% solution of O’Brien’s Christmas Red (4R) (O’Brien’s Ingredients, Dublin, Ireland), which is an effective concentration for avian foods (Marples & Roper 1996). All feeding cups contained a band of filter paper on their inner rim, which was soaked with water (when no odour was required) or the solution of pyrazine.
KELLY & MARPLES: DIETARY CONSERVATISM AND NEOPHOBIA
Statistical Analysis To determine whether latency to incorporate a treatment differed between treatments, we used two-tailed Mann–Whitney U tests, and corrected the results for multiple tests using step-up sequential Bonferroni correction (Hochberg 1988). RESULTS The welfare test described above (birds presented with control food before and after test food) resulted in four of
the 469 trials (i.e. !1%) being discarded. In each instance the birds had sat at the back of the cages and not moved during exposure to the test food. By ignoring these trials, we feel we more accurately reflected the choices of the birds. Some of the birds exposed to the doubly novel treatment had not incorporated the food by the end of the experimental period, so minimum possible values were calculated for them. Minimum possible values needed to be derived only for birds in the doubly novel group (five of the nine individuals). We calculated the values by adding either 1 s or one presentation to the cumulative latency scores of these birds. This gave a value of the shortest possible time any bird could have taken to incorporate a treatment, had the experiment been allowed to continue. These values were guaranteed to be conservative, as none of the birds were sampling the food regularly before we discontinued the trials. Figure 1 shows the mean latencies to peck at and incorporate the four treatments. There were no differences in latency to peck for the control and novel odour groups or for the novel colour and doubly novel groups. Analysis of the time data showed a difference in the latencies to incorporate the four treatments (Kruskal–Wallis test adjusted for ties: c23 Z 30.0, P ! 0.001). There was no main effect of odour (control versus novel odour; Table 1), but there was a main effect of colour (control versus novel colour, novel odour versus doubly novel; Table 1). There was a significant difference between the novel colour and
Latency (presentations)
40 35
(a) Contact
30
Incorporation
25 20 15 10 5 0 50000
C
NO
NC
NCO
C
NO
NC
NCO
(b)
40000 Latency (s)
During testing, birds initially received the control food, then the test treatment, and then the control food again. This protocol allowed the birds to demonstrate their habituation to the test conditions. If a bird refused to eat the control food on the initial presentation within 10 s, it was considered to be ‘uncomfortable’ in the test environment, and was not given the subsequent presentations. Birds display fear by adopting frozen postures known as tonic immobility (Ratner 1967). Birds usually responded immediately to the presentation of the control food, unless they were in this state. We recorded latencies to make contact with (peck at) and to incorporate (eat within 10 s on three successive presentations) the treatments. After testing with a treatment it was removed, and replaced with control food. This acted as a second test on the welfare of the birds. If birds failed to eat either control treatment within 10 s, then we discarded their corresponding test presentation. Birds were allowed up to 1 h with their test foods. This meant that birds were never without food for more than 2.5 h (following EC directive 86/609/EC). As birds were not guaranteed to eat a particular treatment on their first presentation, we added together the latencies from consecutive trials to derive a final total latency score. To prevent inappropriate exposure of test birds to pyrazine odours, we conducted the trials using novel odours after the completion of trials without novel odours. However, this meant that we could still test birds from the control and novel colour groups simultaneously, as well as birds from the novel odour and doubly novel groups. As birds from both the control and novel odour groups incorporated their test foods very quickly, such simultaneous testing lasted for only short periods. When birds had eaten a given treatment on three consecutive presentations in under 10 s (i.e. it was eaten as rapidly as control food), they were considered to have incorporated that food. The point of incorporation was recorded as the first of these three presentations. Some birds (N Z 7) sampled food on single occasions, and subsequently refused that same food. By imposing the criterion that food must be eaten on three successive presentations, we avoided confusing sampling with full acceptance of the food by the bird. Latencies were recorded both as the number of seconds and the number of presentations before contact and incorporation. Bird husbandry and our experimental procedures were approved by the Department of Health and Children.
30000 20000 10000 0
Figure 1. Comparative latencies of zebra finches to peck at (contact) and eat (incorporation) three novel treatments of food: control (C), novel odour (NO), novel colour (NC) and novel colour and odour (NCO). (a) Latency in terms of number of presentations; (b) latency in terms of time (s). Means are shown GSEM where they are significantly greater than zero. N Z 9 for all treatments.
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Table 1. Comparison of incorporation latencies for the four treatment groups using Mann–Whitney U tests Control Novel odour U P Novel colour U P Doubly novel U P
Novel odour
Novel colour
32.0 0.443 0.0 0.0003*
0.0 0.0003*
0.0 0.0003*
0.0 0.0003*
6.0 0.002*
U and P values are given in their uncorrected form. Significant values (following step-up sequential Bonferroni correction using f Z 0.05) are indicated with an asterisk.
doubly novel groups (Table 1), which demonstrates an interaction between novel colour and novel odour. The interaction between novel colour and novel odour can be described as synergistic, as it was greater than the combined effects of these two independent cues. The mean incorporation time for the novel odour treatment GSE was 4 G 0.7 s (N Z 9), and for novel colour 13 262 G 3772 s (N Z 9). This gives an additive incorporation time of 13 266 G 3773 s, but the actual mean incorporation time for the doubly novel treatment was 43 555 G 5711 s (N Z 9). This was an increase from about 3.7 h (novel colour) to about 12.1 h (doubly novel). Some birds were taking over 10 weeks of continuous testing before incorporating the doubly novel treatment, as only one trial was conducted on any individual per day. DISCUSSION The zebra finches showed the same trends towards novel colours and odours of food as has been shown in chicks (Marples & Roper 1996). This result is interesting for a number of reasons. Importantly, it indicates that chicks and adult zebra finches react similarly to the same colour and odour cues independently and in combination, under laboratory conditions. This suggests that results from chick feeding studies are relevant to zebra finches, and probably to a number of other related species. Equally importantly, it indicates that zebra finches possess a sense of smell, and use this sense to determine the acceptability of novel foods. Passerines have similar odour thresholds to those of galliformes (Clark et al. 1993), despite having relatively smaller olfactory bulbs (Bang & Cobb 1968). These thresholds were determined by monitoring pulse rate while the birds were presented with odours from a dilution olfactometer. However, Clark et al. (1993) highlighted an important difference between an individual’s capacity and its tendency to attend to an odour cue. That is, just because a bird has the ability to detect an odour, it does not mean that it will attend to the cue when it encounters it. Our study represents a clear demonstration of a passerine attending to such a cue. Despite the evidence that novel odour does not generate a neophobic effect on its own, the role of odour in food choice should not be underestimated. Odours have been
shown to trigger hidden, unlearned colour preferences in chicks (Rowe & Guilford 1996; Jetz et al. 2001). Pyrazine odour also produces a feeding bias against conspicuous prey (Lindstro¨m et al. 2001a). Clearly the interaction of colour and odour cues is important for these birds. In taste aversion studies with domestic chicks, odour is a preferential learning cue to colour (Roper & Marples 1997b). Roper & Marples (1997b) found that almond odour was a more salient cue than green colour for avoiding quinine-flavoured water. However, vanilla did not show this same overshadowing effect, so the effect appears to be specific to certain odours. Indeed, not all combinations of novel odours and novel colours produce synergistic aversive reactions in domestic chicks (Marples & Roper 1996). The two cues we chose are traditionally associated with aposematic animals. Red is frequently used (sometimes in combination with other colours) as a warning signal (Sille´n-Tullberg 1985; Marples et al. 1994; Exnerova´ et al. 2003; Hagen et al. 2003), and pyrazines are used by a number of aposematic insects (Rothschild & Moore 1987). Perhaps a combination of these two cues elicits an exceptional response. Only further work with other novel colour and odour combinations will answer this question. The latencies of zebra finches to sample the novel colour treatment (red; 13 262 s, N Z 9) were greater than for chicks (21 s, N Z 15; Roper & Marples 1997a), by more than two orders of magnitude. There are many differences between the two groups of animals used, including species, order (Galliformes/Passeriformes), developmental strategy (precocial/altricial), diet (omnivore/granivore), age (juvenile/sexually mature) and perhaps experience (younger/older). It is unclear which of these differences are important in determining the responses of the birds. The literal definition of neophobia is ‘fear of the new’ (Barnett 1958), but this definition is not precise enough for our experimental conditions. Brigham & Sibly (1999) defined neophobia as ‘the initial avoidance of novel objects in an otherwise familiar environment’, where ‘objects’ may include food. Our experimental feeding environment was unmodified once birds began to be tested, so the presence of test food would have been truly novel only on the first presentation. The unfamiliarity of novel objects must decrease with increased exposure to them, and hence neophobia decreases with experience (Coppinger 1969, 1970; Schlenoff 1984; Jones 1987; Mastrota & Mench 1995). It is reasonable to assume that some birds are more neophobic than others, but observations of the zebra finches gave no impression that they were avoiding the area around the novel colour or doubly novel treatments. A number of birds that were not eating the novel colour or doubly novel treatments perched on the presentation cups during their trials. Clearly neophobia (both of the experimental equipment and of the coloured food) was no longer in operation (Brigham & Sibly 1999). The term dietary conservatism (Marples et al. 1998) can be used to describe the stages of incorporation once neophobia has been overcome. Neophobia and dietary conservatism can be considered separately if latency to peck at food is considered as a measure of neophobia, and latency to eat it as a measure of dietary conservatism (Marples & Kelly 1999).
KELLY & MARPLES: DIETARY CONSERVATISM AND NEOPHOBIA
Dietary conservatism may be considered a form of wariness by the birds. Although the novel foods in these trials were not dangerous to be close to (as assessed by the relatively low contact latencies), there was no immediate information available to the birds about the costs or benefits of eating the food. As the natural world contains many aposematic animals (Cott 1940; Edmunds 1974), wariness would appear to be a profitable trait, especially as certain cryptic (or weakly signalling) invertebrates prove to be defended (Codella & Raffa 1996; Krall et al. 1999; Larsson et al. 2000). It is likely that novel foods need to be sampled for some time until they can be assessed as ‘safe’ (Kalat & Rozin 1973). Such a strategy may even lead to less-experienced predators repeatedly sampling aposematic prey before ultimately rejecting them (Lindstro¨m et al. 2001b). We deliberately chose to omit any ‘unprofitable’ aspect to the novel foods from these trials, to investigate the effect of the novel cues on their own. The zebra finches showed increased neophobia (latency to peck at food) towards the coloured treatments (Fig. 1b). However, there was no further increase in neophobia shown towards the doubly novel treatment compared with the novel colour treatment. The zebra finches also showed increased dietary conservatism (latency to eat food) towards the coloured treatments (Fig. 1b). Unlike the neophobic responses of the zebra finches, the dietary conservatism ‘effect’ was much greater for the doubly novel treatment than for the novel colour treatment. This suggests it is the dietary conservatism aspect of a predator’s reaction towards multimodally novel prey that enhances that prey’s protection, rather than the predator’s neophobic responses. Such an analysis is possible only with palatable prey. If the prey were unpalatable, an aversion learning element would also need to be considered. These data represent a clear demonstration of the neophobic and dietary conservative behaviours of an animal being independently modified when confronted with novel foods. It would seem appropriate that neophobia and dietary conservatism should be considered discrete processes, as proposed by Marples & Kelly (1999). An animal’s reaction towards new food can be more fully explained when these two processes are considered individually. If we now consider the evolutionary strategy of an aposematic prey animal, that animal will potentially exploit a predator’s dietary conservatism by advertising itself multimodally. These results suggest that multimodal advertisement would not increase the neophobic response of the predator (i.e. approach), but would increase its latency to sample the prey if it were novel. Such a situation is particularly relevant to avian predators in tropical climates, where there are many potential invertebrate prey (Owen 1977). Many aposematic insects use colour and odour to advertise their unprofitability, which capitalizes on the dietary conservatism response. Indeed, perhaps all multimodal advertisement exploits this behaviour. Acknowledgments We are grateful to the staff of the BioResources Unit in Trinity College for their care of the finches and their patience over the protracted length of the study. We thank Mark
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