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Parasite-induced warning coloration: a novel form of host manipulation
Animal Behaviour 81 (2011) 417e422
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Parasite-induced warning coloration: a novel form of host manipulation Andy Fenton a, *, Lucy Magoolagan a, Zara Kennedy a, Karen A. Spencer b,1 a b
Institute of Integrative Biology, University of Liverpool Division of Ecology and Evolutionary Biology, University of Glasgow
a r t i c l e i n f o Article history: Received 6 July 2010 Initial acceptance 28 September 2010 Final acceptance 8 November 2010 Available online 15 December 2010 MS. number: 10-00469 Keywords: aposematism dietary conservatism entomopathogenic nematode extended phenotype foraging Heterorhabditis bacteriophora neophobia parasite transmission predation
There is currently considerable interest in the evolution of parasite manipulations of hosts that facilitate transmission. The classic examples of such adaptive manipulations typically concern trophically transmitted parasites that increase host conspicuousness or in other ways maximize the likelihood of their intermediate host being consumed by the parasite’s predatory definitive host, thereby completing the parasite’s life cycle. However, examples of manipulations not involving trophically transmitted parasites are rare. Here we present evidence of a novel parasite-induced change in host coloration that we suggest acts as a warning signal to deter predators from consuming infected hosts, which would result in termination of the parasite’s life cycle. When insect larvae are infected by the entomopathogenic nematode Heterorhabditis bacteriophora they become bioluminescent and change to a vivid pink-red colour. We used field trials involving wild European robins, Erithacus rubecula, and showed that infected larvae were rarely handled by these avian predators and, on the rare occasions that infected larvae were handled, they tended to be rejected. Our results suggest that the parasite-induced colour change in the host may act as a visual deterrent or warning signal to avian predators, which may be reinforced by the production of a distasteful chemical that, together, reduce the likelihood of infected prey being consumed. To our knowledge this is the first reported case of a parasite-induced host manipulation involving warning coloration, and so broadens the range of examples of this fascinating adaptive behaviour. Ó 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
The adaptive manipulation of hosts by parasites is one of the most fascinating examples of the extended phenotype (Dawkins 1999). Typically these manipulations involve the parasite altering the host’s behaviour or coloration to maximize transmission success, often by making their intermediate host more conspicuous to predators that act as the definitive host for the parasite (Moore 2002). For example, amphipods infected by the acanthocephalan Polymorphus paradoxus become much more photophilic than uninfected amphipods, making them vulnerable to predation by ducks, the definitive host of the parasite (Bethel & Holmes 1973; Moore 1984). In this case, the parasite benefits by increasing the likelihood of its intermediate host being preyed upon by the definitive host species. However, this form of manipulation only applies to trophically transmitted parasites that require at least two hosts to complete their life cycle: the intermediate host prey species and the definitive host predator species. Other parasites,
* Correspondence: A. Fenton, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, U.K. E-mail address: [email protected] (A. Fenton). 1 K. A. Spencer is at the Division of Ecology and Evolutionary Biology, Graham Kerr Building, University of Glasgow, Glasgow G12 8QQ, U.K.
however, have only one host in their life cycle and, for these species, predation of their host will be detrimental to the parasite if it cannot survive or reproduce within the predator. For these parasites, manipulation of the host to increase conspicuousness may seem unlikely to evolve. However, here we report a novel form of colour-based host manipulation caused by one parasite species, which may reduce the likelihood of predation by acting as a warning signal to deter predators from consuming them. Entomopathogenic nematodes of the genus Heterorhabditis (Nematoda: Rhabditida) are lethal obligate parasites of a range of insect species. The third-stage infective larvae (iL3s) live in the soil, where they actively search for a suitable host, typically grounddwelling larvae of coleopteran, lepidopteran or dipteran species (Kaya & Gaugler 1993). Once located, the nematodes enter the host through the mouth, spiracles or anus, and release a symbiotic bacterium, Photorhabdus luminescens. Strictly speaking, it is this bacterium that is the parasite, and the nematode is merely its vector, but both partners require the other for completion of their life cycle. The bacteria rapidly kill the host and digest its tissues, forming a nutrient-rich broth on which the nematodes feed, develop to hermaphroditic adults, and produce the next generation of nematodes, which mature within the host. The nematodes pass through several generations within a single dead insect until
0003-3472/$38.00 Ó 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2010.11.010
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resources become limiting and the host splits apart, releasing thousands of infective larvae back into the environment. Importantly, whereas dead insect larvae typically rapidly desiccate and shrivel after death, larvae infected with entomopathogenic nematodes remain turgid long after death and, as such, could remain attractive to predators; the potential host species of this parasite form a major component of the diets of many species, including birds, rodents and other insects. We established the potential for predation of infected insect larvae by feeding captive starlings, Sturnus vulgaris (N ¼ 8) with infected third-instar greater waxmoth, Galleria mellonella, larvae (A. Fenton & K. Spencer, unpublished data; birds used in this experiment were closely monitored following exposure and showed no ill effects of the feeding regime. This trial was carried out with the approval of the University of Bristol ethics committee.). However, by examining the faeces of these birds following ingestion of infected hosts, we found no nematodes survived passage through the bird’s gut. Therefore predation of infected hosts results in a dead-end for the parasites and we may expect natural selection to favour any mechanism by which the parasites can reduce the likelihood of an infected host being eaten. A striking feature of insect larvae infected with these nematodes is that they undergo a major colour change, such that within a couple of days after infection the dead insect becomes bioluminescent and changes to a bright pink colour (ffrench-Constant & Bowen 2000; ffrench-Constant et al. 2002). While this bioluminescence is transient, only occurring for a short period at the start of the infection cycle, the change in colour remains for the entire infection period. A number of studies have suggested alternative hypotheses concerning why the parasites should induce such energetically costly and vivid colour changes, primarily relating to the elimination of damaging reactive oxygen species that build up within the host’s body (ffrench-Constant et al. 2002). However, one possibility that has not yet been explored is that these colour changes act to reduce the likelihood of predation; as described above, infected larvae maintain turgidity long after death and may therefore remain viable prey to potential predators, which would terminate the parasite’s life cycle. Avian predators typically rely, at least in part, on visual cues for initial prey selection and they will often avoid novel prey because of an initial neophobic response and a more persistent avoidance of unfamiliar prey known as dietary conservatism (Marples et al. 1998; Marples & Kelly 1999; Thomas et al. 2003; Exnerova et al. 2007; Lee et al. 2009; Thomas et al. 2010). Many potential prey species exploit these behavioural responses, leading to the evolution of aposematic signals, particularly when the visual cues are reinforced by additional predator avoidance mechanisms, such as the production of distasteful chemicals (Thomas et al. 2003, 2004). In this study, we investigated how the appearance of infected hosts changes over the course of infection and, using wild avian predators, whether the induced long-term colour reduces the risk of predation. METHODS Culture of Nematode Parasites Larvae of the greater waxmoth (Livefoods Direct Ltd, Sheffield, U.K.) were used as the host species and were infected with Heterorhabditis bacteriophora nematodes, supplied by D. Clarke and S. Joyce, University of Bath. Ten petri dishes were lined with filter paper and each divided into four compartments using 87 13 mm strips of cardboard. Each compartment contained a single waxmoth larva, allowing repeated spectral measurements of each individual. All individuals underwent spectral measurement prior to any infection at day 0 (for details see next section). Waxmoth larvae in
two of the petri dishes (N ¼ 8) were killed immediately after spectral measurement by inserting a needle diagonally in front of the first thoracic segment; these larvae acted as controls, and were exposed to 250 ml of distilled water dispensed evenly over each compartment. Larvae in the remaining eight petri dishes (N ¼ 32) were infected with the nematode immediately following day 0 spectral measurements by dispensing 250 ml of a solution containing 1 nematode/ml suspended in distilled water over each compartment. All petri dishes were then placed in an incubator and maintained at 20 C throughout the experiment. Spectral Measurements We focused on the spectral colour changes induced by parasitism rather than bioluminescence, since the latter are very short lived, whereas the spectral colour changes are persistent. We measured changes in the reflectance of each waxmoth larva over time using a spectrophotometer with a DeuteriumeHalogen light source (Ocean Optics Inc., Dunedin, FL, U.S.A.). Reflectance was measured relative to the WS-1 white standard (Ocean Optics Inc.) for the wavelength range 300e700 nm. Three separate reflectance measurements were taken from each individual at the second thoracic, third abdominal and seventh abdominal segments on days 0, 3, 5 and 7 after infection. A mean reflectance was then calculated for each individual on each day. Field Trials Training and conditioning We located 16 single-occupancy European robin, Erithacus rubecula, territories within the Ness Botanic Gardens, University of Liverpool, South Wirral between February and March. Initially, birds were fed a mixture of porridge oats and sunflower oil on the ground close to the centre of their territory to encourage individuals to visit the same location to gain access to food. Food was always placed in a relatively open location (e.g. on grass next to an herbaceous border) rather than in close proximity to shaded areas, to maintain a similar light environment across all territory feeding sites. Further training sessions included the addition of mealworms (Livefoods Direct) scattered next to the porridge mixture on a green-painted wooden board (60 40 cm and 2.5 cm high), that would later be used in the experimental trials. Mealworms were used to habituate birds to the presence of larval prey items, without exposing them to the prey to be used in the experimental trials (i.e. infected or uninfected waxmoth larvae). Experimental trials We ran two separate trials. In the first (Trial 1), birds (N ¼ 10) were presented with a choice between infected prey and uninfected, but freshly killed, prey, to determine whether birds preferentially selected uninfected prey above infected. To control for the fact that infected larvae in this first trial would have been dead for different lengths of time from the uninfected control prey, potentially causing differences in both visual and olfactory cues, we conducted a second trial (Trial 2) with a separate group of robins (N ¼ 6). In this trial birds were given a choice between infected and uninfected prey that had been killed at the same time. This allowed us to separate the potential confound of birds simply choosing freshly dead prey in Trial 1. Both trials followed identical protocols, as described below. During each trial, territorial robins were presented with 10 infected and 10 uninfected waxmoth larvae randomly scattered on the same wooden board. Infected larvae were presented at either 3, 5 or 7 days after infection and uninfected controls were either freshly killed (Trial 1), to prevent them moving from the board
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during the trial to ensure that birds were not choosing prey on the basis of movement, or killed at the same time as the infected prey died (Trial 2). The order in which birds experienced infected larvae at each infection stage (day 3, 5 or 7) was randomized across territories within a trial, ensuring that all six possible combinations of infection order were included, and that all birds were exposed to larvae of each stage of infection at some point throughout the trial. Presentations lasted 30 min and, during this time, we recorded the order in which robins chose their prey and the outcome of each encounter (prey eaten or discarded). From this we were able to calculate the number of infected or control prey eaten within the trial. Entomopathogenic nematodes are naturally abundant throughout the U.K. (Hominick & Briscoe 1990a, b) and so wild birds are likely to be exposed to infected insect larvae on a regular basis. Furthermore, these nematodes are known not to be harmful to vertebrates (Kaya & Gaugler 1993); this safety to birds and mammals is one of the factors that make these nematodes attractive for use as biological control agents of insect pests in domestic gardens. All birds involved were monitored closely throughout each experiment; every bird appeared at every feeding session and they were all observed in their territories on the days after the experiments had ended. Statistical Analyses Principal component analyses (PCA) were used to transform the spectral reflectance data (reflectance at 2.6 nm intervals) into orthogonal variables. Data reduction of this kind is standard procedure when analysing spectral reflectance data (Hunt et al. 1998). Linear mixed models (SPSS version 14, SPSS Inc., Chicago, IL, U.S.A.) were then used to compare the reflectance of infected and control larvae and determine the changes in reflectance with infection time. A two-tailed repeated measures model, with larval identity (ID) number entered as a random factor, and infection stage (3, 5 or 7 days) and infection status (infected or control) entered as fixed factors was run for each principal component. Field trial data were analysed using the two-tailed nonparametric Wilcoxon signed-ranks test for paired data (infected versus control larvae within a trial). RESULTS Spectral Reflectance Prior to parasitic infection, waxworm larvae exhibited reflectance across all wavelengths between 300 and 700 nm, with a small
Field Trials Robins were significantly less likely to eat infected caterpillars than controls, regardless of whether the controls were freshly killed (Trial 1) or had been dead for the same duration as the infected ones (Trial 2), and regardless of stage of infection (Trial 1: day 3: W ¼ 43, N ¼ 9, P ¼ 0.011; day 5: W ¼ 55, N ¼ 10, P ¼ 0.002; day 7: W ¼ 55, N ¼ 10, P ¼ 0.002; Fig. 3a; Trial 2: day 3: W ¼ 21, N ¼ 6, P ¼ 0.031; day 5: W ¼ 21, N ¼ 6, P ¼ 0.031; day 7: W ¼ 21, N ¼ 6, P ¼ 0.031; Fig. 4a). Indeed, by 7 days after infection no infected larvae in Trial 1, and only one in Trial 2, were eaten. In addition, birds were more likely to handle uninfected than infected larvae at days 5 and 7 after infection, although the preference for uninfected larvae at day 3 after infection for Trial 1 was not statistically significant (Trial 1: day 3: W ¼ 40, N ¼ 10, P ¼ 0.23; day 5: W ¼ 55, N ¼ 10, P ¼ 0.002; day 7: W ¼ 55, N ¼ 10, P ¼ 0.002; Fig. 3b; Trial 2: day 3: W ¼ 21, N ¼ 6, P ¼ 0.031; day 5: W ¼ 21, N ¼ 6, P ¼ 0.031; day 7: W ¼ 21, N ¼ 6, P ¼ 0.031; Fig. 4b). At 7 days after infection only one infected larva in Trial 1 was handled, and that was rejected; in Trial 2 at 7 days after infection, two larvae were handled, and one of them was rejected.
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peak within the ultraviolet spectrum (300e400 nm), and a more pronounced peak at 600e700 nm (Fig. 1a). As the time after infection increased from day 0 to 7, infected larvae showed a reduction in short wavelength reflectance and an increase in reflectance between 600 and 700 nm (Fig. 1aec). Uninfected larvae measured at the same stages after death showed an overall reduction at all wavelengths (Fig. 1aec). Principal component analysis revealed that three principal components (PC1, PC2 and PC3) accounted for 99% of the variation seen in the spectra data, and so we restricted our analyses to these components. PC1 invariably accounts for most of the betweenspectra variation (typically more than 85%) and is typically associated with variation in mean reflectance (brightness), whereas PC2 and PC3 are normally associated with changes in hue and saturation, and therefore relate to changes in colour (Endler & Thery 1996; Hunt et al. 1998). Here, PC1 accounted for 83% of the variation in our data. The magnitude of PC1 stayed constant across wavelengths (Fig. 2), except within the 600e700 nm range, where it decreased. PC2 showed an increase with wavelength, peaking at 700 nm and PC3 showed two peaks, one within the ultraviolet spectrum (300e400 nm) and another at 700 nm (Fig. 2). We found a significant interaction between infection status and time for all three principal components (PC1: F2,76 ¼ 434.96, P < 0.0001; PC2: F2,76 ¼ 6.96, P < 0.002; PC3: F2,76 ¼ 58.88, P < 0.0001), confirming the changes described by the mixed model results.
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Wavelength (nm) Figure 1. Reflectance spectra of control (black line) and infected (red line) waxmoth larvae over time: (a) prior to infection (day 0); (b) 3 days after infection; and (c) 7 days after infection. Data are presented as mean (solid line) SE of the mean (dotted line).
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There was also an effect of infection duration on the number of birds that first approached an infected larva at the onset of the experiment. For example, in Trial 1, birds presented with larvae early in the infection chose their first prey item seemingly at random, with 50% and 40% of birds initially choosing an infected prey when presented with larvae 3 days and 5 days after infection, respectively. Conversely, all birds presented with larvae at 7 days after infection chose uninfected prey as their first item. Note that since our experimental design was counterbalanced with respect to stage of infection, such that the order in which birds were exposed to infected larvae was randomized with respect to the time after infection of those larvae, this result does not reflect a learned response to avoid infected prey, but rather reflects an inherent repulsion of infected larvae 7 days after infection. To determine predator behaviour following an encounter with an infected larva, we calculated the proportion of prey items taken
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that were infected after a bird handled its first infected larva. In Trial 1, only one bird presented with larvae at 7 days after infection picked up any infected larvae and hence this infection stage was excluded from the analysis. However, at 5 days after infection the choice of prey items was not random, with birds then being significantly less likely to handle an infected larva if they had previously encountered one (one-sample Wilcoxon signed-ranks test: W ¼ 28, N ¼ 6, P ¼ 0.03, based on null hypothesis of 0.5; Fig. 3c). However, there was no significant effect of infection status at 3 days (W ¼ 24, N ¼ 7, P ¼ 0.11); birds were just as likely to pick up infected or uninfected larvae after an initial encounter with an infected one (Fig. 3c). In Trial 2, numbers were too few to conduct any meaningful analyses; at 3 days after infection two of three birds handled infected larvae after handling their first infected larva, whereas for days 5 and 7 after infection only two birds handled an infected larva, with neither handling a subsequent one.
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Days after infection Figure 3. Foraging behaviour of free-living European robins in relation to infection stage in Trial 1, in which uninfected control prey were freshly killed. (a) Proportion of eaten prey that were uninfected or infected after 3, 5 and 7 days of infection. (b) Proportion of handled prey that were uninfected or infected at each infection stage. (c) Proportion of consumed prey that were infected, following initial encounter with an infected prey. Grey: uninfected larvae; white: infected larvae. Data are shown as box plots (medians and interquartile range; whiskers show the data range and asterisks outliers). Number of birds is given at the top of the box plots.
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Days after infection Figure 4. Foraging behaviour of free-living European robins in relation to infection stage in Trial 2, in which uninfected control prey were killed at the same time as infected larvae. (a) Proportion of eaten prey that were uninfected or infected after 3, 5 and 7 days of infection. (b) Proportion of handled prey that were uninfected or infected at each infection stage. Grey: uninfected larvae; white: infected larvae. Data are shown as box plots (medians and interquartile range; whiskers show the data range and asterisks outliers). Number of birds is given at the top of the box plots.
DISCUSSION Our results suggest that the parasite-induced coloration of infected larvae may act to deter avian predators from consuming the infected host. Not only did the predators show an overall preference for uninfected larvae throughout the trials (Figs 3, 4), but infection status also affected the identity of the first prey item approached by the predators. The fact that these results were found with uninfected prey that had died at the same time as the infected prey (Trial 2) suggests that the birds were not preferentially selecting the ‘freshest’ prey, but were showing a genuine aversion to infected prey. There was a general trend in both trials for the magnitude of this effect to increase with the duration of infection (e.g. Figs 3a, 4a), and this reflected the increasing differences in colour between infected and uninfected larvae. For example, at 3 days after infection, infected (Fig. 1b) and uninfected (Fig. 1a) larvae appeared similar in coloration, and there was little difference in the predators’ tendency to approach either type of larva. By day 5 after infection, however, infected larvae had a reduced ultraviolet signal (i.e. between 300 and 400 nm) and increased reddening of the spectrum (i.e. at wavelengths over 600 nm) compared to uninfected larvae (Fig. 1b), and this corresponded with a clear tendency for birds to handle (and consume) uninfected larvae. This suggests that the initial selection of prey is strongly influenced by visual cues, and so the birds showed a greater aversion to infected larvae as the difference in appearance between infected and uninfected larvae grew. If the induced colour change does act to deter predators, such a signal could work in a variety of ways. One possibility is that by altering the appearance of the host, the parasite is able to take advantage of the neophobic responses of avian predators that result in the avoidance of novel prey items, particularly in the presence of familiar prey (Shettleworth 1972; Marples et al. 1998; Marples & Kelly 1999). In this case, infected hosts would be ignored simply because they looked different from the birds’ usual prey. However, neophobia is a relatively short-lived phenomenon, which is unlikely to remain for the 2-week life cycle of the parasite used in this study (Marples et al. 1998; Marples & Kelly 1999). While neophobia describes short-term avoidance behaviour, an analogous but longer-term process called dietary conservatism may provide a mechanism whereby parasite-induced colour change could evolve as a warning signal, by placing these novel colour morphs at
a selective advantage (Marples & Kelly 1999; Thomas et al. 2003; Exnerova et al. 2007; Lee et al. 2009; Thomas et al. 2010). Our results therefore suggest that the induced colour changes act as a genuine warning signal, advertising the prey’s distastefulness to the predator. However, such a signal would be effective only if it was honest; in other words, predator avoidance would occur only if there was an additional mechanism reinforcing the signal (Sherratt 2002; Speed & Ruxton 2005). Observation of the feeding behaviour of the robins showed that on a number of occasions birds would approach infected larvae and peck at them, but then reject them and select uninfected larvae. Indeed, in the majority of trials, once a bird had handled an infected larva, regardless of whether it had consumed it or merely picked it up and discarded it, it was more likely to choose uninfected larvae (e.g. Fig. 3c). These observations suggest that the infected insects contained some distasteful substance that deterred consumption, and that the induced colour changes may act as an honest signal to the predators, which is reinforced by their unpalatable taste. At this stage we cannot rule out nonvisual signals that the birds may use to distinguish between infected and uninfected prey items. In particular, the birds may be using odour cues. However, in our experimental design infected and uninfected larvae were arranged randomly on a board together and it may be unlikely that the birds would have sufficient resolution in their odour detection to discriminate between infected and uninfected larvae in close proximity to each other. Therefore the choice between infected and uninfected prey may be based mainly on visual cues; our analyses show that the induced coloration change of infected larvae is very large compared to uninfected larvae. Previous studies have suggested that passerine birds are primarily visual predators, although they are able to use a combination of senses for final prey choice in the field (Heppner 1965; Sillman 1973; Montgomerie & Weatherhead 1997; Forsman & Appelqvist 1998). A noticeable feature of avian vision is the importance of reflectance in the ultraviolet wavelengths, and the use of these wavelengths in foraging decisions (Church et al. 2001; Kevan et al. 2001; Honkavaara et al. 2002). Since one of the consequences of the parasite-induced colour change is the reduction in reflectance in this range of the spectrum, the infected larvae will appear very distinct from uninfected larvae. Furthermore, red is a common aposematic colour and several studies have shown that this colour is one of the most effective in deterring initial attacks by avian
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predators (Guilford 1990; Gamberale & Tullberg 1996; Lindstrom 1999; Exnerova et al. 2006, 2007). Finally, it is worth considering whether the bioluminescence caused by these infections acts as an additional visual signal to deter predation, particularly early in the infection process when the spectral changes are relatively mild. However, alternative hypotheses have been suggested for this bioluminescence, primarily relating to the bacteria’s metabolic requirements (ffrench-Constant et al. 2002). Owing to the transient nature of the bioluminescence, we focused on the longer-lasting spectral changes following infection, so it remains to be seen whether the bioluminescence also plays a role as an aposematic signal. However, regardless of the potential role of bioluminescence, the observed reduction in ultraviolet and the increased reddening of the host induced by the presence of these parasites will greatly alter the appearance of infected larvae, making them appear particularly novel to birds, possibly exploiting both their neophobia and dietary conservatism (Marples et al. 1998; Marples & Kelly 1999; Thomas et al. 2003; Exnerova et al. 2007). Any distasteful chemical that the predator then encounters on handling the infected larvae will further reinforce this signal, reducing the likelihood of subsequent predation. Future experiments may help to clarify the relationship between host coloration and other potential cues to which the predators may respond. In particular, the experiment could be repeated using infected and uninfected prey dyed the same colour, thereby removing the visual signal. If the two prey types were still selected differentially, then this would suggest that the predators were using some olfactory cue. However, dyeing the larvae would also change their texture and odour, potentially masking any olfactory cues. Given the magnitude of the observed colour changes and the fact that birds are mainly visual predators, it seems most likely that they are primarily selecting their prey on the basis of visual appearance. We have presented compelling evidence of a novel form of parasite-induced host manipulation, causing changes in host colour as a means of deterring predators from consuming infected hosts. This differs from the traditional view of host manipulation, where parasites cause colour changes that increase conspicuousness to favour consumption by their predatory definitive host (Moore 2002). However, both examples show how parasites can exploit predator behaviour, resulting in changes to prey choice and diet composition at the individual level and, potentially, resource competition and abundance at the population level. Acknowledgments Thanks go to Mike Speed, Sarah Clarke and Hannah Rowland for help with project design, field work and use of equipment. We also thank Arthur Goldsmith and Julia Carter for use of their facilities, and Ness Gardens for permission to use their facilities. The field experiments were undertaken following liaison with English Nature. References Bethel, W. M. & Holmes, A. J. 1973. Altered evasive behavior and responses to light in amphipods harboring acanthocephalan cystacanths. Journal of Parasitology, 59, 945e956.
Church, S. C., Merrison, A. S. L. & Chamberlain, T. M. M. 2001. Avian ultraviolet vision and frequency-dependent seed preferences. Journal of Experimental Biology, 204, 2491e2498. Dawkins, R. 1999. The Extended Phenotype. Oxford: Oxford University Press. Endler, J. A. & Thery, M. 1996. Interacting effects of lek placement, display behavior, ambient light, and color patterns in three neotropical forest-dwelling birds. American Naturalist, 148, 421e452. Exnerova, A., Svadova, K., Stys, P., Barcalova, S., Landova, E., Prokopova, M., Fuchs, R. & Socha, R. 2006. Importance of colour in the reaction of passerine predators to aposematic prey: experiments with mutants of Pyrrhocoris apterus (Heteroptera). Biological Journal of the Linnean Society, 88, 143e153. Exnerova, A., Stys, P., Fucikova, E., Vesela, S., Svadova, K., Prokopova, M., Jarosik, V., Fuchs, R. & Landova, E. 2007. Avoidance of aposematic prey in European tits (Paridae): learned or innate? Behavioral Ecology, 18, 148e156. ffrench-Constant, R. H. & Bowen, D. J. 2000. Novel insecticidal toxins from nematode-symbiotic bacteria. Cellular and Molecular Life Sciences, 57, 828e833. ffrench-Constant, R., Waterfield, N., Daborn, P., Joyce, S., Bennett, H., Au, C., Dowling, A., Boundy, S., Reynolds, S. & Clarke, D. 2002. Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen. FEMS Microbiology Reviews, 759, 1e24. Forsman, A. & Appelqvist, S. 1998. Visual predators impose correlational selection on prey color pattern and behavior. Behavioral Ecology, 9, 409e413. Gamberale, G. & Tullberg, B. S. 1996. Evidence for a peak-shift in predator generalization among aposematic prey. Proceedings of the Royal Society B, 263, 1329e1334. Guilford, T. 1990. The secrets of aposematism: unlearned responses to specific colors and patterns. Trends in Ecology & Evolution, 5, 323e348. Heppner, F. 1965. Sensory mechanisms and environmental cues used by the American robin in locating earthworms. Condor, 67, 247e256. Hominick, W. M. & Briscoe, B. R. 1990a. Occurrence of entomopathogenic nematodes (Rhabditida, Steinernematidae and Heterorhabditidae) in British soils. Parasitology, 100, 295e302. Hominick, W. M. & Briscoe, B. R. 1990b. Survey of 15 Sites over 28 months for entomopathogenic nematodes (Rhabditida, Steinernematidae). Parasitology, 100, 289e294. Honkavaara, J., Koivula, M., Korpimaki, E., Siitari, H. & Viitala, J. 2002. Ultraviolet vision and foraging in terrestrial vertebrates. Oikos, 98, 505e511. Hunt, S., Bennett, A. T. D., Cuthill, I. C. & Griffiths, R. 1998. Blue tits are ultraviolet tits. Proceedings of the Royal Society B, 265, 451e455. Kaya, H. K. & Gaugler, R. 1993. Entomopathogenic nematodes. Annual Review of Entomology, 38, 181e206. Kevan, P. G., Chittka, L. & Dyer, A. G. 2001. Limits to the salience of ultraviolet: lessons from colour vision in bees and birds. Journal of Experimental Biology, 204, 2571e2580. Lee, T. J., Marples, N. M. & Speed, M. P. 2009. Can dietary conservatism explain the primary evolution of aposematism? Animal Behaviour, 79, 63e74. Lindstrom, L. 1999. Experimental approaches to studying the initial evolution of conspicuous aposematic signalling. Evolutionary Ecology, 13, 605e618. Marples, N. M. & Kelly, D. J. 1999. Neophobia and dietary conservatism: two distinct processes? Evolutionary Ecology, 13, 641e653. Marples, N. M., Roper, T. J. & Harper, D. G. C. 1998. Responses of wild birds to novel prey: evidence of dietary conservatism. Oikos, 83, 161e165. Montgomerie, R. & Weatherhead, P. J. 1997. How robins find worms. Animal Behaviour, 54, 143e151. Moore, J. 1984. Altered behavioral responses in intermediate hosts: an acanthocephalan parasite strategy. American Naturalist, 123, 572e577. Moore, J. 2002. Parasites and the Behavior of Animals. New York: Oxford University Press. Sherratt, T. N. 2002. The coevolution of warning signals. Proceedings of the Royal Society B, 269, 741e746. Shettleworth, S. J. 1972. Role of novelty in learned avoidance of unpalatable prey by domestic chicks (Gallus gallus). Animal Behaviour, 20, 29e35. Sillman, A. J. 1973. Avian vision. In: Avian Biology Vol. 3 (Ed. by D. S. Farner & J. R. King), pp. 349e387. New York: Academic Press. Speed, M. P. & Ruxton, G. D. 2005. Aposematism: what should our starting point be? Proceedings of the Royal Society B, 272, 431e438. 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, 458e466. Thomas, R. J., Bartlett, L. A., 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, 285e294. Thomas, R. J., King, T. A., Forshaw, H. E., Marples, N. M., Speed, M. P. & Cable, J. 2010. The response of fish to novel prey: evidence that dietary conservatism is not restricted to birds. Behavioral Ecology, 21, 669e675.
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Parasite-induced warning coloration: a novel form of host manipulation Andy Fenton a, *, Lucy Magoolagan a, Zara Kennedy a, Karen A. Spencer b,1 a b
Institute of Integrative Biology, University of Liverpool Division of Ecology and Evolutionary Biology, University of Glasgow
a r t i c l e i n f o Article history: Received 6 July 2010 Initial acceptance 28 September 2010 Final acceptance 8 November 2010 Available online 15 December 2010 MS. number: 10-00469 Keywords: aposematism dietary conservatism entomopathogenic nematode extended phenotype foraging Heterorhabditis bacteriophora neophobia parasite transmission predation
There is currently considerable interest in the evolution of parasite manipulations of hosts that facilitate transmission. The classic examples of such adaptive manipulations typically concern trophically transmitted parasites that increase host conspicuousness or in other ways maximize the likelihood of their intermediate host being consumed by the parasite’s predatory definitive host, thereby completing the parasite’s life cycle. However, examples of manipulations not involving trophically transmitted parasites are rare. Here we present evidence of a novel parasite-induced change in host coloration that we suggest acts as a warning signal to deter predators from consuming infected hosts, which would result in termination of the parasite’s life cycle. When insect larvae are infected by the entomopathogenic nematode Heterorhabditis bacteriophora they become bioluminescent and change to a vivid pink-red colour. We used field trials involving wild European robins, Erithacus rubecula, and showed that infected larvae were rarely handled by these avian predators and, on the rare occasions that infected larvae were handled, they tended to be rejected. Our results suggest that the parasite-induced colour change in the host may act as a visual deterrent or warning signal to avian predators, which may be reinforced by the production of a distasteful chemical that, together, reduce the likelihood of infected prey being consumed. To our knowledge this is the first reported case of a parasite-induced host manipulation involving warning coloration, and so broadens the range of examples of this fascinating adaptive behaviour. Ó 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
The adaptive manipulation of hosts by parasites is one of the most fascinating examples of the extended phenotype (Dawkins 1999). Typically these manipulations involve the parasite altering the host’s behaviour or coloration to maximize transmission success, often by making their intermediate host more conspicuous to predators that act as the definitive host for the parasite (Moore 2002). For example, amphipods infected by the acanthocephalan Polymorphus paradoxus become much more photophilic than uninfected amphipods, making them vulnerable to predation by ducks, the definitive host of the parasite (Bethel & Holmes 1973; Moore 1984). In this case, the parasite benefits by increasing the likelihood of its intermediate host being preyed upon by the definitive host species. However, this form of manipulation only applies to trophically transmitted parasites that require at least two hosts to complete their life cycle: the intermediate host prey species and the definitive host predator species. Other parasites,
* Correspondence: A. Fenton, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, U.K. E-mail address: [email protected] (A. Fenton). 1 K. A. Spencer is at the Division of Ecology and Evolutionary Biology, Graham Kerr Building, University of Glasgow, Glasgow G12 8QQ, U.K.
however, have only one host in their life cycle and, for these species, predation of their host will be detrimental to the parasite if it cannot survive or reproduce within the predator. For these parasites, manipulation of the host to increase conspicuousness may seem unlikely to evolve. However, here we report a novel form of colour-based host manipulation caused by one parasite species, which may reduce the likelihood of predation by acting as a warning signal to deter predators from consuming them. Entomopathogenic nematodes of the genus Heterorhabditis (Nematoda: Rhabditida) are lethal obligate parasites of a range of insect species. The third-stage infective larvae (iL3s) live in the soil, where they actively search for a suitable host, typically grounddwelling larvae of coleopteran, lepidopteran or dipteran species (Kaya & Gaugler 1993). Once located, the nematodes enter the host through the mouth, spiracles or anus, and release a symbiotic bacterium, Photorhabdus luminescens. Strictly speaking, it is this bacterium that is the parasite, and the nematode is merely its vector, but both partners require the other for completion of their life cycle. The bacteria rapidly kill the host and digest its tissues, forming a nutrient-rich broth on which the nematodes feed, develop to hermaphroditic adults, and produce the next generation of nematodes, which mature within the host. The nematodes pass through several generations within a single dead insect until
0003-3472/$38.00 Ó 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2010.11.010
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resources become limiting and the host splits apart, releasing thousands of infective larvae back into the environment. Importantly, whereas dead insect larvae typically rapidly desiccate and shrivel after death, larvae infected with entomopathogenic nematodes remain turgid long after death and, as such, could remain attractive to predators; the potential host species of this parasite form a major component of the diets of many species, including birds, rodents and other insects. We established the potential for predation of infected insect larvae by feeding captive starlings, Sturnus vulgaris (N ¼ 8) with infected third-instar greater waxmoth, Galleria mellonella, larvae (A. Fenton & K. Spencer, unpublished data; birds used in this experiment were closely monitored following exposure and showed no ill effects of the feeding regime. This trial was carried out with the approval of the University of Bristol ethics committee.). However, by examining the faeces of these birds following ingestion of infected hosts, we found no nematodes survived passage through the bird’s gut. Therefore predation of infected hosts results in a dead-end for the parasites and we may expect natural selection to favour any mechanism by which the parasites can reduce the likelihood of an infected host being eaten. A striking feature of insect larvae infected with these nematodes is that they undergo a major colour change, such that within a couple of days after infection the dead insect becomes bioluminescent and changes to a bright pink colour (ffrench-Constant & Bowen 2000; ffrench-Constant et al. 2002). While this bioluminescence is transient, only occurring for a short period at the start of the infection cycle, the change in colour remains for the entire infection period. A number of studies have suggested alternative hypotheses concerning why the parasites should induce such energetically costly and vivid colour changes, primarily relating to the elimination of damaging reactive oxygen species that build up within the host’s body (ffrench-Constant et al. 2002). However, one possibility that has not yet been explored is that these colour changes act to reduce the likelihood of predation; as described above, infected larvae maintain turgidity long after death and may therefore remain viable prey to potential predators, which would terminate the parasite’s life cycle. Avian predators typically rely, at least in part, on visual cues for initial prey selection and they will often avoid novel prey because of an initial neophobic response and a more persistent avoidance of unfamiliar prey known as dietary conservatism (Marples et al. 1998; Marples & Kelly 1999; Thomas et al. 2003; Exnerova et al. 2007; Lee et al. 2009; Thomas et al. 2010). Many potential prey species exploit these behavioural responses, leading to the evolution of aposematic signals, particularly when the visual cues are reinforced by additional predator avoidance mechanisms, such as the production of distasteful chemicals (Thomas et al. 2003, 2004). In this study, we investigated how the appearance of infected hosts changes over the course of infection and, using wild avian predators, whether the induced long-term colour reduces the risk of predation. METHODS Culture of Nematode Parasites Larvae of the greater waxmoth (Livefoods Direct Ltd, Sheffield, U.K.) were used as the host species and were infected with Heterorhabditis bacteriophora nematodes, supplied by D. Clarke and S. Joyce, University of Bath. Ten petri dishes were lined with filter paper and each divided into four compartments using 87 13 mm strips of cardboard. Each compartment contained a single waxmoth larva, allowing repeated spectral measurements of each individual. All individuals underwent spectral measurement prior to any infection at day 0 (for details see next section). Waxmoth larvae in
two of the petri dishes (N ¼ 8) were killed immediately after spectral measurement by inserting a needle diagonally in front of the first thoracic segment; these larvae acted as controls, and were exposed to 250 ml of distilled water dispensed evenly over each compartment. Larvae in the remaining eight petri dishes (N ¼ 32) were infected with the nematode immediately following day 0 spectral measurements by dispensing 250 ml of a solution containing 1 nematode/ml suspended in distilled water over each compartment. All petri dishes were then placed in an incubator and maintained at 20 C throughout the experiment. Spectral Measurements We focused on the spectral colour changes induced by parasitism rather than bioluminescence, since the latter are very short lived, whereas the spectral colour changes are persistent. We measured changes in the reflectance of each waxmoth larva over time using a spectrophotometer with a DeuteriumeHalogen light source (Ocean Optics Inc., Dunedin, FL, U.S.A.). Reflectance was measured relative to the WS-1 white standard (Ocean Optics Inc.) for the wavelength range 300e700 nm. Three separate reflectance measurements were taken from each individual at the second thoracic, third abdominal and seventh abdominal segments on days 0, 3, 5 and 7 after infection. A mean reflectance was then calculated for each individual on each day. Field Trials Training and conditioning We located 16 single-occupancy European robin, Erithacus rubecula, territories within the Ness Botanic Gardens, University of Liverpool, South Wirral between February and March. Initially, birds were fed a mixture of porridge oats and sunflower oil on the ground close to the centre of their territory to encourage individuals to visit the same location to gain access to food. Food was always placed in a relatively open location (e.g. on grass next to an herbaceous border) rather than in close proximity to shaded areas, to maintain a similar light environment across all territory feeding sites. Further training sessions included the addition of mealworms (Livefoods Direct) scattered next to the porridge mixture on a green-painted wooden board (60 40 cm and 2.5 cm high), that would later be used in the experimental trials. Mealworms were used to habituate birds to the presence of larval prey items, without exposing them to the prey to be used in the experimental trials (i.e. infected or uninfected waxmoth larvae). Experimental trials We ran two separate trials. In the first (Trial 1), birds (N ¼ 10) were presented with a choice between infected prey and uninfected, but freshly killed, prey, to determine whether birds preferentially selected uninfected prey above infected. To control for the fact that infected larvae in this first trial would have been dead for different lengths of time from the uninfected control prey, potentially causing differences in both visual and olfactory cues, we conducted a second trial (Trial 2) with a separate group of robins (N ¼ 6). In this trial birds were given a choice between infected and uninfected prey that had been killed at the same time. This allowed us to separate the potential confound of birds simply choosing freshly dead prey in Trial 1. Both trials followed identical protocols, as described below. During each trial, territorial robins were presented with 10 infected and 10 uninfected waxmoth larvae randomly scattered on the same wooden board. Infected larvae were presented at either 3, 5 or 7 days after infection and uninfected controls were either freshly killed (Trial 1), to prevent them moving from the board
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during the trial to ensure that birds were not choosing prey on the basis of movement, or killed at the same time as the infected prey died (Trial 2). The order in which birds experienced infected larvae at each infection stage (day 3, 5 or 7) was randomized across territories within a trial, ensuring that all six possible combinations of infection order were included, and that all birds were exposed to larvae of each stage of infection at some point throughout the trial. Presentations lasted 30 min and, during this time, we recorded the order in which robins chose their prey and the outcome of each encounter (prey eaten or discarded). From this we were able to calculate the number of infected or control prey eaten within the trial. Entomopathogenic nematodes are naturally abundant throughout the U.K. (Hominick & Briscoe 1990a, b) and so wild birds are likely to be exposed to infected insect larvae on a regular basis. Furthermore, these nematodes are known not to be harmful to vertebrates (Kaya & Gaugler 1993); this safety to birds and mammals is one of the factors that make these nematodes attractive for use as biological control agents of insect pests in domestic gardens. All birds involved were monitored closely throughout each experiment; every bird appeared at every feeding session and they were all observed in their territories on the days after the experiments had ended. Statistical Analyses Principal component analyses (PCA) were used to transform the spectral reflectance data (reflectance at 2.6 nm intervals) into orthogonal variables. Data reduction of this kind is standard procedure when analysing spectral reflectance data (Hunt et al. 1998). Linear mixed models (SPSS version 14, SPSS Inc., Chicago, IL, U.S.A.) were then used to compare the reflectance of infected and control larvae and determine the changes in reflectance with infection time. A two-tailed repeated measures model, with larval identity (ID) number entered as a random factor, and infection stage (3, 5 or 7 days) and infection status (infected or control) entered as fixed factors was run for each principal component. Field trial data were analysed using the two-tailed nonparametric Wilcoxon signed-ranks test for paired data (infected versus control larvae within a trial). RESULTS Spectral Reflectance Prior to parasitic infection, waxworm larvae exhibited reflectance across all wavelengths between 300 and 700 nm, with a small
Field Trials Robins were significantly less likely to eat infected caterpillars than controls, regardless of whether the controls were freshly killed (Trial 1) or had been dead for the same duration as the infected ones (Trial 2), and regardless of stage of infection (Trial 1: day 3: W ¼ 43, N ¼ 9, P ¼ 0.011; day 5: W ¼ 55, N ¼ 10, P ¼ 0.002; day 7: W ¼ 55, N ¼ 10, P ¼ 0.002; Fig. 3a; Trial 2: day 3: W ¼ 21, N ¼ 6, P ¼ 0.031; day 5: W ¼ 21, N ¼ 6, P ¼ 0.031; day 7: W ¼ 21, N ¼ 6, P ¼ 0.031; Fig. 4a). Indeed, by 7 days after infection no infected larvae in Trial 1, and only one in Trial 2, were eaten. In addition, birds were more likely to handle uninfected than infected larvae at days 5 and 7 after infection, although the preference for uninfected larvae at day 3 after infection for Trial 1 was not statistically significant (Trial 1: day 3: W ¼ 40, N ¼ 10, P ¼ 0.23; day 5: W ¼ 55, N ¼ 10, P ¼ 0.002; day 7: W ¼ 55, N ¼ 10, P ¼ 0.002; Fig. 3b; Trial 2: day 3: W ¼ 21, N ¼ 6, P ¼ 0.031; day 5: W ¼ 21, N ¼ 6, P ¼ 0.031; day 7: W ¼ 21, N ¼ 6, P ¼ 0.031; Fig. 4b). At 7 days after infection only one infected larva in Trial 1 was handled, and that was rejected; in Trial 2 at 7 days after infection, two larvae were handled, and one of them was rejected.
40 (b)
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peak within the ultraviolet spectrum (300e400 nm), and a more pronounced peak at 600e700 nm (Fig. 1a). As the time after infection increased from day 0 to 7, infected larvae showed a reduction in short wavelength reflectance and an increase in reflectance between 600 and 700 nm (Fig. 1aec). Uninfected larvae measured at the same stages after death showed an overall reduction at all wavelengths (Fig. 1aec). Principal component analysis revealed that three principal components (PC1, PC2 and PC3) accounted for 99% of the variation seen in the spectra data, and so we restricted our analyses to these components. PC1 invariably accounts for most of the betweenspectra variation (typically more than 85%) and is typically associated with variation in mean reflectance (brightness), whereas PC2 and PC3 are normally associated with changes in hue and saturation, and therefore relate to changes in colour (Endler & Thery 1996; Hunt et al. 1998). Here, PC1 accounted for 83% of the variation in our data. The magnitude of PC1 stayed constant across wavelengths (Fig. 2), except within the 600e700 nm range, where it decreased. PC2 showed an increase with wavelength, peaking at 700 nm and PC3 showed two peaks, one within the ultraviolet spectrum (300e400 nm) and another at 700 nm (Fig. 2). We found a significant interaction between infection status and time for all three principal components (PC1: F2,76 ¼ 434.96, P < 0.0001; PC2: F2,76 ¼ 6.96, P < 0.002; PC3: F2,76 ¼ 58.88, P < 0.0001), confirming the changes described by the mixed model results.
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Wavelength (nm) Figure 1. Reflectance spectra of control (black line) and infected (red line) waxmoth larvae over time: (a) prior to infection (day 0); (b) 3 days after infection; and (c) 7 days after infection. Data are presented as mean (solid line) SE of the mean (dotted line).
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There was also an effect of infection duration on the number of birds that first approached an infected larva at the onset of the experiment. For example, in Trial 1, birds presented with larvae early in the infection chose their first prey item seemingly at random, with 50% and 40% of birds initially choosing an infected prey when presented with larvae 3 days and 5 days after infection, respectively. Conversely, all birds presented with larvae at 7 days after infection chose uninfected prey as their first item. Note that since our experimental design was counterbalanced with respect to stage of infection, such that the order in which birds were exposed to infected larvae was randomized with respect to the time after infection of those larvae, this result does not reflect a learned response to avoid infected prey, but rather reflects an inherent repulsion of infected larvae 7 days after infection. To determine predator behaviour following an encounter with an infected larva, we calculated the proportion of prey items taken
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that were infected after a bird handled its first infected larva. In Trial 1, only one bird presented with larvae at 7 days after infection picked up any infected larvae and hence this infection stage was excluded from the analysis. However, at 5 days after infection the choice of prey items was not random, with birds then being significantly less likely to handle an infected larva if they had previously encountered one (one-sample Wilcoxon signed-ranks test: W ¼ 28, N ¼ 6, P ¼ 0.03, based on null hypothesis of 0.5; Fig. 3c). However, there was no significant effect of infection status at 3 days (W ¼ 24, N ¼ 7, P ¼ 0.11); birds were just as likely to pick up infected or uninfected larvae after an initial encounter with an infected one (Fig. 3c). In Trial 2, numbers were too few to conduct any meaningful analyses; at 3 days after infection two of three birds handled infected larvae after handling their first infected larva, whereas for days 5 and 7 after infection only two birds handled an infected larva, with neither handling a subsequent one.
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Days after infection Figure 3. Foraging behaviour of free-living European robins in relation to infection stage in Trial 1, in which uninfected control prey were freshly killed. (a) Proportion of eaten prey that were uninfected or infected after 3, 5 and 7 days of infection. (b) Proportion of handled prey that were uninfected or infected at each infection stage. (c) Proportion of consumed prey that were infected, following initial encounter with an infected prey. Grey: uninfected larvae; white: infected larvae. Data are shown as box plots (medians and interquartile range; whiskers show the data range and asterisks outliers). Number of birds is given at the top of the box plots.
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Days after infection Figure 4. Foraging behaviour of free-living European robins in relation to infection stage in Trial 2, in which uninfected control prey were killed at the same time as infected larvae. (a) Proportion of eaten prey that were uninfected or infected after 3, 5 and 7 days of infection. (b) Proportion of handled prey that were uninfected or infected at each infection stage. Grey: uninfected larvae; white: infected larvae. Data are shown as box plots (medians and interquartile range; whiskers show the data range and asterisks outliers). Number of birds is given at the top of the box plots.
DISCUSSION Our results suggest that the parasite-induced coloration of infected larvae may act to deter avian predators from consuming the infected host. Not only did the predators show an overall preference for uninfected larvae throughout the trials (Figs 3, 4), but infection status also affected the identity of the first prey item approached by the predators. The fact that these results were found with uninfected prey that had died at the same time as the infected prey (Trial 2) suggests that the birds were not preferentially selecting the ‘freshest’ prey, but were showing a genuine aversion to infected prey. There was a general trend in both trials for the magnitude of this effect to increase with the duration of infection (e.g. Figs 3a, 4a), and this reflected the increasing differences in colour between infected and uninfected larvae. For example, at 3 days after infection, infected (Fig. 1b) and uninfected (Fig. 1a) larvae appeared similar in coloration, and there was little difference in the predators’ tendency to approach either type of larva. By day 5 after infection, however, infected larvae had a reduced ultraviolet signal (i.e. between 300 and 400 nm) and increased reddening of the spectrum (i.e. at wavelengths over 600 nm) compared to uninfected larvae (Fig. 1b), and this corresponded with a clear tendency for birds to handle (and consume) uninfected larvae. This suggests that the initial selection of prey is strongly influenced by visual cues, and so the birds showed a greater aversion to infected larvae as the difference in appearance between infected and uninfected larvae grew. If the induced colour change does act to deter predators, such a signal could work in a variety of ways. One possibility is that by altering the appearance of the host, the parasite is able to take advantage of the neophobic responses of avian predators that result in the avoidance of novel prey items, particularly in the presence of familiar prey (Shettleworth 1972; Marples et al. 1998; Marples & Kelly 1999). In this case, infected hosts would be ignored simply because they looked different from the birds’ usual prey. However, neophobia is a relatively short-lived phenomenon, which is unlikely to remain for the 2-week life cycle of the parasite used in this study (Marples et al. 1998; Marples & Kelly 1999). While neophobia describes short-term avoidance behaviour, an analogous but longer-term process called dietary conservatism may provide a mechanism whereby parasite-induced colour change could evolve as a warning signal, by placing these novel colour morphs at
a selective advantage (Marples & Kelly 1999; Thomas et al. 2003; Exnerova et al. 2007; Lee et al. 2009; Thomas et al. 2010). Our results therefore suggest that the induced colour changes act as a genuine warning signal, advertising the prey’s distastefulness to the predator. However, such a signal would be effective only if it was honest; in other words, predator avoidance would occur only if there was an additional mechanism reinforcing the signal (Sherratt 2002; Speed & Ruxton 2005). Observation of the feeding behaviour of the robins showed that on a number of occasions birds would approach infected larvae and peck at them, but then reject them and select uninfected larvae. Indeed, in the majority of trials, once a bird had handled an infected larva, regardless of whether it had consumed it or merely picked it up and discarded it, it was more likely to choose uninfected larvae (e.g. Fig. 3c). These observations suggest that the infected insects contained some distasteful substance that deterred consumption, and that the induced colour changes may act as an honest signal to the predators, which is reinforced by their unpalatable taste. At this stage we cannot rule out nonvisual signals that the birds may use to distinguish between infected and uninfected prey items. In particular, the birds may be using odour cues. However, in our experimental design infected and uninfected larvae were arranged randomly on a board together and it may be unlikely that the birds would have sufficient resolution in their odour detection to discriminate between infected and uninfected larvae in close proximity to each other. Therefore the choice between infected and uninfected prey may be based mainly on visual cues; our analyses show that the induced coloration change of infected larvae is very large compared to uninfected larvae. Previous studies have suggested that passerine birds are primarily visual predators, although they are able to use a combination of senses for final prey choice in the field (Heppner 1965; Sillman 1973; Montgomerie & Weatherhead 1997; Forsman & Appelqvist 1998). A noticeable feature of avian vision is the importance of reflectance in the ultraviolet wavelengths, and the use of these wavelengths in foraging decisions (Church et al. 2001; Kevan et al. 2001; Honkavaara et al. 2002). Since one of the consequences of the parasite-induced colour change is the reduction in reflectance in this range of the spectrum, the infected larvae will appear very distinct from uninfected larvae. Furthermore, red is a common aposematic colour and several studies have shown that this colour is one of the most effective in deterring initial attacks by avian
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predators (Guilford 1990; Gamberale & Tullberg 1996; Lindstrom 1999; Exnerova et al. 2006, 2007). Finally, it is worth considering whether the bioluminescence caused by these infections acts as an additional visual signal to deter predation, particularly early in the infection process when the spectral changes are relatively mild. However, alternative hypotheses have been suggested for this bioluminescence, primarily relating to the bacteria’s metabolic requirements (ffrench-Constant et al. 2002). Owing to the transient nature of the bioluminescence, we focused on the longer-lasting spectral changes following infection, so it remains to be seen whether the bioluminescence also plays a role as an aposematic signal. However, regardless of the potential role of bioluminescence, the observed reduction in ultraviolet and the increased reddening of the host induced by the presence of these parasites will greatly alter the appearance of infected larvae, making them appear particularly novel to birds, possibly exploiting both their neophobia and dietary conservatism (Marples et al. 1998; Marples & Kelly 1999; Thomas et al. 2003; Exnerova et al. 2007). Any distasteful chemical that the predator then encounters on handling the infected larvae will further reinforce this signal, reducing the likelihood of subsequent predation. Future experiments may help to clarify the relationship between host coloration and other potential cues to which the predators may respond. In particular, the experiment could be repeated using infected and uninfected prey dyed the same colour, thereby removing the visual signal. If the two prey types were still selected differentially, then this would suggest that the predators were using some olfactory cue. However, dyeing the larvae would also change their texture and odour, potentially masking any olfactory cues. Given the magnitude of the observed colour changes and the fact that birds are mainly visual predators, it seems most likely that they are primarily selecting their prey on the basis of visual appearance. We have presented compelling evidence of a novel form of parasite-induced host manipulation, causing changes in host colour as a means of deterring predators from consuming infected hosts. This differs from the traditional view of host manipulation, where parasites cause colour changes that increase conspicuousness to favour consumption by their predatory definitive host (Moore 2002). However, both examples show how parasites can exploit predator behaviour, resulting in changes to prey choice and diet composition at the individual level and, potentially, resource competition and abundance at the population level. Acknowledgments Thanks go to Mike Speed, Sarah Clarke and Hannah Rowland for help with project design, field work and use of equipment. We also thank Arthur Goldsmith and Julia Carter for use of their facilities, and Ness Gardens for permission to use their facilities. The field experiments were undertaken following liaison with English Nature. References Bethel, W. M. & Holmes, A. J. 1973. Altered evasive behavior and responses to light in amphipods harboring acanthocephalan cystacanths. Journal of Parasitology, 59, 945e956.
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