Behaviour 140 (2018) 93e98

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Animal Behaviour

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Colour plasticity alters thermoregulatory behaviour in caterpillars by modifyin g the cue received

Matthew E. Nielsen a, *, Eran Levin b, Goggy Davidowitz b, Daniel R. Papaj a a Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, USA b Department of Entomology, University of Arizona, Tucson, AZ, USA a r t i c l e i n f o Behaviour is an important way for to rapidly respond to changes in their current environment; Article history: however, over extended periods animals can also respond to environmental change via slower, devel- Received 8 December 2017 opmental plasticity in other traits. This developmental plasticity could itself alter the animal's behaviour Initial acceptance 19 February 2018 in two ways: it could change the state of the aspect of the animal's current environment that induces the Final acceptance 26 March 2018 behaviour (the cue), or it could change the physiology underlying production of that behaviour (the Available online 16 May 2018 behavioural reaction norm). We tested these alternatives for two responses to temperature, colour MS. number: A17-00971 plasticity and refuge-seeking behaviour, in pipevine swallowtail, Battus philenor, caterpillars. Prior research found that black caterpillars seek thermal refuges at lower ambient temperatures than red Keywords: caterpillars in the field. Here, we found that the effect of colour on behaviour in the laboratory depended Battus philenor on how we heated the caterpillars. When warmed by radiant heat, black caterpillars sought refuge colour sooner than red caterpillars, as occurs in nature. In contrast, when warmed by conduction of heat, black cue caterpillars no longer sought refuges sooner than red caterpillars. Both colour morphs began see king developmental plasticity temperature refuges at the same body temperature in both experiments, and the sensitivity of their metabolic rate to thermoregulatory behaviour temperature was also the same. Taken together, our findings indicate that while colour does change the cue for refuge seeking, it does not change the behaviour's reaction norm. Similar cue-mediated in- teractions may often occur for thermoregulatory behaviour in other species. © 2018 The Association for t he Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Behaviour is a key means by which animals respond to rapid canutus, is associated with a change in preferred foraging grounds changes in their environment, particularly because most behav- towards those with harder molluscs as prey (van Gils, Dekinga, iours can be started or stopped almost immediately in response to Spaans, Vahl, & Piersma, 2005). environmental change (Duckworth, 2009; Snell-Rood, 2013). The mechanisms by which developmental plasticity influences Behaviour is not, however, the only way for animals to respond to behaviour has, however, received little previous attention. Here, we environmental change, particularly over extended periods. In propose two general mechanisms by which developmental plas- addition to behaviour, animals often respond to environmental ticity can alter a behavioural response: developmental plasticity change with developmental plasticity in other traits, such as can change the cue for the behaviour, or it can change the behav- morphology or life history (Boersma, Spaak, & De Meester, 1998; iour itself (the behavioural reaction norm). First, developmental Foster et al., 2015; Relyea, 2004). While such plastic responses are plasticity could change the information used by the animal as a cue often slower, they can nevertheless still be adaptive in a stable for the state of the environment. Behaviour, like developmental environment where some aspect of the environment provides a plasticity, uses cues about the environment to determine an reliable cue for predicting future conditions (Levins, 1968; Moran, appropriate response, and if a change in another trait alters the 1992). Furthermore, slower developmental plasticity can poten- perceived value of one of these cues, it will subsequently change tially alter the expression of a more rapid behavioural response. For the animal's behaviour (Fig. 1a). Environmental cues can be either example, a plastic increase in gizzard size of red knots, Calidris external to the organism, such as photoperiod, or internal to the organism but still related to the environment, such as nutritional state or body temperature (Moczek, 2015; Schlichting & Smith, 2002). * Correspondence: M. Nielsen, Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA. Alternatively, developmental plasticity could change how E-mail address: [email protected] (M. E. Nielsen). behaviour responds to the environment. A diverse set of https://doi.org/10.1016/j.anbehav.2018.04.009 0003-3472/© 2018 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. 94 M. E. Nielsen et al. / Animal Behaviour 140 (2018) 93e98

(a) (b) g g e e n n g g i i u u k k f f e e e e e e R s R s r u o i v a h e B t t s s e e r r

t t A A Cue (body temperature)

Figure 1. Hypothetical diagram of two mechanisms by which developmental plasticity could alter the expression of a behaviour, using colour plasticity and refuge-seeking behaviour in Battus philenor as an example. The X axis shows the value of the cue for the behaviour (body temperature for refuge seeking in B. philenor), and the Y axis shows the behavioural response, in this case modelled as simple one off dichotomy with two discreet states (either at rest or refuge seeking). The curve represents the behaviour's reaction norm, whose key feature in this case is the temperature threshold at which the behaviour starts. Arrows are used to show the behavioural response associated with a particular value of the cue. (a) Hypothesis 1: developmental plasticity changes the intensity of the cue, the information used by the animal to determine whether to perform a behaviour. This is shown by two different arrows (solid and dashed), representing the different values of the cue received by animals with different developmentally plastic phenotypes, leading to different behaviours. For B. philenor, this would manifest as colour plasticity altering body temperature, the cue for refuge-seeking behaviour. (b) Hypothesis 2: developmental plasticity changes the reaction norm for the behaviour, changing how the animal responds to an unchanged value of the cue. This is shown by two different reaction norms (solid and dashed), representing the different response thresholds of animals raised under different conditions, leading to different responses to the same value of the cue. For B. philenor, this would manifest as the developmental temperature variation producing colour change also producing a linked shift in the body temperature response threshold for refuge- seeking behaviour. physiological systems and processes produce behaviour, including example, changes in metabolic rate and enzyme activity following neural, hormonal and muscular systems, which can themselves be exposure to different temperatures have been associated with developmentally plastic and change alongside other traits. The changes in preferred temperatures for behavioural thermoregula- reaction norm for the behaviourda function that relates the tion in multiple ectothermic vertebrates (Berner & Puckett, 2010; phenotype to the state of the environment (Scheiner, 1993)d Glanville & Seebacher, 2006). Alternatively, developmental plas- summarizes all of these processes. Developmental plasticity in the ticity in another trait could have side-effects on the animal's ner- systems underlying a behaviour will change its reaction norm, and vous or sensory systems, leading to a change in how they integrate thus the behaviour itself, independent of the immediate environ- sensory information on temperature, and thus their behavioural ment and its cues (Fig. 1b). response. In both cases, the animal experiences the same body Temperature and thermoregulation provide an especially good temperature (cue), but responds differently, indicating a change in ecological context for studying the relationship between behaviour the reaction norm. and plasticity in other traits. Behaviour is arguably the most impor- To test how developmental plasticity changes thermoregulatory tant class of traits for thermoregulation, particularly in ectotherms, behaviour, we used the caterpillar of the pipevine swallowtail, due to both its speed and effectiveness (Stevenson, 1985), but plastic Battus philenor L., which responds to temperature in two ways: changes in other traits such as physiology and morphology are also colour change (developmental plasticity) and refuge-seeking often involved in thermoregulation (Angilletta, 20 09; Kingsolver & (behaviour). Battus philenor caterpillars develop red body colora- Huey, 1998; Stevenson, 1985). This creates the opportunity for tion when raised at warm ambient temperatures (about 36 C or slower plastic changes in other traits to affect thermoregulatory above), and black coloration at cooler temperatures (Nice & behaviour via either of the two mechanisms described above. Tem- Fordyce, 2006). Placed in sunlight, red caterpillars warm more perature of all or part of the body is a common cue for thermoreg- slowly and reach asymptotic body temperatures about 3  C lower ulatory behaviour (May, 1979), in which case developmental than black caterpillars (Nice & Fordyce, 2006). At particularly high plasticity of other traits that affect body temperature, such as colour, temperatures, caterpillars also seek cooler locations. These ‘thermal size and shape, should in turn alter the expression of thermoregu- refuges’ are typically off their low-growing host plant, on nonhost latory behaviour. vegetation higher above the ground where air temperature is Alternatively, the thermoregulatory behaviour's reaction norm cooler (Nice & Fordyce, 2006; Nielsen & Papaj, 2015, 2017). could be changed by developmental plastic responses to temper- Nielsen and Papaj (2017) demonstrated that body coloration ature. Temperature affects behaviour through both ‘kinetic’ changes refuge-seeking behaviour: black caterpillars in field ex- effectsdthe direct physical influence of temperature on the periments moved to refuges at lower environmental temperatures biochemical, metabolic and physiological processes that underlie than red ones and thus spent more time on refuges overall. Here, in behaviourdand ‘integrated’ effectsdbehavioural responses to laboratory experiments, we tested whether the effect of colour on thermal information made using integrated sensory and nervous refuge-seeking behaviour occurs because of the direct effect of systems (Abram, Boivin, Moiroux, & Brodeur, 2017). Behavioural body colour on body temperature (Fig. 1a) or because develop- reaction norms depend on both of these effects, and developmental mental plasticity changes the behaviour's reaction norm (Fig. 1b). plasticity in other traits could alter the reaction norm by changing Based on previous work (Nielsen & Papaj, 2015), we hypothesized either of them. Thermal acclimation (temperature-induced change that body temperature was either the cue for refuge seeking, or in an organism's physiology and biochemistry that alters its sub- strongly correlated to the cue. We experimentally heated caterpil- sequent sensitivity to temperature; Angilletta, 2009; Huey, lars in two ways, using either radiant heat (a strong light) or con- Berrigan, Gilchrist, & Herron, 1999) provides an example where duction (a hot plate), and assessed behaviour of the different colour plasticity alters the kinetic effects of temperature on behaviour. For morphs. Body coloration affects body temperature by changing M. E. Nielsen et al. / Animal Behaviour 140 (2018) 93e98 95 absorption of visible and near-infrared radiation, with darker col- the absorption of radiant heat in the visible to near-infrared range. ours absorbing more radiation (Clusella Trullas, van Wyk, & Spotila, In a second experiment, we warmed the set-up by placing it on a 2007; Gates, 1980). If body colour changes the refuge-seeking cue, hot plate. This protocol warmed the caterpillar primarily through body temperature, this interaction should only occur in caterpillars conduction, minimizing the effect of colour on body temperature. warmed by radiant heat and not conduction, and we should only Under these conditions, colour could affect the caterpillar's detect an effect of body colour on behaviour under these condi- behaviour only if it changed its response to body temperature. tions. Alternatively, if body colour is associated with a change in the Before a trial, caterpillars were held at room temperature for at reaction norm for thermal refuge seeking, we should detect an least 30 min to allow for short-term acclimation. At the start of the effect of body colour on behaviour regardless of heating method trial, a red or black caterpillar was weighed and then placed in the used. Finally, to assess whether developmental colour plasticity is centre of the leaf. If the caterpillar never stopped moving after associated with physiological acclimation, we measured the placement on the leaf or left the leaf, we discarded it as this typi- metabolic rate of caterpillars at two temperatures and looked for an cally indicated movement for other reasons, such as seeking a effect of colour on metabolic rate. nonhost substrate on which to moult. After a caterpillar stopped moving (usually within 20 min), the heating device for that METHODS experiment was turned on. A trial ended when a caterpillar had crawled completely onto the wooden dowel. We excluded from Study Organism analysis five caterpillars that crawled onto the sand (four heated by hot plate, one by halogen lamp). During the experiment, we used a The pipevine swallowtail, B. philenor L., is a papilionid butterfly thermal imaging camera (FLIR T-300) to record caterpillar tem- whose larvae feed exclusively on species in the genus . peratures. We took a thermal image of the caterpillar each time it In southern Arizona, there is a single host species, Aristolochia started moving until it was fully on the refuge and recorded the watsonii Woot. Standl. Aristolochia watsonii is a small perennial, time at which movement began. We specifically used the last time deciduous, ground-hugging vine with multiple stems found at low the caterpillar started moving before climbing the wooden dowel to mid-elevation, often in or near washes. This low growth pattern as the onset of refuge-seeking behaviour because earlier bouts of frequently places caterpillars near the hot ground, on leaves whose movement (typically associated with eating) were evidently not temperatures frequently exceed 40  C (Nielsen & Papaj, 2015). To associated with stressful enough temperatures to cause the cater- avoid these high temperatures on the host plant, caterpillars typi- pillars to leave their host. We capped the duration of the experi- cally seek refuges on the surrounding grasses and other herbaceous ment at 30 min, in which case we treated the caterpillar's time until plants. Both red and black body colours also serve as a warning refuge sought as 30 min and excluded it from analysis for body signal of the toxic aristolochic acids sequestered from their hosts temperature (since it would be an underestimate). This occurred (Sime, Feeny, & Haribal, 2000), and thus deter the majority of only once, for a red individual during the radiant heating trial. predators. In southern Arizona, B. philenor has four broadly over- During the trial, we also recorded whether the caterpillar ate any of lapping broods from March to September; the timing and the leaf. In trials using a halogen light, we tested 20 red and 19 black discreteness of broods varies substantially from year to year and caterpillars, with each trial lasting between 3 and 30 min. In trials site to site. This exposes the larvae to a wide range of temperatures using a hot plate, we tested 25 red, 24 dark red and 26 black cat- between and within generations. erpillars, with each trial lasting between 4 and 19 min. To estimate body temperature from thermal images, we first The Effect of Body Coloration on Thermoregulatory Behaviour converted the images to greyscale using FLIR Quick Report (v.1.2, FLIR Systems, Inc., Wilsonville, OR, U.S.A.). Next, we estimated mean We raised caterpillars for our experiments from eggs laid by surface temperature as precisely as possible by averaging across the butterflies caught at the University of Arizona Santa Rita Experi- area of the caterpillar, as selected freehand in ImageJ (v.1.45s, Na- mental Range in Pima County, Arizona (3147. 0490N, 110 49.5240W) tional Institutes of Health, Bethesda, MD, U.S.A.) while taking care to and the first generation of descendants from these butterflies. Eggs avoid selecting areas outside the caterpillar. All statistical analyses were initially kept at 30  C, but upon hatching they were split be- were conducted in R (v.3.1.4, R Foundation for Statistical Computing, tween 30 C (which produced a black colour) and 38  C (which Vienna, Austria). We used an accelerated failure model with a produced a red colour). When conducting the behavioural experi- Weibull distribution (survival package) to test whether body colour ment using a hot plate (see below), we also tested caterpillars affected the time until a caterpillar began refuge seeking, and an raised at a third temperature, 34 C, which produces very dark red ANOVA to test whether body colour affected the body temperature caterpillars. At all temperatures, caterpillars were kept in plastic at which it did so. Independent tests were performed for each cups and boxes in stand-alone growth chambers with a 12:12 h experiment, heating with light and heating with the hot plate. For all light:dark cycle and fed freshly collected leaves of Aristolochia analyses, we also tested the following covariates using a hierarchical mbriata ad libitum. Fourth-instar caterpillars were used in the (type II sum of squares) approach: the caterpillar's mass, whether or experiments. not it ate during the trial, and the interactions of these factors with To distinguish whether a change in body temperature or the colour. However, only one of these factors was significant: when a reaction norm for body temperature caused the effect of body hot plate was used for heating, whether or not the caterpillar ate coloration on thermoregulatory behaviour, we performed two ex- during the trial (a binary factor) affected the body temperature at periments using a protocol based on a previous study (Nielsen & which a refuge was sought. All others were nonsignificant (P>0.1) Papaj, 2015). In a 28  18 cm aluminium pan filled with 2.0e2.5 and thus excluded from the final analysis. cm of dry sand, we placed a fresh A. mbriata leaf flat, in the centre of the sand. Immediately adjacent to the leaf, we inserted a wooden The Effect of Colour on Response of Metabolic Rate to Temperature dowel (0.6 cm diameter, 30 cm long) into the sand to serve as a thermal refuge. We heated this set-up using a different method for Caterpillars for this experiment were raised under the same each experiment. For one experiment, we placed a 500 W halogen conditions as for the previous experiments, except that they were work light 42 cm from the leaf, at a 30 angle. Under these condi- tested during the fifth instar instead of the fourth. As before, black tions, colour would change body temperature through its effect on caterpillars were produced by development at 30  C and red 96 M. E. Nielsen et al. / Animal Behaviour 140 (2018) 93e98 caterpillars by development at 38 C. Metabolic rate of the larvae Nevertheless, some of the variance in body temperature when was measured using a flow-through respirometry system. Larvae seeking a refuge was due to the occurrence of feeding during the (all fasted for at least 12 h) were weighed using an electronic balance hot plate trial: caterpillars that ate part of the provided leaf began (Mettler Toledo ML54, precision: 0.1 mg) and placed in an envi- to seek a refuge at a body temperature 1.81 C higher than those ronmental cabinet that used a Peltier device to control temperature that did not, a significant difference (F1,71¼11.22, P¼0.0013). via convection (PTC-1 Temperature Cabinet; Sable Systems Inter- national, Las Vegas, NV, U.S.A.). During the first set of measurements, Effect of Body Coloration on Response of Metabolic Rate to temperature was maintained at 42  C, af ter which the chamber was Temperature held at 30  C for a second round of measurements of the same caterpillars. Before each round of measurements, caterpillars had at Metabolic rate, measured as oxygen consumption, was 60%   2¼ least 20 min for short-term acclimation to the current temperature. higher for caterpillars tested at 42 C than at 30 C ( 1 25.15, To measure metabolic rate, we placed each caterpillar individ- P<0.0001; Fig. 3). Colour, however, neither significantly interacted 2 ¼ ¼ ually into a metabolic chamber (20 ml syringe). Caterpillars were with temperatures' effect on metabolic rate ( 1 0.426, P 0.51) nor fi 2¼ ¼ then left undisturbed for at least 5 min before metabolic rate data had any signi cant independent effect ( 1 0.405, P 0.52; Fig. 3). were collected. Oxygen consumption was measured by gas analyzer (FoxBox, Sable Systems Int., Las Vegas, CA, U.S.A.). Dry, carbon DISCUSSION dioxide-free air (from a cylinder, chemically scrubbed of remaining carbon dioxide and water using Ascarite and Drierite, Sigma- The effects of developmental plasticity in other traits on the Aldrich, St Louis, MO, U.S.A.) was passed through two parallel expression of behavioural traits have rarely been investigated, and flow controllers (Alicat Scientific Inc., Tucson, AZ, U.S.A.) at a con- no previous study has fully distinguished the degree to which these stant flow of 30 ml/min. One flow controller fed a constant stream interactions are caused by immediate changes in the intensity of of air to the metabolic chamber and the other flow controller fed air the behaviour's cues versus developmental changes in the behav- directly to the analyzer for calibration and use as a baseline reading iour's reaction norm that responds to those cues. In B. philenor between measurements. The air source for analysis (chamber or caterpillars, we found that the effect of developmental colour baseline) was manually operated using a three-directional valve. plasticity on thermoregulatory behaviour is mediated entirely by The air from the metabolic chamber was scrubbed of water, but not the effects of body coloration on body temperature, with no evi- carbon dioxide, before measurement. dence for an effect of developmental plasticity on the shape of the After at least 5 min of measuring baseline concentrations, O2 behavioural reaction norm. Body temperature, or something very concentrations from the metabolic chamber (with the caterpillar) strongly correlated, is the cue for refuge-seeking behaviour, as were recorded at a rate of 1 Hz using a PC and Expedata software indicated by the relatively low overall variation in body tempera- (Sable Systems Inc.). For analysis, we averaged the first up to 500 s ture at which refuge seeking began in our study. Our results indi- of stable measurements of metabolic rate at each temperature, cated that black caterpillars sought refuges sooner than red excluding any measurements with less than 200 s of stable O2 caterpillars only when warmed by radiant heat and not when consumption. This lead to some caterpillars only being considered heated via conduction. Black animals generally absorb more radiant for one of the two treatments. We then calculated VO2 (ml of O2 per heat (i.e. light) than lighter coloured ones, thus reaching higher g per h) according to Withers (2001). Data were analysed with a body temperatures faster under the same external conditions general linear mixed model fit by maximum likelihood (‘lme’ in the (Clusella Trullas et al., 2007; Gates, 1980), and this is the case for ‘nlme’ package in R), using temperature, caterpillar colour, and B. philenor (Fordyce & Nice, 2006; Nielsen & Papaj, 2017). their interaction as fixed effects, and individual caterpillar as a At the same time, we found evidence against developmental random effect. Significance of factors was established using hier- plasticity in the behaviour's reaction norm: body colour did not archical likelihood ratio tests. change the body temperature threshold at which caterpillars star- ted refuge seeking in either experiment. Previous work on Ethical Note B. philenor caterpillars using similar methods has shown that as caterpillars grow larger, the body temperature at which they start No ethical or other permits are required for rearing or experi- seeking refuges decreases (Nielsen & Papaj, 2015). Thus, even if ments on B. philenor. This research complied with all legal and developmental plasticity of body colour does not change the ethical guidelines of the United States of America. behavioural response to body temperature, we have evidence that developmental changes associated with other aspects of RESULTS morphology can change the behavioural reaction norm. Addition- ally, manipulating developmental temperature and body colour did Time and Body Temperature at Onset of Thermal Refuge Seeking not affect metabolic rate (measured as O2 consumption) and neither did it alter the effect of current temperature on metabolic When warmed using radiant heat, red caterpillars sought ther- rate. Thus, we found no evidence of long-term acclimation associ- 2¼ ¼ mal refuges 47.9% later than black caterpillars ( 1 5.42, P 0.020; ated with colour, further arguing against developmental plasticity Fig. 2a). Importantly, we failed to detect a significant effect of body in the behavioural reaction norm. coloration on the caterpillars' body temperature at the time they The cue-mediated interaction between morphology and ther- began to seek a refuge (F1,36¼0.097, P¼0.758; Fig. 2b). When moregulatory behaviour we have found in B. philenor should be warmed using a hot plate rather than radiant heat, black caterpil- widespread, potentially existing in all ectotherms. Change in any lars did not seek thermal refuges significantly sooner than lighter trait (due to developmental plasticity or other causes) that alters an ones, and the pattern was even in the opposite direction, with dark animal's body temperature in a given environment (e.g. size, colour, red and red caterpillars seeking thermal refuges 10.5% and 2.9% shape) should also change any behaviour that uses body temper- 2¼ ¼ fi sooner, respectively ( 2 1.91, P 0.385; Fig. 2c). As with radiant ature or something closely related as its cue. Completely arti cial heat, we found no significant effect of body coloration on the body manipulation of colour with marker or paint can change thermo- temperature at which caterpillars sought a thermal refuge when regulatory behaviour in other , such as adult butterflies and warmed with a hot plate (F2,71¼1.67, P¼0.195; Fig. 2d). grasshoppers, showing some of the potential breadth of the effect M. E. Nielsen et al. / Animal Behaviour 140 (2018) 93e98 97

Heated with light Heated without light )

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Figure 2. Effect of colour (as determined by developmental temperature) on refuge-seeking behaviour when heated by a halogen light in the laboratory (a, b) or using a hot plate (c, d). Error bars represent 95% confidence intervals, and sample sizes are given at the base of each bar. (a, c) Effect of colour on mean time until the start of refuge seeking. (b, d) Effect of colour on mean body temperature at the start of refuge seeking. *P¼0.020.

1 b other organisms, particularly because the two mechanisms are not b mutually exclusive. For example, although not necessarily as dra-

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f 0.4 n using experiments similar to ours in a variety of species. Which o

a l e mechanism underlies an interaction matters because it can change m M ( 0.2 the conditions under which the interaction occurs. For example, the cue-mediated effect of colour on behaviour we found in B. philenor =7 =11 =10 =10 depended on the light environment, which we manipulated arti- 0 30 °C/Black 30 °C/Red 42 °C/Black 42 °C/Red ficially, but the light environment also varies considerably within Test temperature (°C)/Colour and among natural environments (Gates, 1980). If, alternatively, the effect of colour on thermoregulatory behaviour had been caused by Figure 3. Mean metabolic rate, measured as the rate of oxygen consumption (VO2, ml developmental plasticity of the behaviour's reaction norm, the ef- of O2 per g per h) of red and black caterpillars at two different environmental test fect would have been independent of the light environment, and temperatures. Error bars represent 95% confidence intervals and sample sizes are given at the base of each bar. Different letters above bars denote significant (P<0.0001) changes in behavioural reaction norms may generally be less sen- differences between temperature treatments. sitive to the current environment than cue-mediated interactions. The idea that developmental plasticity in other traits can alter behaviour by changing its cues potentially applies to many of colour on thermoregulatory behaviour (Karpestam, Wennersten, ecological contexts beyond just temperature. For example, both & Forsman, 2012; Kingsolver, 1987). In our study, we show that the behaviourally and morphologically induced defences to predation same type of interaction can occur because of natural plasticity in are widespread in aquatic animals, where their ecological conse- colour, but still without any simultaneous change in reaction norm. quences have been well studied (Kishida, Trussell, Mougi, & The change in a behaviour's cue caused by plasticity in another trait Nishimura, 2010; Relyea, 2004), but whether these responses (colour) is entirely sufficient to change the behaviour's expression. affect each other's expression has received little investigation, Although we expect many cases exist where developmental much less any potential causes of those interactions. In addition to plasticity alters the cues for thermoregulatory behaviour, we the effects of developmental plasticity on the immediate environ- cannot rule out the possibility of developmental plasticity also ment that behaviour responds to, researchers could also investigate changing the reaction norm of thermoregulatory behaviour in the inverse situation: whether repeated or prolonged use of a 98 M. E. Nielsen et al. / Animal Behaviour 140 (2018) 93e98 behaviour can affect the perceived intensity of the cues used to Duckworth, R. A. (2009). The role of behavior in evolution: a search for mechanism. e determine a developmentally plastic phenotype. Evolutionary Ecology, 23, 513 531. https://doi.org/10.1007/s10682-008-9252-6. Foster, S. A., Wund, M. A., Graham, M. A., Earley, R. L., Gardiner, R., Kearns, T., et al. Although behavioural ecology has traditionally focused on how (2015). Iterative development and the scope for plasticity: Contrasts among animals use information from their environment to make behav- trait categories in an adaptive radiation. Heredity, 115, 335e348. https://doi.org/ ioural decisions, our work shows that these cues can also depend 10.1038/hdy.2015.66. fi Gates, D. M. (1980). Biophysical ecology. New York, NY: Springer-Verlag. on the animal itself, speci cally developmental plasticity in other Glanville, E. J., & Seebacher, F. (2006). Compensation for environmental change by traits. At the same time, the ability of these other traits to affect complementary shifts of thermal sensitivity and thermoregulatory behavior in behaviour's expression may itself depend on the environment. We an ectotherm. Journal of Experimental Biology, 209, 4869e4877. https://doi.org/ 10.1242/jeb.02585. see this in our study of B. philenor, where the effect of colour on Goulson, D. (1994). Determination of larval melanization in the moth, Mamestra behaviour depended on whether light was a primary source of heat, brassicae, and the role of melanin in thermoregulation. Heredity, 73, 471e479. a factor that can also vary in nature. Ultimately, our research sug- https://doi.org/10.1038/hdy.1994.145. Hazel, W. N. (2002). The environmental and genetic control of seasonal poly- gests that to fully understand behaviour and phenotypic plasticity, phenism in larval color and its adaptive significance in a swallowtail butterfly. we need to consider the organism, its various traits and its envi- Evolution, 56, 342e348. https://doi.org/10.1111/j.0014-3820.2002.tb01344.x. ronment not as separate elements but as an integrated whole Huey, R. B., Berrigan, D., Gilchrist, G. W., & Herron, J. C. (1999). Testing the adaptive significance of acclimation: A strong inference approach. American Zoologist, 39, (Moczek, 2015). 135e148. https://doi.org/10.1093/icb/39.2.323. Karpestam, E., Wennersten, L., & Forsman, A. (2012). Matching habitat choice by Data Accessibility experimentally mismatched phenotypes. Evolutionary Ecology, 26, 893e907. https://doi.org/10.1007/s10682-011-9530-6. Kingsolver, J. G. (1987). Evolution and coadaptation of thermoregulatory behavior Data are available from the Dryad Digital Depository (Nielsen, and wing pigmentation pattern in pierid butterflies. Evolution, 41, 472e490. Levin, Davidowitz, & Papaj, 2018): doi: https://doi.org/10.5061/ https://doi.org/10.1111/j.1558-5646.1987.tb05819.x. dryad.m4p474d. Kingsolver, J. G., & Huey, R. B. (1998). Evolutionary analyses of morphological and physiological plasticity in thermally variable environments. American Zoologist, 38, 545e560. https://doi.org/1093/icb/38.3.545. Authors' Contributions Kishida, O., Trussell, G. C., Mougi, A., & Nishimura, K. (2010). Evolutionary ecology of inducible morphological plasticity in predatoreprey interaction: Toward the practical links with population ecology. Population Ecology, 52, 37e46. https:// M.N. conceived and designed the study, conducted the experi- doi.org/10.1007/s10144-009-0182-0. ments and statistical analyses and drafted the manuscript; E.L. Levins, R. (1968). Evolution in changing environments. Princeton, NJ: Princeton assisted with gathering and analysing the metabolic data; G.D. University Press. May, M. L. (1979). thermoregulation. Annual Review of Entomology, 24, assisted with the conception and design of the study and drafting 313e349. https://doi.org/10.1146/annurev.en.24.010179.001525. the manuscript; D.P. assisted with the conception and design of the Moczek, A. P. (2015). Developmental plasticity and evolution: Quo vadis? Heredity, study and drafting the manuscript. 115 , 302e305. https://doi.org/10.1038/hdy.2015.14. Moran, N. A. (1992). The evolutionary maintenance of alternative phenotypes. American Naturalist, 139, 971e989. https://doi.org/10.1086/285369. Competing Interests Nice, C. C., & Fordyce, J. A. (20 06). How caterpillars avoid overheating: Behavioral and phenotypic plasticity of pipevine swallowtail larvae. Oecologia, 146, e We have no competing interests. 541 548. https://doi.org/10.1007/s00442-005-0229-7. Nielsen, M. E., Levin, E., Davidowitz, G., & Papaj, D. (2018). Dryad. Data from: Colour plasticity alters thermoregulatory behaviour in Battus philenor caterpillars by Acknowledgments modifying the cue received. https://doi.org/10.5061/dryad.m4p474d. Nielsen, M. E., & Papaj, D. R. (2015). Effects of developmental change in body size on ectotherm body temperature and behavioral thermoregulation: Caterpillars in a We thank Dr Larry Venable for use of equipment. Kim Byers heat-stressed environment. Oecologia, 177, 171e179. https://doi.org/10.1007/ assisted with the metabolic data collection. 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