Spectral mouth colour of nestlings changes with

Functional Ecology 2008, 22, 1044 1051 doi: 10.1111/j.1365-2435.2008.01455.x Spectral mouth colour of nestlings changes with Blackwell Publishing Ltd carotenoid availability R. Thorogood 1 *, R. M. Kilner 1, F. Karada1 2 and J. G. Ewen 3 1 Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, UK; 2 Department of Animal Science, University of Yüzüncü Yil, Van, Turkey; and 3 Institute of Zoology, Zoological Society of London, Regents Park NW1 4RY, London, UK Summary 1. Despite the proliferation of studies on the role of nestling mouth colour in parent offspring communication, there has been very little work regarding the proximate mechanism for mouth pigmentation. 2. Carotenoids, a class of phytochemicals important for immune function and gained by birds only through their diet, also serve as pigments for yellow orange colours. Carotenoids have been shown to be responsible for the colouration of adult plumage and integuments (e.g. bills, legs, combs of adults of several species), but their role in colouring nestling gapes, and the surrounding fleshy rictal flanges, remains hypothetical. 3. Here, we present direct evidence for the importance of carotenoid availability in determining nestling mouth colouration. In field experiments on Hihi (Notiomystis cincta), a passerine endemic to New Zealand, we experimentally supplemented carotenoids directly to nestlings, and indirectly by provisioning parents, and measured the effect on nestling mouth colouration with spectrometer. 4. We found that increased carotenoid availability in the diet enhanced circulating blood plasma carotenoid concentrations, and that this in turn influenced mouth palate and rictal flange colouration. High carotenoid availability increased saturation of the yellow wavelengths of the spectrum reflected by both the palate and flanges, but reduced the reflectance of ultraviolet (UV) wavelengths of the rictal flanges. 5. We suggest that carotenoids influence the appearance of nestling gapes both by increasing pigmentation and as a filter of UV-reflecting structures. Key-words: gape, parent offspring communication, rictal flange, signalling, UV colouration Introduction In altricial birds, parents are confronted with a profusion of potential signals when they visit the nest and encounter the begging display of their nestlings. While most interest has been paid to the vocal and postural signals given during this display (reviewed in Kilner & Johnstone 1997), the potential for mouth colouration to act as an important visual signal is receiving ever-increasing attention (reviewed in Kilner 2006). Mouth colouration may act simply to improve detectability within the nest (Ficken 1965), by providing a target for parental feeding (Pycraft 1907; Ingram 1920; Kilner & Davies 1998; Heeb, Schwander & Faoro 2003), and/or serving as an honest signal of individual offspring need (Kilner 1997), or quality (Saino et al. 2000). While there is an evidence to suggest that mouth colour can indicate various phenotypic qualities (e.g. hunger in the Common Canary Serinus canaria (Kilner 1997), temperature *Correspondence author. E-mail: rt303@cam.ac.uk in the dark-eyed junco Junco hyemalis (Clotfelter et al. 2003), and health status (Saino et al. 2003) and body size (De Ayala et al. 2007) in the Barn Swallow Hirundo rustica), and influence parental provisioning (e.g. Great Tit Parus major parents preferentially fed nestlings with gapes artificially dyed red (Götmark & Ahlström 1997; Heeb et al. 2003); as did parent Barn Swallows (Saino et al. 2000)), very little is known of the mechanism behind this signal. Carotenoids were first suggested to be the pigments responsible for yellow orange mouth colours by Wetherbee (1961). Carotenoids are organic pigments that cannot be synthesized by animals but must be obtained from their diet (Fox 1979). Nevertheless, carotenoids are widely used by animals, serving key physiological functions (reviewed in Olson & Owens 1998) as well as being the biochemicals responsible (either directly as pigments, or via metabolism) for colouration of most yellow, orange or red feathers (Fox & Vevers 1960; McGraw 2006a; but see McGraw 2004). Perhaps more importantly, carotenoids have also been demonstrated to pigment fleshy integuments of adults from a range of species 2008 The Authors. Journal compilation 2008 British Ecological Society

Nestling mouth colour and carotenoids 1045 (e.g bill colour of European Blackbird Turdus merula (Faivre et al. 2003) and Mallard duck Anas platyrhynchos (Peters et al. 2004), and the areas of bare skin on the head, the ceres and lores, of the American Kestrel Falco sparverius (Bortolotti et al. 1996; Bortolotti, Fernie & Smits 2003)). Integument colour may be more dynamic than plumage colour, and therefore able to more rapidly signal condition of an individual (Bortolotti et al. 1996). For example, evidence suggests that the yellow bill of adult male blackbirds responds quickly to immunostimulation (Faivre et al. 2003), while colour of the ceres and lores of the American Kestrel changes seasonally (Negro et al. 1998), particularly when immunochallenged (Bortolotti et al. 2003). Although the importance of the antioxidant actions of carotenoids for birds has been recently questioned (Constantini & Møller 2008), and that the role of carotenoids as signalling antioxidant activity is a red herring (Hartley & Kennedy 2004), there is strong evidence for their importance for enhancing immune function in a range of species (Blount et al. 2003; Faivre et al. 2003; McGraw & Ardia 2003). As carotenoids must be used for health, and assuming that this is of greater importance than pigmentation, then carotenoid availability may provide a mechanism for maintaining the honesty of the signals that rely on these pigments. Although the role of carotenoids in pigmenting plumage and adult integuments has been extensively studied, to date there have been no studies directly assessing the contribution of carotenoids in the diet to the spectral colouration of mouth integuments of nestlings. Carotenoids are often assumed to be the pigment responsible, but this has never been demonstrated experimentally (McGraw 2004; McGraw 2006b). Previous studies inferring a role for carotenoids have been limited by either not directly measuring carotenoid availability (e.g. Hunt et al. 2003; De Ayala et al. 2007), or by measuring mouth colouration using human-biased perception techniques (e.g. Kilner 1997; Kilner & Davies 1998; Ewen et al. 2008; Loiseau et al. 2008). A study conducted by Saino et al. (2000) where nestlings were fed carotenoids is generally referred to as providing the evidence that carotenoids are responsible for mouth colouration. However, this study did not measure the success of their carotenoid supplementation, and also relied upon ranking of flange colours by observers to conclude that carotenoid availability reversed the effects of an immunechallenge on mouth colour. Perhaps better evidence for a role of carotenoids in colouring nestlings mouths comes from a recent study of House Sparrows Passer domesticus (Loiseau et al. 2008). Nestlings supplemented with carotenoids had higher levels of circulating plasma carotenoids, and yellower flanges. However, this study did not consider the effects of carotenoids on the mouth gape, and did not measure the full range of spectrum visible to birds (instead, relying on digital photography). Avian vision differs from that of humans (reviewed in Cuthill et al. 2000), allowing birds to see a wider spectral range (320 700 nm) including the ultraviolet (UV). Traditional methods of colour measurement (using photography or colour ranking) therefore do not allow for analysis of the full spectrum visible of birds. The purpose of our study was to test the hypothesis that increased availability of carotenoids to nestlings leads to a concomitant change in circulating carotenoids, and a change in the reflectance spectra of mouth palate and rictal flange colour. Previous research on nestling colouration in Hihi (an endemic New Zealand passerine, Notiomystis cincta) has suggested a link between dietary carotenoid availability and the yellow orange colour of nestlings flanges (Ewen et al. 2008). Adult Hihi breeding pairs were supplemented with carotenoids from time of egg lay until nestlings fledged, and an increase in the orangeness of nestlings flanges (by comparison with colour charts) was noted. However, similar to previous studies, this work was limited in that colour measurement and was based on human perception, and therefore could not provide information on the UV component of this potential signal. In addition, because carotenoids were provided to nestlings solely by their parents (either in the yolk, or by feeding), the effects of carotenoids could have been further modulated by maternal effects and/or differences in parental behaviour. Therefore, this study provided limited information regarding the relationship between carotenoids and colouration of the mouth. As a result, in our study we experimentally manipulated carotenoid availability to nestling Hihi both directly by hand-feeding and via parental supplementation. To analyse the success of this manipulation, we measured the concentration of carotenoids circulating in the blood plasma. These were then related to mouth colouration of the nestlings, measured using spectrometer. Materials and methods STUDY SPECIES Hihi are a medium sized (30 40 g) passerine that is restricted to one natural island population, and several recently translocated populations (Higgins, Peter & Steele 2001). The introduced population on Tiritiri Matangi Island nest in provided boxes, and have access to supplementary sugar water (20% by weight) throughout the year. All c. 150 individuals are uniquely identifiable by leg ring combinations, and their ages are known. Hihi take nectar, fruits, and invertebrates and regurgitate all food types to their young (R. Thorogood, unpublished data). Sexes are dimorphic in adult plumage and body size. Hihi are socially monogamous, although they exhibit high rates of extra-pair paternity (Ewen, Armstrong & Lambert 1999). Clutches of three to five eggs are incubated solely by the female, while both parents care for the young during a nestling period of 30 days. All nesting attempts on the island, during the 2006 2007 breeding season were monitored, with initiation of nest building, clutch size, date of completion, and hatching recorded. CAROTENOID SUPPLEMENTATION At 4 days of age, all nesting Hihi pairs with broods of at least three chicks were assigned to one of the three experimental treatments: 1. Control (N = 11): sugar water provisioned to adults and hand-fed to nestlings (placebo for control of hand-feeding). 2. Hand-fed (N = 10): nestlings directly fed carotenoid-enhanced sugar water by hand, sugar water provisioned to adults.

1046 R. Thorogood et al. 3. Parentally-fed (N = 10): carotenoids provided for adults, nestlings fed extra carotenoids by parents, and individually hand-fed sugar water to control for hand-feeding. All nestlings within a brood were hand-fed to satiation once daily from day 4. Nestlings were stimulated to beg by tapping the nesting box and fed until begging was no longer elicited. Sugar water was enhanced by the addition of the carotenoids lutein and zeaxanthin (available in liquid form, OroGLO, Kemin Industries) at a final concentration of 100 μg ml 1. Lutein and zeaxanthin are the main carotenoids circulated by adult Hihi, and the ratio of these carotenoids in OroGLO is very similar to that circulating in their blood plasma (see Ewen et al. 2006b for details). The carotenoid-enhanced food was no different to that used in previous successful manipulations of carotenoid availability in adult Hihi (Ewen et al. 2006c). The food received by hand-fed nestlings was identical to that which adults received and fed to their nestlings. In each treatment, adults were provided with additional supplementary food, 10 m from the active nesting box. Food supplements were changed every 2 days, and presented in hummingbird-type feeders familiar to the birds because of their permanent use on the island (Taylor & Castro 2000). The use of feeders after establishment was monitored, with all pairs finding and taking the food within 30 min. Furthermore, previous research of the same population found no difference in the use of sugar and carotenoid-enhanced supplementary food (Ewen et al. 2006c). Supplementary food was provided to both adults and nestlings for 7 days, ending when nestlings were 10 days of age. Nests were allocated to feeding treatments sequentially in order of hatching date, with the exception of a small number of nests that occurred in close proximity to each other. These had to be assigned to the same adult feeding treatment. In order to address the possibility that groups varied prior to treatment, small blood samples (c. 150 μl, using brachial venipuncture) were collected from a random sample of males (N = 24) and females (N = 10) prior to supplementary feeding (at the end of egg laying). Blood was immediately centrifuged to separate plasma which was then stored at 20 C until subsequent carotenoid analyses were performed (see below). There were no significant differences in parental circulating plasma carotenoid levels (mean μg ml 1 ± SE, control (N = 13): 8 68 ± 1 08, hand-fed (N = 11): 9 45 ± 1 59, Parentally-fed (N = 10): 9 33 ± 1 64, F (2,31) = 0 14, P = 0 87), parental experience (mean combined parental ages in years ± SE, control (N = 11): 3 91 ± 0 71, hand-fed (N = 10): 5 9 ± 1 03, parentally-fed (N = 10): 4 89 ± 1 06, F (2,27) = 1 86, P = 0 18), or timing of breeding (mean days from first nest found ± SE, control (N = 11): 40 55 ± 3 95, hand-fed (N = 10): 39 3 ± 5 72, Parentally-fed (N = 10): 33 56 ± 5 89, F (2,27) = 0 78, P = 0 47) between the experimental groups. Neither sex, nor its interaction with experimental group was significant for any variable. In addition, there were no significant differences in mean brood mass (g ± SE, Control: 8 57 ± 0 44, handfed: 8 88 ± 0 6, parentally-fed: 9 07 ± 0 6, F (2,23) = 0 35, P = 0 71) at the start of the experimental treatment. MOUTH COLOURATION MEASUREMENTS At 11 days post-hatching (and 1 day after cessation of supplementary feeding), we measured spectral mouth colouration following a standardized period of food deprivation. The largest and smallest nestlings from each brood were temporarily removed from the nest (for less than 2 h, always leaving at least one nestling within the nest) and taken to the field laboratory (always less than 15 min away by transport). While there is some sexual size dimorphism of Hihi nestlings, this is not sufficient to be used discriminatively (R. Thorogood, unpublished data). As we did not have the resources to genetically sex all individuals, we chose to measure the largest and smallest nestlings to ensure that we would have individuals of both sexes represented in our sample. After transport to the field laboratory, nestlings were stimulated to beg, and fed a standard sized meal (0 5 ml) of Wombaroo, a balanced honeyeater food containing a mix of protein, carbohydrates, and minerals (but not carotenoids, available from Wombaroo Food Products of Glen Osmond, South Australia). Following this, nestlings were placed in heated artificial nests (a plastic nest pan containing fleece material over a heat-retaining flask containing hot water) and maintained at 30 35 C using a temperature sensor fitted within the nest cup. Nestlings were then stimulated to beg every 20 min for a different experiment. Following 70 min of food deprivation, nestlings were again stimulated to beg and then fed to satiation. Following this standardization of hunger and temperature, we recorded reflectance spectra three times with a USB2000 plug and play spectrometer, DT-Mini Lamp (Deuterium Tungsten Halogen source) and a 100 μm premium grade reflectance probe (Ocean Optics Inc., Netherlands) from each of two locations on both left and right rictal flanges (measurements were later pooled for flanges), and mouth palate from each focal nestling. The probe illuminated an area of c. 1 5 mm, and was held perpendicular to the surface at 90, closely following the technique used by Hunt et al. (2003). Each measurement was an average of 10 scans, and was calculated relative to a diffuse reflectance standard (WS-1, Ocean Optics Inc., Netherlands). The spectrometer was calibrated before each brood was measured. All measurements were made by one researcher (RT). From these reflectance spectra, we calculated four physical colour measurements (following Johnsen et al. 2003; Montgomerie 2006; Peters et al. 2007). Reflectance spectra of colours visible as yellow to red typically have peaks in both the ultraviolet (UV) wavelengths and above 550 nm, making them difficult to describe using the common measures of hue and chroma (Cuthill et al. 1999). Therefore, we measured the proportion of relative reflectance in the wavelength region generally regarded as giving a yellow hue (Endler 1990) across the total spectrum (Yellow chroma, R 550 625 /R 300 700 ). This is analogous to the Carotenoid-Chroma recommended as the most appropriate objective colorimetric for quantifying spectral purity (Andersson & Prager 2006). The spectral intensity or brightness of this yellow region was calculated using average reflectance (Yellowbrightness, R 550 625 ). Because of the bimodality of yellow colours, we also calculated chroma and brightness measures of the wavelengths of 300 400 nm in order to quantify variation in the UV (UVchroma, R 300 400 /R 300 700 and UV-brightness, R 300 400 ). All our measures of colouration were significantly repeatable (Table 1; see Lessells & Boag 1987), with repeatabilities within the two locations measured for each mouth part very highly repeatable (all > 0 85, not presented). PLASMA CAROTENOIDS ANALYSIS Immediately following mouth colouration measurements we collected a small blood sample (c. 150 μl) from focal nestlings via brachial venipuncture. Blood was immediately centrifuged to separate and remove plasma. This was stored at 20 C until subsequent carotenoid analyses were performed. Carotenoids were extracted and determined by high performance liquid chromatography as described previously (Ewen et al. 2006c). In brief, 20 μl of plasma was homogenized with 40 μl of ethanol, then extracted by vortexing with 400 μl of hexane, twice. After centrifugation, hexane extracts were pooled and evaporated at 60 65 C under flow of nitrogen, and residue dissolved in 100 μl of a 50 : 50 mix of dichloromethane and methanol. Total carotenoid

Nestling mouth colour and carotenoids 1047 Table 1. Repeatabilities for colour measurements of (a) mouth palate and (b) rictal flanges. Two locations for each mouth part were measured three times each. Repeatabilities were calculated for all flange measures combined (both left and right flanges) r F P (a) Mouth palate: Yellow chroma 0 79 23 34 < 0 0001*** Yellow brightness 0 65 11 97 < 0 0001*** UV chroma 0 80 24 66 < 0 0001*** UV brightness 0 64 11 54 < 0 0001*** (b) Rictal flanges: Yellow chroma 0 59 18 60 < 0 0001*** Yellow brightness 0 62 20 62 < 0 0001*** UV chroma 0 51 13 78 < 0 0001*** UV brightness 0 44 10 53 < 0 0001*** d.f. = 62, 305. d.f. = 62, 699. concentration of each sample was quantified using a Spherisorb type S5NH2 reverse-phase column (250 4 6 mm, Phase Separation Ltd., Clwyd, UK) with a mobile phase of methanol distilled water (97 : 3) delivered at a flow rate of 1 5 ml min 1 (using Thermo Spectra System P1000 isocratic pumps), and detected with a UV/Vis absorbance detector at 445 nm. STATISTICAL ANALYSES We analysed plasma carotenoid concentrations and spectral parameters using linear mixed models with restricted maximum likelihood (REML) estimation in R2.4.1 (R Development Core Team, 2006). For tests of the effect of experimental treatment, nest was entered as a random term to account for multiple nestlings sampled from each brood. Analyses of spectral parameters included nestling identity, nested within nest, and the entire term treated as a random effect to account for multiple measurements of individuals and nests. Brood size, nestling mass (measured on day of spectral measurement) and date (days from first nest found) were initially entered into all models as covariates to control for potential confounding variation, and their interactions with the fixed carotenoid-treatment factor were also included to assess validity of the model. No significant covariate interactions were identified, and covariates were only retained in models where their contribution to the model fit was significant (using the AIC). All spectral variables were log-transformed to normalise the residuals, and the validity of models was checked by visual inspection of residual plots and normal probability plots of withingroup errors and random effects (Pinheiro & Bates 2000). To control for multiple comparisons, we used the Benjamini and Hochberg (1995) False Discovery Rate technique as this is less conservative than the Bonferroni correction, the use of which in bird colouration studies has been criticized (Montgomerie 2006). As we used mixed models, means ± SEs as predicted by the final models are presented. Results PLASMA CAROTENOIDS AND EXPERIMENTAL TREATMENT Provisioning of carotenoids both directly to nestlings, and via their parents, resulted in increased circulating plasma Fig. 1. Mean (± SE, model estimates from REML mixed model with nest as a random effect) plasma carotenoids (μg ml 1 ) of nestlings from nests provided with sugar water (control), hand-fed carotenoids, or parentally-fed supplementary carotenoids. All three groups were significantly different from each other in a priori Treatment Contrast tests (all P < 0 001). carotenoid levels which were significantly greater than those of control nestlings (Fig. 1, F (2,30) = 81 28, P < 0 0001, no covariates contributed significantly to the model). This indicates that the experimental provisioning of carotenoids via both hand-feeding and parental supplementation successfully increased carotenoid availability to nestlings when compared to controls. MOUTH COLOURATION AND AVAILABILITY OF CAROTENOIDS The reflectance spectra of both mouth palate and rictal flanges of nestlings were characterized by a large peak of reflectance in the UV (300 400 nm), a depression from 400 to 500 nm, and a sharp increase in reflectance from 500 nm (Fig. 2). Therefore, not only do the mouth palate and flanges appear yellow to humans, but they also have a striking peak in the UV, visible to birds. As well as the gross differences detectable from the mean reflectance spectra, there were large differences in the spectral parameters of the mouth palate (Fig. 2a) and rictal flanges (Fig. 2b) between carotenoid treatment groups (Table 2). Nestlings with greater access to carotenoids, either directly or because of parental feeding, expressed greater yellow chromaticity of the flanges while only carotenoid supplementation by parents correlated with greater yellow chroma of the mouth palate. The chroma and brightness of UV wavelengths however, decreased with availability of carotenoids (Table 2). This negative relationship was strongly significant for the rictal flanges, while the results for the mouth palate displayed a similar negative trend but was not statistically significant. Yellow brightness of both palate and flanges remained similar between treatment groups. Therefore, increased carotenoid availability resulted in a more saturated yellow palate and rictal flange and decreased reflectance of UV wavelengths from the rictal flanges.

1048 R. Thorogood et al. Discussion Fig. 2. Reflectance spectra (mean ± SE) of (a) mouth palate and (b) rictal flanges of nestlings from nests provided with either sugar water (control, dashed line), hand-fed carotenoids (grey line), or parentally-provided supplementary carotenoids (black line). Means calculated from all individuals, irrespective of nest, and curves smoothed by binning readings at 7 5 nm intervals. The yellow orange colour of the mouth palate and flanges of nestling birds has been suggested to act as a signal to parents, either of some phenotypic condition, or for detectability in a dark environment (see Introduction). While most attention has been given to exploring the potential role of mouth colouration, far less work has been conducted on the physiological mechanism behind mouth colour. In this study, we have shown that carotenoid supplementation, either by hand-feeding or by parental supplementation, leads to an increase in circulating plasma carotenoids, and changes in palate and rictal flange colouration. Importantly, this is not limited to only the human-visible wavelengths, but also affects the amount of UV that is reflected. Carotenoid availability therefore can influence mouth colour, potentially changing nestling detectability (Kilner & Davies 1998; Heeb et al. 2003) and influencing any information that parents may gather from the colour of the gape (Kilner 1997; Saino et al. 2000; Clotfelter et al. 2003; Ewen et al. 2008). Carotenoid provisioning increased the chroma of the yellow-colour of both the mouth palate and flanges, and decreased the UV-brightness and UV-chroma of the flanges in particular. Carotenoids are subtractive colorants (Andersson & Prager 2006), absorbing light most strongly between 400 and 500 nm (Burkhardt 1989). This is exemplified by the deep trough of the spectral reflectance curves between these wavelengths (Fig. 2). Because of this absorbance, carotenoids directly affect the saturation of colours. Increasing amounts of carotenoid pigment increases the spectral absorbance and changes the shape of the spectral reflectance curve. Andersson and Prager (2006) demonstrated this relationship via Table 2. Spectral parameters (mean ± SE, model estimates from REML mixed models with nest included as a random effect) of (a) mouth palate and (b) rictal flanges of two nestlings from each nest from experimental groups either hand- or parentally-fed carotenoids or sugar water as a control. Bolded, capital letters denote significant differences (P < 0 05) between groups following a priori Treatment contrasts with shared letters denoting non-significant differences Control (N = 11) Hand-fed (N = 10) Parentally-fed (N = 10) F P (a) Mouth palate: Yellow chroma 1 574 ± 0 025 A 1 498 ± 0 026 B 1 414 ± 0 026 C 9 95 < 0 0001*** Yellow brightness 7 064 ± 0 213 7 048 ± 0 196 7 000 ± 0 187 0 22 0 807 UV chroma 0 991 ± 0 052 0 978 ± 0 07 1 085 ± 0 061 2 37 0 113 UV brightness 6 912 ± 0 074 6 820 ± 0 077 6 654 ± 0 079 2 89 0 072 (b) Rictal flanges: Yellow chroma 1 240 ± 0 056 A 1 166 ± 0 051 B 1 119 ± 0 049 B 10 48 < 0 0001*** Yellow brightness 7 745 ± 0 126 7 804 ± 0 116 7 750 ± 0 111 0 64 0 535 UV chroma 1 279 ± 0 036 A 1 307 ± 0 047 A 1 380 ± 0 041 B 4 18 0 026* UV brightness 7 448 ± 0 041 A 7 394 ± 0 042 A 7 250 ± 0 044 B 5 80 0 008** All P values 0 03 were not significant after false discovery rate (FDR) control for multiple testing. Nestling mass and brood size when colour measured were entered into all models as covariates, but did not contribute significantly to models (using AIC). d.f. = 2, 28. d.f. = 2, 27. Controlling for date (F (1,27) = 9 15, P = 0 005). Controlling for parental age (F (1,27) = 10 71, P = 0 003). Controlling for date (F (1,27) = 10 39, P = 0 003). Controlling for date (F (1,27) = 9 78, P = 0 004). Controlling for parental age (F (1,27) = 4 54, P = 0 04).

Nestling mouth colour and carotenoids 1049 a simulation of lutein concentration and measured brightness, hue and carotenoid-chroma (analogous to our measure of yellow-chroma). As lutein (the main carotenoid circulated by, and present in adult Hihi males yellow feathers (Ewen et al. 2006b), and one of the xanthophyll carotenoids that most commonly colours yellow integuments (McGraw 2006a)) deposition increased, only carotenoid-chroma varied. This relationship is also empirically common, with carotenoids often correlating with the chroma of yellow orange plumage colour (e.g. Saks, McGraw & Hoark 2003; McGraw & Gregory 2004; Tschirren, Fitze & Richner 2005) and integuments (e.g. beak colour in adult zebra finches Taeniopygia guttata Mcgraw et al. 2003). Our results demonstrate that increasing the availability of carotenoids, either directly to nestlings, or indirectly via parental supplementation, leads to an increase in the chroma of the yellow orange colour of both the mouth palate and the rictal flanges. While the role of human-visible mouth colours has received some attention, avian-visible UV mouth colouration has been only recently noticed. Flanges and gapes of all species thus far measured highly reflect UV wavelengths, with the flanges reflecting more than gapes (e.g. Barn swallow, European Blackbird, Blue Tit Parus caeruleus, House Sparrow (Hunt et al. 2003); Starling Sturnus vulgaris (Jourdie et al. 2004)). To the best of our knowledge, our measures are the first published example of the reflectance spectra of the mouth of a non-european species, and show that Hihi are no different. This is of particular interest not only because of their spatial distance from the species previously investigated, but because they are phylogenetically distant from these northern hemisphere species (Ewen, Flux & Ericson 2006a; Driskell et al. 2007). Both the rictal flanges and the inside of the mouth reflected strongly in the UV region of the spectrum. Although UV colouration is most likely structural rather than pigment-based, with the colour created by the interaction of light wavelengths with micro structures of the object s surface (Prum & Torres 2003), there is some potential for carotenoids to contribute to the amount of UV visible signals (Bleiweiss 2005). In this study, we found that increasing the availability of carotenoids strongly resulted in a decrease in UV reflectance of rictal flanges, with a similar, but nonsignificant relationship within the mouth. From studies of plumage, it has been suggested that increased carotenoids may actually obscure UV colours by acting as a filter (Shawkey & Hill 2005). The interaction between structural UV and carotenoid-based colouration of nestling mouth parts may be no different. For example, when the strongly carotenoid-laden epidermis of the integumentary comb ornament of the Red Grouse Lagopus lagopus was removed, it was found that the dermis reflected more in the UV than the intact comb (Mougeot et al. 2007). Carotenoid availability may similarly interact with UV light reflected from the dermis of the flanges (and possibly the gape), creating a more complex signal than that which has been previously assumed. Reduced UV reflectance coupled with a more saturated carotenoid-based colour may be the signal that parents respond to. The synergy of these aspects of the spectral reflectance of the mouthparts is very important to understand and we should understand whether, how, and why parents may respond to differences in UV (e.g. Jourdie et al. 2004; De Ayala et al. 2007) and other coloured signals (e.g. Saino et al. 2000). Our study clearly shows that pigmentation of nestling mouths and flanges is undoubtedly affected by carotenoid availability and circulating levels in the plasma. Despite this, in the context of nestling mouth colouration it remains to be determined by what biochemical pathways carotenoids are transported, how pigments are assimilated into mouths and flanges, and how this in turn may be modulated by other physiological mechanisms. Carotenoids are transported in the blood via lipoproteins, with increased cholesterol availability shown to raise levels of circulating carotenoids in adult zebra finches, resulting in a more colourful bill (McGraw & Parker 2006). This synergy between lipoprotein and carotenoid availability is further modulated by testosterone, which upregulates cholesterol (in both male and female Red-legged Partridge Alectoris rufa (Blas et al. 2006) and male Zebra Finch (McGraw, Correa & Adkins-Regan 2006), but with a different effect in females (McGraw 2006c)). In nestlings, it appears that the relationship between carotenoid availability and mouth colouration may be mediated in part by stresslevels, with corticosterone recently demonstrated to negatively affect flange colouration in the House Sparrow (Loiseau et al. 2008). Future work is required to determine what role testosterone may play in the transportation and deposition of carotenoid pigments in nestlings. Here, we have experimentally demonstrated that dietary carotenoids are responsible for nestling mouth colouration. However, elucidating exactly which carotenoids are acting as pigments and how this expression is mediated by other physiological systems requires further research. Acknowledgements The authors thank Patricia Brekke, Rebecca Gribble, Renske Kwikkel and Claudia Carraro for assistance in the field and laboratory. This manuscript was greatly improved by helpful discussion and review by Mark Hauber, and the comments of three anonymous referees. Kemin Industries and New Zealand Sugar Company donated OroGlo and sugar, respectively, while accommodation and further logistical support was given by the Supporters of Tiritiri Matangi Island Inc. Carotenoid extraction and high performance liquid chromatography were conducted with kind support of Hülya Özdemir in the laboratory of the Department of Pharmacy, Yüzüncü Yıl University Research Hospital. 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