-Discussion -Did the author(s) immediately state the study’s conclusions? (e.g. Was their hypothesis supported or not?) Did they provide adequate interpretation/explanation of their results? Did effect sizes indicate biological significance? Were results interpreted in the context of previous studies? Was it clear what the study contributes to ornithology? -Did the authors identify weaknesses/limitations of their study? Were avenues for further research suggested? Facial display and blushing: Means of visual communication in blue-and-yellow macaws (Ara Ararauna)? Aline Bertin , Arielle Beraud12, Léa Lansade’, Marie-Claire Blache’, Amandine Diot? Baptiste Mulot?, Cécile Arnould 1 PRC, CNRS, IFCE, INRA, Université de Tours, Nouzily, France, 2 ZooParc de Beauval & Beauval Nature, Saint-Aignan, France aline.bertin @inra.fr Abstract Mainly recognizet for their cognitive performance, the visual communication system and, particularly, the potential function of facial displays in parrots remain thus far unexplored. Here, we provide the first descriptive study of facial display use in captive blue-and-yellow macaws. We observed the feather position (sleeked or ruffled) on the crown, nape and cheek at the group level during the macaws’ daily routine and individually while interacting with a familiar animal caretaker. In the latter context, blushing was also assessed on the bare skin of the cheek. Group level observations showed that crown, nape and cheek feath- ers ruffling was more frequent in activities requiring no locomotion than in activities requiring locomotion. With the animal caretaker, crown ruffling was significantly more frequent when the caretaker was actively engaging with the parrot than during a control phase with no mutual interaction. In addition, a significantly higher proportion of naïve observers judged blushing as being present on photographs taken during the mutual interaction phase than during the control phase. We thus showed significant variations in facial displays and ba skin colour based on the birds’ social context and activity. Our results broaden the scope f further studies to determine whether parrots’ faces provide visual social signals Discusslon We provided the first descriptive study on facial feather display and bare skin colour variations in macaws. Our data show that feather ruffling varied with the activity in which the bird was engaged and that crown feathers ruffling varied significantly with the presence or absence of mutual interaction between the parrot and caretaker. Furthermore, blushing occurred signif- cantly more frequently during mutual interaction with the caretaker than in the control context. During the birds’ daily aviary routine, our data show a higher occurrence of feathér ruffling in activities requiring no locomotion than in activities requiring locomotion. In addition, the high frequency of feathers ruffling in activities like “rest” or “chew enrichments” suggests that head feathers ruffling (crown, nape and cheek) may be associated with states of low arousal level and positive valence (e-g. relaxed, calm) [28]. Head feathers ruffling was also associated with positive social interactions and preening. In finches, rufling the crown and nape feathers independently of the body feathers is considered a signal to concentrate mutual preening orn this specific region, which is less accessible when birds preen themselves (20). In addition, the spheroid posture engendered by ruffled body and nape feathers, is thought to stimulate clump- ing in social groups and to trigger the same body posturing in approaching birds (20.22). In macaws, when ruffled, head feathers may contribute to a more spheroid posture of the head (cf. Eig 5). So far, the receiver responses to these visual displays have not been explored in avian species. In spice finches (Lonchura punctulata), crown-ruffing is involved in agonistic interactions (29]. In crested birds, crest rising is well described as being involved in body threat displays or sexual displays (eg. [21.30,31l): however, in our study, no agonistic interactions were observed between parrots, and birds were non-breeding juveniles (no expression of sex ual displays). The high proportion of scans with nape ruffing during low arousal levels is also consistent with observations in passerine birds. When at rest, body and nape ruffling is thought to compensate for heat loss and lower heat production [20) In the second part of the study, we found significantly more secking behaviours towards the caretaker during the control phase than during the mutual interaction phase. As predicted Facial display and blushing: Means of visual communication in blue-and-yell ual displays). The high proportion of scans with nape ruffling during low arousal levels is also consistent with observations in passerine birds. When at rest, body and nape ruffing is thought to compensate for heat loss and lower heat production [20). In the second part of the study, we found significantly more seeking behaviours towards the caretaker during the control phase than during the mutual interaction phase. As predicted, Fig 5. Examples of head displays. A) Photograph of a macaw with all feathers sleeked B) The same macaw with all feathers ruffled. (All photographs taken by C. Arnould) 8/12 August 22 2018 load PDF Facial display and blushing Means of visual communication in blue-and-yell In conclusion, although caution must be exercised when interpreting these data due to the small sample size, we argue that crown ruffling and skin colour variation may provide facial inner subjective feelings. They may provide indicators of arousal level and/ or emotional valence, two key components of animal affective states 128]. Additional investiga- tions would help to better understand this communication system. Particularly, analysing the potential responses of receivers to visual displays is now required to determine whether birds do use variations in facial display as visual signals. We also hope to see future investigations in PLOS ONE Ib/dolorg/10 1371 ournal pone 0201782 August 22, 2018 9/12 PLOSNE Facial displays in parrots natural populations since captivity and human interactions may alter the way birds use facial displays. From an applied point of view, understanding visual communication in parrots may help to assess their well-being in captive conditions. Parrots are popular companion animals, with millions of parrots being kept as pets, but they are particularly vulnerable to being stereo- typed LA3dil. Facial expression of emotion is well described in several mammalian species (e-g 1124546) but not in avian species. How birds use facial displays and whether they com- municate their inner subjective feelings is a question that is crucial to deepening our under ling of bird sentience