The Making of a Flight Feather: Bio-architectural Principles and Adaptation.

The evolution of flight in feathered dinosaurs and early birds over millions of years required flight feathers whose architecture features hierarchical branches. While barb-based feather forms were investigated, feather shafts and vanes are understudied. Here, we take a multi-disciplinary approach to study their molecular control and bio-architectural organizations. In rachidial ridges, epidermal progenitors generate cortex and medullary keratinocytes, guided by Bmp and transforming growth factor ß (TGF-ß) signaling that convert rachides into adaptable bilayer composite beams. In barb ridges, epidermal progenitors generate cylindrical, plate-, or hooklet-shaped barbule cells that form fluffy branches or pennaceous vanes, mediated by asymmetric cell junction and keratin expression. Transcriptome analyses and functional studies show anterior-posterior Wnt2b signaling within the dermal papilla controls barbule cell fates with spatiotemporal collinearity. Quantitative bio-physical analyses of feathers from birds with different flight characteristics and feathers in Burmese amber reveal how multi-dimensional functionality can be achieved and may inspire future composite material designs. VIDEO ABSTRACT.

Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars.

Parrot feathers contain red, orange, and yellow polyene pigments called psittacofulvins. Budgerigars are parrots that have been extensively bred for plumage traits during the last century, but the underlying genes are unknown. Here we use genome-wide association mapping and gene-expression analysis to map the Mendelian blue locus, which abolishes yellow pigmentation in the budgerigar. We find that the blue trait maps to a single amino acid substitution (R644W) in an uncharacterized polyketide synthase (MuPKS). When we expressed MuPKS heterologously in yeast, yellow pigments accumulated. Mass spectrometry confirmed that these yellow pigments match those found in feathers. The R644W substitution abolished MuPKS activity. Furthermore, gene-expression data from feathers of different bird species suggest that parrots acquired their colors through regulatory changes that drive high expression of MuPKS in feather epithelia. Our data also help formulate biochemical models that may explain natural color variation in parrots. VIDEO ABSTRACT.

Cyclic growth of dermal papilla and regeneration of follicular mesenchymal components during feather cycling.

How dermis maintains tissue homeostasis in cyclic growth and wounding is a fundamental unsolved question. Here, we study how dermal components of feather follicles undergo physiological (molting) and plucking injury-induced regeneration in chickens. Proliferation analyses reveal quiescent, transient-amplifying (TA) and long-term label-retaining dermal cell (LRDC) states. During the growth phase, LRDCs are activated to make new dermal components with distinct cellular flows. Dermal TA cells, enriched in the proximal follicle, generate both peripheral pulp, which extends distally to expand the epithelial-mesenchymal interactive interface for barb patterning, and central pulp, which provides nutrition. Entering the resting phase, LRDCs, accompanying collar bulge epidermal label-retaining cells, descend to the apical dermal papilla. In the next cycle, these apical dermal papilla LRDCs are re-activated to become new pulp progenitor TA cells. In the growth phase, lower dermal sheath can generate dermal papilla and pulp. Transcriptome analyses identify marker genes and highlight molecular signaling associated with dermal specification. We compare the cyclic topological changes with those of the hair follicle, a convergently evolved follicle configuration. This work presents a model for analyzing homeostasis and tissue remodeling of mesenchymal progenitors.

Changes in body surface temperature reveal the thermal challenge associated with catastrophic moult in captive gentoo penguins.

Once a year, penguins undergo a catastrophic moult, replacing their entire plumage during a fasting period on land or on sea-ice during which time individuals can lose 45% of their body mass. In penguins, new feather synthesis precedes the loss of old feathers, leading to an accumulation of two feather layers (double coat) before the old plumage is shed. We hypothesized that the combination of the high metabolism required for new feather synthesis and the potentially high thermal insulation linked to the double coat could lead to a thermal challenge requiring additional peripheral circulation to thermal windows to dissipate the extra heat. To test this hypothesis, we measured the surface temperature of different body regions of captive gentoo penguins (Pygoscelis papua) throughout the moult under constant environmental conditions. The surface temperature of the main body trunk decreased during the initial stages of the moult, suggesting greater thermal insulation. In contrast, the periorbital region, a potential proxy of core temperature in birds, increased during these same early moulting stages. The surface temperature of the bill, flipper and foot (thermal windows) tended to initially increase during the moult, highlighting the likely need for extra heat dissipation in moulting penguins. These results raise questions regarding the thermoregulatory capacities of penguins in the wild during the challenging period of moulting on land in the current context of global warming.

Differential expression of ASIP transcripts reveals genetic mechanism underpinning black-tail independence from body plumage in yellow-bodied chickens.

The genetic foundation of chicken body plumage color has been extensively studied. However, little attention has been paid to the inheritance patterns and molecular mechanisms underlying the formation of distal feather colors (tail and wingtip). Differences in these colors are common; for example, the Chinese Huiyang Beard chicken has black tail feathers, but yellow body plumage. Here, the hybrid offspring of Huiyang Beard and White Leghorn chickens were used to study the inheritance patterns of tail-feather color. The expression levels of pigment genes in differently colored feather follicles were analyzed using quantitative real-time PCR. The results showed that genetic regulation of tail-feather color was independent of body-plumage color. The Dominant White locus inhibited eumelanin synthesis in tail feathers without affecting the formation of yellow body plumage, whereas the Silver locus had the opposite effect. The expression of agouti signaling protein (ASIP) gene class 1 transcripts was significantly lower in black tail-feather follicles than in yellow body follicles, whereas tyrosinase-related protein 1 (TYRP1) gene expression was significantly higher in black tail feathers. These differentially expressed genes were confirmed to exert an effect on eumelanin and pheomelanin formation in feathers, thus influencing the regulation of chicken tail-feather color. In conclusion, this study lays the foundation for further research on the genetic mechanisms of regional differences in feather color, contributing to a better understanding of plumage pigmentation in chickens.

Cis-acting mutation affecting GJA5 transcription is underlying the Melanotic within-feather pigmentation pattern in chickens.

Melanotic (Ml) is a mutation in chickens that extends black (eumelanin) pigmentation in normally brown or red (pheomelanin) areas, thus affecting multiple within-feather patterns [J. W. Moore, J. R. Smyth Jr, J. Hered. 62, 215-219 (1971)]. In the present study, linkage mapping using a back-cross between Dark Cornish (Ml/Ml) and Partridge Plymouth Rock (ml+/ml+ ) chickens assigned Ml to an 820-kb region on chromosome 1. Identity-by-descent mapping, via whole-genome sequencing and diagnostic tests using a diverse set of chickens, refined the localization to the genomic region harboring GJA5 encoding gap-junction protein 5 (alias connexin 40) previously associated with pigmentation patterns in zebrafish. An insertion/deletion polymorphism located in the vicinity of the GJA5 promoter region was identified as the candidate causal mutation. Four different GJA5 transcripts were found to be expressed in feather follicles and at least two showed differential expression between genotypes. The results showed that Melanotic constitutes a cis-acting regulatory mutation affecting GJA5 expression. A recent study established the melanocortin-1 receptor (MC1R) locus and the interaction between the MC1R receptor and its antagonist agouti-signaling protein as the primary mechanism underlying variation in within-feather pigmentation patterns in chickens. The present study advances understanding the mechanisms underlying variation in plumage color in birds because it demonstrates that the activity of connexin 40/GJA5 can modulate the periodic pigmentation patterns within individual feathers.

Sonic hedgehog specifies flight feather positional information in avian wings.

Classical tissue recombination experiments performed in the chick embryo provide evidence that signals operating during early limb development specify the position and identity of feathers. Here, we show that Sonic hedgehog (Shh) signalling in the embryonic chick wing bud specifies positional information required for the formation of adult flight feathers in a defined spatial and temporal sequence that reflects their different identities. We also reveal that Shh signalling is interpreted into specific patterns of Sim1 and Zic transcription factor expression, providing evidence of a putative gene regulatory network operating in flight feather patterning. Our data suggest that flight feather specification involved the co-option of the pre-existing digit patterning mechanism and therefore uncovers an embryonic process that played a fundamental step in the evolution of avian flight.

Urbanisation impacts plumage colouration in a songbird across Europe: Evidence from a correlational, experimental and meta-analytical approach.

Urbanisation is accelerating across the globe, transforming landscapes, presenting organisms with novel challenges, shaping phenotypes and impacting fitness. Urban individuals are claimed to have duller carotenoid-based colouration, compared to their non-urban counterparts, the so-called 'urban dullness' phenomenon. However, at the intraspecific level, this generalisation is surprisingly inconsistent and often based on comparisons of single urban/non-urban populations or studies from a limited geographical area. Here, we combine correlational, experimental and meta-analytical data on a common songbird, the great tit Parus major, to investigate carotenoid-based plumage colouration in urban and forest populations across Europe. We find that, as predicted, urban individuals are paler than forest individuals, although there are large population-specific differences in the magnitude of the urban-forest contrast in colouration. Using one focal region (Malmö, Sweden), we reveal population-specific processes behind plumage colouration differences, which are unlikely to be the result of genetic or early-life conditions, but instead a consequence of environmental factors acting after fledging. Finally, our meta-analysis indicates that the urban dullness phenomenon is well established in the literature, for great tits, with consistent changes in carotenoid-based plumage traits, particularly carotenoid chroma, in response to anthropogenic disturbances. Overall, our results provide evidence for uniformity in the 'urban dullness' phenomenon but also highlight that the magnitude of the effect on colouration depends on local urban characteristics. Future long-term replicated studies, covering a wider range of species and feeding guilds, will be essential to further our understanding of the eco-evolutionary implications of this phenomenon.

Integration of GWAS and eGWAS to screen candidate genes underlying green head traits in male ducks.

Sexually dimorphic plumage coloration is widespread in birds. The male possesses more brightly colored feathers than the female. Dark green head feathers comprise one of the most typical appearance characteristics of the male Ma duck compared with the female. However, there are noticeable individual differences observed in these characteristics. Herein, genome-wide association studies (GWAS) were employed to investigate the genetic basis of individual differences in male duck green head-related traits. Our results showed that 165 significant SNPs were associated with green head traits. Meanwhile, 71 candidate genes were detected near the significant SNPs, including four genes (CACNA1I, WDR59, GNAO1 and CACNA2D4) related to the individual differences in the green head traits of male ducks. Additionally, the eGWAS identified three SNPs located within two candidate genes (LOC101800026 and SYNPO2) associated with TYRP1 gene expression, and might be important regulators affecting the expression level of TYRP1 in the head skin of male ducks. Our data also suggested that transcription factor MXI1 might regulate the expression of TYRP1, thereby causing differences in the green head traits among male ducks. This study provided primary data for further analysis of the genetic regulation of duck feather color.

Star finches Neochmia ruficauda have a visual preference for white dot patterns: a possible case of trypophilia.

Many animals have polka dot patterns on their body surface, some of which are known to have signalling functions; however, their evolutionary origins remain unclear. Dot patterns can trigger a fear response (trypophobia) in humans and are known to function as aposematic signals in non-human animals, suggesting that dots may deserve attention for biological reasons. Interestingly in many birds, plumage dot patterns serve for social/sexual signalling. To understand their evolution, we have focused on the sensory bias hypothesis, which predicts the role of pre-existing sensory preference driven by natural selection in shaping signal design. Our previous phylogenetic comparative study supported the hypothesis and showed that diet-driven visual preference promoted the evolution of plumage patterns, as there was an evolutionary correlation between termite-eating (white roundish gregarious prey) and the presence of plumage dot patterns in species of the family Estrildidae. This suggests that these species possess an intrinsic preference for dots. To test this, we compared the responses of an Estrildid species with dot plumage pattern (star finch Neochmia ruficauda) towards simultaneously presented monochrome-printed white dot vs white stripe patterns under both food-deprived and -supplied conditions. Overall, star finches preferred dots to stripes. They showed foraging-like behaviours almost only toward dots when hungry and gazed at dots frequently even when food was available, suggesting both hunger-related and hunger-neutral dot preferences. These results are rather surprising, given how strongly the subjects were attracted to abstract dot patterns without organic structure, but provided good support for the sensory bias hypothesis.

Practices and issues of moulting programs for laying hens: a review.

1. Moulting is a natural physiological process in birds when they shed their old feathers and replace them with new ones, and it is followed by reproductive quiescence resulting in reduced egg production. Different birds undergo moulting at different points in their life. Some birds have seasonal moulting while some moult at the end of their breeding cycle. This review will mainly focus on moulting practices associated with commercial layer birds because, in all other bird types, this is not managed.2. Commercial farms commonly analyse the cost-benefit ratio to decide the time and method to adopt for moulting. Commercial layer farms adopt different practices to force birds out of moult and restart the production cycle. Animal welfare groups consider this as stressful and against animal welfare, raising questions about the ethics of this practice.3. Many studies have been conducted using complete or partial feed withdrawal and non-feed withdrawal programs to measure their effectiveness in maintaining animal welfare, economy, and post-moult performance in mind.4. Animal welfare should not be compromised during moulting. The United States Egg Producers and other such groups from the United Kingdom and Europe have decided to sell eggs produced only through a non-feed withdrawal moulting programs.

Transcriptome profiling of morphogenetic differences between contour and flight feathers in duck.

1. This study examined the transcriptomic profiles of contour and flight feather follicles from two duck breeds to determine the molecular network and the candidate genes associated with contour and flight feather morphogenesis.2. High-throughput RNA sequencing was performed to compare differences in feather follicles between contour and flight feathers in two duck breeds (Heiwu and Nonghua duck).3. Comparing the contour feather follicles with flight feather follicles, 4,757 and 4,820 differentially expressed genes (DEGs) were identified in Heiwu and Nonghua duck respectively. Weighted gene co-expression network analysis (WGCNA) was used to construct a gene co-expression network of all DEGs and identify the key modules and hub genes associated with feather morphogenesis.4. Two key modules were enriched in many pathways involved in feather morphogenesis, such as the Wnt signalling pathway, anatomical structure morphogenesis, and focal adhesion. The CCNA2, TTK, NUF2, ECT2 and INCENP (in one module), and PRSS23, LAMC1, IGFBP3, SHISA5, and APLP2 (in another module) may be essential candidate genes for influencing feather morphology. Moreover, seven transcription factors (TFs) (UBP1, MBD2, ZNF512B, SMAD1, CAPN15, JDP2, KLF10, and MEF2A) were predicted to regulate the essential genes that contribute to feather morphogenesis.5. This work demonstrated gene expression changes of contour and flight feather follicles and is beneficial for further understanding of the complex structure of feathers.

Long non-coding RNAs regulate Wnt signaling during feather regeneration.

Long non-coding RNAs (lncRNAs) are non-protein coding transcripts that are involved in a broad range of biological processes. Here, we examine the functional role of lncRNAs in feather regeneration. RNA-seq profiling of the regenerating feather blastema revealed that Wnt signaling is among the most active pathways during feather regeneration, with Wnt ligands and their inhibitors showing distinct expression patterns. Co-expression analysis identified hundreds of lncRNAs with similar expression patterns to either the Wnt ligands (the Lwnt group) or their downstream target genes (the Twnt group). Among these, we randomly picked two lncRNAs in the Lwnt group and three lncRNAs in the Twnt group to validate their expression and function. Members in the Twnt group regulated feather regeneration and axis formation, whereas members in the Lwnt group showed no obvious phenotype. Further analysis confirmed that the three Twnt group members inhibit Wnt signal transduction and, at the same time, are downstream target genes of this pathway. Our results suggest that the feather regeneration model can be utilized to systematically annotate the functions of lncRNAs in the chicken genome.

Coupling of apical-basal polarity and planar cell polarity to interpret the Wnt signaling gradient in feather development.

Sensing a global directional cue to orient cell growth is crucial in tissue morphogenesis. An anterior-posterior gradient of Wnt signaling controls the helical growth of feather branches (barbs), and thus the formation of bilateral feathers. However, it remains unclear how the keratinocytes sense this gradient and orient barb growth. Here, we show that in chicken, owing to feather branching, the global Wnt gradient is subdivided into periodic barbs. Within each barb, the anterior barbule plate cells tilt before the posterior cells. The core planar cell polarity gene Prickle1 is involved, as knockdown of its expression resulted in no cell shape change and no barb tilting. Furthermore, perturbation of the Wnt gradient leads to diffusive Prickle1 expression and loss of barb orientation. Finally, the asymmetric distribution of Wnt6/Fzd10 is coordinated by the apical-basal polarity of the barbule plate keratinocytes, which is in turn regulated by the Par3/aPKC machinery. Our data elucidate a new mechanism through which the global Wnt signaling gradient is interpreted locally to construct complex spatial forms.

Mechanisms involved in the production of differently colored feathers in the structurally colored swallow tanager (Tersina viridis; Aves: Thraupidae).

Non-iridescent, structural coloration in birds originates from the feather's internal nanostructure (the spongy matrix) but melanin pigments and the barb's cortex can affect the resulting color. Here, we explore how this nanostructure is combined with other elements in differently colored plumage patches within a bird. We investigated the association between light reflectance and the morphology of feathers from the back and belly plumage patches of male swallow tanagers (Tersina viridis), which look greenish-blue and white, respectively. Both plumage patches have a reflectance peak around 550 nm but the reflectance spectrum is much less saturated in the belly. The barbs of both types of feathers have similar spongy matrices at their tips, rendering their reflectance spectra alike. However, the color of the belly feather barbs changes from light green at their tips to white closer to the rachis. These barbs lack pigments and their morphology changes considerably throughout. Toward the rachis, the barb is almost hollow, with a reduced area occupied by spongy matrix, and has a flattened shape. By contrast, the blue back feathers' barbs have melanin underneath the spongy matrix resulting in a much more saturated coloration. The color of these barbs is also even along the barbs' length. Our results suggest that the color differences between the white and greenish-blue plumage are mostly due to the differential deposition of melanin and a reduction of the spongy matrix near the rachis of the belly feather barbs and not a result of changes in the characteristics of the spongy matrix.

The effects of life history and sexual selection on male and female plumage colouration.

Classical sexual selection theory provides a well-supported conceptual framework for understanding the evolution and signalling function of male ornaments. It predicts that males obtain greater fitness benefits than females through multiple mating because sperm are cheaper to produce than eggs. Sexual selection should therefore lead to the evolution of male-biased secondary sexual characters. However, females of many species are also highly ornamented. The view that this is due to a correlated genetic response to selection on males was widely accepted as an explanation for female ornamentation for over 100 years and current theoretical and empirical evidence suggests that genetic constraints can limit sex-specific trait evolution. Alternatively, female ornamentation can be the outcome of direct selection for signalling needs. Since few studies have explored interspecific patterns of both male and female elaboration, our understanding of the evolution of animal ornamentation remains incomplete, especially over broad taxonomic scales. Here we use a new method to quantify plumage colour of all ~6,000 species of passerine birds to determine the main evolutionary drivers of ornamental colouration in both sexes. We found that conspecific male and female colour elaboration are strongly correlated, suggesting that evolutionary changes in one sex are constrained by changes in the other sex. Both sexes are more ornamented in larger species and in species living in tropical environments. Ornamentation in females (but not males) is increased in cooperative breeders--species in which female-female competition for reproductive opportunities and other resources related to breeding may be high. Finally, strong sexual selection on males has antagonistic effects, causing an increase in male colouration but a considerably more pronounced reduction in female ornamentation. Our results indicate that although there may be genetic constraints to sexually independent colour evolution, both female and male ornamentation are strongly and often differentially related to morphological, social and life-history variables.

Hybrid speciation leads to novel male secondary sexual ornamentation of an Amazonian bird.

Hybrid speciation is rare in vertebrates, and reproductive isolation arising from hybridization is infrequently demonstrated. Here, we present evidence supporting a hybrid-speciation event involving the genetic admixture of the snow-capped (Lepidothrix nattereri) and opal-crowned (Lepidothrix iris) manakins of the Amazon basin, leading to the formation of the hybrid species, the golden-crowned manakin (Lepidothrix vilasboasi). We used a genome-wide SNP dataset together with analysis of admixture, population structure, and coalescent modeling to demonstrate that the golden-crowned manakin is genetically an admixture of these species and does not represent a hybrid zone but instead formed through ancient genetic admixture. We used spectrophotometry to quantify the coloration of the species-specific male crown patches. Crown patches are highly reflective white (snow-capped manakin) or iridescent whitish-blue to pink (opal-crowned manakin) in parental species but are a much less reflective yellow in the hybrid species. The brilliant coloration of the parental species results from nanostructural organization of the keratin matrix feather barbs of the crown. However, using electron microscopy, we demonstrate that the structural organization of this matrix is different in the two parental species and that the hybrid species is intermediate. The intermediate nature of the crown barbs, resulting from past admixture appears to have rendered a duller structural coloration. To compensate for reduced brightness, selection apparently resulted in extensive thickening of the carotenoid-laden barb cortex, producing the yellow crown coloration. The evolution of this unique crown-color signal likely culminated in premating isolation of the hybrid species from both parental species.

Archaeorhynchus preserving significant soft tissue including probable fossilized lungs.

We describe a specimen of the basal ornithuromorph Archaeorhynchus spathula from the Lower Cretaceous Jiufotang Formation with extensive soft tissue preservation. Although it is the fifth specimen to be described, unlike the others it preserves significant traces of the plumage, revealing a pintail morphology previously unrecognized among Mesozoic birds, but common in extant neornithines. In addition, this specimen preserves the probable remnants of the paired lungs, an identification supported by topographical and macro- and microscopic anatomical observations. The preserved morphology reveals a lung very similar to that of living birds. It indicates that pulmonary specializations such as exceedingly subdivided parenchyma that allow birds to achieve the oxygen acquisition capacity necessary to support powered flight were present in ornithuromorph birds 120 Mya. Among extant air breathing vertebrates, birds have structurally the most complex and functionally the most efficient respiratory system, which facilitates their highly energetically demanding form of locomotion, even in extremely oxygen-poor environments. Archaeorhynchus is commonly resolved as the most basal known ornithuromorph bird, capturing a stage of avian evolution in which skeletal indicators of respiration remain primitive yet the lung microstructure appears modern. This adds to growing evidence that many physiological modifications of soft tissue systems (e.g., digestive system and respiratory system) that characterize living birds and are key to their current success may have preceded the evolution of obvious skeletal adaptations traditionally tracked through the fossil record.

Plumage development and environmental factors influence surface temperature gradients and heat loss in wandering albatross chicks.

Young birds in cold environments face a range of age-specific thermal challenges. Studying the thermal biology of young birds throughout ontogeny may further our understanding of how such challenges are met. We investigated how age and environmental parameters influenced surface temperature gradients across various body regions of wandering albatross (Diomedea exulans) chicks on Bird Island, South Georgia. This study was carried out over a 200 d period during the austral winter, from the end of the brood-guard period until fledging, bridging a gap in knowledge of surface temperature variation and heat loss in developing birds with a long nestling stage in severe climatic conditions. We found that variation in surface temperature gradients (i.e. the difference between surface and environmental temperature) was strongly influenced by chick age effects for insulated body regions (trunk), with an increase in the surface temperature gradient that followed the progression of plumage development, from the second set of down (mesoptiles), to final chick feathers (teleoptiles). Environmental conditions (primarily wind speed and relative humidity) had a stronger influence on the gradients in uninsulated areas (eye, bill) than insulated regions, which we interpret as a reflection of the relative degree of homeothermy exhibited by chicks of a given age. Based on biophysical modelling, total heat loss of chicks was estimated to increase linearly with age. However, mass specific heat loss decreased during the early stages of growth and then subsequently increased. This was attributed to age-related changes in feather growth and activity that increased surface temperature and, hence, metabolic heat loss. These results provide a foundation for further work on the effects of environmental stressors on developing chicks, which are key to understanding the physiological responses of animals to changes in climate in polar regions.

Effects of poor plumage conditions on egg production, antioxidant status and gene expression in laying hens.

The objective of this study was to investigate the effects of poor plumage conditions on production performance, antioxidant status and gene expression in laying hens. Two hundred ten 54-week-old laying hens with similar body weights were assigned into two groups based on plumage conditions (the poor plumage conditions (PPC) group and the control group). All the birds had free access to water and crumbled feed, and received the same management in step cages. Compared with hens in the control group, the hens in the PPC group consumed more feed and produced lighter eggs (P < 0.05). Hens in the PPC group showed lower serum concentrations of glutathione peroxidase and total antioxidant capacity and higher malondialdehyde content than those in the control group. The eggshell breaking strength was lower in the PPC group than in the control group (P < 0.05). The eggshell shape index and yolk colour in the PPC group were significantly higher than those in the control group. The mRNA expression level of HTR2C in the neck skin and that of IL-2 in the liver and breast muscle were higher in the PPC group than in the control group (P < 0.05). The results indicated that PPC may increase feed consumption and influence egg quality, antioxidant status and gene expression in laying hens.