Captivity affects mitochondrial aerobic respiration and carotenoid metabolism in the house finch (Haemorhous mexicanus).

In many species of animals, red carotenoid-based coloration is produced by metabolizing yellow dietary pigments, and this red ornamentation can be an honest signal of individual quality. However, the physiological basis for associations between organism function and the metabolism of red ornamental carotenoids from yellow dietary carotenoids remains uncertain. A recent hypothesis posits that carotenoid metabolism depends on mitochondrial performance, with diminished red coloration resulting from altered mitochondrial aerobic respiration. To test for an association between mitochondrial respiration and red carotenoids, we held wild-caught, molting male house finches in either small bird cages or large flight cages to create environmental challenges during the period when red ornamental coloration is produced. We predicted that small cages would present a less favorable environment than large flight cages and that captivity itself would decrease both mitochondrial performance and the abundance of red carotenoids compared with free-living birds. We found that captive-held birds circulated fewer red carotenoids, showed increased mitochondrial respiratory rates, and had lower complex II respiratory control ratios - a metric associated with mitochondrial efficiency - compared with free-living birds, though we did not detect a difference in the effects of small cages versus large cages. Among captive individuals, the birds that circulated the highest concentrations of red carotenoids had the highest mitochondrial respiratory control ratio for complex II substrate. These data support the hypothesis that the metabolism of red carotenoid pigments is linked to mitochondrial aerobic respiration in the house finch, but the mechanisms for this association remain to be established.

Testosterone Coordinates Gene Expression Across Different Tissues to Produce Carotenoid-Based Red Ornamentation.

Carotenoid pigments underlie most of the red, orange, and yellow visual signals used in mate choice in vertebrates. However, many of the underlying processes surrounding the production of carotenoid-based traits remain unclear due to the complex nature of carotenoid uptake, metabolism, and deposition across tissues. Here, we leverage the ability to experimentally induce the production of a carotenoid-based red plumage patch in the red-backed fairywren (Malurus melanocephalus), a songbird in which red plumage is an important male sexual signal. We experimentally elevated testosterone in unornamented males lacking red plumage to induce the production of ornamentation and compared gene expression in both the liver and feather follicles between unornamented control males, testosterone-implanted males, and naturally ornamented males. We show that testosterone upregulates the expression of CYP2J19, a gene known to be involved in ketocarotenoid metabolism, and a putative carotenoid processing gene (ELOVL6) in the liver, and also regulates the expression of putative carotenoid transporter genes in red feather follicles on the back, including ABCG1. In black feathers, carotenoid-related genes are downregulated and melanin genes upregulated, but we find that carotenoids are still present in the feathers. This may be due to the activity of the carotenoid-cleaving enzyme BCO2 in black feathers. Our study provides a first working model of a pathway for carotenoid-based trait production in free-living birds, implicates testosterone as a key regulator of carotenoid-associated gene expression, and suggests hormones may coordinate the many processes that underlie the production of these traits across multiple tissues.

A mechanism for red coloration in vertebrates.

Red coloration is a salient feature of the natural world. Many vertebrates produce red color by converting dietary yellow carotenoids into red ketocarotenoids via an unknown mechanism. Here, we show that two enzymes, cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L), are sufficient to catalyze this conversion. In birds, both enzymes are expressed at the sites of ketocarotenoid biosynthesis (feather follicles and red cone photoreceptors), and genetic evidence implicates these enzymes in yellow/red color variation in feathers. In fish, the homologs of CYP2J19 and BDH1L are required for ketocarotenoid production, and we show that these enzymes are sufficient to produce ketocarotenoids in cell culture and when ectopically expressed in fish skin. Finally, we demonstrate that the red-cone-enriched tetratricopeptide repeat protein 39B (TTC39B) enhances ketocarotenoid production when co-expressed with CYP2J19 and BDH1L. The discovery of this mechanism of ketocarotenoid biosynthesis has major implications for understanding the evolution of color diversity in vertebrates.

Methods for extracting and analyzing carotenoids from bird feathers.

Carotenoid pigments serve many endogenous functions in organisms, but some of the more fascinating are the external displays of carotenoids in the colorful red, orange and yellow plumages of birds. Since Darwin, biologists have been curious about the selective advantages (e.g., mate attraction) of having such ornate features, and, more recently, advances in biochemical methods have permitted researchers to explore the composition and characteristics of carotenoid pigments in feathers. Here we review contemporary methods for extracting and analyzing carotenoids in bird feathers, with special attention to the difficulties of removal from the feather keratin matrix, the possibility of feather carotenoid esterification and the strengths and challenges of different analytical methods like high-performance liquid chromatography and Raman spectroscopy. We also add an experimental test of current common extraction methods (e.g., mechanical, thermochemical) and find significant differences in the recovery of specific classes of carotenoids, suggesting that no single approach is best for all pigment or feather types.

Development and genetics of red coloration in the zebrafish relative Danio albolineatus.

Animal pigment patterns play important roles in behavior and, in many species, red coloration serves as an honest signal of individual quality in mate choice. Among Danio fishes, some species develop erythrophores, pigment cells that contain red ketocarotenoids, whereas other species, like zebrafish (D. rerio) only have yellow xanthophores. Here, we use pearl danio (D. albolineatus) to assess the developmental origin of erythrophores and their mechanisms of differentiation. We show that erythrophores in the fin of D. albolineatus share a common progenitor with xanthophores and maintain plasticity in cell fate even after differentiation. We further identify the predominant ketocarotenoids that confer red coloration to erythrophores and use reverse genetics to pinpoint genes required for the differentiation and maintenance of these cells. Our analyses are a first step toward defining the mechanisms underlying the development of erythrophore-mediated red coloration in Danio and reveal striking parallels with the mechanism of red coloration in birds.

Avian color expression and perception: is there a carotenoid link?

Carotenoids color many of the red, orange and yellow ornaments of birds and also shape avian vision. The carotenoid-pigmented oil droplets in cone photoreceptors filter incoming light and are predicted to aid in color discrimination. Carotenoid use in both avian coloration and color vision raises an intriguing question: is the evolution of visual signals and signal perception linked through these pigments? Here, we explore the genetic, physiological and functional connections between these traits. Carotenoid color and droplet pigmentation share common mechanisms of metabolic conversion and are both affected by diet and immune system challenges. Yet, the time scale and magnitude of these effects differ greatly between plumage and the visual system. Recent observations suggest a link between retinal carotenoid levels and color discrimination performance, but the mechanisms underlying these associations remain unclear. Therefore, we performed a modeling exercise to ask whether and how changes in droplet carotenoid content could alter the perception of carotenoid-based plumage. This exercise revealed that changing oil droplet carotenoid concentration does not substantially affect the discrimination of carotenoid-based colors, but might change how reliably a receiver can predict the carotenoid content of an ornament. These findings suggest that, if present, a carotenoid link between signal and perception is subtle. Deconstructing this relationship will require a deeper understanding of avian visual perception and the mechanisms of color production. We highlight several areas where we see opportunities to gain new insights, including comparative genomic studies of shared mechanisms of carotenoid processing and alternative approaches to investigating color vision.

A genetic mechanism for sexual dichromatism in birds.

Sexual dichromatism, a difference in coloration between males and females, may be due to sexual selection for ornamentation and mate choice. Here, we show that carotenoid-based dichromatism in mosaic canaries, a hybrid phenotype that arises in offspring of the sexually dichromatic red siskin and monochromatic canaries, is controlled by the gene that encodes the carotenoid-cleaving enzyme ß-carotene oxygenase 2 (BCO2). Dichromatism in mosaic canaries is explained by differential carotenoid degradation in the integument, rather than sex-specific variation in physiological functions such as pigment uptake or transport. Transcriptome analyses suggest that carotenoid degradation in the integument might be a common mechanism contributing to sexual dichromatism across finches. These results suggest that differences in ornamental coloration between sexes can evolve through simple molecular mechanisms controlled by genes of major effect.

Genetic Basis of De Novo Appearance of Carotenoid Ornamentation in Bare Parts of Canaries.

Unlike wild and domestic canaries (Serinus canaria), or any of the three dozen species of finches in genus Serinus, the domestic urucum breed of canaries exhibits bright red bills and legs. This novel trait offers a unique opportunity to understand the mechanisms of bare-part coloration in birds. To identify the mutation producing the colorful phenotype, we resequenced the genome of urucum canaries and performed a range of analyses to search for genotype-to-phenotype associations across the genome. We identified a nonsynonymous mutation in the gene BCO2 (beta-carotene oxygenase 2, also known as BCDO2), an enzyme involved in the cleavage and breakdown of full-length carotenoids into short apocarotenoids. Protein structural models and in vitro functional assays indicate that the urucum mutation abrogates the carotenoid-cleavage activity of BCO2. Consistent with the predicted loss of carotenoid-cleavage activity, urucum canaries tended to have increased levels of full-length carotenoid pigments in bill tissue and reduced levels of carotenoid-cleavage products (apocarotenoids) in retinal tissue compared with other breeds of canaries. We hypothesize that carotenoid-based bare-part coloration might be readily gained, modified, or lost through simple switches in the enzymatic activity or regulation of BCO2 and this gene may be an important mediator in the evolution of bare-part coloration among bird species.

Thyroid hormone regulates distinct paths to maturation in pigment cell lineages.

Thyroid hormone (TH) regulates diverse developmental events and can drive disparate cellular outcomes. In zebrafish, TH has opposite effects on neural crest derived pigment cells of the adult stripe pattern, limiting melanophore population expansion, yet increasing yellow/orange xanthophore numbers. To learn how TH elicits seemingly opposite responses in cells having a common embryological origin, we analyzed individual transcriptomes from thousands of neural crest-derived cells, reconstructed developmental trajectories, identified pigment cell-lineage specific responses to TH, and assessed roles for TH receptors. We show that TH promotes maturation of both cell types but in distinct ways. In melanophores, TH drives terminal differentiation, limiting final cell numbers. In xanthophores, TH promotes accumulation of orange carotenoids, making the cells visible. TH receptors act primarily to repress these programs when TH is limiting. Our findings show how a single endocrine factor integrates very different cellular activities during the generation of adult form.

Testing the resource trade-off hypothesis for carotenoid-based signal honesty using genetic variants of the domestic canary.

Carotenoid-based coloration in birds is widely considered an honest signal of individual condition, but the mechanisms responsible for condition dependency in such ornaments remain debated. Currently, the most common explanation for how carotenoid coloration serves as a reliable signal of condition is the resource trade-off hypothesis, which proposes that use of carotenoids for ornaments reduces their availability for use by the immune system or for protection from oxidative damage. However, two main assumptions of the hypothesis remain in question: whether carotenoids boost the performance of internal processes such as immune and antioxidant defenses, and whether allocating carotenoids to ornaments imposes a trade-off with such benefits. In this study, we tested these two fundamental assumptions using types of domestic canary (Serinus canaria domestica) that enable experiments in which carotenoid availability and allocation can be tightly controlled. Specifically, we assessed metrics of immune and antioxidant performance in three genetic variants of the color-bred canary that differ only in carotenoid phenotype: ornamented, carotenoid-rich yellow canaries; unornamented, carotenoid-rich 'white dominant' canaries; and unornamented, carotenoid-deficient 'white recessive' canaries. The resource trade-off hypothesis predicts that carotenoid-rich individuals should outperform carotenoid-deficient individuals and that birds that allocate carotenoids to feathers should pay a cost in the form of reduced immune function or greater oxidative stress compared with unornamented birds. We found no evidence to support either prediction; all three canary types performed equally across measures. We suggest that testing alternative mechanisms for the honesty of carotenoid-based coloration should be a key focus of future studies of carotenoid-based signaling in birds.

A non-coding region near Follistatin controls head colour polymorphism in the Gouldian finch.

Discrete colour morphs coexisting within a single population are common in nature. In a broad range of organisms, sympatric colour morphs often display major differences in other traits, including morphology, physiology or behaviour. Despite the repeated occurrence of this phenomenon, our understanding of the genetics that underlie multi-trait differences and the factors that promote the long-term maintenance of phenotypic variability within a freely interbreeding population are incomplete. Here, we investigated the genetic basis of red and black head colour in the Gouldian finch (Erythrura gouldiae), a classic polymorphic system in which naturally occurring colour morphs also display differences in aggressivity and reproductive success. We show that the candidate locus is a small (approx. 70 kb) non-coding region mapping to the Z chromosome near the Follistatin (FST) gene. Unlike recent findings in other systems where phenotypic morphs are explained by large inversions containing hundreds of genes (so-called supergenes), we did not identify any structural rearrangements between the two haplotypes using linked-read sequencing technology. Nucleotide divergence between the red and black alleles was high when compared to the remainder of the Z chromosome, consistent with their maintenance as balanced polymorphisms over several million years. Our results illustrate how pleiotropic phenotypes can arise from simple genetic variation, probably regulatory in nature.

No evidence that carotenoid pigments boost either immune or antioxidant defenses in a songbird.

Dietary carotenoids have been proposed to boost immune system and antioxidant functions in vertebrate animals, but studies aimed at testing these physiological functions of carotenoids have often failed to find support. Here we subject yellow canaries (Serinus canaria), which possess high levels of carotenoids in their tissue, and white recessive canaries, which possess a knockdown mutation that results in very low levels of tissue carotenoids, to oxidative and pathogen challenges. Across diverse measures of physiological performance, we detect no differences between carotenoid-rich yellow and carotenoid-deficient white canaries. These results add further challenge to the assumption that carotenoids are directly involved in supporting physiological function in vertebrate animals. While some dietary carotenoids provide indirect benefits as retinoid precursors, our observations suggest that carotenoids themselves may play little to no direct role in key physiological processes in birds.

Evolution, Development and Function of Vertebrate Cone Oil Droplets.

To distinguish colors, the nervous system must compare the activity of distinct subtypes of photoreceptors that are maximally sensitive to different portions of the light spectrum. In vertebrates, a variety of adaptations have arisen to refine the spectral sensitivity of cone photoreceptors and improve color vision. In this review article, we focus on one such adaptation, the oil droplet, a unique optical organelle found within the inner segment of cone photoreceptors of a diverse array of vertebrate species, from fish to mammals. These droplets, which consist of neutral lipids and carotenoid pigments, are interposed in the path of light through the photoreceptor and modify the intensity and spectrum of light reaching the photosensitive outer segment. In the course of evolution, the optical function of oil droplets has been fine-tuned through changes in carotenoid content. Species active in dim light reduce or eliminate carotenoids to enhance sensitivity, whereas species active in bright light precisely modulate carotenoid double bond conjugation and concentration among cone subtypes to optimize color discrimination and color constancy. Cone oil droplets have sparked the curiosity of vision scientists for more than a century. Accordingly, we begin by briefly reviewing the history of research on oil droplets. We then discuss what is known about the developmental origins of oil droplets. Next, we describe recent advances in understanding the function of oil droplets based on biochemical and optical analyses. Finally, we survey the occurrence and properties of oil droplets across the diversity of vertebrate species and discuss what these patterns indicate about the evolutionary history and function of this intriguing organelle.

Cambrian origin of the CYP27C1-mediated vitamin A1-to-A2 switch, a key mechanism of vertebrate sensory plasticity.

The spectral composition of ambient light varies across both space and time. Many species of jawed vertebrates adapt to this variation by tuning the sensitivity of their photoreceptors via the expression of CYP27C1, an enzyme that converts vitamin A1 into vitamin A2, thereby shifting the ratio of vitamin A1-based rhodopsin to red-shifted vitamin A2-based porphyropsin in the eye. Here, we show that the sea lamprey (Petromyzon marinus), a jawless vertebrate that diverged from jawed vertebrates during the Cambrian period (approx. 500 Ma), dynamically shifts its photoreceptor spectral sensitivity via vitamin A1-to-A2 chromophore exchange as it transitions between photically divergent aquatic habitats. We further show that this shift correlates with high-level expression of the lamprey orthologue of CYP27C1, specifically in the retinal pigment epithelium as in jawed vertebrates. Our results suggest that the CYP27C1-mediated vitamin A1-to-A2 switch is an evolutionarily ancient mechanism of sensory plasticity that appeared not long after the origin of vertebrates.

High-density lipoprotein receptor SCARB1 is required for carotenoid coloration in birds.

Yellow, orange, and red coloration is a fundamental aspect of avian diversity and serves as an important signal in mate choice and aggressive interactions. This coloration is often produced through the deposition of diet-derived carotenoid pigments, yet the mechanisms of carotenoid uptake and transport are not well-understood. The white recessive breed of the common canary (Serinus canaria), which carries an autosomal recessive mutation that renders its plumage pure white, provides a unique opportunity to investigate mechanisms of carotenoid coloration. We carried out detailed genomic and biochemical analyses comparing the white recessive with yellow and red breeds of canaries. Biochemical analysis revealed that carotenoids are absent or at very low concentrations in feathers and several tissues of white recessive canaries, consistent with a genetic defect in carotenoid uptake. Using a combination of genetic mapping approaches, we show that the white recessive allele is due to a splice donor site mutation in the scavenger receptor B1 (SCARB1; also known as SR-B1) gene. This mutation results in abnormal splicing, with the most abundant transcript lacking exon 4. Through functional assays, we further demonstrate that wild-type SCARB1 promotes cellular uptake of carotenoids but that this function is lost in the predominant mutant isoform in white recessive canaries. Our results indicate that SCARB1 is an essential mediator of the expression of carotenoid-based coloration in birds, and suggest a potential link between visual displays and lipid metabolism.

Specialized photoreceptor composition in the raptor fovea.

The retinae of many bird species contain a depression with high photoreceptor density known as the fovea. Many species of raptors have two foveae, a deep central fovea and a shallower temporal fovea. Birds have six types of photoreceptors: rods, active in dim light, double cones that are thought to mediate achromatic discrimination, and four types of single cones mediating color vision. To maximize visual acuity, the fovea should only contain photoreceptors contributing to high-resolution vision. Interestingly, it has been suggested that raptors might lack double cones in the fovea. We used transmission electron microscopy and immunohistochemistry to evaluate this claim in five raptor species: the common buzzard (Buteo buteo), the honey buzzard (Pernis apivorus), the Eurasian sparrowhawk (Accipiter nisus), the red kite (Milvus milvus), and the peregrine falcon (Falco peregrinus). We found that all species, except the Eurasian sparrowhawk, lack double cones in the center of the central fovea. The size of the double cone-free zone differed between species. Only the common buzzard had a double cone-free zone in the temporal fovea. In three species, we examined opsin expression in the central fovea and found evidence that rod opsin positive cells were absent and violet-sensitive cone and green-sensitive cone opsin positive cells were present. We conclude that not only double cones, but also single cones may contribute to high-resolution vision in birds, and that raptors may in fact possess high-resolution tetrachromatic vision in the central fovea.

Complementary shifts in photoreceptor spectral tuning unlock the full adaptive potential of ultraviolet vision in birds.

Color vision in birds is mediated by four types of cone photoreceptors whose maximal sensitivities (λmax) are evenly spaced across the light spectrum. In the course of avian evolution, the λmax of the most shortwave-sensitive cone, SWS1, has switched between violet (λmax > 400 nm) and ultraviolet (λmax < 380 nm) multiple times. This shift of the SWS1 opsin is accompanied by a corresponding short-wavelength shift in the spectrally adjacent SWS2 cone. Here, we show that SWS2 cone spectral tuning is mediated by modulating the ratio of two apocarotenoids, galloxanthin and 11',12'-dihydrogalloxanthin, which act as intracellular spectral filters in this cell type. We propose an enzymatic pathway that mediates the differential production of these apocarotenoids in the avian retina, and we use color vision modeling to demonstrate how correlated evolution of spectral tuning is necessary to achieve even sampling of the light spectrum and thereby maintain near-optimal color discrimination.

Genetic Basis for Red Coloration in Birds.

The yellow and red feather pigmentation of many bird species [1] plays pivotal roles in social signaling and mate choice [2, 3]. To produce red pigments, birds ingest yellow carotenoids and endogenously convert them into red ketocarotenoids via an oxidation reaction catalyzed by a previously unknown ketolase [4-6]. We investigated the genetic basis for red coloration in birds using whole-genome sequencing of red siskins (Spinus cucullata), common canaries (Serinus canaria), and "red factor" canaries, which are the hybrid product of crossing red siskins with common canaries [7]. We identified two genomic regions introgressed from red siskins into red factor canaries that are required for red coloration. One of these regions contains a gene encoding a cytochrome P450 enzyme, CYP2J19. Transcriptome analysis demonstrates that CYP2J19 is significantly upregulated in the skin and liver of red factor canaries, strongly implicating CYP2J19 as the ketolase that mediates red coloration in birds. Interestingly, a second introgressed region required for red feathers resides within the epidermal differentiation complex, a cluster of genes involved in development of the integument. Lastly, we present evidence that CYP2J19 is involved in ketocarotenoid formation in the retina. The discovery of the carotenoid ketolase has important implications for understanding sensory function and signaling mediated by carotenoid pigmentation.

Human cytochrome P450 27C1 catalyzes 3,4-desaturation of retinoids.

In humans, a considerable fraction of the retinoid pool in skin is derived from vitamin A2 (all-trans 3,4-dehydroretinal). Vitamin A2 may be locally generated by keratinocytes, which can convert vitamin A1 (all-trans retinol) into vitamin A2 in cell culture. We report that human cytochrome P450 (hP450) 27C1, a previously 'orphan' enzyme, can catalyze this reaction. Purified recombinant hP450 27C1 bound and desaturated all-trans retinol, retinal, and retinoic acid, as well as 11-cis-retinal. Although the physiological role of 3,4-dehydroretinoids in humans is unclear, we have identified hP450 27C1 as an enzyme capable of efficiently mediating their formation.

Cyp27c1 Red-Shifts the Spectral Sensitivity of Photoreceptors by Converting Vitamin A1 into A2.

Some vertebrate species have evolved means of extending their visual sensitivity beyond the range of human vision. One mechanism of enhancing sensitivity to long-wavelength light is to replace the 11-cis retinal chromophore in photopigments with 11-cis 3,4-didehydroretinal. Despite over a century of research on this topic, the enzymatic basis of this perceptual switch remains unknown. Here, we show that a cytochrome P450 family member, Cyp27c1, mediates this switch by converting vitamin A1 (the precursor of 11-cis retinal) into vitamin A2 (the precursor of 11-cis 3,4-didehydroretinal). Knockout of cyp27c1 in zebrafish abrogates production of vitamin A2, eliminating the animal's ability to red-shift its photoreceptor spectral sensitivity and reducing its ability to see and respond to near-infrared light. Thus, the expression of a single enzyme mediates dynamic spectral tuning of the entire visual system by controlling the balance of vitamin A1 and A2 in the eye.