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.

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.

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.

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.

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.

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.

Ketocarotenoid circulation, but not retinal carotenoid accumulation, is linked to eye disease status in a wild songbird.

Pathogenic or parasitic infections pose numerous physiological challenges to organisms. Carotenoid pigments have often been used as biomarkers of disease state and impact because they integrate multiple aspects of an individual's condition and nutritional and health state. Some diseases are known to influence carotenoid uptake from food (e.g. coccidiosis) and carotenoid use (e.g. as antioxidants/immunostimulants in the body, or for sexually attractive coloration), but there is relatively little information in animals about how different types of carotenoids from different tissue sources may be affected by disease. Here we tracked carotenoid accumulation in two body pools (retina and plasma) as a function of disease state in free-ranging house finches (Haemorhous mexicanus). House finches in eastern North America can contract mycoplasmal conjunctivitis (Mycoplasma gallisepticum, or MG), which can progress from eye swelling to eye closure and death. Previous work showed that systemic immune challenges in house finches lower carotenoid levels in retina, where they act as photoprotectors and visual filters. We assessed carotenoid levels during the molt period, a time of year when finches uniquely metabolize ketocarotenoids (e.g. 3-hydroxy-echinenone) for acquisition of sexually selected red plumage coloration, and found that males infected with MG circulated significantly lower levels of 3-hydroxy-echinenone, but no other plasma carotenoid types, than birds exhibiting no MG symptoms. This result uncovers a key biochemical mechanism for the documented detrimental effect of MG on plumage redness in H. mexicanus. In contrast, we failed to find a relationship between MG infection status and retinal carotenoid concentrations. Thus, we reveal differential effects of an infectious eye disease on carotenoid types and tissue pools in a wild songbird. At least compared to retinal sources (which appear somewhat more temporally stable than other body carotenoid pools, even to diseases of the eye evidently), our results point to either a high physiological cost of ketocarotenoid synthesis (as is argued in models of sexually selected carotenoid coloration) or high benefit of using this ketocarotenoid to combat infection.

Mate choice for a male carotenoid-based ornament is linked to female dietary carotenoid intake and accumulation.

BACKGROUND: The coevolution of male traits and female mate preferences has led to the elaboration and diversification of sexually selected traits; however the mechanisms that mediate trait-preference coevolution are largely unknown. Carotenoid acquisition and accumulation are key determinants of the expression of male sexually selected carotenoid-based coloration and a primary mechanism maintaining the honest information content of these signals. Carotenoids also influence female health and reproduction in ways that may alter the costs and benefits of mate choice behaviours and thus provide a potential biochemical link between the expression of male traits and female preferences. To test this hypothesis, we manipulated the dietary carotenoid levels of captive female house finches (Carpodacus mexicanus) and assessed their mate choice behavior in response to color-manipulated male finches. RESULTS: Females preferred to associate with red males, but carotenoid supplementation did not influence the direction or strength of this preference. Females receiving a low-carotenoid diet were less responsive to males in general, and discrimination among the colorful males was positively linked to female plasma carotenoid levels at the beginning of the study when the diet of all birds was carotenoid-limited. CONCLUSIONS: Although female preference for red males was not influenced by carotenoid intake, changes in mating responsiveness and discrimination linked to female carotenoid status may alter how this preference is translated into choice. The reddest males, with the most carotenoid rich plumage, tend to pair early in the breeding season. If carotenoid-related variations in female choice behaviour shift the timing of pairing, then they have the potential to promote assortative mating by carotenoid status and drive the evolution of carotenoid-based male plumage coloration.

Ontogenetic immune challenges shape adult personality in mallard ducks.

Consistent individual differences in behaviour are widespread in animals, but the proximate mechanisms driving these differences remain largely unresolved. Parasitism and immune challenges are hypothesized to shape the expression of animal personality traits, but few studies have examined the influence of neonatal immune status on the development of adult personality. We examined how non-pathogenic immune challenges, administered at different stages of development, affected two common measures of personality, activity and exploratory behaviour, as well as colour-dependent novel object exploration in adult male mallard ducks (Anas platyrhynchos). We found that individuals that were immune-challenged during the middle (immediately following the completion of somatic growth) and late (during the acquisition of nuptial plumage) stages of development were more active in novel environments as adults relative to developmentally unchallenged birds or those challenged at an earlier developmental time point. Additionally, individuals challenged during the middle stage of development preferred orange and avoided red objects more than those that were not immune-challenged during development. Our results demonstrate that, in accordance with our predictions, early-life immune system perturbations alter the expression of personality traits later in life, emphasizing the role that developmental plasticity plays in shaping adult personality, and lending support to recent theoretical models that suggest that parasite pressure may play an important role in animal personality development.

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.

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.

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.

The effects of dietary carotenoid intake on carotenoid accumulation in the retina of a wild bird, the house finch (Carpodacus mexicanus).

Carotenoid pigments accumulate in the retinas of many animals, including humans, where they play an important role in visual health and performance. Recently, birds have emerged as a model system for studying the mechanisms and functions of carotenoid accumulation in the retina. However, these studies have been limited to a small number of domesticated species, and the effects of dietary carotenoid access on retinal carotenoid accumulation have not been investigated in any wild animal species. The purpose of our studies was to examine how variation in dietary carotenoid types and levels affect retinal accumulation in house finches (Carpodacus mexicanus), a common and colorful North American songbird. We carried out three 8-week studies with wild-caught captive birds: (1) we tracked the rate of retinal carotenoid depletion, compared to other body tissues, on a very low-carotenoid diet, (2) we supplemented birds with two common dietary carotenoids (lutein + zeaxanthin) and measured the effect on retinal accumulation, and (3) we separately supplemented birds with high levels of zeaxanthin--an important dietary precursor for retinal carotenoids--or astaxanthin--a dominant retinal carotenoid not commonly found in the diet (i.e. a metabolic derivative). We found that carotenoids depleted slowly from the retina compared to other tissues, with a significant (~50%) decline observed only after 8 weeks on a very low-carotenoid diet. Supplementation with lutein + zeaxanthin or zeaxanthin alone significantly increased only retinal galloxanthin and ε-carotene levels, while other carotenoid types in the retina remained unaffected. Concentrations of retinal astaxanthin were unaffected by direct dietary supplementation with astaxanthin. These results suggest highly specific mechanisms of retinal carotenoid metabolism and accumulation, as well as differential rates of turnover among retinal carotenoid types, all of which have important implications for visual health maintenance and interventions.

Immune-system activation depletes retinal carotenoids in house finches (Carpodacus mexicanus).

The costs of developing, maintaining, and activating the immune system have been cited as an important force shaping life-history evolution in animals. Immunological defenses require energy, nutrients and time that might otherwise be devoted to other life-history traits like sexual displays or reproduction. Carotenoid pigments in animals provide a unique opportunity to track the costs of immune activation, because they are diet-derived, modulate the immune system, and are used to develop colorful signals of quality. Carotenoids also accumulate in the retinas of birds, where they tune spectral sensitivity and provide photoprotection. If carotenoid accumulation in the retina follows the patterns of other tissues, then immune activation may deplete retinal carotenoid levels and impact visual health and function. To test this hypothesis, we challenged molting wild-caught captive house finches (Carpodacus mexicanus) with weekly injections of lipopolysaccharide (LPS) and phytohaemagglutinin (PHA) over the course of 8 weeks. Immunostimulated adult males and females produced significant antibody responses and molted more slowly than uninjected control birds. After 8 weeks, immune-challenged birds had significantly lower levels of specific retinal carotenoid types (galloxanthin and zeaxanthin), but there were no significant differences in the plasma, liver or feather carotenoid levels between the treatment groups. These results indicate that immune-system activation can specifically deplete retinal carotenoids, which may compromise visual health and performance and represent an additional somatic and behavioral cost of immunity.

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.

Iridescence: views from many angles.

Iridescent colours have been fascinating to humans throughout history; they are flashy, shimmering, dynamic, and examples surround us, from the commonly seen iridescent sheen of oily street puddles to the exotic, gaudy displays of birds-of-paradise featured in nature documentaries. Iridescent colours and the structures that produce them have unique properties in comparison with other types of colourants found in nature. Scientists from a variety of disciplines study the optics, development, heritability, chemical make-up, origin, evolution, functions and biomimetic technological applications of naturally occurring iridescent colours. For the first time, graduate students at Arizona State University brought together these scientists, along with educators and artists, at 'Iridescence: more than meets the eye', a conference to promote interdisciplinary communication and collaboration in the study of iridescent coloration from all of these perspectives. Here, we summarize the outcomes of this conference, introduce the papers that follow in this special journal issue and briefly review the current status of our understanding of iridescence.

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.

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.

Modified saponification and HPLC methods for analyzing carotenoids from the retina of quail: implications for its use as a nonprimate model species.

PURPOSE: To investigate carotenoid content in the retina of Japanese quail (Coturnix japonica), for comparison with carotenoids in human retina, and to assess the effects of different saponification procedures on the recovery of quail retinal carotenoids. METHODS: Extracted retinal carotenoids were saponified with methods adapted from recent studies, then identified and quantified with reverse-phase high-performance liquid chromatography (HPLC). To assess the effects of saponification conditions on carotenoid recovery from quail retina, we varied base concentration and the total time of saponification across a wide range and again used HPLC to compare carotenoid concentrations among conditions. RESULTS: Astaxanthin and galloxanthin were the dominant carotenoids recovered in the quail retina, along with smaller amounts of five other carotenoids (lutein, zeaxanthin, 3'-epilutein, epsilon-carotene, and an unidentified carotenoid). Astaxanthin was sensitive to saponification conditions; recovery was poor with strong bases (0.2 and 0.5 M KOH) and best with weak bases (0.01 and 0.2 M KOH). In contrast, xanthophyll carotenoids (galloxanthin, zeaxanthin, lutein, 3'-epilutein, and the unknown) were best recovered with strong base after 6 hours of saponification at room temperature. The recovery of epsilon-carotene was not affected by saponification conditions. CONCLUSIONS: Separate chemical hydrolysis procedures--using a strong base to recover xanthophylls and a weak base to recover astaxanthin--should be used for maximizing recovery of quail retinal carotenoids. Because the dominant carotenoids in quail retina are absent in human retina, and because of their different packaging (e.g., esterified in oil droplets) and light-absorbance properties compared with xanthophylls in the human eye, use of the quail as a model organism for studying human retinal carotenoids should be approached with caution.