Dietary canthaxanthin reduces xanthophyll uptake and red coloration in adult red-legged partridges.

Carotenoids give color to conspicuous animal signals that are often the product of sexual selection. Knowledge of the mechanisms involved in carotenoid-based signaling is critical to understanding how these traits evolve. However, these mechanisms remain only partially understood. Carotenoids are usually viewed as scarce dietary antioxidants whose allocation to ornaments may trade off against health. This trade-off would ensure its reliability as a signal of individual quality. In the case of red (keto)carotenoids, the literature suggests that some species may show constraints in their uptake. Canthaxanthin is one of the most common ketocarotenoids in red ornaments of animals. It is often commercially used as a dietary supplement to obtain redder birds (e.g. poultry). We increased the dietary canthaxanthin levels in captive red-legged partridges (Alectoris rufa). This species shows red non-feathered parts mostly pigmented by another common ketocarotenoid: astaxanthin. We studied the impact on the uptake of carotenoids and vitamins and, finally, on coloration. We also tested the potential protective effect of canthaxanthin when exposing birds to a free radical generator (diquat). Canthaxanthin did not apparently protect birds from oxidative stress, but interfered with the absorption of yellow carotenoids (lutein and zeaxanthin). Zeaxanthin is a precursor of astaxanthin in enzymatic pathways, and their levels in tissues and eggs were lower in canthaxanthin-supplied birds. This led to lower astaxanthin levels in ornaments and paler coloration. As far as we know, this is the first report of a carotenoid supplementation decreasing animal coloration. The results have implications for understanding carotenoid-based signaling evolution, but also for improving husbandry/experimental procedures.

Specific carotenoid pigments in the diet and a bit of oxidative stress in the recipe for producing red carotenoid-based signals.

Colorful ornaments have been the focus of sexual selection studies since the work of Darwin. Yellow to red coloration is often produced by carotenoid pigments. Different hypotheses have been formulated to explain the evolution of these traits as signals of individual quality. Many of these hypotheses involve the existence of a signal production cost. The carotenoids necessary for signaling can only be obtained from food. In this line, carotenoid-based signals could reveal an individual's capacity to find sufficient dietary pigments. However, the ingested carotenoids are often yellow and became transformed by the organism to produce pigments of more intense color (red ketocarotenoids). Biotransformation should involve oxidation reactions, although the exact mechanism is poorly known. We tested the hypothesis that carotenoid biotransformation could be costly because a certain level of oxidative stress is required to correctly perform the conversion. The carotenoid-based signals could thus reveal the efficiency of the owner in successfully managing this challenge. In a bird with ketocarotenoid-based ornaments (the red-legged partridge; Alectoris rufa), the availability of different carotenoids in the diet (i.e. astaxanthin, zeaxanthin and lutein) and oxidative stress were manipulated. The carotenoid composition was analyzed and quantified in the ornaments, blood, liver and fat. A number of oxidative stress biomarkers were also measured in the same tissues. First, we found that color and pigment levels in the ornaments depended on food levels of those carotenoids used as substrates in biotransformation. Second, we found that birds exposed to mild levels of a free radical generator (diquat) developed redder bills and deposited higher amounts of ketocarotenoids (astaxanthin) in ornaments. Moreover, the same diquat-exposed birds also showed a weaker resistance to hemolysis when their erythrocytes were exposed to free radicals, with females also enduring higher oxidative damage in plasma lipids. Thus, higher color production would be linked to higher oxidative stress, supporting the biotransformation hypothesis. The recent discovery of an avian oxygenase enzyme involved in converting yellow to red carotenoids may support our results. Nonetheless, the effect could also depend on the abundance of specific substrate carotenoids in the diet. Birds fed with proportionally higher levels of zeaxanthin showed the reddest ornaments with the highest astaxanthin concentrations. Moreover, these birds tended to show the strongest diquat-mediated effect. Therefore, in the evolution of carotenoid-based sexual signals, a biotransformation cost derived from maintaining a well-adjusted redox machinery could coexist with a cost linked to carotenoid acquisition and allocation (i.e. a resource allocation trade-off).

Adverse effects of thiram-treated seed ingestion on the reproductive performance and the offspring immune function of the red-legged partridge.

Pesticide research traditionally has focused on compounds with high acute toxicity or persistence, but the adverse sublethal effects of pesticides with different properties also may have important consequences on exposed wildlife. The authors studied the effects of thiram, a fungicide used for seed coating with known effects as endocrine disruptor. Red-legged partridges (Alectoris rufa; n = 15 pairs per treatment group) were fed wheat treated with 0%, 20%, or 100% of the thiram application rate used in autumn (25 d) and late winter (10 d) to mimic cereal sowing periods. The authors studied the effects on reproductive performance, carotenoid-based ornamentation and cellular immune responsiveness of adult partridges, and their relationship with changes in oxidative stress biomarkers and plasma biochemistry. The authors also studied the effect of parental exposure on egg antioxidant content and on the survival, growth, and cellular immune response of offspring. Exposure to thiram-coated seeds delayed egg laying, reduced clutch size, and affected egg size and eggshell thickness. Partridges exposed to the 20% thiram dose exhibited reduced egg fertility and brood size (55% and 28% of controls, respectively). Chick survival was unaffected by parental exposure to treated seeds, but adverse effects on their growth rate and cellular immune response were apparent. These effects on reproduction and immune function may have important demographic consequences on farmland bird populations.

Accumulation of dietary carotenoids, retinoids and tocopherol in the internal tissues of a bird: a hypothesis for the cost of producing colored ornaments.

Carotenoid-based ornaments may have evolved as a consequence of their costs of production, which would assure the reliability of the traits as signals of individual quality. Different costs due to carotenoid allocation to the signal have been proposed, considering the scarcity of these pigments at the environment (ecological cost) and their physiological properties that would trade against the maintenance of the organism. Carotenoids of many red ornaments (ketocarotenoids) are often the result of biotransformation of those pigments abundant in the diet (usually lutein and zeaxanthin). Some authors have suggested that such a conversion implies a cost relevant for signaling because it requires high levels of antioxidant vitamins in the tissues where biotransformation takes place. We explore this hypothesis in red-legged partridges (Alectoris rufa) by analyzing ketocarotenoids in the ornaments (bare parts) and carotenoids, vitamin A in different forms (free and esterified) and vitamin E in blood, liver and fat. Ketocarotenoids in ornaments (astaxanthin and papilioerythrinone) were not found in internal tissues, suggesting that they were directly transformed in the bare parts. However, ketocarotenoid levels where positively correlated with the levels of their precursors (zeaxanthin and lutein, respectively) in internal tissues. Interestingly, ketocarotenoid levels in bare parts negatively and positively correlated with vitamin A and E in the liver, respectively, the same links only being positive in blood. Moreover, retinyl and zeaxanthin levels in liver were negatively related. We hypothesize that storing substrate carotenoids in the main storage site (the liver) implies a cost in terms of regulating the level of vitamin A.

Astaxanthin and papilioerythrinone in the skin of birds: a chromatic convergence of two metabolic routes with different precursors?

Carotenoids are organic pigments involved in several important physiological functions and may serve as indicators of individual quality in animals. These pigments are only obtained by animals from the diet, but they can be later transformed into other carotenoids by specific enzymatic reactions. The diet of farm-reared and probably wild red-legged partridges (Alectoris rufa) is mainly based on cereals that contain high levels of lutein and zeaxanthin. These two carotenoids are also predominant in internal tissues and blood of red-legged partridges. However, in their integuments, astaxanthin and papilioerythrinone (the last one identified in this work) are mainly present in their free form and esterified with fatty acids. According to available literature about carotenoid metabolism in animals, we propose that astaxanthin (λ max = 478 nm) and papilioerythrinone (λ max = 452-478 nm) are the result of a chromatic convergence of the transformation of dietary zeaxanthin and lutein, respectively. Moreover, the results obtained in this work provide the first identification by liquid chromatography coupled to accurate mass quadrupole time-of-flight mass spectrometer system of papilioerythrinone (m/z 581.3989 [M + H](+)) in the skin (i.e., not feathers) of a vertebrate. Astaxanthin and papilioerythrinone are very close in terms of chemical structure and coloration, and the combination of these two keto-carotenoids is responsible for the red color of the ornaments in red-legged partridges.

Free and esterified carotenoids in ornaments of an avian species: the relationship to color expression and sources of variability.

Many animal species show ornaments with yellow-orange-red colors produced by carotenoid pigments. Such traits have evolved as reliable signals of individual quality because of the costs inherent to their production or maintenance. In animal tissues, carotenoids are often found combined with free fatty acids, as carotenoid esters, which may confer more stability to coloration than free carotenoids. Surprisingly, the potential relevance of carotenoid esterification in the expression of animal sexual signals has been virtually ignored. Moreover, the sources of variability of esterified carotenoid levels are barely known, because most studies have not quantified their concentrations. Here, carotenoids in the ornaments (bill, eye rings, and legs) of red-legged partridges Alectoris rufa were quantified in their free and esterified forms. Carotenoid ester levels were the best predictors of leg color, whereas the redness of the other traits was better explained by free carotenoids. Nonetheless, total carotenoid levels (the sum of free and esterified forms) were always significantly correlated to redness. Young partridges had lower levels of free and esterified carotenids in the legs than did older individuals. Also, wild animals had higher ester levels and a higher proportion of carotenoids in esterified forms in all traits than did captive partridges. Probable physiological mechanisms explaining these patterns are discussed.

Identification of carotenoid pigments and their fatty acid esters in an avian integument combining HPLC-DAD and LC-MS analyses.

Yellow-orange-red ornaments present in the integuments (feathers, bare parts) of birds are often produced by carotenoid pigments and may serve to signal the quality of the bearer. Although carotenoid esterification in tissues is a common phenomenon, most of the work on avian carotenoids has been focused on the identification of free forms or have been done after sample saponification. Here we determined free and esterified carotenoid composition in a bird species with red ornaments: the red-legged partridge (Alectoris rufa). Carotenoids from leg integument were extracted and processed by TLC to separate three major carotenoid groups (free form, mono- and diesters with fatty acids), whereas saponified extracts gave only free forms of carotenoids. TLC fractions were then analyzed by HPLC-DAD with C18 phase column for a preliminary identification of carotenoid groups. The final characterization of free carotenoids and its esters with fatty acids was performed with direct extracts analyzed by LC-MS and LC-MS/MS with a C30 phase, always with a system coupled to DAD. The main carotenoid (λ(max) 478 nm and [M+H](+) at m/z 597.2) was identified as astaxanthin by comparison with standards. A second carotenoid (λ(max) between 440 and 480 nm and [M+H](+) at m/z 581.3) was not identified among any of the commercially available carotenoid standards, although it could correspond to pectenolone according to its fragmentation pattern. Both the unidentified carotenoid and astaxanthin formed monoesters with fatty acids, but only astaxanthin was in its diesterified form. Monoesters were mainly formed with palmitic, stearic, oleic and linoleic acids. Complementary analyses of fatty acid composition in partridge integument by GC-MS revealed high amounts of these and other fatty acids, such as myristic, arachidic and docosanoic acids. The combination of HPLC-DAD and LC-MS/MS spectra was especially useful to identify the carotenoids present in the esterified forms and the probable masses of the fatty acids included in them, respectively.