Many of the colour displays of animals are proposed to have evolved in response to female mate choice for honest signals of quality, but such honest signalling requires mechanisms to prevent cheating. The most widely accepted and cited mechanisms for ensuring signal honesty are based on the costly signalling hypothesis, which posits that costs associated with ornamentation prevent low-quality males from being highly ornamented. Alternatively, by the index hypothesis, honesty can be achieved via cost-free mechanisms if ornament production is causally linked to core physiological pathways. In this essay, we review how a costly signalling framework has shaped empirical research in mate choice for colourful male ornaments and emphasize that alternative interpretations are plausible under an index signalling framework. We discuss the challenges in both empirically testing and distinguishing between the two hypotheses, noting that they need not be mutually exclusive. Finally, we advocate for a comprehensive approach to studies of colour signals that includes the explicit consideration of cost-free mechanisms for honesty.This article is part of the themed issue 'Animal coloration: production, perception, function and application'.

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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 wellunderstood. 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.coloration | carotenoids | lipid metabolism | Serinus canaria T he yellow, orange, and red coloration of the feathers, skin, and beaks of birds is most commonly produced through the deposition of carotenoid pigments (1). Carotenoid coloration of birds has been a focus of study in the fields of behavior, evolution, and physiology because it plays a key role in mate assessment in many species. In addition, it is frequently an indicator of individual quality, and can signal species identity (2-4). Birds cannot synthesize carotenoids de novo and must acquire them through their diet (1), potentially linking coloration to the acquisition of pigments from the environment (3). Thus, key hypotheses related to honest signaling and sexual selection have been shaped by and are currently being tested in carotenoidornament systems (5, 6). Ultimately, the information content and evolutionary trajectories of carotenoid ornaments are a function of the physiological mechanisms underlying color expression, yet our understanding of these mechanisms is limited.The expression of carotenoid coloration in birds involves four distinct physiological steps: uptake in the gut, transport in circulatory and lymphatic systems, metabolism either at the site of deposition or in the liver, and deposition in the integument (7). Recent progress has been made in understanding how carotenoids are metabolized to novel forms. In 2016, two studies independently identified a key carotenoid metabolism enzyme,...

Understanding the mechanisms that link ornamental displays and individual condition is key to understanding the evolution and function of ornaments. Immune function is an aspect of individual quality that is often associated with the expression of ornamentation, but a general explanation for why the expression of some ornaments seems to be consistently linked to immunocompetence remains elusive. We propose that condition-dependent ornaments may be linked to key aspects of immunocompetence through co-dependence on mitochondrial function. Mitochondrial involvement in immune function is rarely considered outside of the biomedical literature, but the role of mitochondria as the primary energy producers of the cell and the centres of biosynthesis, the oxidative stress response, and cellular signalling place them at the hub of a variety of immune pathways. A promising new mechanistic explanation for correlations between a wide range of ornamental traits and the properties of individual quality is that mitochondrial function may be the 'shared pathway' responsible for links between ornament production and individual condition. Herein, we first review the role of mitochondria as both signal transducers and metabolic regulators of immune function. We then describe connections between hormonal pathways and mitochondria, with implications for both immune function and the expression of ornamentation. Finally, we explore the possibility that ornament expression may link directly to mitochondrial function. Considering condition-dependent traits within the framework of mitochondrial function has the potential to unify central tenets within the study of sexual selection, eco-immunology, oxidative stress ecology, stress and reproductive hormone biology, and animal physiology.

Summary1. Physiological ecologists require techniques for controlled oxidative challenges in live animals to facilitate the study of oxidative stress. 2. Techniques for manipulating oxidative stress include agents that increase generation of pro-oxidants, such as paraquat, diquat, radiation, heavy metals, dietary oxidized lipids, and tert-butyl-hydroperoxide, as well as genetic (RNAi) and chemical (buthionine sulfoximine) knock-downs that target specific antioxidants. 3. We critically assess both currently used and potentially useful methods for inducing systemic oxidative challenge in animals. We provide a resource for biologists to select the most robust methods for oxidative challenge in their study system, to improve interpretation of results within the context of cellular mechanisms and to maximize effectiveness of experiments while minimizing unintended side effects.