Mitochondria-targeted molecules determine the redness of the zebra finch bill

Abstract

The evolution and production mechanisms of red carotenoid-based ornaments in animals are poorly understood. Recently, it has been suggested that enzymes transforming yellow carotenoids to red pigments (ketolases) in animal cells may be positioned in the inner mitochondrial membrane (IMM) intimately linked to the electron transport chain. These enzymes may mostly synthesize coenzyme Q₁₀ (coQ₁₀), a key redox-cycler antioxidant molecularly similar to yellow carotenoids. It has been hypothesized that this shared pathway favours the evolution of red traits as sexually selected individual quality indices by revealing a well-adjusted oxidative metabolism. We administered mitochondria-targeted molecules to male zebra finches (Taeniopygia guttata) measuring their bill redness, a trait produced by transforming yellow carotenoids. One molecule included coQ₁₀ (mitoquinone mesylate, MitoQ) and the other one (decyl-triphenylphosphonium; dTPP) has the same structure without the coQ₁₀ aromatic ring. At the highest dose, the bill colour of MitoQ and dTPP birds strongly differed: MitoQ birds' bills were redder and dTPP birds showed paler bills even compared to birds injected with saline only. These results suggest that ketolases are indeed placed at the IMM and that coQ₁₀ antioxidant properties may improve their efficiency. The implications for evolutionary theories of sexual signalling are discussed.

1. Introduction

Why colourful ornaments in animals evolve is a recurrent question in evolutionary biology. In 1957, an ornithologist [1] argued that red birds become yellow in captivity because the oxidative metabolism required to perform transformations on some dietary pigments (carotenoids) is disabled due to flying restrictions. Although apparently peripheral, the cited mechanism may contribute to explaining how red traits in many species evolve as signals of individual quality under sexual selection.

Red carotenoid-based colours in animals can be obtained from the diet, which may imply costs in terms of acquisition (e.g. [2]), but often require conversion by oxidization of dietary yellow carotenoids, especially in birds [3]. Recently, Geoffrey Hill and colleagues suggested that enzymes responsible for yellow-to-red carotenoid transformations (ketolases) are located in the inner mitochondrial membrane (IMM) and are directly linked to the electron transport chain (ETC) [4,5]. They proposed that yellow carotenoids (e.g. zeaxanthin in figure 1) are oxidized in the enzymatic complex devoted to coenzyme Q₁₀ (coQ₁₀) biosynthesis, which they proposed to be located between complexes I and III of the ETC (figure 1; [3,4]). Here, the oxidized and reduced forms of coQ₁₀ (ubiquinol [QH2] and ubiquinone [Q]) constitute an important redox couple that can be considered molecularly similar to yellow and red carotenoids, respectively [4]. The main function of coQ₁₀ is electron shunting from complex I to III (figure 1), but QH2 also acts as an antioxidant protecting IMM from oxidative damage to lipids (e.g. [6]). Red carotenoid production could thus compete with coQ₁₀ synthesis, interfering with ETC and IMM protection [3]. This shared pathway implies that red ornaments should obligatorily reveal the quality of the oxidative metabolism (they would act as ‘indices’ of quality) [7]. This ‘Inner Mitochondrial Membrane Carotenoid Oxidation’ hypothesis (IMMCOH; [3]) is a particular case of the broader ‘shared pathway’ hypothesis [8], which challenges the traditional view of reliable signals due to costs generated by allocation trade-offs between investing limited resources (here carotenoids) in reproduction (sexual signalling) or instead self-maintenance (carotenoids may act as antioxidants).

Figure 1.

MitoQ and dTPP are located in the IMM driven by their membrane potential. Both molecules hold a triphenylphosphonium (TPP) cation that is bound at the fatty acid carbonyls and an alkyl chain inserted in the lipid bilayer, but only MitoQ includes the aromatic ring. Ubiquinone (Q) has both the aromatic ring and the alkyl chain (isoprenoid tail). MitoQ derived Q is reduced to ubiquinol (QH2) in complex II (see §1) to move two electrons to complex III of the respiratory chain. The location of a yellow pigment (zeaxanthin) is also shown.

We analysed variability in bill redness (generated by red carotenoids biotransformed from dietary yellow carotenoids [9]) of male zebra finches injected with one of two different mitochondria-targeted molecules: decyl-triphenylphosphonium (dTPP) or mitoquinone mesylate (MitoQ; [10]). The latter has the same structure as dTPP plus the Q aromatic ring (figure 1). MitoQ accesses complex II stimulating QH2 production that is transferred to complex III (figure 1; [11]; also Michael Murphy 2017, personal communication). We tentatively predict that these high QH2 levels interfere with carotenoid conversion, with MitoQ birds producing paler beaks. Alternatively, an hypothetical antioxidant protection due to QH2 could stabilize the IMM [10,11] and improve the ketolase function, leading to redder bills. Given the current lack of knowledge on these metabolic pathways, this experiment constitutes the first approach towards testing a mitochondrial-based mechanism for the expression of red carotenoid-based coloration.

2. Material and methods

(a). Experimental design

The study was performed on 81 unpaired adult male zebra finches. Birds were housed randomly in twos in cages (see electronic supplementary material). Birds were randomly assigned to a treatment group and injected subcutaneously into the back every 2 days for 20 days with 50 µl of MitoQ or dTPP in saline solution at a dose of 0.25 (n = 12 and 11, respectively), 0.5 (n = 11 and 12), or 1 mM (n = 11 and 10). Additionally, a saline-only control group (50 µl; n = 14) was included. All birds were weighed and bill colour was measured at days 1 and 21 (initial and final values).

(b). Colour measurements

The bills were photographed with a Nikon D-3100 in a standardized position and light conditions (electronic supplementary material). Digital photographs were analysed using ‘SpotEgg’ [12], a new software that solves the need for linearizing the camera's response and corrects for subtle light intensity changes. For each animal, we calculated the average of red, green and blue components of the bill surface. We then determined hue values by means of the Foley & van Dam algorithm (electronic supplementary material).

(c). Statistical analyses

Mixed models (PROC MIXED in SAS v. 9.3 software) were used to include cage identity and the origin of the birds (the supplier) as random factors (electronic supplementary material). Random terms were always non-significant (p > 0.20), and their removal did not affect the results. The treatment (MitoQ versus dTPP-control) and dosages (0.25, 0.5 and 1 mM) were tested as fixed factors. The initial hue and body mass (also alternatively size-corrected body mass [body condition] residuals or body mass change; electronic supplementary material) values were covariates. The interactions between the two factors and body mass covariates were tested. A backward stepwise procedure was used to remove non-significant terms. Nonetheless, when similar tests provided similar results the lowest Akaike criterion was chosen to select the best-fitted model. Least significant difference (LSD) post-hocs and least square mean ± s.e. are shown (but see electronic supplementary material, figure S3 for raw data differences). Effect sizes were provided as Cohen's d in pairwise tests with p < 0.10 (also electronic supplementary material). Satterthwaite DFs were used. Since saline serum was the vehicle and saline-control birds constituted a single level, this group could not be included in the interactions. To solve this, pairwise comparisons from an alternative model including a factor with seven levels (treatments × dosage plus saline-control group) were also tested.

3. Results

A significant treatment × dosage interaction was detected in final bill hue values (F_2,58 = 5.05, p = 0.0095; figure 2; also electronic supplementary material, table S1). The pairwise comparisons revealed that birds receiving the highest MitoQ dose differed from those receiving lower doses of any compound (all p-values < 0.024; d range: 0.94–1.10). dTPP treated groups did not differ from each other (p > 0.180). However, when saline-control birds were compared, this group significantly differed from the highest dTPP dose (p = 0.033; d = 0.99) and showed a trend toward significance when compared to the highest MitoQ dose (p = 0.096; d = 0.68). Other comparisons of the saline group were non-significant (all p-values > 0.24; also electronic supplementary material, figure S3).

Figure 2.

Final values of bill hue in male zebra finches subcutaneously treated with MitoQ or dTPP at different doses or receiving the vehicle (saline) only. Least squared means ± s.e. from mixed models. Inset: bill colour example of a male zebra finch treated with MitoQ (left) and dTPP (right).

The best-fitted model included an interaction between initial body mass and treatment (F_2,58 = 14.59, p = 0.0003). Heavier MitoQ-treated birds generated redder bills (lower hue; slope ± s.e.: −0.226 ± 0.039, p < 0.0001), with the effect being absent among dTPP birds (0.012 ± 0.030, p = 0.688). This interaction was also present when the saline-control group was added in an alternative model (three groups × initial body mass: F_2,73.2 = 6.04, p = 0.0037; electronic supplementary material, figure S2), excluding the dose factor and its interaction with treatments. Similar to dTPP birds, saline-control birds did not show a significant relationship (slope ± s.e.: −0.045 ± 0.073, p = 0.56; also electronic supplementary material). In all the models, the treatment × dose interaction remained significant (p < 0.05) when initial body mass × treatment interaction was removed (electronic supplementary material).

4. Discussion

Our results support the assumption of the ‘Inner Mitochondrial Membrane Carotenoid Oxidation’ hypothesis (IMMCOH) that the IMM is the site of transforming yellow to red carotenoids. The two mitochondrial-targeted molecules affected red coloration in different directions. This may also support the second prediction in which coQ₁₀ antioxidant activity probably contributes to improving the activity of ketolases transforming yellow to red carotenoids. Nonetheless, dTPP clearly exerted the opposite effect (below). The results apparently contradict the idea of competitive interference of coQ₁₀ on carotenoid transformation (a shared pathway; [3]), but we must be very cautious in drawing definitive conclusions, as this is an initial step in understanding the full mechanism.

Interestingly, among MitoQ-treated birds only, heavier birds produced redder bills than lighter birds. This suggests context dependency in the response (e.g. [13]), but also indicates individual quality differences (the same is found when testing body condition instead; electronic supplementary material). This supports the role of coQ₁₀ in a mechanism assuring the reliability of red carotenoid-based ornaments as signals.

With regard to dTPP, recent work showed reduced activity of IMM enzymatic complexes and increased production of mitochondrial reactive oxygen species (mROS) in mammalian cells exposed to this molecule [14]. The fact that the greatest colour difference was observed when comparing birds exposed to the highest doses of dTPP and MitoQ (figure 2) suggests that coQ₁₀ can reverse these hypothetical dTPP-linked effects. This may at least partially support a positive effect of coQ₁₀ on ketolases via QH2 antioxidant properties.

A gene coding a monooxygenase enzyme involved in transforming yellow carotenoids to red pigments in animals was recently discovered in zebra finches and canaries (Serinus canaria; [9,15]). This enzyme is CYP2J19, a member of the cytochrome P450 family of enzymes. Ubiquinone biosynthesis enzymes are not CYPs, which contradicts the enzymatic mechanism as proposed in the IMMCOH [3]. Moreover, CYP2 members are usually considered as microsomal enzymes (e.g. [16]). Nonetheless, it has also been shown that some CYP2 have a bimodal location, also being present in the mitochondria [16]. Moreover, high levels of red carotenoids have been detected in the liver mitochondria of birds [17]. These two findings and our results thus support the IMMCOH assumption that ketolases are located in that organelle.

Further work deciphering the role of coQ₁₀ in mitochondrial physiology is now required to verify the pathways involved in the mechanism of red colour expression in animals. In any event, the results suggest that we are close to disentangling the machinery, which will provide understanding of the evolution of red signals.

Ethics

This study was approved by the Bioethical Committees of CSIC (Ref. 404/2016) and the local government (Castilla-La Mancha; Ref. 486728).

Data accessibility

All data are available in the electronic supplementary material.

Authors' contributions

A.C. carried out the experiment, analysed data and drafted the manuscript. C.A.-A. designed the study, supervised the analyses, and drafted the manuscript. All authors approve publication and are accountable for this work.

Competing interests

We have no competing interests.

Funding

A.C. was funded by a postdoctoral grant (FJCI-2015-23536; Ministerio de Economía, Industria y Competitividad, MINECO, Spanish Government). Financial support was obtained from project CGL2015-69338-C2-2-P (MINECO).