Chemical manipulation of mitochondrial function affects metabolism of red carotenoids in a marine copepod (Tigriopus californicus)

Ornamental traits can provide reliable information about the condition or quality of an organism (Bonduriansky, 2007; Hill, 2011; Rowe and Houle, 1996). The mechanism by which such honest signaling is achieved remains a focus of debate (Higham, 2014; Searcy and Nowicki, 2010; Smith and Harper, 2003). The shared-pathway hypothesis proposes that some ornaments act as condition-dependent signals of quality because their production is intimately tied to the same vital cellular processes that give rise to individual performance, regardless of the influence of sexual selection (Hill, 2011, 2014a,b; Powers and Hill, 2021; Weaver et al., 2017). The hypothesized consequence of such a link is that condition-dependent traits serve as indices of individual quality, such that expression of the ornamental trait reflects the function of internal processes required for the maintenance of life (Biernaskie et al., 2014; Hill, 2011). Specifically, the dynamics of the mitochondrial organelle, a critical, cellular hub of organismal function and condition (Havird et al., 2019; Heine and Hood, 2020; Picard and Sandi, 2020), has been identified as a candidate regulator in the expression of condition-dependent traits (Hill, 2014a,b; Koch et al., 2017).

A primary and well-studied example of a condition-dependent ornamental trait is bright red coloration that arises from the accumulation of carotenoid pigments in animal tissues (Blount and McGraw, 2008; Hill, 2002; Svensson and Wong, 2011). With few exceptions, animals cannot synthesize carotenoid pigments de novo; they must acquire carotenoids through their diet in a trophic manner (Svensson and Wong, 2011). Further, once ingested, many animals metabolize dietary carotenoids into other carotenoid forms in order to produce pigments with new functional roles or new colors (Maoka, 2011; Von Lintig, 2010). This process can occur through a series of oxidation reactions, such as hydroxylations and/or ketolations, that may cause a color change from the yellow hues of dietary carotenoids to the red hues of metabolized carotenoids (Meléndez-Martínez et al., 2007). For example, many crustaceans convert yellow dietary xanthophylls found in algae to red ketolated carotenoids (ketocarotenoids) such as astaxanthin. Many animals display astaxanthin-based coloration, including birds (Hill and McGraw, 2006), insects (Kayser, 1982), reptiles (Czeczuga, 1980), amphibians (Baruah et al., 2012), fish (Seabra and Pedrosa, 2010) and crustaceans (Maoka, 2011). Astaxanthin is also often recognized as a potent antioxidant and potential immune system regulator (Dose et al., 2016; Naguib, 2000; Weaver et al., 2018b).

The shared-pathway hypothesis, as it applies to ketocarotenoid-based coloration, posits that ketocarotenoid accumulation is intrinsically linked to individual condition through metabolic pathways shared with oxidative phosphorylation (OXPHOS) in the mitochondria (Hill, 2014a,b; Koch et al., 2017; Powers and Hill, 2021). Under this hypothesis, the bioconversion of red ketocarotenoid pigments is hypothesized to share a biochemical environment with the enzyme complexes in the electron transport system (Hill et al., 2019; Powers and Hill, 2021). These complexes are positioned within the inner mitochondria membrane (IMM) and produce a proton gradient across the membrane needed for the synthesis of ATP. This activity requires a series of reduction–oxidation (redox) reactions driven by coupled electron donors and acceptors, such as NADH/NAD+. The enzymes responsible for the production of ketocarotenoids also metabolize dietary carotenoids using coupled redox reactions, likely utilizing similar electron donor/acceptor systems (Hill and Johnson, 2012; Lopes et al., 2016; Munro et al., 2007; Twyman et al., 2018). Indeed, ketocarotenoids have been observed at the IMM in red birds, and molecular modeling of the avian carotenoid bioconversion enzyme, CYP2J19, suggests it may localize either in the IMM or mitochondria-associated membranes (MAMs) (Ge et al., 2015; Hill et al., 2019). Moreover, positive relationships between resting metabolic rate, mitochondrial damage, lower rates of mitochondrial replacement and ketocarotenoid-based coloration have been observed in red birds, suggesting a potential for ketocarotenoid coloration to reflect metabolic capacity in the face of ongoing cellular stress (Hill et al., 2019). Observations in arthropod systems corroborate these observations in vertebrate systems. In the marine copepod Tigriopus californicus, interpopulation hybrids show a significant positive relationship between oxygen consumption and ketocarotenoid accumulation (Powers and Hill, 2021) and a significant negative relationship between ATP production and ketocarotenoid coloration (Powers et al., 2021), thus indicating that ketocarotenoid accumulation may reliably indicate the capacity to withstand stress and exert energy in invertebrate species as well.

To test the shared-pathway hypothesis, we experimentally exposed T. californicus copepods to a mitochondrially targeted compound, 2,4-dinitrophenol (DNP), and measured their accumulation of ketocarotenoid pigmentation. This copepod, native to splash-pools along the west coast of North America, accumulates astaxanthin to produce its vibrant, red coloration, using many of the same carotenoid substrates preferred by red birds and other terrestrial organisms (Prado-Cabrero et al., 2020; Weaver et al., 2018a). Moreover, T. californicus ketocarotenoid-based coloration has also been shown to be dependent on individual condition (Powers et al., 2021; Weaver et al., 2016, 2018b). In birds, mitochondria-targeted compounds, such as synthetic ubiquinones and superoxide dismutase mimetics, have been observed to alter ketocarotenoid metabolism (Cantarero and Alonso-Alvarez, 2017; Cantarero et al., 2020a,b). However, these compounds may also increase ROS levels as they alter oxidative state in the mitochondria, and this may, in turn, influence ketocarotenoid levels given their possible role in ROS scavenging (Dose et al., 2016; Naguib, 2000). In contrast, the ionophore DNP does not incur this complication (Goldgof et al., 2014; Korde et al., 2005; Leverve et al., 1998; Lozinsky et al., 2013; Rognstad and Katz, 1969; Stier et al., 2014). This compound functions as a mitochondrial ‘uncoupler’, acting as a protonophore in the IMM of living mitochondria to dissipate the proton gradient required to make ATP (Fig. 1A) (Pinchot, 1967). Therefore, at sublethal, acute doses, DNP increases the rate of respiration in animals, including crustaceans (Caldeira da Silva et al., 2008; Cantelmo et al., 1978; Geisler et al., 2017). We predicted that, if carotenoid metabolizing enzymes share an oxidative environment with mitochondrial OXPHOS enzymes, DNP should simultaneously increase respiration rate and ketocarotenoid metabolism in T. californicus copepods. We tested this prediction using copepods with natural levels of astaxanthin and copepods deficient of carotenoids to test their ability to restore pigmentation from a colorless state (Fig. 1B).

Tigriopus californicus (Baker 1912) cultures were maintained in a climate-controlled incubator under the following conditions: 32 psu artificial seawater (ASW) at 20°C on a 12 h:12 h light:dark cycle and fed live microalgae (Tetraselmis chuii). The cultures used in this experiment were maintained for approximately 3 years as continuous cultures. Cultures were a mix of commercially obtained copepods (Reef Nutrition, Campbell, CA, USA) and admixed wild populations collected from San Diego, Bird Rock, Abalone Cove, Santa Cruz and Bodega Bay, CA, USA. The cultures used in this experiment were admixed once a month (approximately once per generation) via transfer of water and individuals amongst all culture vessels in attempt to maintain homogeneity among culture vessels. However, we only sampled from two specific culture vessels as a source of stock copepods and one vessel as a source of colorless copepods, in an attempt to reduce variability among copepods from different sources. Regardless, the heterogeneity in the sourcing of copepods that make up our stock cultures could contribute to variability in results. Lab-reared copepods fed Tetraselmis retain their red coloration similar to wild populations (Davenport et al., 2004; Weaver et al., 2018a) and the Tetraselmis diet approximates the algae-based diet found in natural splash-pools (Powlik et al., 1997; Vittor, 1971). Tetraselmis algae provides dietary carotenoid precursors which T. californicus metabolize to synthesize astaxanthin, specifically β-carotene and hydroxyechinenone (Ahmed et al., 2014; Powers et al., 2021; Prado-Cabrero et al., 2020; Weaver et al., 2018a). We refer to copepods cultured under these conditions as ‘red stock’ copepods.

‘Colorless’ T. californicus cultures were established by isolating gravid female copepods from the stock conditions described above and gently removing their eggs. Eggs were placed in a separate container containing only clean artificial saltwater and after hatch, were reared on ground nutritional yeast powder, but no algae (and thus, no dietary carotenoids). These nearly clear copepod cultures were kept in the dark 24 h of the day to minimize the chance of algae growth in the culture containers. As these copepods did not have access to dietary carotenoids, they remained almost entirely clear in color throughout their development (Powers et al., 2020, 2021; Weaver et al., 2018a).

Before experimentation, we performed survival assays to identify a maximum concentration of DNP (TCI, Portland, OR, USA) that did not produce lethality. DNP was dissolved in 32 psu ASW to make the following stock concentrations: 100, 50, 25, 10, 2, 1, 0.5 and 0 μmol l⁻¹ (control). To assay survival at each of these concentrations, we placed five copepods in a single well of a 24-well plate with 50 μl of concentrated Tetraselmis algae and monitored for individual death every day for 4 days. This was repeated five times each for each concentration, for a total of 25 copepods per DNP concentration. The highest concentration of DNP tested, 100 μmol l⁻¹, was more than 80% lethal after just 24 h (Fig. S1). The next highest concentration, 50 µmol l⁻¹, reached 50% lethality after 48 h (Fig. S1). The concentration of 25 µmol l⁻¹ DNP neared 65% lethality after 4 days exposure. The concentration of 10 µmol l⁻¹ DNP saw less than 15% lethality after 4 days, was comparable to the death observed in the control group (0 μmol l⁻¹; Fig. S1). The lowest concentrations of DNP tested, 2, 1 and 0.5 µmol l⁻¹, all showed minimal or no lethality after 4 days (Fig. S1). This was less than the death observed in the control group (0 µmol l⁻¹ DNP) after 4 days (Fig. S1). Based on these results, 10 and 2 μmol l⁻¹ DNP were chosen as the maximum acute dosages for the experiment. These two concentrations were chosen to maximize the effect of DNP on respiration over a short period of time, without reaching 50% lethality during experimental exposure. Thus, the lowest concentrations, 1 and 0.5 μmol l⁻¹, and highest concentrations, 25 μmol l⁻¹ and higher, were not tested in the subsequent respiration and carotenoid metabolism assays to minimize the risk of observing no effect on respiration and the risk of cytotoxicity, respectively.

We exposed adult copepods (three individuals per replicate) to either 0 (control), 2 or 10 μmol l⁻¹ DNP by placing them in 5 ml test solutions of ASW in six-well plates. Each replicate of three copepods was maintained through respiration and HPLC analyses (i.e. the three individuals were isolated as one unit). We separated adult copepods by sex (using morphology of forward antennules) owing to observed differences in carotenoid ketolation in previous experiments (Powers et al., 2021) and maintained them under the standard rearing conditions described above for 3 or 7 days (Table 1). At the beginning of the experiments, we added 50 μl of concentrated, live Tetraselmis algae as food and a source of dietary carotenoids (Table 1). This serving of algae was ad libitum: at the end of the DNP exposure time, the water was still green with algal cells. We exposed red stock copepods to DNP under the following conditions: 10 μmol l⁻¹ DNP for 3 days, 10 μmol l⁻¹ DNP for 7 days and 2 μmol l⁻¹ DNP for 7 days. We first started with only 3 days exposure to the higher dose of DNP, 10 μmol l⁻¹, in order to minimize the risk of death. When this period of time did not result in elevated death compared with controls, we extended the exposure time to 7 days to test whether oxygen consumption remained elevated at a slightly longer exposure period. We then exposed red stock copepods to 2 μmol l⁻¹ DNP for the same week-long period in order to facilitate a comparison with colorless copepods exposed to DNP under the same regime (see further rationale below).

We exposed colorless copepods to DNP in a single concentration and exposure time, 2 μmol l⁻¹ DNP for 7 days, in order to test carotenoid metabolism from a colorless state during acute exposure to DNP. The concentration of 2 μmol l⁻¹ DNP was chosen over 10 μmol l⁻¹ for the experiment with colorless copepods owing to previous observations that yeast-fed, colorless copepods may be more susceptible to oxidative stress than algae-fed, red stock copepods (Powers et al., 2021). The exposure time of 7 days was chosen to ensure copepods had time to (1) respond to mitochondrial uncoupling and (2) metabolize enough astaxanthin to produce a strong signal during HPLC analyses of copepod body tissue. At a minimum, T. californicus needs 48 h to produce detectible amounts of astaxanthin from a colorless state (Weaver et al., 2018a), and takes about 2 weeks to fully restore astaxanthin concentrations to that of red stock cultures when feeding on Tetraselmis algae (Davenport et al., 2004). At the end of DNP exposure, algae feeding and carotenoid restoration, the colorless copepods regained visible red coloration; thus, we refer to these as ‘color-restored’ in figures displaying the carotenoid content of these copepods.

We measured whole-animal oxygen consumption rates of control and DNP-exposed copepods using a 24-well microplate respirometer (Loligo Systems, Copenhagen, Denmark). Prior to respiration measurement, copepods were moved to a six-well plate with clean ASW and allowed to clear their gut contents for a minimum of 1 h. After this period, the three copepods from each replicate of the DNP-exposure experiment were added to separate 80 μl wells of the microrespirometer filled with 32 psu ASW. Copepods were left undisturbed in the wells for 30 min prior to recording oxygen consumption. We then sealed the wells using a gel seal and weighted block. Oxygen consumption was recorded over 30 min after an initial burn-in period of 5 min. Throughout the measurement process, the temperature was 22°C. Final oxygen consumption was calculated as millimoles oxygen consumed per minute per milligram of copepod body mass. This was calculated by calculating the slope of the raw oxygen values over time in minutes and subtracting the average slope of empty blank wells (minimum three per plate). Copepod body mass was measured using a precision microscale (accurate to 0.001 mg) following oxygen consumption. Prior to weighing, the copepods were dried under air flow in a fume hood for 30 min to obtain dry body mass. The copepods were then placed in microtubes and frozen in a −80°C freezer until HPLC analysis.

Carotenoids were extracted from dried copepod tissues using sonication in acetone. We extracted carotenoids from the three copepods per replicate as a single pooled sample. Centrifugation was used to pellet and remove cellular debris, saving the carotenoid extract suspended in acetone. The carotenoid extract was evaporated to dryness and resuspended in 50 μl acetone for HPLC analysis. We separated and quantified copepod carotenoids using HPLC following the methods of Weaver et al. (2018a) and Wright et al. (1991). We injected 10 µl of carotenoid extract in acetone onto a Sonoma C18 column (10 µm, 250×4.6 mm, ES Technologies, NJ, USA) fitted with a C18 guard cartridge. Carotenoids were separated using a Shimadzu Prominence HPLC system with mobile phases A (80:20 methanol:0.5 mol l⁻¹ ammonium acetate), B (90:10 acetonitrile:water) and C (ethyl acetate) in a tertiary gradient of 100% A to 100% B over 4 min, then to 80% C:20% B over 14 min, back to 100% B over 3 min, and returning to 100% A over 5 min and held for 6 min. We visualized and detected carotenoid absorbance using a Prominence UV/Vis detector set to a wavelength of 450 nm. We identified and quantified carotenoids by comparison with authentic standards that included: astaxanthin, zeaxanthin, β-carotene, lutein, hydroxyechinenone and canthaxanthin (Fig. S2). We normalized carotenoid concentration of each carotenoid detected by the dry mass of each copepod sample (reported as µg carotenoid mg⁻¹ copepod tissue).

All statistical analyses were performed in R (https://www.r-project.org/) using the following packages: lme4 (Bates et al., 2015), Hmisc (https://CRAN.R-project.org/package=Hmisc), lmerTest (Kuznetsova et al., 2017), agricolae (https://CRAN.R-project.org/package=agricolae), MASS (Venables and Ripley, 2013), emmeans (https://CRAN.R-project.org/package=emmeans), car (Fox and Weisberg, 2018), sf (Pebesma, 2018), MuMIn (https://CRAN.R-project.org/package=MuMIn) and bestNormalize (Peterson, 2021). Data wrangling was performed in R with the help of the follow packages: tidyverse (https://tidyverse.tidyverse.org/), dplyr (https://CRAN.R-project.org/package=dplyr), reshape2 (Wickham, 2007), plotrix (Lemon, 2006) and HH (Heiberger et al., 2015). Figures were produced using the following packages: ggpubr (https://CRAN.R-project.org/package=ggpubr), cowplot (Wilke, 2016), RColorBrewer (https://CRAN.R-project.org/package=RColorBrewer) and ggjoy (https://CRAN.R-project.org/package=ggjoy). For a full review of our statistical analyses, please refer to the annotated R code included in the Supplementary Materials and Methods; we provide below a brief description of each analysis and the type of modeling used.

To test for statistically significant differences in respiration rate between DNP-treated and control copepods, we encoded a linear model with treatment (DNP versus control) as a fixed effect, an interaction with sex, and copepod mass (dry body mass) as a fixed effect. We inspected pairwise comparisons between DNP-treated and control copepods, blocked by sex, using a Tukey correction for multiple pairwise comparisons. In these models, mass-independent respiration rate was used as the dependent variable instead of mass-corrected respiration rate. As noted above, the effect of mass was controlled by including this variable as a fixed effect (Lighton, 2018; Niitepõld, 2019; Schmidt-Nielsen and Knut, 1984). We included mass a covariate for all models with respiration rate as the dependent variable. For models where respiration rate was included as a fixed effect (i.e. independent variable), we used mass-corrected respiration rate as a covariate (see below).

To test for differences in carotenoid concentration, we fit a linear model with treatment, sex and their interaction as fixed effects. We then inspected pairwise comparisons between DNP-treated and control copepods, blocked by sex, using a Tukey correction for multiple pairwise comparisons. To model the effect of increasing respiration on carotenoid concentrations across all samples, we specified a linear mixed-effects model with mass-corrected respiration rate and sex as fixed-effect covariates and diet (red stock versus colorless) as a random effect. We subset our data by control and DNP-treated copepods and used the same model for each subset to inspect the relationship between respiration rate and carotenoid concentration. Prior to analyzing the effect of respiration rate on dietary carotenoid concentration, we transformed dietary carotenoid concentrations using an ordered quantile normalization (chosen using the bestNormalize package) owing to observed non-normality in the data frequency distribution. To test for any statistical relationship between dietary carotenoid and astaxanthin concentrations in our control and DNP-treated samples, we fit a mixed-effects model with astaxanthin concentration as a dependent variable, transformed dietary carotenoid concentration and sex as fixed effects, and diet as a random effect.

To test whether DNP treatment altered the body mass of copepods compared with controls, we fit a linear mixed-effects model with treatment and sex encoded as fixed effects and a random effect of diet (i.e. red stock or colorless).

We found no significant difference in the mass of copepods exposed to DNP and control copepods across all treatments, after controlling for differences owing to sex and diet (mean±s.e.m. tissue mass: DNP, 0.0453±0.0031 mg; control, 0.0451±0.0031 mg; Fig. S3).

We detected three primary carotenoids across all copepod samples: astaxanthin, β-carotene and hydroxyechinenone (Fig. S2). Astaxanthin was the most abundant carotenoid present in the T. californicus tissues, on average making up 97.6% of the quantifiable carotenoid content in red stock copepods and 95.3% in color-restored copepods (mean±s.d. μg astaxanthin mg⁻¹ tissue; red stock: 1.29±0.45; color-restored: 0.51±0.14; Fig. S4). The second-most abundant carotenoid in both red stock and color-restored copepods was hydroxyechinenone (mean±s.d. μg hydroxyechinenone mg⁻¹ tissue; red stock: 0.020±0.023; color-restored: 0.024±0.011; Fig. S4). Finally, β-carotene was present and quantifiable in only some, but not all, samples and was the least abundant precursor to astaxanthin (mean±s.d. μg β-carotene mg⁻¹ tissue; red stock: 0.012±0.018; color-restored: 0.0095±0.0038; Fig. S4). In both red stock and color-restored copepods, the concentration of hydroxyechinenone was statistically greater than the concentration of β-carotene (β₁=model estimate; red stock: β₁=0.0076 μg mg⁻¹ tissue, P=0.0184; color-restored: β₁=0.023 μg mg⁻¹ tissue, P<0.001).

After 3 days exposure to 10 μmol l⁻¹ DNP, the respiration of red stock copepods was significantly elevated in both sexes (mean±s.e.m. mmol O₂ min⁻¹; DNP male: 2.14±0.20; control male: 1.17±0.19; DNP female: 2.06±0.17; control female: 1.54±0.18; Fig. 2A). Males treated with DNP consumed on average 0.53 mmol O₂ min⁻¹ more than control males [confidence interval (CI)=0.036–1.01], and females treated with DNP consumed on average 0.97 mmol O₂ min⁻¹ more than control females (CI=0.40–1.53). When 10 μmol l⁻¹ DNP exposure was extended to 7 days, respiration was again elevated in male copepods given DNP, but similar to control levels in females (mean±s.e.m. mmol O₂ min⁻¹; DNP male: 1.30±0.17; control male: 0.89±0.21; DNP female: 1.29±0.20; control female: 1.30±0.18; Fig. 2B). However, the difference was not statistically significant in either sex (Fig. 2B). When the exposure time was kept at 7 days, but the concentration of DNP was lowered to 2 μmol l⁻¹, female copepod respiration dropped significantly below that of control females (mean±s.e.m. mmol O₂ min⁻¹; DNP female: 0.81±0.09; control female: 1.12±0.11; β₁=−0.311 mmol O₂ min⁻¹, CI=−0.60 to −0.023; Fig. 2C). At this concentration and exposure time, male respiration was significantly elevated (mean±s.e.m. mmol O₂ min⁻¹; DNP male: 0.98±0.11; control male: 0.63±0.11; β₁=0.35 mmol O₂ min⁻¹, CI=0.026–0.67; Fig. 2C).

After 3 days exposure to 10 μmol l⁻¹ DNP, the astaxanthin concentration in male and female copepod tissues was not significantly different from that in control copepod tissues (mean±s.e.m. μg astaxanthin mg⁻¹ tissue; DNP male: 1.50±0.12; control male: 1.52±0.12; DNP female: 1.24±0.13; control female: 1.35±0.12; Fig. 3A). However, after 7 days exposure to 2 μmol l⁻¹ DNP, astaxanthin concentrations were increased in both sexes, significantly in males (mean±s.e.m. μg astaxanthin mg⁻¹ tissue; DNP male: 1.64±0.16; control male: 0.97±0.15; DNP female: 1.13±0.13; control female: 0.97±0.14; Fig. 3B). Male copepods exposed to DNP contained 0.67 μg astaxanthin mg⁻¹ tissue more than control copepods (CI=0.23–1.10).

After 3 days exposure to 10 μmol l⁻¹ DNP, we observed no clear or significant difference between the concentration of dietary carotenoids in the tissues of DNP-treated copepods compared with tissues of control copepods (mean±s.e.m. μg dietary carotenoids mg⁻¹ tissue; DNP male: 0.050±0.012; control male: 0.040±0.12; DNP female: 0.047±0.013; control female: 0.046±0.012; Fig. S5A). Similarly, we found no significant difference between the concentration of dietary carotenoids in copepods treated with 2 μmol l⁻¹ DNP for 3 days compared with control copepods (mean±s.e.m. μg dietary carotenoids mg⁻¹ tissue; DNP male: 0.028±0.0067; control male: 0.018±0.0063; DNP female: 0.0081±0.0054; control female: 0.0095±0.0059; Fig. S5B).

In colorless copepods, DNP increased the rate of respiration after 7 days in both males and females (mean±s.e.m. mmol O₂ min⁻¹; DNP male: 1.02±0.062; control male: 0.82±0.073; DNP female: 0.84±0.076; control female: 0.68±0.080; Fig. 4A). This was statistically significant in males (β₁=0.20 mmol O₂ min⁻¹, CI=0.014–0.39).

When allowed to restore red coloration through supplementation with Tetraselmis algae, male copepods treated with DNP produced only slightly more astaxanthin than controls (mean±s.e.m. μg astaxanthin mg⁻¹ tissue; DNP male: 0.54±0.025; control male: 0.52±0.028; Fig. 4B). However, female copepods treated with DNP accumulated significantly more astaxanthin compared with controls (mean±s.e.m. μg astaxanthin mg⁻¹ tissue; DNP female: 0.53±0.032; control female: 0.43±0.032; β₁=0.098 μg astaxanthin mg⁻¹ tissue, CI=0.0091–0.19; Fig. 4B). There were no significant differences between the average concentration of dietary carotenoids in DNP-treated and control copepods (mean±s.e.m. μg dietary carotenoids mg⁻¹ tissue; DNP male: 0.021±0.0020; control male: 0.020±0.0022; DNP female: 0.033±0.0025; control female: 0.030±0.0025; Fig. S6). Both DNP-treated and control copepods of both sexes contained significantly more astaxanthin than colorless males and females never given access to algae to restore their coloration (P<0.0001 for each comparison; mean±s.e.m. μg astaxanthin mg⁻¹ tissue; colorless males: 0.133±0.052; colorless females; 0.128±0.052; Fig. 4B).

Across all experiments, there was a positive relationship between respiration rate and astaxanthin concentration after controlling for sex and diet (Fig. 5A,B), significant only in DNP-treated copepods (d.f.=74, t=0.52; β₁=4.4e⁻³ μg astaxanthin mg⁻¹ tissue, CI=6.1e⁻⁴–8.5e⁻³; Fig. 5B). This same effect was observed between dietary carotenoids and respiration rate in DNP-treated copepods (Fig. 5C,D), again controlling for sex and diet (d.f.=55, t=2.49; β₁=0.014 ordered quantile of dietary carotenoid concentration, CI=7.8e⁻⁴–0.024; Fig. 5D). However, there was a significant relationship between the concentration of dietary carotenoids and the concentration of astaxanthin in the tissues of control copepods only (d.f.=79, t=3.41; β₁=0.12 μg astaxanthin mg⁻¹ tissue, CI=0.050–0.18; Fig. 6A). We observed no significant effect of increasing dietary carotenoid concentration on astaxanthin concentration in the tissues in DNP-exposed copepods (Fig. 6B).

When treated with the mitochondria uncoupler and protonophore DNP, T. californicus copepods produced more of the red carotenoid pigment astaxanthin after 7 days, but not after 3 days. These experiments provide further support for the hypothesis that metabolism of ketocarotenoids from yellow dietary pigments is intimately linked to energetic processes in the mitochondria. Here, we observed that acute exposure to DNP increased the metabolic rate in T. californicus copepods similar to observations in other taxa (Caldeira da Silva et al., 2008; Cantelmo et al., 1978; Geisler et al., 2017; Goldgof et al., 2014; Pinchot, 1967). As predicted if carotenoid metabolism is tied to mitochondrial function, we then observed that DNP-treated copepods produced more astaxanthin than did control copepods. This study provides the first experimental demonstration of a relationship between DNP-induced changes to mitochondrial metabolism and the accumulation of metabolized carotenoid pigments. Critically, it is the first chemical manipulation of mitochondrial function to affect metabolism of ketocarotenoids where results should not be confounded by an increase in the production of reactive oxygen species (ROS) in the mitochondria.

An alternative hypothesis is that carotenoid-based coloration is a signal of condition because there are trade-offs in the allocation of carotenoid resources between coloration and stress responses (Alonso-Alvarez et al., 2007, 2008; Moller et al., 2000; Simons et al., 2012). This is the resource trade-off hypothesis, which proposes that carotenoids such as astaxanthin may serve a defensive role against oxidative stress such that carotenoid pigments may be a key, but limited, metabolic resource (Hill, 1999; Lozano, 1994; Moller et al., 2000). The shared-pathway hypothesis and the resource trade-off hypothesis are not mutually exclusive; carotenoid resource availability and mitochondrial energetics might both affect coloration (see fig. 2 of Powers and Hill, 2021). For instance, our observation that copepods increased the concentration of astaxanthin in their tissues could indicate that the DNP, in stabilizing the mitochondrial membranes via uncoupling, freed up carotenoid resources that would normally be consumed during antioxidant activities (Caramujo et al., 2012; Davenport et al., 2004; Weaver et al., 2018b).

Our experiments cannot fully refute the resource trade-off hypothesis. However, for several reasons we think a carotenoid resource trade-off alone cannot fully explain our results. Differences in astaxanthin concentrations between DNP-treated and control copepods did not appear to be due to differences in a greater uptake of dietary carotenoids. In both red stock copepods and colorless copepods and for both males and females, individuals exposed to DNP did not accumulate more dietary carotenoids than control copepods (Figs S6, S7). Moreover, we observed no relationship between dietary carotenoid concentration and astaxanthin concentrations in DNP-treated copepods (Fig. 6). Thus, the positive relationship between dietary carotenoids and respiration rate (Fig. 5D) may have been driven by hydroxyechinenone, which is also a metabolic intermediate between β-carotene and astaxanthin (Weaver et al., 2018a). Further, DNP exposure did not result in an increase in mass in DNP-treated copepods compared with controls, countering the idea that differences in astaxanthin concentrations might simply be due to a difference in algae consumption and, by extension, dietary carotenoid resources.

Several lines of evidence support the hypothesis that DNP treatment stimulated increased metabolism of astaxanthin via increased mitochondrial metabolism. We observed an increase in the rate of oxygen consumption, and hence respiration, in copepods exposed to DNP compared with controls (Figs 2 and 4). This effect was present in both sexes after 3 days exposure to a moderate dose of 10 μmol l⁻¹ DNP. Excluding red stock female copepods (addressed below), the effect persisted in both red stock and colorless copepods when exposure was extended to 7 days and the DNP dosage was lowered to 2 μmol l⁻¹ (Figs S2 and S4). We observed that, on average, copepods exposed to DNP over 7 days accumulated more astaxanthin in their tissues compared with control copepods (Figs S3 and S4), even though this effect was statistically significant in red stock males and colorless females only. This indicates that the red stock copepods exposed to DNP increased astaxanthin concentrations in their tissues beyond the normal levels found in copepods in routine culture conditions. Again, this effect was strongest in male red stock copepods (Fig. 3B). In comparison, colorless copepods exposed to DNP replenished their tissue concentrations of astaxanthin at a faster rate than control individuals, significantly in the case of the colorless females tested. We suspect that we did not see a significant increase in astaxanthin concentrations after 3 days exposure to DNP (Fig. 3A) owing to the base carotenoid conversion rate in the species requiring a full 2 weeks to reach levels comparable to those in wild individuals (Davenport et al., 2004). Three days may not have been enough time to detect measurable differences owing to DNP exposure, a limitation addressed through the inclusion of 7-day exposure trials.

It previously was shown that hybridization had a greater negative impact on astaxanthin metabolism in colorless male T. californicus than in colorless females (Powers et al., 2021). Colorless copepods supplemented with DNP also replenished more astaxanthin over 7 days than did colorless copepods not provided DNP. However, this effect was observed to be stronger in colorless females (Fig. 4B). Colorless females – but not red stock females – supplemented with 2 μmol l⁻¹ DNP for 7 days showed an elevated oxygen consumption on average compared with controls (Figs 3B,C and 4). However, red stock females exposed to DNP showed oxygen consumption rates that had, on average, fallen below that of controls (Fig. 2C). This creates an interesting pattern, because at higher concentrations of DNP (10 μmol l⁻¹) over a shorter period (3 days), red stock females showed significantly elevated respiration rates compared with controls (Fig. 2A). When exposure to 10 μmol l⁻¹ DNP was extended to 1 week, the respiration rate of red stock females was not different from that of controls (Fig. 2B). These observations could indicate a hormetic effect of DNP in red stock females fed algae or may also be explained by the observations that female T. californicus copepods are more resistant to changes owing to oxidative stress (Foley et al., 2019; Harada et al., 2019; Willett, 2010; Willett and Son, 2018). It is possible that colorless females raised on yeast were less able to compensate for the effects of DNP, possibly owing to the relatively poor nutrition provided by yeast compared with algae (Powers et al., 2021). We think it unlikely that this result was due to DNP degrading on its own over the course of 7 days. The half-life of DNP in water is over a year (Tratnyek and Hoigne, 1991), and even powerful bacterial degradation of DNP can still take up to 68 days (Capel and Larson, 1995).

It has been observed that in low doses or relatively short exposures, DNP acts to increase the rate of respiration during OXPHOS, speeding up the rate at which redox reactions occur to compensate for the lack of ATP produced (Goldgof et al., 2014; Leverve et al., 1998; Rognstad and Katz, 1969; Stier et al., 2014). This uncoupling does not result in an increase of ROS (Goldgof et al., 2014; Korde et al., 2005; Lozinsky et al., 2013; Stier et al., 2014), and thus should not have required the consumption of astaxanthin to combat oxidative stress, as has been observed in T. californicus exposed to pro-oxidants (Weaver et al., 2018b). We propose that the acute doses of DNP that we tested uncoupled OXPHOS from ATP production, briefly increasing the rate at which redox reactions occurred in copepod mitochondria in compensation. In turn, this shift in redox environment may have also stimulated an increase in the rate of redox reactions required to metabolize β-carotene and hydroxyechinenone to astaxanthin. In hybrid inbred lines of T. californicus, we also observed an inverse relationship between astaxanthin accumulation and ATP production in colorless copepods (Powers et al., 2021); although in that previous study, respiration rate was not measured. In a separate analysis of T. californicus hybrids, however, increased respiration was associated with increased ketocarotenoid accumulation (Powers and Hill, 2021).

It appears that across diverse animal taxa, the enzymes responsible for carotenoid metabolism belong to the cytochrome p450 superfamily of oxygenases (Lopes et al., 2016; Mojib et al., 2014; Mundy et al., 2016; Twyman et al., 2018, 2016; Weaver et al., 2020). The predicted cellular location of carotenoid oxygenases places them either directly in the IMM, or in the MAMs in the cytosol (Hill et al., 2019). However, a full description of the mechanism and subcellular location of carotenoid metabolizing enzymes in T. californicus is needed before we can fully understand the role of mitochondrial redox state in influencing the expression of ketocarotenoid-based coloration in copepods (Mojib et al., 2014; Weaver et al., 2020). These oxygenases perform their functions via redox reactions with coupled reductases that utilize electron donors such as NADH, NADPH or FADH in the same fashion as the enzymes of the mitochondrial electron transport system (Cojocaru et al., 2007; Munro et al., 2007; Strohmaier et al., 2019). Low doses of DNP result in an increase in electron donors, such as NADH or NADPH (Leverve et al., 1998; Rognstad and Katz, 1969). This increase is observed to occur first in the cytosol, and then subsequently in the mitochondria itself via the influx of NADH and ATP back into the mitochondria (Leverve et al., 1998; Rognstad and Katz, 1969). These changes lead to a decrease in the ratios of electron donors to acceptors in both the cytosol and mitochondria but an increase in reducing power (Leverve et al., 1998). An increase in the redox reaction rate of the mitochondria in the copepods is critically important given the predicted biochemistry of the enzymatic reactions required to metabolize dietary carotenoids to ketocarotenoids.

Recent evidence suggests that changes to mitochondrial energetics can affect carotenoid metabolism. It has been demonstrated that other mitochondria-targeted compounds, besides DNP, can affect the expression of ketocarotenoid-based coloration or the expression of carotenoid metabolizing enzymes. Cantarero et al. (2020a) demonstrated that exposure to the IMM-targeted superoxide dismutase mimetic, mitoTEMPO, successfully altered avian ketolase expression in zebra finches (Taeniopygia guttata). These same researchers also successfully documented an effect of stabilizing or destabilizing the mitochondrial redox environment with mitoQ and dTTP, respectively, on the visible redness of bills of these same birds (Cantarero and Alonso-Alvarez, 2017). These compounds may have altered the normal ratios of ubiquinol and ubiquinone, redox cyclers responsible for the shuttling of electrons in the electron transport system, thereby altering the redox state of carotenoid metabolizing enzymes (Cantarero and Alonso-Alvarez, 2017). Caution should be taken to extrapolate these results to our observations in T. californicus considering the biological differences between terrestrial and aquatic organism physiology. Moreover, the effect of mitochondria-targeted compounds is likely highly contextual and, perhaps, species specific. Indeed, when red crossbills (Loxia curvirostra) were exposed to mitoQ and mitoTEMPO, each compound affected the levels of ketocarotenoids in the blood and feathers differently (Cantarero et al., 2020b).

Our observations of an association between aerobic respiration and metabolism of carotenoids in copepods supports the hypothesis of an intimate link between ketocarotenoid metabolism and mitochondrial function and corroborates previous studies. In wild-caught house finches (Haemorhous mexicanus), Hill et al. (2019) observed a positive relationship between state 4 respiration (resting rate) and ketocarotenoid-based coloration as well as an inverse relationship between lipid damage, mitochondrial replacement and red coloration. In that study, the authors hypothesized that redder individuals that accumulated more ketocarotenoids in their feathers may have been more capable of withstanding higher levels of metabolic damage and energetic output before needing to replace old mitochondria with new copies. Similarly, in T. californicus hybrids fed a restrictive diet supplemented with ad libitum carotenoids, tissue concentrations of ketocarotenoids was negatively correlated with ATP production and not significantly related to offspring development time (Powers et al., 2021), indicating that redder individuals may have needed less ATP to maintain homeostasis. When the dietary stress was alleviated in this copepod study via reintroduction of algal nutrients, the negative correlation between ATP production and ketocarotenoid accumulation disappeared, and there was a positive correlation between ketocarotenoid concentrations and offspring development times (Powers et al., 2021). As noted previously, experimentation with T. californicus fed algae ad libitum also resulted in a significant positive relationship between ketocarotenoid biosynthesis and respiration rate (Powers and Hill, 2021).

Observations in the house finch and T. californicus lend support to an evolving understanding of how metabolic rate relates to ‘quality’ or ‘fitness’, and an increasing understanding that metabolic rate itself may be highly context dependent (Koch et al., 2021). Indeed, it has been suggested that under conditions in which food is not limiting, it may be beneficial to operate at a higher metabolic rate (Norin and Metcalfe, 2019). Likewise, quantitative analyses of over 100 published studies have revealed that it is common to find positive relationships between metabolic rate and many components of fitness (Arnold et al., 2021).

In the house finch and T. californicus studies discussed above, it was unclear how the interaction between ROS produced via normal OXPHOS might influence the relationship between ketocarotenoid concentrations and mitochondrial function at a mechanistic level. In our study, however, we observed an overall relationship between respiration rate and ketocarotenoid biosynthesis without the confounding effects of increased ROS, despite a potential additional effect of sex on the relationship. A critical point is that we did not measure natural differences in metabolic efficiency in this study; the chemically induced uncoupling that we used forced an increase in respiration regardless of natural efficiency. With this design, we did not test for a correlation between mitochondrial efficiency and carotenoid ketolation; rather, we tested for a correlation between the rates of mitochondrial redox reactions and carotenoid ketolation, regardless of ATP production. As we extend conclusions to systems such as the wild house finches or the interpopulation hybrids of T. californicus, we make the assumption that ATP production scales with respiratory rate naturally, such that faster respiring individuals generally make more ATP. In this experiment, we did not measure ATP production. Thus, we cannot make explicit conclusions about the ratio of energy produced to oxygen consumed or the incurrence of any compensatory mechanisms to restore ATP production in response to DNP exposure in the copepods. Future studies may measure both ATP production and respiration rate to assess relationships between carotenoid ketolation and mitochondrial efficiency. Regardless, the results presented in this study suggest that a propensity for ketocarotenoid coloration to signal metabolic capacity is independent of a mediating influence of ROS, even though we cannot conclude that ROS has no effect on tissue carotenoid concentrations in wild populations.

Our observations of potential links between cellular respiration and carotenoid coloration in copepods hold important implications for the evolution of condition-dependent traits. Unlike with house finches, T. californicus copepods do not choose mates based on red carotenoid coloration (Powers et al., 2020). Thus, the condition-dependent carotenoid signal in these copepods appears to exist without a receiver, and it seems unlikely to have evolved in copepods because it is a condition-dependent signal. Rather, our observations with copepods may speak to a fundamental biochemical link between ketocarotenoid-based coloration and mitochondrial function: red carotenoid coloration in copepods evolved for UV protection (Weaver et al., 2018b), and its condition dependency is simply a property of the enzymatic pathways that produce the red pigments. The implications of this observation are that in some systems, such as songbirds, metabolically modified carotenoids have been co-opted as a social signal of individual condition (Twyman et al., 2016) and that condition dependency of red carotenoid coloration does not evolve via sexual selection.

We thank Dr Paul Cobine for continued help with carotenoid analysis. We also thank Dr Matthew Wolak, Dr Tonia Schwartz and Dr Andreas Kavazis for valuable feedback on our experiment and early versions of our figures, as well as Dr Ryan Weaver and members of the Hill and Hood lab groups for feedback on an early version of this manuscript.