Redox-regulation and life-history trade-offs: scavenging mitochondrial ROS improves growth in a wild bird

It has been proposed that animals usually restrain their growth because fast growth leads to an increased production of mitochondrial reactive oxygen species (mtROS), which can damage mitochondrial DNA and promote mitochondrial dysfunction. Here, we explicitly test whether this occurs in a wild bird by supplementing chicks with a mitochondria-targeted ROS scavenger, mitoubiquinone (mitoQ), and examining growth rates and mtDNA damage. In the yellow-legged gull Larus michahellis, mitoQ supplementation increased the early growth rate of chicks but did not reduce mtDNA damage. The level of mtDNA damage was negatively correlated with chick mass, but this relationship was not affected by the mitoQ treatment. We also found that chick growth was positively correlated with both mtDNA copy number and the mitochondrial enzymatic activity of citrate synthase, suggesting a link between mitochondrial content and growth. Additionally, we found that MitoQ supplementation increased mitochondrial content (in males), altered the relationship between mtDNA copy number and damage, and downregulated some transcriptional pathways related to cell rejuvenation, suggesting that scavenging mtROS during development enhanced growth rates but at the expense of cellular turnover. Our study confirms the central role of mitochondria modulating life-history trade-offs during development by other mechanisms than mtROS-inflicted damage.

. Summary of the linear mixed model testing the effect of mitoQ supplementation on body mass in yellow-legged gull chicks at age eight days. Interactions between fixed factors were not significant.   Table S2. Summary of the linear mixed models testing the effects of mitoQ supplementation on ROMs level in plasma and mtDNA damage and mtDNA copy number in blood cells in yellow-legged gull chicks at age eight days. Only significant interactions were retained in the models 1 ROMs, mtDNA copy number and mtDNA damage at one day of age, in their respective analyses.

Reactive oxygen metabolites (ROMs)
We estimated the level of reactive oxygen metabolites (ROMs) in plasma using the method described in 1 . In gull chicks, high levels of plasma ROMs are associated with stressful growing conditions 2 . This assay mainly measures secondary metabolites, as the alkoxy and peroxy radicals derived from the hydroperoxides formed by ROS attack on free unsaturated fatty acids 3 , so a possible indirect proxy of ROS production Briefly, ROMs in plasma (5μL) were reacted with N,Ndiethyl-p-phenylenediamine to produce a coloured complex that can be measured were highly repeatable when estimated for each replicate (r ICCC = 0.991, P < 0.001). Importantly, relative mtDNA copy number was significantly correlated with citrate synthase activity (see results), a proxy of mitochondrial density.

Mitochondrial DNA damage
We estimated mtDNA damage using a quantitative 'long' PCR-based assay based on the principle that DNA damage slows down or block DNA polymerase advance [7][8][9] . Thus, the levels of lesions were quantified by the amplification of large genomic fragment and normalized by a short fragment, which is less likely to be affected by the random damage. This assay has been previously validated in several species to detect mitochondrial DNA damage induced by a large number of damaging agents (see 10 ). For DNA quantification, we used the PicoGreen dsDNA assay kit Invitrogen) and a Synergy HT BioTek microplate reader (excitation wavelength 480 nm, emission wavelength 520 nm).
We designed primers to amplify a large mtDNA target (almost the entire mitochondrial genome, but excluding the D-loop to avoid bias from this mutation hotspot) by using the known complete mitochondrial genomes of five gull species (AN: AY293619.1; JX155863.1; KJ507782.1; KM507782.1; KT943749.1). We optimized the primer combination (Supplementary Table S5) and PCR conditions to produce a bright and unique band of predicted amplicon size (13 kbp) without any secondary product. We also selected four digestion enzymes (ApaI, Eco52I, SacII XBaI, Thermo Scientific) that produce a consistent digestion pattern (number and size of digested fragments) in the expected mitochondrial fragment across the five gull species used to design the primers (see above). Thus, the identity of the product amplified by the designed primers in our study species was confirmed by restriction digestion with these enzymes and gel electrophoresis of cut products. We used the primers for COI gene to amplify a short mtDNA target (see above; Supplementary Table S5). from the same individual chicks were always allocated in duplicate in the same plate, and with similar numbers of different experimental groups. We selected a sample with high levels of damage as a reference to inter-run normalization. Thus, the reference and 50% reference and no template (blank) control were included in duplicate in each microplate. PCR products were quantified by PicoGreen assay and corrected by blank fluorescence values. We optimized the amplification linearity that was considered acceptable if the 50% reference showed a 40-60% reduction of amplification signal. Duplicates were highly repeatable (large mtDNA target, r ICCC = 0.971, P < 0.001; small mtDNA target, r ICCC = 0.901, P < 0.001, n = 89). Relative DNA lesion frequencies were normalized to reference as previously described 11 . Briefly, we estimated the relative damage per DNA strand as the ratio of fluorescence values of large and small mtDNA target in each sample (RS) and in the reference (RR). Normalized mtDNA damage was determined as -ln(RS/RR) and these values were highly repeatable when estimated for each replicate (r ICCC = 0.899, P < 0.001).

Citrate synthase activity
Citrate synthase activity was measured in blood samples at age eight days. Citrate synthase is a mitochondrial matrix enzyme typically used as a marker of mitochondria abundance 12,13 . In a preliminary study of gull chicks, we found that the activities of citrate synthase and cytochrome c oxidase (Complex IV, a respiratory chain enzyme) were highly correlated (r=0.62, P<0.001, n = 45; unpublished data). Thus, citrate synthase activity may be a good proxy of the abundance of functional mitochondria. We followed the assays previously described in 13

Gene expression
The