Inbreeding negatively affects various life-history traits, with inbred individuals typically having lower ﬁt- ness than outbred individuals (¼ inbreeding depression). Inbreeding depression is often emphasized under environmental stress, but the underlying mechanisms and potential long-lasting consequences of such inbreeding–environment interactions remain poorly understood. Here, we hypothesize that inbreeding–environment interactions that occur early in life have long-term physiological effects, in partic- ular on the adult oxidative balance. We applied a unique experimental design to manipulate early life conditions of inbred and outbred songbirds (Serinus canaria) that allowed us to separate prenatal and postnatal components of early life conditions and their respective importance in inbreeding–environment interactions. We measured a wide variety of markers of oxidative status in adulthood, resulting in a com- prehensive account for oxidative balance. Using a Bayesian approach with Markov chain Monte Carlo, we found clear sex-speciﬁc effects and we also found only in females small yet signiﬁcant long-term effects of inbreeding–environment interactions on adult oxidative balance. Postnatal components of early life conditions were most persuasively reﬂected on adult oxidative balance, with inbred females that experienced disadvantageous postnatal conditions upregulating enzymatic antioxidants in adulthood. Our study provides some evidence that adult oxidative balance can reﬂect inbreeding–environment inter- actions early in life, but given the rather small effects that were limited to females, we conclude that oxida- tive stress might have a limited role as mechanism underlying inbreeding–environment interactions. Key words: canary, maternal effects, gene–environment interactions, hatching asynchrony. There is ample empirical evidence that inbreeding (mating between interactions remain largely unknown, which prohibits a thorough related individuals) negatively affects fitness-related traits like fecund- understanding of both the causes and consequences of these phenom- ity and survival (Keller and Waller 2002; Fox and Reed 2011). ena (Kristensen et al. 2010). Inbreeding depression is often more pronounced in stressful compared Preventing oxidative stress [defined as an imbalance between the to benign environments, indicating that inbred individuals show production of highly reactive substances and the capacity to neutral- increased susceptibility to stressful environmental conditions in com- ize such compounds with antioxidant defenses (Halliwell and parison to outbred individuals (Armbruster and Reed 2005; Fox and Gutteridge 2007)] is an important component of stress resistance. Reed 2011). Such inbreeding–environment interactions are important Interestingly, genome-wide analyses show that inbreeding affects a in many areas of biology, among others in conservation biology. wide variety of genes but has a disproportionate impact on genes However, the mechanisms that underlie inbreeding–environment involved in stress resistance and metabolism (Kristensen et al. 2005; V C The Author(s) (2017). Published by Oxford University Press. 631 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org Downloaded from https://academic.oup.com/cz/article-abstract/64/5/631/4683472 by Ed 'DeepDyve' Gillespie user on 18 October 2018 632 Current Zoology, 2018, Vol. 64, No. 5 Kristensen et al. 2006; Ayroles et al. 2009; Menzel et al. 2015), and Here, we describe long-term effects of inbreeding in interaction that a number of these genes are also linked to the regulation of oxi- with early life conditions and test if baseline values of oxidative damage and antioxidant protection in blood are affected at adult- dative stress (Pletcher et al. 2002; Landis et al. 2004; Kristensen hood. To this end, we kept the birds that were reared in the experi- et al. 2005; Kristensen et al. 2010). Therefore, oxidative stress could mental design under laboratory conditions after fledging. We then be an important factor that underlies the detrimental effects of collected a sample of blood when the individuals were adult (i.e., inbreeding (Okada et al. 2011; Freitak et al. 2014). Moreover, oxi- fully sexually mature), but before they were mated. We measured a dative stress has been associated with early environmental stress range of antioxidants and oxidative damage markers [damage to (Haussmann et al. 2012; Marasco et al. 2013; Giordano et al. 2015; proteins (carbonyls), nitric oxide (NO)], enzymatic antioxidants Zimmer and Spencer, 2015; Marasco et al. 2016). This suggests that (GPX, CAT, and SOD), and glutathione [ratio: GSH (reduced inbreeding–environment interactions might affect the oxidative bal- form)/GSSG (oxidized form)] that were designed to give us a com- ance, which remains largely unexplored. prehensive picture of the individual’s oxidative status. However, inbreeding–environment interactions and especially We expect that disadvantageous conditions early in life could their consequences are by no means restricted to the moment at have long-term effects on oxidative balance in adulthood (Marasco which environmental conditions are experienced. Individuals are et al. 2013; Zimmer and Spencer, 2015). Additionally, we expect particularly sensitive early in life when development occurs, and the that inbred birds have higher oxidative stress in comparison to out- environmental conditions that an individual experiences during this bred birds (Okada et al. 2011; Freitak et al. 2014), and that this time frame may therefore have a major impact on the adult pheno- should be especially noticeable when they were reared under disad- type (Metcalfe and Monaghan 2001; Monaghan 2008). Inbreeding– vantaged conditions. That is, a size disadvantaged position in the environment interactions that occur early in life can therefore have within-brood hierarchy (i.e. low food availability due to high levels potentially long-lasting consequences, and an inbreeding–environ- of sibling competition) (Forbes 2010), and/or mismatched prenatal ment interaction caused by early life stress may be reflected in stress and postnatal conditions (i.e., the laying position of the egg is mis- sensitivity and levels of oxidative stress at adulthood. matched with the position in the brood hierarchy) (Muller and Early life conditions that have the potential for life-long conse- Groothuis 2013). quences can be separated in prenatal and postnatal conditions depending on during which period they act. In birds, prenatal condi- tions are formed by the conditions experienced within the egg. This Materials and Methods often refers to differential maternal investment in egg size (Williams Inbreeding and early life conditions 1994; Royle et al. 1999), and egg components such as antioxidants The parental generation of the birds used in this study originates (Royle et al. 1999; Royle et al. 2001; Blount et al. 2002; Royle et al. from an outbred population kept at the University of Antwerp. In 2003) and hormones (Schwabl, 1993; Gil et al. 1999; Royle et al. March 2014, two types of breeding pairs were created: full-sibling 2001; Groothuis and Schwabl 2002). The early postnatal environ- breeding pairs (¼ inbred offspring) and unrelated breeding pairs ment, on the other hand, consists of different factors such as sibling (¼ outbred offspring). For the here described study, a total of 140 competition or the quality of parental care (at least in altricial birds (70 males, 70 females) nestlings were used. Inbred and outbred nest- that raise more than one chick at a time) (Forbes 2010; Mainwaring lings were combined in duos, based on body mass (< 0.2 g differ- et al. 2010). Specifically, larger siblings are generally advantaged in ence), and on the position in the laying sequence (¼ prenatal food acquisition in comparison to smaller nestlings (Oddie, 2000; conditions, early laid eggs: first or second egg, or late laid eggs: Royle et al. 2002), which is even noticeable under ad libitum condi- third, fourth or fifth egg), and were then placed in a foster nest tions (de Boer et al. 2015). Therefore, sibling size hierarchies are a (¼“experimental nest”) at maximum 2 days after hatching. major determinant of the early environment of developing nestlings Approximately half of these nests were reared by full sibling parents (Forbes 2010), which has been shown to interact with inbreeding and half by unrelated parents. However, couple composition did not (de Boer et al. 2015). Prenatal and postnatal conditions can not only have an effect on nestling growth (de Boer et al. 2016). In each have separate effects but also interactive effects, since prenatal experimental nest, two duos of inbred and outbred nestlings were (maternal) effects are thought to adjust offspring phenotype to the combined based on age, in such a way that each experimental nest expected post-hatching conditions (Muller and Groothuis 2013). contained an older, size advantaged duo, and a younger, size disad- In this study, we applied an experimental design that enabled us vantaged duo. The difference between the older/size advantaged and to examine the effects of inbreeding and prenatal and postnatal con- younger/size disadvantaged nestlings was 2 days in age, or if this ditions simultaneously. We weight-matched inbred and outbred was impossible at least 0.5 g difference in body mass at time of nestlings and placed them in either a size advantaged or size disad- cross-fostering. The difference in age/size between the nestlings cre- vantaged position in the within-brood hierarchy, mimicking hatch- ated an artificial sibling hierarchy within the nest, manipulating ing asynchrony (¼ postnatal conditions). The laying position of the postnatal conditions (Figure 1). egg from which they hatched (¼ prenatal conditions) either matched Approximately half of the duos that were reared in a size advan- or mismatched with their position in the within-brood hierarchy. taged position in the hierarchy hatched from early laid eggs This experimental manipulation revealed substantial effects of post- (¼ matched prenatal and postnatal conditions; laying order of the natal conditions on early growth, with size advantaged nestlings egg corresponded to the position in the hierarchy), and half from growing faster than size disadvantaged nestlings. Birds that hatched late laid eggs (¼ mismatched prenatal and postnatal conditions), and from late laid eggs obtained a larger size at fledging than those vice versa for duos that were reared in size disadvantaged positions. hatched from early laid eggs, showing the importance of prenatal Thus, four different experimental treatments in the early life conditions too. Inbred birds grew slower than outbred birds, but (¼ prenatal and postnatal conditions) of the inbred and outbred this was independent of (dis)advantageous prenatal or postnatal focal birds can be distinguished: size advantaged/early laid egg conditions (see de Boer et al. 2016 for more details). (¼matched conditions; inbred female N ¼ 10, inbred male N ¼ 8, Downloaded from https://academic.oup.com/cz/article-abstract/64/5/631/4683472 by Ed 'DeepDyve' Gillespie user on 18 October 2018 de Boer et al. Inbreeding, early life conditions, and oxidative balance 633 Inc., Vermont, USA) was used for the activity measurements. Specifically, GPX activity was measured as the decrease in NADPH absorbance measured at 340 nm (Drotar et al. 1985), SOD activity was measured as the inhibition of nitroblue tetrazolium (NBT) reduction at 560 nm (Dhindsa et al. 1981) and CAT activity was measured as the rate of decomposition of H O at 240 nm (Aebi 2 2 1984). Analyses were run in duplicate and inter-assay and intra- assay variation was <16%. Further, the concentration of both reduced glutathione (GSH) and oxidized glutathione (GSSG) was measured in 2.5 mL of red blood cells using reversed-phase high-performance liquid chroma- tography (HPLC) with electrochemical detection (Shimadzu, Hai Zhonglu, Shanghai) according to AbdElgawad et al. (2015). The concentrations of GSH and GSSG were expressed as micromole per gram fresh weight of red blood cells. We used the GSH/GSSG ratio, which is a conventional index of redox state with higher values indi- cating lower oxidative stress (Jones, 2006). Finally, we measured in Figure 1. A schematic representation of our experimental set-up. Each experi- plasma the production of the free radical nitric oxide with a spectro- mental nest consisted of two pairs of weight-matched inbred (¼ dark grey) photometric assay (see for more details: Sild and Horak ~ 2009; and outbred (¼ light grey) birds, that were placed either in a size advantaged Vermeulen et al. 2016). position (¼ dark grey beaks), or in a size disadvantaged (¼ light grey beaks) position. Each pair of inbred and outbred birds hatched from either early laid Statistical analyses eggs (¼ﬁrst or second laid egg) or late laid eggs (¼ all later laid eggs). Hatching from an early laid egg matched a size advantaged position in the A total of six markers of oxidative balance [damage to proteins (car- nest, whereas hatching from a late laid egg mismatched a size advantage, bonyls), nitric oxide (NO)], enzymatic antioxidants (GPX, CAT, and vice versa for birds reared in size disadvantaged positions. and SOD), and glutathione [ratio: GSH (reduced form)/GSSG (oxi- dized form)] were analyzed with generalized linear mixed effects outbred female N ¼ 9, outbred male N ¼ 12), size advantaged/late models using a Bayesian approach with Markov chain Monte Carlo laid egg (¼mismatched conditions; inbred female N ¼ 8, inbred male (MCMC) algorithms implemented in the MCMCglmm R package N ¼ 10, outbred female N ¼ 9, outbred male N ¼ 6), size disadvan- (Hadfield 2010). We ran a preliminary model on data of all birds taged/late laid egg (¼ matched conditions; inbred female N ¼ 8, which included a fixed effect for sex in addition to the below men- inbred male N ¼ 14, outbred female N ¼ 15, outbred male N ¼ 9), tioned fixed effects. This model revealed strong effects of sex on the and size disadvantaged/early laid egg (¼mismatched conditions; oxidative balance (see results) and males and females were analysed inbred female N ¼ 7, inbred male N ¼ 4, outbred female N ¼ 4, out- separately to account for sex-specific resistance to oxidative stress. bred male N ¼ 7) (Figure 1). At fledging (6 25 days after hatching), The fixed effects included in the models were: prenatal condition a blood sample was taken from the brachial vein to determine sex (hatched from early or late laid eggs), postnatal condition (size with the use of PCR, and birds were from that time onwards housed advantaged or size disadvantaged), and inbreeding status (inbred or in two groups separated by sex in large aviaries, with food and outbred). The interactions between these effects were included, and water available ad libitum. the potential three-way interaction between all factors was also taken into account. Additionally, the weight of the birds at time of Analyses of adult blood oxidative balance blood sampling was included as a fixed effect. Brothers and sisters The birds were exposed to a long light regime (14 h light: 10 h dark) were included in this study, as well as individuals that were reared from December 2014 onwards. This set off reproductive state, and together in an experimental nest. To control for potential confound- when the birds were 1-year old (March 2015) they were weighed, ing effects of these factors we included both the nest of origin and and a blood sample was taken from the brachial vein. Blood samples the rearing nest as random effects in the model. were stored in a freezer (80 C) until laboratory analyses were All response variables had a Gaussian distribution and were conducted. scaled before the sex-specific models were run. MCMC chains were Blood markers of oxidative damage, antioxidant protection, and run for 505,000 iterations with a burn-in phase of 5000 iterations, reactive nitrogen species production were measured according to and 1000 independent samples were taken from the posterior at established protocols. Specifically, we measured in red blood cells intervals of 500 iterations. Convergence was determined by visual the concentration of protein carbonyls (marker of oxidative damage inspection of the traces and autocorrelation plots. The results are to proteins) according to Levine et al. (1990). Extracting buffer was presented as the estimates of the sampled iterations with a 95% con- used to dilute the red blood cells in order to obtain a concentration fidence interval. Statistical significance of the estimate can be of 2 mg proteins/ml which was measured with the use of the assumed when confidence intervals do not overlap with zero. The Bradford protein assay (Bradford 1976). The concentration of pro- correlation between parameters (X, Y) of the adult oxidative bal- tein carbonyls was noted as nanomoles per milligram proteins. ance was calculated by dividing the covariance (cov(X, Y)) between The activity of three antioxidant enzymes [glutathione peroxi- parameters by the product of the standard deviations of the variance dase (GPX), superoxide dismutase (SOD) and catalase (CAT)] was (SD(X)*SD(Y)). assessed in 5 mL of red blood cells that were homogenized in 250 mL The effect size of differences between inbred and outbred birds of extracting buffer (pH 7.4, 1.15% KCL and 0.02 M EDTA in within each experimental group was calculated with the R package 0.01 M PBS) with the use of a MagNALyser (Roche, Vilvoorde, “compute.es” (Del Re 2013) using the mean, standard deviation, Belgium). A micro-plate reader (Synergy Mx, Biotek Instruments and sample size. Downloaded from https://academic.oup.com/cz/article-abstract/64/5/631/4683472 by Ed 'DeepDyve' Gillespie user on 18 October 2018 634 Current Zoology, 2018, Vol. 64, No. 5 All analyses were performed in R (R Core Development Team Table 1. Differences in body mass and markers of the adult oxida- tive balance between non-breeding male and female Canaries 2014). In the analysis of CAT in females, and in the analysis of nitric oxide and protein carbonyls in males, we excluded one individual Parameter Mean6 SE females Mean6 SE males from the data that was considered an outlier (>0.5 Cook’s distance). All above described procedures have been approved by the ethical Body mass (g) 19.376 0.2 17.906 0.2 Protein carbonyls 3.006 0.20 0.406 004 committee for animal experimentation at the University of Antwerp (nmol/mg proteins) (file number 2011–86), and carried out in accordance with the rele- NO (mmol/L) 0.0046 0.0002 0.0066 0.0006 vant rules and guidelines. GPX (mmol NADPH/ 0.0096 0.0002 0.0036 0.0003 mg prot/min) CAT (mmol H2O2/ 0.156 0.008 0.256 0.02 Results mg prot/min) Baseline levels of all markers for oxidative balance except for nitric SOD (U/mg prot/min) 0.276 0.009 1.036 0.04 oxide significantly differed between males and females (estimate GSH/GSSG (each in mmol/ 4.436 0.37 2.516 0.32 g fresh weight) [95% CI]: carbonyls: 2.59 [3.00; 2.11], P< 0.001; nitric oxide: 0.001 [0.004; 0.006], P ¼ 0.6; GPX: 0.006 [0.011;-0.001], P ¼ 0.016; CAT: 0.09 [0.04; 0.15], P< 0.001; SOD: 0.77 [0.68; In males, the activity of CAT and SOD, and GPX and nitric oxide 0.86], P< 0.001; GSH/GSSG: 2.22 [3.41; 1.18], P< 0.001) showed significant co-variation (Table 3). (Table 1). In males, we find no effects of early life conditions and/or inbreeding in any of the parameters of oxidative balance (Table 2). In females, the effects of inbreeding on protein carbonyls Discussion (marker of oxidative damage to proteins) differed according to pre- In this study, we investigated the long-term effects of inbreeding, pre- natal and postnatal conditions (Table 2). In Figure 2, it can be natal and early postnatal conditions, and their potential interactive observed that inbred females tended to have higher levels of oxida- effects on adult oxidative balance. Our findings show that the effects tive damage in comparison to outbred females when hatched from of inbreeding were mediated by early postnatal conditions for the con- an early laid egg and raised in a size disadvantaged position, and to centration of enzymatic antioxidants, and by prenatal and postnatal a lesser extent when hatched from late laid eggs but raised in a size early life conditions for the damage to proteins and the nitric oxide advantaged position, which are both conditions where the post- concentration in females. Specifically, inbred females seemed to upre- hatching environment mismatched with the laying position of the gulate enzymatic antioxidants in adulthood when they had been egg they had hatched from. Inbred females tended to have less oxi- reared under disadvantageous postnatal conditions. Inbred females dative damage to proteins than outbred females when hatched from that were reared under disadvantageous conditions also suffered more an early laid egg and raised in size-advantaged position and to a oxidative damage in adulthood than outbred females that were reared lesser extent when hatched from late laid eggs and raised in size- under disadvantageous conditions, but this effect was restricted to disadvantaged position, which is under matched conditions. In females that hatched from early laid eggs, i.e., were reared under mis- Figure 3, it can be seen that the effect sizes are rather small and con- matched pre- and postnatal conditions. Inbred females also had lower fidence intervals overlap with zero indicating non-significant differ- levels of nitric oxide than outbred females in this experimental group, ences between inbred and outbred females within the experimental which may be directly related to the increased levels of enzymatic groups. antioxidants (Price et al. 2000). Nitric oxide is a very potent oxidant, The effects of inbreeding on the nitric oxide concentration were and can have deleterious effects such as tissue damage when it is pro- also mediated by prenatal and postnatal conditions in females duced prolonged and excessively (Price et al. 2000; Vajdovich, 2008; (Table 2). The negative effect of inbreeding was most noticeable in Pamplona and Costantini, 2011). However, nitric oxide also has vari- females that hatched from an early-laid egg and were placed in a ous important functions, such as protection against infection, and low size disadvantaged position. Further, inbred and outbred females levels of nitric oxide could therefore also relate to an impaired did not show noteworthy differences in nitric oxide concentrations in other early life conditions (Figures 2 and 3). immune system (Vajdovich, 2008; Bichet et al. 2012). The activity of the enzymatic antioxidants GPX, CAT and SOD There was no consistent effect of either prenatal or postnatal depended on inbreeding status in interaction with postnatal condi- conditions or their combination on the adult oxidative balance, tions in females. In Figures 3 and 4, it can be observed that inbred which makes it difficult to pinpoint what aspect of early life condi- females that had been reared with a size disadvantage tended to tions caused the observed inbreeding–environment interactions in have more enzymatic antioxidants than outbred females. On the females. However, the inbreeding–environment interactions always other hand, inbred females tended to have less of the enzymatic anti- included the postnatal conditions if significant, which suggests that oxidant CAT when reared with a size advantage. These effects were the postnatal component of early life conditions may have been an important factor in inbreeding-environment interactions. Indeed, rather small and for most of the experimental groups the confidence our experimental manipulation of the postnatal conditions had intervals of differences between inbred and outbred females over- more pronounced effects on early growth and size at fledging than lapped with zero indicating non-significant effects (Figure 3). There prenatal conditions (de Boer et al. 2016). Yet, neither postnatal nor were no significant effects of inbreeding and/or early life conditions on the GSH/GSSG ratio in females (Table 2). In both females and prenatal conditions per se were clearly reflected in the adult oxida- males, there were no significant effects of body mass on any of the tive balance. This is in contrast with previous studies showing effects parameters of oxidative balance (Table 2). of prenatal and/or postnatal conditions on the adult oxidative bal- The correlation between the various parameters of the adult oxi- ance in birds (Marasco et al. 2013; Zimmer v Spencer, 2015) and dative balance is reported for males and females in Table 3.In the more general concept that conditions affecting early growth females, the activity of GPX, CAT and SOD co-varied significantly. should be reflected in the adult phenotype, including physiology Downloaded from https://academic.oup.com/cz/article-abstract/64/5/631/4683472 by Ed 'DeepDyve' Gillespie user on 18 October 2018 de Boer et al. Inbreeding, early life conditions, and oxidative balance 635 Table 2. Parameter estimates of Markov Chain Monte Carlo generalized linear mixed models for the effects of inbreeding, prenatal and post- natal early life conditions on different parameters of the adult oxidative balance Parameter Fixed effects Estimate 95% CI P Estimate 95% CI P Females Females Females Males Males Males Damage (proteins) Carbonyls Inbreeding 0.55 [1.81; 0.69] 0.37 0.35 [1.63; 1.02] 0.61 Postnatal 0.11 [1.06; 0.89] 0.81 0.39 [1.73; 0.81] 0.55 Postnatal: Inbreeding 1.38 [0.09; 2.90] 0.06 1.04 [0.77; 2.62] 0.23 Prenatal 0.24 [1.28; 0.73] 0.68 0.09 [1.17; 1.21] 0.88 Prenatal: Inbreeding 1.35 [0.15; 2.85] 0.08 0.29 [1.21; 1.96] 0.73 Prenatal: Postnatal 0.24 [1.11; 1.60] 0.75 0.05 [1.55; 1.46] 0.92 Prenatal: Postnatal: Inbreeding 2.65 [4.71; 0.77] 0.014 0.15 [2.29; 1.97] 0.90 Weight 0.06 [0.21; 0.08] 0.45 0.11 [0.07; 0.26] 0.19 Nitric oxide NO Inbreeding 1.62 [0.43; 2.99] 0.016 0.26 [1.12; 1.57] 0.70 Postnatal 1.10 [0.17; 2.07] 0.07 0.72 [0.59; 1.95] 0.26 Postnatal: Inbreeding 1.75 [3.29; 0.20] 0.032 0.72 [2.45; 0.86] 0.37 Prenatal 1.08 [0.08; 2.21] 0.05 0.07 [1.16; 1.17] 0.92 Prenatal: Inbreeding 2.24 [3.61; 0.70] 0.012 0.35 [2.06; 1.12] 0.65 Prenatal: Postnatal 1.49 [3.17; 0.03] 0.07 0.42 [1.00; 2.02] 0.57 Prenatal: Postnatal: Inbreeding 2.25 [0.38; 4.49] 0.040 0.06 [1.84; 2.60] 0.94 Weight 0.00 [0.18; 0.16] 1.00 0.05 [0.13; 0.19] 0.57 Enzymatic antioxidants GPX Inbreeding 1.82 [3.13; 0.51] 0.006 0.07 [1.05; 1.03] 0.90 Postnatal 0.43 [1.46; 0.55] 0.39 0.22 [1.29; 0.95] 0.72 Postnatal: Inbreeding 1.89 [0.21; 3.25] 0.026 0.14 [1.01; 1.45] 0.86 Prenatal 0.03 [1.04; 1.13] 0.93 0.24 [0.74; 1.25] 0.65 Prenatal: Inbreeding 1.26 [0.23; 2.78] 0.12 0.48 [1.67; 0.77] 0.44 Prenatal: Postnatal 0.20 [1.61; 1.32] 0.80 0.09 [1.41; 1.63] 0.93 Prenatal: Postnatal: Inbreeding 1.08 [3.01; 0.84] 0.26 0.33 [1.52; 1.77] 0.67 Weight 0.00 [0.16; 0.14] 0.94 0.06 [0.07; 0.22] 0.42 CAT Inbreeding 1.21 [2.43; 0.21] 0.070 0.42 [0.86; 1.77] 0.49 Postnatal 1.06 [2.19; 0.003] 0.050 0.03 [1.19; 1.40] 0.96 Postnatal: Inbreeding 2.29 [0.62; 3.71] 0.006 0.95 [2.48; 0.76] 0.23 Prenatal 0.49 [1.49; 0.72] 0.38 0.38 [1.51; 0.73] 0.50 Prenatal: Inbreeding 0.83 [0.74; 2.38] 0.30 0.35 [1.89; 1.18] 0.63 Prenatal: Postnatal 1.14 [0.53; 2.61] 0.18 0.41 [2.16; 0.97] 0.56 Prenatal: Postnatal: Inbreeding 1.83 [3.78; 0.31] 0.086 1.57 [0.54; 3.55] 0.14 Weight 0.10 [0.24; 0.06] 0.21 0.06 [0.22; 0.11] 0.47 SOD Inbreeding 1.23 [2.41; 0.11] 0.058 0.38 [0.99; 1.66] 0.60 Postnatal 0.34 [1.32; 0.67] 0.50 0.18 [1.23; 1.57] 0.80 Postnatal: Inbreeding 1.92 [0.44; 3.60] 0.022 0.30 [2.00; 1.35] 0.73 Prenatal 0.08 [1.20; 0.87] 0.89 0.01 [1.19; 1.21] 0.99 Prenatal: Inbreeding 1.28 [0.39; 2.77] 0.13 0.17 [1.51; 1.76] 0.84 Prenatal: Postnatal 0.15 [1.50; 1.31] 0.81 0.35 [2.03; 1.26] 0.67 Prenatal: Postnatal: Inbreeding 1.60 [3.72; 0.40] 0.13 0.23 [2.04; 2.56] 0.87 Weight 0.01 [0.14; 0.16] 0.86 0.03 [0.15; 0.20] 0.76 Glutathione GSH/GSSG Inbreeding 0.10 [1.28; 1.49] 0.88 0.60 [0.71; 1.78] 0.38 Postnatal 0.44 [0.54; 1.69] 0.43 0.24 [0.89; 1.53] 0.72 Postnatal: Inbreeding 0.23 [2.10; 1.33] 0.79 0.21 [1.77; 1.30] 0.79 Prenatal 0.42 [0.81; 1.53] 0.45 0.73 [0.42; 1.93] 0.22 Prenatal: Inbreeding 0.23 [1.96; 1.26] 0.78 0.65 [2.33; 0.82] 0.45 Prenatal: Postnatal 0.82 [2.46; 0.81] 0.30 0.93 [2.37; 0.52] 0.20 Prenatal: Postnatal: Inbreeding 0.96 [1.33; 2.94] 0.42 0.10 [2.09; 2.06] 0.90 Weight 0.03 [0.20; 0.14] 0.75 0.08 [0.25; 0.07] 0.34 Signiﬁcant effects in which conﬁdence intervals do not overlap with zero are indicated in bold and P < 0.1 are indicated with italics. Males without 1 outlier. Females without 1 outlier. (Metcalfe and Monaghan, 2001; Monaghan, 2008). After fledging, developmental strategy of the precocial species used in those studies all birds had ad libitum access to food with only limited competi- likely differs from that of the canary, this suggests that the lack of a tion, which may have alleviated any long-lasting effects of early life clear effect of early life conditions in our study may not necessarily conditions, and such effects might be more apparent in the wild. relate to the benign food conditions in captivity. It may also be that However, both above-mentioned studies showing long-lasting a different type of manipulation of early life conditions (e.g., expo- effects of early life stress, which was manipulated via prenatal sure to stress hormones) than what we have done here has more pro- administration of corticosterone (Marasco et al. 2013; Zimmer and nounced effects on the adult oxidative balance (Marasco et al. 2013; Spencer, 2015), were performed in captivity too. Although the Zimmer and Spencer, 2015). Downloaded from https://academic.oup.com/cz/article-abstract/64/5/631/4683472 by Ed 'DeepDyve' Gillespie user on 18 October 2018 636 Current Zoology, 2018, Vol. 64, No. 5 Figure 2. Differences in protein carbonyls (nmol/mg proteins) (top) and nitric oxide (NO, mmol/L) (bottom) between inbred (gray boxes) and outbred (white boxes) females canaries depended on prenatal and postnatal early life conditions. These early life conditions consisted of four experimental treatments: size advantaged/early laid eggs (¼ matched conditions; inbred females: N ¼ 10, outbred females: N ¼ 9), size advantaged/late laid eggs (¼ mismatched conditions; inbred females: N ¼ 8, outbred females: N ¼ 9), size disadvantaged/late laid eggs (¼ matched conditions; inbred females: N ¼ 8, outbred females: N ¼ 15), size dis- advantaged/early laid eggs (¼ mismatched conditions; inbred females: N ¼ 7, outbred females: N ¼ 4). Figure 3. Effect sizes (6 95%CI) of the differences in markers of oxidative balance between inbred and outbred females in relation to early life conditions: size advantaged/early laid eggs (¼ matched conditions; inbred females: N ¼ 10, outbred females: N ¼ 9), size advantaged/late laid eggs (¼ mismatched conditions; inbred females: N ¼ 8, outbred females: N ¼ 9), size disadvantaged/late laid eggs (¼ matched conditions; inbred females: N ¼ 8, outbred females: N ¼ 15), size dis- advantaged/early laid eggs (¼ mismatched conditions; inbred females: N ¼ 7, outbred females: N ¼ 4). Negative effect sizes represent higher values for outbred females in comparison to inbred females, and vice versa for positive effect sizes. Downloaded from https://academic.oup.com/cz/article-abstract/64/5/631/4683472 by Ed 'DeepDyve' Gillespie user on 18 October 2018 de Boer et al. Inbreeding, early life conditions, and oxidative balance 637 Figure 4. Differences in the activity of enzymatic antioxidants GPX (mmol NADPH/mg prot/min), CAT (mmol H2O2/mg prot/min), and SOD (U/mg prot/min) between inbred (gray boxes) and outbred (white boxes) female canaries depended on the postnatal early life conditions. Postnatal conditions in the nest were manipulated by rearing birds with a size advantage (inbred females: N ¼ 18, outbred females: N ¼ 18) or with a size disadvantage (inbred females: N ¼ 15, outbred females: N ¼ 19). Our study shows a clear effect of sex on the adult oxidative bal- Table 3. The correlation between parameters of adult oxidative bal- ance (X, Y) was calculated by dividing the covariance (cov(X.Y)) ance. Oxidative damage to proteins, in particular, was on average between parameters by the product of the standard deviations of 7.5 times higher in females than in males. Moreover, in females the variance (SD(X)*SD(Y)) there was some evidence of inbreeding-environment interactions but in males there were no effects of early life conditions and/or inbreed- Parameter Carbonyls NO GPX CAT SOD GSH/GSSG ing at all. Sex-specific effects on the oxidative balance have been pre- Females Carbonyls x 0.04 0.02 0.04 0.03 0.15 viously reported in birds (Bize et al. 2008; Isaksson 2013; Marasco NO x x 0.02 0.13 0.22 0.03 et al. 2013; Giordano et al. 2015). Females were, although the birds GPX x x x 0.43 0.65 0.06 were not mated at the time of blood sampling, likely developing CAT x x x x 0.49 0.02 their ovaries, which is an energetically demanding process SOD x x x x x 0.24 (Monaghan and Nager 1997; Nilsson and Ra ˚ berg 2001). Thus, it GSH/GSSG x x x x x x Males Carbonyls x 0.09 0.10 0.04 0.10 0.28 could be that females were more constrained than males by costs NO x x 0.21 0.17 0.03 0.01 associated with this process, causing the large differences in markers GPX x x x 0.69 0.22 0.03 of oxidative balance between males and females. In addition, the CAT x x x x 0.22 0.16 oxidative balance is not necessarily the same across tissues SOD x x x x x 0.31 (Speakman et al. 2015). We focused here on a variety of blood GSH/GSSG x x x x x x markers of oxidative balance, but perhaps the effects of inbreeding Conﬁdence intervals of the covariance values that did not overlap with zero and/or early life conditions targeted other tissues in males than in are indicated in bold. females. Alternatively, the oxidative balance can vary with age which does not necessarily show the same pattern in males and Inbreeding affected the female oxidative balance always in inter- females (Bize et al. 2008). Thus, there is also a possibility that the action with early life conditions, suggesting that inbreeding per se effects of inbreeding or early life conditions become apparent at a does not necessarily relate to decreased resistance to oxidative stress. different life phase in males than in females, which could be an inter- Unfortunately, studies that link inbreeding with oxidative stress are esting topic for future studies. as yet extremely rare and restricted to insects (Okada et al. 2011; Our study provides some evidence that inbreeding–environment Freitak et al. 2014), which limits further conclusions. Inbreeding interactions have long-term effects on adult oxidative balance. Our negatively affected growth in early life (de Boer et al. 2016), but intricate experimental design allowed to test for the individual and those effects were transient and no more present in body mass at combined effects of prenatal and postnatal components of such fledging and in adulthood, at which time we measured oxidative inbreeding–environment interactions simultaneously. However, this balance. Differences in the oxidative balance between inbred and complex design did not allow us to obtain a large sample size and outbred birds may therefore have been more pronounced early in indeed the effects were rather small and also limited to females. life than in adulthood. Based on these data, we conclude that oxidative stress might have a Downloaded from https://academic.oup.com/cz/article-abstract/64/5/631/4683472 by Ed 'DeepDyve' Gillespie user on 18 October 2018 638 Current Zoology, 2018, Vol. 64, No. 5 Fox CW, Reed DH, 2011. Inbreeding depression increases with environmental limited role as a mechanism underlying inbreeding–environment stress: an experimental study and meta-analysis. Evolution 65:246–258. interactions. Freitak D, Bos N, Stucki D, Sundstro ¨ m L, 2014. Inbreeding-related trade-offs in stress resistance in the ant Formica exsecta. Biol Lett 10:20140805. Gil D, Graves J, Hazon N, Wells A, 1999. Male attractiveness and differential Author contributions statement testosterone investment in zebra ﬁnch eggs. Science 286:126–128. R.B., D.C., M.E., W.M. designed the experiment. R.B., D.C., G.C., Giordano M, Costantini D, Tschirren B, 2015. Sex-speciﬁc effects of prenatal H.E., H.A. contributed to the collection and analysis of the data. and postnatal nutritional conditions on the oxidative status of great tit nest- R.B. wrote the first draft. R.B., D.C., G.C., M.E., W.M. helped to lings. Oecologia 177:123–131. Groothuis TG, Schwabl H, 2002. Determinants of within- and among-clutch improve the article, and all authors approved the final version for variation in levels of maternal hormones in black-headed gull eggs. Funct submission to the journal. Ecol 16:281–289. Halliwell B, Gutteridge J, 2007. Cellular responses to oxidative stress: adapta- tion, damage, repair, senescence and death. In: Free Radicals in Biology and Acknowledgments Medicine.Vol. 4. Oxford: Oxford University Press. 187–267. We thank Peter Scheys, Josie Meaney, and Geert Eens for their invaluable Haussmann MF, Longenecker AS, Marchetto NM, Juliano SA, Bowden RM, practical support, the members of the BECO group for useful discussions and 2012. Embryonic exposure to corticosterone modiﬁes the juvenile stress Danny Huybrecht for helping with the HPLC analyses. We thank two anony- response, oxidative stress and telomere length. P Roy Soc B-Biol Sci 279: mous reviewers for their helpful comments on an earlier version of this 1447–1456. manuscript. Isaksson C, 2013. Opposing effects on glutathione and reactive oxygen metab- olites of sex, habitat, and spring date, but no effect of increased breeding density in great tits Parus major. Ecol Evol 3:2730–2738. Funding Jones DP, 2006. Redeﬁning oxidative stress. Antioxid Redox Sign 8: This study was funded by Research Foundation Flanders (11O5914N and 1865–1879. 11O5916N granted to R.A.dB. and project G.0102.12 N granted to W.M. Keller LF, Waller DM, 2002. Inbreeding effects in wild populations. Trends Ecol Evol 17:230–241. and M.E.). Kristensen T, Sørensen P, Kruhøffer M, Pedersen K, Loeschcke V, 2005. Genome-wide analysis on inbreeding effects on gene expression in References Drosophila melanogaster. Genetics 171:157–167. Kristensen T, Sørensen P, Pedersen K, Kruhøffer M, Loeschcke V, 2006. AbdElgawad H, Farfan-Vignolo ER, de Vos D, Asard H, 2015. Elevated CO2 Inbreeding by environmental interactions affect gene expression in mitigates drought and temperature-induced oxidative stress differently in Drosophila melanogaster. Genetics 173:1329–1336. grasses and legumes. Plant Sci 231:1–10. Kristensen TN, Pedersen KS, Vermeulen CJ, Loeschcke V, 2010. Research on Aebi H, 1984. Catalase in vitro. Method Enzymol 105:121–126. inbreeding in the ‘omic’ era. Trends Ecol Evol 25:44–52. Armbruster P, Reed DH, 2005. Inbreeding depression in benign and stressful Landis GN, Abdueva D, Skvortsov D, Yang J, Rabin BE et al., 2004. Similar environments. Heredity 95:235–242. gene expression patterns characterize aging and oxidative stress in Ayroles JF, Hughes KA, Rowe KC, Reedy MM, Rodriguez-Zas SL et al., Drosophila melanogaster. Proc Natl Acad Sci USA 101:7663–7668. 2009. A genomewide assessment of inbreeding depression: gene number, Levine RL, Garland D, Oliver CN, Amici A, Climent I et al., 1990. function, and mode of action. Conserv Biol 23:920–930. Determination of carbonyl content in oxidatively modiﬁed proteins. Bradford MM, 1976. A rapid and sensitive method for the quantitation of Methods Enzymol 186:464–478. microgram quantities of protein utilizing the principle of protein-dye bind- Mainwaring MC, Dickens M, Hartley IR, 2010. Environmental and not ing. Anal Biochem 72:248–254. maternal effects determine variation in offspring phenotypes in a passerine Bichet C, Cornet S, Larcombe S, Sorci G, 2012. Experimental inhibition of bird. J Evol Biol 23:1302–1311. nitric oxide increases Plasmodium relictum (lineage SGS1) parasitaemia. Marasco V, Spencer KA, Robinson J, Herzyk P, Costantini D, 2013. Exp Parasitol 132:417–423. Developmental post-natal stress can alter the effects of pre-natal stress on Bize P, Devevey G, Monaghan P, Doligez B, Christe P, 2008. Fecundity and the adult redox balance. Gen Comp Endocr 191:239–246. survival in relation to resistance to oxidative stress in a free-living bird. Marasco V, Herzyk P, Robinson J, Spencer KA, 2016. Pre-and post-natal Ecology 89:2584–2593. stress programming: developmental exposure to glucocorticoids causes Blount JD, Surai PF, Nager RG, Houston DC, Møller AP et al., 2002. long-term brain-region speciﬁc changes to transcriptome in the precocial Carotenoids and egg quality in the lesser blackbacked gull Larus fuscus:a Japanese Quail. J Neuroendocrinol 28:1–17. supplemental feeding study of maternal effects. P Roy Soc B-Biol Sci 269: Menzel M, Sletvold N, Agren J, Hansson B, 2015. Inbreeding affects gene 29–36. expression differently in two self-incompatible Arabidopsis lyrata popula- de Boer RA, Eens M, Fransen E, Mu ¨ ller W, 2015. Hatching asynchrony aggra- tions with similar levels of inbreeding depression. Mol Biol Evol 32: vates inbreeding depression in a songbird Serinus canaria:an 2036–2047. inbreeding-environment interaction. Evolution 69:1063–1068. Metcalfe NB, Monaghan P, 2001. Compensation for a bad start: grow now, de Boer RA, Eens M, Mu ¨ ller W, 2016. A loss of heterozygosity, a loss in com- pay later? Trends Ecol Evol 16:254–260. petition? The effects of inbreeding, prenatal, and postnatal conditions on Monaghan P, Nager RG, 1997. Why don’t birds lay more eggs? Trends Ecol nestling development. Ecol Evol 6:7921–7930. Evol 12:270–274. Del Re A, 2013. compute.es: Compute Effect Sizes. R package version 0.2–2. Monaghan P, 2008. Early growth conditions, phenotypic development and Dhindsa RS, Plump-Dhindsa P, Thorpe TA, 1981. Leaf senescence: correlated environmental change. Philos T Roy Soc B 363:1635–1645. with increased levels of membrane permeability and lipid peroxidation, and Muller M, Groothuis TGG, 2013. Within-clutch variation in yolk testosterone decreased levels of superoxide dismutase and catalase. J Exp Bot 32: as an adaptive maternal effect to modulate avian sibling competition: evi- 93–101. dence from a comparative study. Am Nat 181:125–136. Drotar A, Phelps P, Fall R, 1985. Evidence for glutathione peroxidase activ- Nilsson J-A,Ra ˚ berg L, 2001. The resting metabolic cost of egg laying and nest- ities in cultured plant cells. Plant Sci 42:35–40. ling feeding in great tits. Oecologia 128:187–192. Forbes S, 2010. Social rank governs the effective environment of siblings. Biol Oddie KR, 2000. Size matters: competition between male and female great tit Lett 7:346–348. offspring. J Anim Ecol 69:903–912. Downloaded from https://academic.oup.com/cz/article-abstract/64/5/631/4683472 by Ed 'DeepDyve' Gillespie user on 18 October 2018 de Boer et al. Inbreeding, early life conditions, and oxidative balance 639 Okada K, Blount JD, Sharma MD, Snook RR, Hosken DJ, 2011. Male attrac- Royle NJ, Surai PF, Hartley IR, 2003. The effect of variation in dietary intake tiveness, fertility and susceptibility to oxidative stress are inﬂuenced by on maternal deposition of antioxidants in zebra ﬁnch eggs. Funct Ecol 17: inbreeding in Drosophila simulans. J Evol Biol 24:363–371. 472–481. Pamplona R, Costantini D, 2011. Molecular and structural antioxidant Schwabl H, 1993. Yolk is a source of maternal testosterone for developing defenses against oxidative stress in animals. Am J Physiol-Reg I 301: birds. Proc Natl Acad Sci USA 90:11446–11450. R843–R863. Sild E, Horak ~ P, 2009. Nitric oxide production: an easily measurable condi- Pletcher SD, Macdonald SJ, Marguerie R, Certa U, Stearns SC et al., 2002. tion index for vertebrates. Behav Ecol Sociobiol 63:959–966. Genome-wide transcript proﬁles in aging and calorically restricted Speakman JR, Blount JD, Bronikowski AM, Buffenstein R, Isaksson C et al., Drosophila melanogaster. Curr Biol 12:712–723. 2015. Oxidative stress and life histories: unresolved issues and current Price DT, Vita JA, Keaney JF, Jr., 2000. Redox control of vascular nitric oxide needs. Ecol Evol 5:5745–5757. bioavailability. Antioxid Redox Sign 2:919–935. Vajdovich P, 2008. Free radicals and antioxidants in inﬂammatory processes R Core Development Team, 2014. R: A Language and Environment for and ischemia-reperfusion injury. Vet Clin N Am-Small 38:31–123. Statistical Computing. Vienna: R Foundation for Statistical Computing. Vermeulen A, Mu ¨ ller W, Eens M, 2016. Vitally important: does early innate Royle NJ, Surai PF, McCartney RJ, Speake BK, 1999. Parental investment and immunity predict recruitment and adult innate immunity? Ecol Evol 6: egg yolk lipid composition in gulls. Func Ecol 13:298–306. 1799–1808. Royle NJ, Surai PF, Hartley IR, 2001. Maternally derived androgens and anti- Williams TD, 1994. Intraspeciﬁc variation in egg size and egg composition in oxidants in bird eggs: complementary but opposing effects? Behav Ecol 12: birds: effects on offspring ﬁtness. Biol Rev 69:35–59. 381–385. Zimmer C, Spencer KA, 2015. Reduced resistance to oxidative stress Royle NJ, Hartley IR, Parker GA, 2002. Begging for control: when are off- during reproduction as a cost of early-life stress. Comp Biochem Phys A spring solicitation behaviours honest? Trends Ecol Evol 17:434–440. 183:9–13.
Current Zoology – Oxford University Press
Published: Oct 1, 2018
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