Background: The amount of resources provided by the mother before birth has important and long-lasting effects on offspring fitness. Despite this, there is a large amount of variation in maternal investment seen in natural populations. Life-history theory predicts that this variation is maintained through a trade-off between the benefits of high maternal investment for the offspring and the costs of high investment for the mother. However, the proximate mechanisms underlying these costs of reproduction are not well understood. Here we used artificial selection for high and low maternal egg investment in a precocial bird, the Japanese quail (Coturnix japonica) to quantify costs of maternal reproductive investment. Results: We show that females from the high maternal investment lines had significantly larger reproductive organs, which explained their overall larger body mass, and resulted in a higher resting metabolic rate (RMR). Contrary to our expectations, this increase in metabolic activity did not lead to a higher level of oxidative damage. Conclusions: This study is the first to provide experimental evidence for metabolic costs of increased per offspring investment.

Conditions experienced by individuals during prenatal development can have long-term effects on their phenotype. Maternally transmitted resources are important mediators of such prenatal effects, but the potential interactive effects among them in shaping offspring phenotype have never been studied. Maternally derived testosterone is known to stimulate growth, but these benefits may be counterbalanced by an increase in the production of reactive oxygen species (ROS). Maternally transmitted carotenoids might have the capacity to scavenge ROS and thereby buffer an increase in oxidative stress caused by prenatal exposure to high testosterone levels. Here, we experimentally tested for such interactive effects between maternal yolk testosterone and carotenoid in Japanese quail (Coturnix japonica). We found that hatching mass was reduced and reactive oxygen metabolites (ROMs) levels at the end of the period of maximal growth increased in chicks from eggs injected with either testosterone or carotenoid (only a tendency in chicks from testosterone-injected eggs). However, when both egg compounds were manipulated simultaneously, hatching mass and ROM levels were not affected, showing that both carotenoid and testosterone lose their detrimental effects when the ratio between the 2 compounds is balanced. Our study provides the first experimental evidence for interactive effects of 2 maternally derived egg compounds on offspring phenotype and suggests that developmental cues are tightly coadjusted within an egg. INTERACTIVE EFFECTS OF YOLK TESTOSTERONE AND CAROTENOID ON 10 PRE-NATAL GROWTH AND OFFSPRING PHYSIOLOGY IN A PRECOCIAL BIRD

The large-scale impact of urbanization on wildlife is rather well documented, however the mechanisms underlying the effects of urban environments on animal physiology and behaviour are still poorly understood. Here, we focused on one major urban pollutant - artificial light at night (ALAN) - and its effects on the capacity to mount an innate immune response in wild great tit Parus major nestlings. Exposure to ALAN alters circadian rhythms of physiological processes, by disrupting the nocturnal production of the hormone melatonin. Nestlings were exposed to a light source emitting 3 lux for seven consecutive nights. Subsequently, nestlings were immune-challenged with a lipopolysaccharide injection, and we measured haptoglobin and nitric oxide levels pre- and post-injection. Both haptoglobin and nitric oxide are important markers for innate immune function. We found that ALAN exposure altered the innate immune response, with ALAN nestlings having lower haptoglobin and higher nitric oxide levels after the immune-challenge compared to dark-night nestlings. Unexpectedly, nitric oxide levels were overall, lower after the immune-challenge than before. These effects were likely mediated by melatonin, since ALAN-treated birds had on average 49% lower melatonin levels than the dark-night birds. ALAN exposure did not have any clear effects on nestling growth. This study provides a potential physiological mechanism underlying the documented differences in immune function between urban and rural birds observed in other studies. Moreover, it gives evidence that ALAN exposure affects nestling physiology, potentially causing long-term effects on physiology and behaviour, which ultimately can affect their fitness.

Conditions experienced during prenatal development can have long-lasting organizational effects on offspring. Maternal carotenoids deposited in the eggs of birds and other oviparous species play an important role during fast embryonic growth and chick development through their antioxidant properties. However, the long-term consequences of variation in maternal carotenoid transfer for the offspring have seldom been considered. Since plasma carotenoid levels at adulthood are known to influence testis size and yolk carotenoid levels influence the ability to extract carotenoids later in life, we hypothesized that maternally transmitted carotenoids might influence gonad size at adulthood. Here, we showed that male Japanese quail (Coturnix japonica) originating from a carotenoid-enriched egg had smaller testes than control individuals at adulthood. This result shows that yolk carotenoids have long-term organizational effects. In addition, given that carotenoid intake at sexual maturity increases sperm quality and that a decreased testis size is associated with a lower sperm production, we propose that carotenoid exposure during embryo development might influence a trade-off between ejaculate size and sperm quality.

Yolk androgens of maternal origin are important mediators of prenatal maternal effects. Although in many species short-term benefits of exposure to high yolk androgen concentrations for the offspring have been observed, females differ substantially in the amount of androgens they transfer to their eggs. It suggests that costs for the offspring or the mother constrain the evolution of maternal hormone transfer. However, to date, the nature of these costs remains poorly understood. Unlike most previous work that focused on potential costs for the offspring, we here investigated whether high yolk testosterone transfer is associated with metabolic costs (i.e., a higher metabolic rate) for the mother. We show that Japanese quail (Coturnix japonica) females that deposit higher testosterone concentrations into their eggs have a higher resting metabolic rate. Because a higher metabolic rate is often associated with a shorter life span, this relationship may explain the negative association between yolk testosterone transfer and female longevity observed in the wild. Our results suggest that metabolic costs for the mother can balance the short-term benefits of yolk testosterone exposure for the offspring, thereby contributing to the maintenance of variation in maternal yolk hormone transfer in natural populations. for the offspring, we here show that yolk hormone transfer is associated with metabolic 28 costs for the mother. These costs for the mother can balance beneficial effects on 29 offspring and thereby contribute to the maintenance of variation in maternal yolk 30 hormone transfer in animal populations. 31 32 3 Abstract 33Yolk androgens of maternal origin are important mediators of prenatal maternal effects. 34Although in many species short-term benefits of exposure to high yolk androgen 35 concentrations for the offspring have been observed, females differ substantially in the 36 amount of androgens they transfer to their eggs. It suggests that costs for the offspring or 37 the mother constrain the evolution of maternal hormone transfer. However, to date the 38 nature of these costs remains poorly understood. Unlike most previous work that focused 39 on potential costs for the offspring, we here investigated if high yolk testosterone transfer 40 is associated with metabolic costs (i.e. a higher metabolic rate) for the mother. We show 41 that Japanese quail (Coturnix japonica) females that deposit higher testosterone 42 concentrations into their eggs have a higher resting metabolic rate (RMR). Because a 43 higher metabolic rate is often associated with a shorter lifespan, this relationship may 44 explain the negative association between yolk testosterone transfer and female longevity 45 observed in the wild. Our results suggest that metabolic costs for the mother can balance 46 the short-term benefits of yolk testosterone exposure for the offspring, thereby 47 contributing to the maintenance of variation in maternal yolk hormone transfer in natural 48 populations. 49 50