Suvi Ruuskanen

Yolk hormones have sex-specific long-term effects on behavior in the pied flycatcher (Ficedula hypoleuca).

The hormonal environment during early development, such as maternally derived androgens in bird eggs, shapes the development and phenotype of the offspring in ways that may have important long-term consequences for behaviour. We studied the effects of yolk androgens on multiple behavioural traits in female and male pied flycatchers (Ficedula hypoleuca) by experimentally elevating androgen levels (testosterone and androstenedione) in the eggs. The birds were housed in a common-garden environment in captivity until full independence, after which their behaviour was tested. We found that androgen-treated males were more likely than control males to explore a novel environment and showed higher activity in the presence of a novel object. In response to a simulated predator attack, androgen-treated males mainly showed freezing behaviour, while control males showed escape behaviour. Females from the androgen treatment and control group showed no differences in these behaviours. Androgen treatment did not affect neophobia (latency to approach the novel object) or dominance behaviour in either sex. Behaviour in the novel environment and towards a novel object was repeatable, but behaviours in the different experiments were mostly not inter-correlated. These results indicate that yolk androgens have various long-lasting effects on behaviour, especially in males, but that they do not induce a distinct behavioural syndrome. As behaviour is strongly linked with fitness, our results suggest that yolk androgens may play a role in determining fitness, and thus play a potentially adaptive role.

Geographical variation in egg mass and egg content in a passerine bird.

Reproductive, phenotypic and life-history traits in many animal and plant taxa show geographic variation, indicating spatial variation in selection regimes. Maternal deposition to avian eggs, such as hormones, antibodies and antioxidants, critically affect development of the offspring, with long-lasting effects on the phenotype and fitness. Little is however known about large-scale geographical patterns of variation in maternal deposition to eggs. We studied geographical variation in egg components of a passerine bird, the pied flycatcher (Ficedula hypoleuca), by collecting samples from 16 populations and measuring egg and yolk mass, albumen lysozyme activity, yolk immunoglobulins, yolk androgens and yolk total carotenoids. We found significant variation among populations in most egg components, but ca. 90% of the variation was among individuals within populations. Population however explained 40% of the variation in carotenoid levels. In contrast to our hypothesis, we found geographical trends only in carotenoids, but not in any of the other egg components. Our results thus suggest high within-population variation and leave little scope for local adaptation and genetic differentiation in deposition of different egg components. The role of these maternally-derived resources in evolutionary change should be further investigated.

Yolk androgens do not appear to mediate sexual conflict over parental investment in the collared flycatcher Ficedula albicollis

Males and females are in conflict over parental care, as it would be favourable for one parent to shift labour to the other. Yolk hormones may offer a mechanism through which female birds could influence offspring traits in ways that increase the relative investment by the male. We studied the role of yolk androgens in mediating sexual conflict over parental care in the collared flycatcher (Ficedula albicollis). In a cross-fostering experiment, the males proportion of total feeding visits increased with increasing androgen levels in the foster eggs. This could suggest that sexual conflict over parental care may be influenced by the females differential allocation of yolk androgens or a maternal effect associated with yolk androgens. However, when we experimentally elevated yolk androgen levels, male feeding rates did not differ between control and androgen-manipulated nests. This suggests that other egg components correlated with yolk androgen levels, rather than yolk androgen levels per se, may influence male parental effort. In conclusion, yolk androgens per se do not appear to mediate sexual conflict over parental investment in the collared flycatcher.

Long-lasting effects of yolk androgens on phenotype in the pied flycatcher (Ficedula hypoleuca)

The hormonal environment during early development, such as maternally derived androgens in bird eggs, shapes the development of the offspring in ways that may have important long-term consequences for phenotype and behavior and, ultimately, fitness. We studied the long-term effects of yolk androgens on several phenotypic and physiological traits in male and female pied flycatchers (Ficedula hypoleuca) by experimentally elevating yolk androgen levels and rearing birds in common-garden environment in captivity. We found that high yolk androgen levels increased the basal metabolic rates in both females and males in adulthood. High yolk androgen levels did not affect male melanin coloration or plumage ornaments, or timing or speed of moult in either sex. No effect of androgen treatment on cell-mediated or humoral immune response was found in either sex. Covariation among the measured phenotypic traits was further not altered by androgen treatment. Our results suggest that exposure to high androgen levels can have long-lasting effects on some offspring traits, but do not seem to lead to different phenotypes. Furthermore, the role of yolk androgens affecting sexually selected male traits in our study species seems to be minor. The fitness consequences of yolk androgen-induced higher metabolic rates remain to be studied.

Long-term effects of yolk androgens on phenotype and parental feeding behavior in a wild passerine

Early growth conditions, such as exposure to maternally derived androgens in bird eggs, have been shown to shape offspring in ways that may have important long-term consequences for phenotype and behavior. Using an experimental approach, we studied the long-term effects of yolk androgens on several phenotypic traits and parental behavior in adult and female collared flycatchers (Ficedula albicollis). We elevated yolk androgen levels and monitored the experimental recruits the following breeding seasons. Androgen treatment had a sex-dependent effect on adult body condition, yolk androgen-treated males being heavier than control males when controlling for size, a result which may be caused potentially by selective mortality, physiological differences, or different life-history strategies. Androgen treatment did not however affect the expression of sexually selected plumage ornaments (forehead and wing patch size), UV coloration, or parental feeding rate in either sex. Our results suggest that yolk androgens are unlikely to affect sexual selection via plumage characteristics or contribute to breeding success via altered parental care. Yolk androgens do not seem to act as a means for female collared flycatchers to enhance the attractiveness of their sons. The lower return rate previously observed for androgen-treated male offspring compared to controls may therefore not be due to lower mating or breeding success, but may rather reflect lower survival or higher dispersal propensity of yolk androgen-treated males.

Geographical trends in the yolk carotenoid composition of the pied flycatcher (Ficedula hypoleuca)

Carotenoids in the egg yolks of birds are considered to be important antioxidants and immune stimulants during the rapid growth of embryos. Yolk carotenoid composition is strongly affected by the carotenoid composition of the female’s diet at the time of egg formation. Spatial and temporal differences in carotenoid availability may thus be reflected in yolk concentrations. To assess whether yolk carotenoid concentrations or carotenoid profiles show any large-scale geographical trends or differences among habitats, we collected yolk samples from 16 European populations of the pied flycatcher, Ficedula hypoleuca. We found that the concentrations and proportions of lutein and some other xanthophylls in the egg yolks decreased from Central Europe northwards. The most southern population (which is also the one found at the highest altitude) also showed relatively low carotenoid levels. Concentrations of β-carotene and zeaxanthin did not show any obvious geographical gradients. Egg yolks also contained proportionally more lutein and other xanthophylls in deciduous than in mixed or coniferous habitats. We suggest that latitudinal gradients in lutein and xanthophylls reflect the lower availability of lutein-rich food items in the northern F. hypoleuca populations and in montane southern populations, which start egg-laying earlier relative to tree phenology than the Central European populations. Similarly, among-habitat variation is likely to reflect the better availability of lutein-rich food in deciduous forests. Our study is the first to indicate that the concentration and profile of yolk carotenoids may show large-scale spatial variation among populations in different parts of the species’ geographical range. Further studies are needed to test the fitness effects of this geographical variation.

Digit ratios have poor indicator value in a wild bird population

Early androgen exposure is known to have long-lasting effects on phenotype, behaviour and even fitness, but difficulties in measuring the exposure hinders the study of its importance in evolutionary context. Digit ratios have been highlighted as a potential easy-to-measure indicator of early steroid exposure, as they have been suggested to reflect steroid, mainly testosterone levels during prenatal development. However, evidence for digit ratios reflecting early steroid levels is weak, as experimental studies, especially in wild populations, are scarce. We studied the association between maternally derived yolk androgens and digit ratios (2D:4D, 2D:3D and 3D:4D) using both correlative data and a rather high level of experimental elevation of yolk androgens in a passerine bird, the pied flycatcher (Ficedula hypoleuca). We also examined whether digit ratios have indicator value in an evolutionary context by studying correlations between digit ratios and reproductive traits, secondary sexual traits and exploratory behaviour. We did not find any association between digit ratios and yolk androgen level either in correlative or experimental data. Digit ratios were neither related to any of the reproductive and secondary sexual traits or exploratory behaviour measured. There was, however, a sex difference in 2D:3D and 3D:4D of adult birds (due to second and fourth digits being shorter in females), which was not apparent in fledglings or captivity-raised juveniles. This suggests that either the sex difference may develop as late as during the sexual maturation for breeding. These results indicate that, in this species, digit ratios are not reliable markers of maternally derived yolk androgen exposure and that they bear little relevance as correlates of the adaptive traits we measured.

Effects of early-life lead exposure on oxidative status and phagocytosis activity in great tits (Parus major).

Lead is a highly poisonous metal with a very long half-life, distributing throughout the body in blood and accumulating primarily in bones and kidney. We studied the short and long-term effects of early-life lead exposure on antioxidant defense and phagocytosis activity in a small passerine bird, the great tit (Parus major) by manipulating dietary lead levels of the nestlings. We had three experimental groups, exposed to environmentally relevant lead concentrations; high (4 μg/g body mass), low (1 μg/g body mass) and control (0 μg/g body mass) group. As a comparison, a great tit population breeding in the vicinity of a metal smelter was included to the experimental set-up. We measured glutathione, the ratio of reduced and oxidized glutathione, and the antioxidant enzymes: glutathione peroxidase, glutathione-S-transferase, catalase and superoxide dismutase together with protein carbonylation and phagocytosis activity to study the effects of lead on the oxidative status and immune function of birds. We found differences in enzyme activities between the study groups, but in most cases the smelter group differed from the other groups. Despite the differences observed in antioxidant enzymes, our results indicate only minor short-term effects of lead exposure on oxidative status, since either glutathione ratio or protein carbonylation were not affected by lead. Phagocytosis activity was not linked to higher lead concentrations either. Interestingly, protein carbonylation was positively associated with enzyme activities and glutathione level. Our results did not show major long-term effects of lead on the oxidative status of great tits.

The proteomics of feather development in pied flycatchers (Ficedula hypoleuca) with different plumage coloration

The genetic theory of morphological evolution postulates that form evolves largely by changing the expression proteins that are functionally conserved. It follows that understanding the function of proteins during different phases of development as well as the mechanisms by which the functions are modified is a prerequisite for understanding evolutionary change. Male pied flycatchers exhibit marked phenotypic variation in their breeding plumage. This variation has repeatedly been shown to have adaptive significance, but the molecular basis of this variation is not known. Here, we characterize the proteome of developing pied flycatcher feathers from differently pigmented males and also introduce a new method for examining the effect sizes of expression differences in protein interaction networks. Approximately 300 proteins were identified in the developing feathers of males. Gene products associated with cellular transport, cell metabolism and protein synthesis formed a large part of the developing feather proteome. Sixty‐five proteins associated with the development of the epidermis and/or pigmentation were detected in the data. The examination of expression level differences of protein–protein interaction networks revealed an immunological signalling–related network to exhibit significantly higher expression in black compared to brown males. Additionally, indications of differences in energy balance and oxidative stress related characteristics were detected. Together, these results provide new insight into the molecular mechanisms and evolutionary significance of plumage colour variation.

Effects of dietary lead exposure on vitamin levels in great tit nestlings – An experimental manipulation ☆

Exposure to metal pollution negatively affects animal physiology, including nutrient metabolism, but in the wild an effect can seldom be attributed to a single metal. Moreover, little is known about how the metabolism of vitamins, essential micronutrients for developing juveniles, is affected by toxic metals. Therefore we experimentally investigated the effects of lead (Pb), a widespread toxic metal, on four fat-soluble vitamins A (total and retinol), D3, E (total and α-tocopherol) and K and carotenoids (lutein, zeaxanthin and unidentified) in great tit (Parus major) nestlings. In addition to a control group where no Pb was provided, two Pb-dosed groups were compared to a metal exposed group in the vicinity of a Ni-Cu smelter. We examined whether Pb treatment affects vitamin homeostasis and how the response of Pb-treated birds relates to that of a population under industrial exposure of Pb and other metals. For this purpose, vitamin and carotenoid levels were quantified with UPLC-MS from plasma of 7 days-old nestlings. All metal exposed groups showed increased vitamin A and retinol levels. However, vitamin levels were not directly associated with fecal Pb levels, with the exception of retinol, which was positively correlated with fecal Pb. Alpha-tocopherol, lutein and zeaxanthin levels were positively associated with body mass and wing growth rate. To conclude, Pb exposure increased plasma vitamin A and retinol levels while the levels of other vitamins and carotenoids rather reflected secondary pollution effects via differences in habitat and diet quality at the smelter site. Our findings suggest Pb exposed nestlings may allocate the vitamins needed for growth and development to fight the physiological stress thus compromising their fitness.