Are offspring begging levels exaggerated beyond the parental optimum? Evidence from a bidirectional selection experiment.

Parental care involves elaborate behavioural interactions between parents and their offspring, with offspring stimulating their parents via begging to provision resources. Thus, begging has direct fitness benefits as it enhances offspring growth and survival. It is nevertheless subject to a complex evolutionary trajectory, because begging may serve as a means for the offspring to manipulate parents in the context of evolutionary conflicts of interest. Furthermore, it has been hypothesized that begging is coadapted and potentially genetically correlated with parental care traits as a result of social selection. Further experiments on the causal processes that shape the evolution of begging are therefore essential. We applied bidirectional artificial selection on begging behaviour, using canaries (Serinus canaria) as a model species. We measured the response to selection, the consequences for offspring development, changes in parental care traits, here the rate of parental provisioning, as well as the effects on reproductive success. After three generations of selection, offspring differed in begging behaviour according to our artificial selection regime: nestlings of the high begging line begged significantly more than nestlings of the low begging line. Intriguingly, begging less benefitted the nestlings, as reflected by on average significantly higher growth rates, and increased reproductive success in terms of a higher number of fledglings in the low selected line. Begging could thus represent an exaggerated trait, possibly because parent-offspring conflict enhanced the selection on begging. We did not find evidence that we co-selected on parental provisioning, which may be due to the lack of power, but may also suggest that the evolution of begging is probably not constrained by a genetic correlation between parental provisioning and offspring begging.

Perineuronal nets and vocal plasticity in songbirds: A proposed mechanism to explain the difference between closed-ended and open-ended learning.

Perineuronal nets (PNN) are aggregations of chondroitin sulfate proteoglycans surrounding the soma and proximal processes of neurons, mostly GABAergic interneurons expressing parvalbumin. They limit the plasticity of their afferent synaptic connections. In zebra finches PNN develop in an experience-dependent manner in the song control nuclei HVC and RA (nucleus robustus arcopallialis) when young birds crystallize their song. Because songbird species that are open-ended learners tend to recapitulate each year the different phases of song learning until their song crystallizes at the beginning of the breeding season, we tested whether seasonal changes in PNN expression would be found in the song control nuclei of a seasonally breeding species such as the European starling. Only minimal changes in PNN densities and total number of cells surrounded by PNN were detected. However, comparison of the density of PNN and of PNN surrounding parvalbumin-positive cells revealed that these structures are far less numerous in starlings that show extensive adult vocal plasticity, including learning of new songs throughout the year, than in the closed-ended learner zebra finches. Canaries that also display some vocal plasticity across season but were never formally shown to learn new songs in adulthood were intermediate in this respect. Together these data suggest that establishment of PNN around parvalbumin-positive neurons in song control nuclei has diverged during evolution to control the different learning capacities observed in songbird species. This differential expression of PNN in different songbird species could represent a key cellular mechanism mediating species variation between closed-ended and open-ended learning strategies. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 975-994, 2017.

Genetic Basis for Red Coloration in Birds.

The yellow and red feather pigmentation of many bird species [1] plays pivotal roles in social signaling and mate choice [2, 3]. To produce red pigments, birds ingest yellow carotenoids and endogenously convert them into red ketocarotenoids via an oxidation reaction catalyzed by a previously unknown ketolase [4-6]. We investigated the genetic basis for red coloration in birds using whole-genome sequencing of red siskins (Spinus cucullata), common canaries (Serinus canaria), and "red factor" canaries, which are the hybrid product of crossing red siskins with common canaries [7]. We identified two genomic regions introgressed from red siskins into red factor canaries that are required for red coloration. One of these regions contains a gene encoding a cytochrome P450 enzyme, CYP2J19. Transcriptome analysis demonstrates that CYP2J19 is significantly upregulated in the skin and liver of red factor canaries, strongly implicating CYP2J19 as the ketolase that mediates red coloration in birds. Interestingly, a second introgressed region required for red feathers resides within the epidermal differentiation complex, a cluster of genes involved in development of the integument. Lastly, we present evidence that CYP2J19 is involved in ketocarotenoid formation in the retina. The discovery of the carotenoid ketolase has important implications for understanding sensory function and signaling mediated by carotenoid pigmentation.

Hatching late in the season requires flexibility in the timing of song learning.

Most songbirds learn their songs from adult tutors, who can be their father or other male conspecifics. However, the variables that control song learning in a natural social context are largely unknown. We investigated whether the time of hatching of male domesticated canaries has an impact on their song development and on the neuroendocrine parameters of the song control system. Average age difference between early- and late-hatched males was 50 days with a maximum of 90 days. Song activity of adult tutor males decreased significantly during the breeding season. While early-hatched males were exposed to tutor songs for on average the first 99 days, late-hatched peers heard adult song only during the first 48 days of life. Remarkably, although hatching late in the season negatively affected body condition, no differences between both groups of males were found in song characteristics either in autumn or in the following spring. Similarly, hatching date had no effect on song nucleus size and circulating testosterone levels. Our data suggest that late-hatched males must have undergone accelerated song development. Furthermore, the limited tutor song exposure did not affect adult song organization and song performance.

Hatching asynchrony aggravates inbreeding depression in a songbird (Serinus canaria): an inbreeding-environment interaction.

Understanding how the intensity of inbreeding depression is influenced by stressful environmental conditions is an important area of enquiry in various fields of biology. In birds, environmental stress during early development is often related to hatching asynchrony; differences in age, and thus size, impose a gradient in conditions ranging from benign (first hatched chick) to harsh (last hatched chick). Here, we compared the effect of hatching order on growth rate in inbred (parents are full siblings) and outbred (parents are unrelated) canary chicks (Serinus canaria). We found that inbreeding depression was more severe under more stressful conditions, being most evident in later hatched chicks. Thus, consideration of inbreeding-environment interactions is of vital importance for our understanding of the biological significance of inbreeding depression and hatching asynchrony. The latter is particularly relevant given that hatching asynchrony is a widespread phenomenon, occurring in many bird species. The exact causes of the observed inbreeding-environment interaction are as yet unknown, but may be related to a decrease in maternal investment in egg contents with laying position (i.e. prehatching environment), or to performance of the chicks during sibling competition and/or their resilience to food shortage (i.e. posthatching environment).

Sources of variation in yolk hormone deposition: consistency, inheritance and developmental effects.

Maternal effects occur when the phenotype of the mother affects the phenotype of their offspring. They are thought to have evolved to translate the environmental conditions experienced by the mother into adaptive phenotypic variation of the offspring. However, the integration of environmental cues allowing adaptive responses requires some form of plasticity that depends on the interaction of the maternal phenotype and her environment. In birds, maternal yolk hormones represent such a pathway for maternal effects, and their adaptive significance depends thus on the plasticity in maternal yolk hormone deposition. We studied sources of variation in yolk testosterone deposition, focusing on the often neglected contribution of the (partly heritable) maternal phenotype. We investigated consistency and heritability of yolk testosterone deposition in captive canaries of which the F(1) generation was raised in foster nests and analyzed the potential effects of the early developmental conditions. We found significant female consistency across years in egg mass, yolk mass and total amount of yolk testosterone but not in yolk testosterone concentrations. Females varied the yolk testosterone concentrations of their eggs across years mainly via changes in yolk mass. The heritable variation in egg mass, yolk mass and amount of yolk testosterone but not yolk testosterone concentrations was within the range of previous studies, but not significantly different from zero. Finally, the growth of the daughters as nestling had a significant effect on their yolk testosterone deposition at adulthood indicating the transgenerational potential for environmental effects - via the effects of yolk hormones on offspring development.

Negative effects of yolk testosterone and ticks on growth in canaries.

Maternal yolk hormones in bird eggs are thought to adjust the offspring to the post-hatching environment. This implies that the effects of maternal yolk hormones should vary with the post-hatching environment, but to date such context-dependency has largely been ignored. We experimentally increased yolk testosterone concentrations in canary eggs and simultaneously manipulated the post-hatching context via an experimental tick-infestation of the chicks. This allows us to evaluate the context-dependency of hormone-mediated maternal effects, as it has previously been shown that ectoparasites alter the maternal yolk androgen deposition. The experimental tick infestation reduced growth in chicks from sham-treated eggs, indicating harmful effects of this ectoparasite in canaries. Chicks from testosterone-treated eggs were not affected in their development by ticks, suggesting lower ectoparasite vulnerability. But this may also be due to the fact that experimentally elevated yolk testosterone levels impaired growth even under parasite-free conditions. This contrasts previous studies, but these studies often manipulated first laid eggs, while we used eggs of subsequent laying positions. Later laid eggs are presumably of lower quality and contain higher yolk testosterone concentrations. Thus, the effects of elevated yolk testosterone on growth may be dose-dependent or vary with the egg quality, suggesting prenatal context-dependency.

Electrophysiological and morphological development of the inner ear in Belgian Waterslager canaries.

Belgian Waterslager (BW) canaries have an inherited hearing loss due to missing and abnormal hair cells, but it is unclear whether the loss is congenital or developmental. We used auditory brainstem responses and scanning electron microscopy to describe the development of auditory sensitivity and hair cell abnormalities in BW and non-BW canaries. In both strains, adult ABR thresholds were higher than behavioral thresholds, but BW canaries exhibited higher thresholds than non-BW canaries across all frequencies. Immediately post-hatch, ABR thresholds and hair cell numbers were similar in both strains. Two weeks later, thresholds were significantly higher in BW canaries, and hair cell number progressively decreased as the birds aged. These data show that in BW canaries: the peripheral auditory system is functionally similar to non-BW canary from hatch to 2 weeks, ABR thresholds improve during this developmental period, actually becoming better than those of adults, but then worsen as the bird continues to age. Hair cell number and appearance is similar to non-BW canaries at hatch but progressively declines after 30 days of age. These data show that the hearing loss characteristic of BW canaries is, at least in part, developmental and is established by the time song learning begins.

Parent-offspring conflict and coadaptation.

The evolution of family life has traditionally been studied in parallel by behavioral ecologists and quantitative geneticists. The former focus on parent-offspring conflict and whether parents or offspring control provisioning, whereas the latter concentrate on the coadaptation of parental supply and offspring demand. Here we show how prenatal effects on offspring begging can link the two different approaches. Using theoretical and experimental analyses, we show that when offspring control provisioning, prenatal effects primarily serve the parent's interests: Selection on parents drives coadaptation of parent and offspring traits. In contrast, when parents control provisioning, prenatal effects primarily serve the offspring's interests: Selection on the offspring drives coadaptation of parent and offspring traits. Parent-offspring conflict may thus be responsible for the selective forces that generate parent-offspring coadaptation.

Maternal effects and beta-carotene assimilation in Canary chicks.

Carotenoids are pigments responsible for the red, orange and yellow coloration of plants and animals. They may be beneficial in two ways; they have a powerful antioxidant activity, and they can behave as an immunostimulant. Animals however cannot synthesize carotenoids de novo, they must obtain them through their diet. In our experiments on Canaries, we investigated how mothers transfer their dietary carotenoid-related benefits to their offspring; either through the egg, or through the diet (during chicks' feeding). Female Canaries were allowed to access beta-carotene enriched food during egg formation and/or chicks' feeding. We sorted the chicks into four groups using the period when they assimilated the beta-carotene as a variable. The four groups were: (i) before hatching (from yolk), (ii) after hatching (from maternal feeding), (iii) before and after hatching, or (iv) never. Colorimetry and HPLC analysis from sub-samples of yolks confirmed the maternal transfer of dietary carotenoids to the yolk. Our results show that benefits from maternal dietary carotenoids are transferred to the chicks, but according to the period when they are assimilated by the chicks, the physiological effects are different. It was found that the chicks' growth was enhanced when carotenoids were assimilated both before and after hatching. However an increase in cellular immunity efficiency only occurs when the assimilation takes place after hatching.

Yolk testosterone, postnatal growth and song in male canaries.

Avian eggs contain substantial amounts of maternal yolk androgens, which have been shown to modulate offspring phenotype. The first studies on the functional consequences of maternal yolk androgens have focused on early life stages and their role in sibling competition. However, recent longitudinal studies reported long-lasting effects of maternal yolk androgens on offspring phenotype, mostly concerning traits that are sensitive to androgens. This suggests that maternal yolk androgens could play an important role in sexual selection, since the expression of many male sexual characters is testosterone-dependent. Using male canaries as a model, we examined the consequences of an experimental elevation of yolk testosterone concentrations on early development as well as long-lasting effects particularly on song, which is one of the most important sexual characters in male songbirds. Elevated yolk testosterone concentrations inhibited male growth, possibly in interaction with an existent ectoparasite exposure. Males hatched from testosterone-treated eggs (T-males) did not have enhanced competitive skills, in contrast to previous studies. The elevation of yolk testosterone concentrations delayed song development but did not affect adult song phenotype. This is intriguing, as yolk testosterone possibly induced developmental stress, which is known to reduce song quality. We hypothesize that yolk testosterone has either no direct effect on adult song phenotype, or that positive effects are merged by the negative effects of developmental stress. Finally, females mated with T-males invested more in their clutch indicating that females either assess T-males as more attractive (differential allocation hypothesis) or compensated for lower offspring viability (compensation hypothesis).

Does testosterone mediate the trade-off between nestling begging and growth in the canary (Serinus canaria)?

Nestling birds solicit food from their parents with vigorous begging displays, involving posturing, jostling and calling. In some species, such as canaries, begging is especially costly because it causes a trade off against nestling growth. Fitness costs of begging like this are predicted by evolutionary theory because they function to resolve conflicts of interest within the family over the provision of parental investment. However, the mechanism that links these costs with nestling behaviour remains unclear. In the present study, we determine if the relationships between nestling androgen levels, nestling begging intensities and nestling growth rates are consistent with the hypothesis that testosterone is responsible for the trade-off between begging and growth. We test this idea with a correlational study, using fecal androgens as a non-invasive method for assaying nestling androgen levels. Our results show that fecal androgen levels are positively correlated with nestling begging intensity, and reveal marked family differences in each trait. Furthermore, changes in fecal androgen levels between 5 and 8 days after hatching are positively associated with changes in nestling begging intensity, and negatively associated with nestling growth during this time. Although these correlational results support our predictions, we suggest that that experimental manipulations are now required to test the direct or indirect role of testosterone in mediating the trade-off between begging and growth.

Song and brain development in canaries raised under different conditions of acoustic and social isolation over two years.

Early isolation experiments indicate that male songbirds learn their songs during an early sensitive period, although later work has shown that some open-ended learners modify songs in later years. Recent isolation experiments suggest that in some species song has a stronger genetic basis than previously thought. This study raised domestic canaries under different combinations of acoustic and social isolation and followed song development into the second year. Males raised alone in acoustic isolation developed songs with normal syllables, but larger repertoires and also produced syllables with lower repetition rates when compared to controls. The smallest repertoire occurred in males raised in a peer group. Isolate males had a smaller song control nucleus HVC than controls, but there was no effect on nucleus RA or on brain weight in general. In the second year, after introduction into a large normal colony, isolate and peer group males adjusted their syllable repertoire to normal size. In particular, the isolates reduced their repertoire even though the size of HVC showed a significant increase in volume. However, songs of isolate and peer group males still differ in repetition rate and number of single syllables in the common aviary. In contrast, control males showed low syllable turnover and no significant change in repertoire size. Nor did they show any significant change in the volumes of song control nuclei. It seems that complete isolation affects only some aspects of song and brain development, and later socialization corrects some but not all of these in the second year.

Genomic resources for songbird research and their use in characterizing gene expression during brain development.

Vocal learning and neuronal replacement have been studied extensively in songbirds, but until recently, few molecular and genomic tools for songbird research existed. Here we describe new molecular/genomic resources developed in our laboratory. We made cDNA libraries from zebra finch (Taeniopygia guttata) brains at different developmental stages. A total of 11,000 cDNA clones from these libraries, representing 5,866 unique gene transcripts, were randomly picked and sequenced from the 3' ends. A web-based database was established for clone tracking, sequence analysis, and functional annotations. Our cDNA libraries were not normalized. Sequencing ESTs without normalization produced many developmental stage-specific sequences, yielding insights into patterns of gene expression at different stages of brain development. In particular, the cDNA library made from brains at posthatching day 30-50, corresponding to the period of rapid song system development and song learning, has the most diverse and richest set of genes expressed. We also identified five microRNAs whose sequences are highly conserved between zebra finch and other species. We printed cDNA microarrays and profiled gene expression in the high vocal center of both adult male zebra finches and canaries (Serinus canaria). Genes differentially expressed in the high vocal center were identified from the microarray hybridization results. Selected genes were validated by in situ hybridization. Networks among the regulated genes were also identified. These resources provide songbird biologists with tools for genome annotation, comparative genomics, and microarray gene expression analysis.

Vitamin A requirements of alipochromatic ('recessive-white') and coloured canaries (Serinus canaria) during the breeding season.

Six pairs of alipochromatic ('recessive-white') canaries (Serinus canaria) and six pairs of coloured canaries were kept through a complete breeding cycle while being fed a diet providing 12,000 iu vitamin A/kg. The eggs of three pairs (one recessive-white and two coloured) were all unfertilised and there were only 23 hatchlings (14 recessive-white and nine coloured), of which 14 (10 recessive-white and four coloured) were alive after the first moult. However, there was no clinical, biochemical or pathological evidence that the recessive-white canaries were suffering from vitamin A deficiency or that the coloured canaries were suffering from vitamin A toxicity, suggesting that the diet met the vitamin A requirements of both groups.

Freedom and rules: the acquisition and reprogramming of a bird's learned song.

Canary song is hierarchically structured: Short stereotyped syllables are repeated to form phrases, which in turn are arranged to form songs. This structure occurs even in the songs of young isolates, which suggests that innate rules govern canary song development. However, juveniles that had never heard normal song imitated abnormal synthetic songs with great accuracy, even when the tutor songs lacked phrasing. As the birds matured, imitated songs were reprogrammed to form typical canary phrasing. Thus, imitation and innate song constraints are separate processes that can be segregated in time: freedom in youth, rules in adulthood.

Investigations on feathering, feather growth and potential influences of nutrient supply on feathers' regrowth in small pet birds (canaries, budgerigars and lovebirds).

The aim of this study was to quantitate feathering in several companion birds. Besides the ratio of feathers to whole body mass, feather length as well as feather weight were of interest. Furthermore, data on feather loss and growth rates were estimated. In general, it could be observed that the proportion of feathers relative to body mass varied between 14 (canaries) and 7.4% (lovebirds). Feather losses (outside the moult period) amounted to an average of 6.65 (canaries), 8.98 (budgerigars), and 8.43 (lovebirds) mg/bird/day respectively or 37 (canaries), 20 (budgerigars), and 17 (lovebirds) mg/100 g body weight/day (values of interest in calculating of protein requirements for maintenance). In canaries, the average growth rate of the developing feathers amounted to 2 mm/day. In contrast to the onset of feather regeneration, the growth rate of new feathers leaving the follicle was not influenced by the supplements used here. The regeneration period (first measurable feather growth) of a plucked pinion can be used as an indicator and objective parameter to test potential nutritional influences. Parallel to the improvement of nutrient supply the rates of feather losses and also replacement increased, whereas the rates decreased when seed mixtures without any addition of minerals, sulphurous amino acids, and vitamins were fed.

A growth cost of begging in captive canary chicks.

Nestling birds solicit food from adults by using begging displays that appear paradoxically costly and wasteful. Theoretical work suggests that the evolution of such exuberant offspring behavior reflects parent-offspring conflict over the supply of parental investment. Originally, extravagant begging was seen as a means of psychological trickery by which offspring could wheedle additional resources from resistant parents. Subsequently, costly begging came to be viewed as the hallmark of resolved parent-offspring conflict, serving either to prevent escalated scramble competition or to enforce honest signaling. However, the theoretical assumption of costly solicitation has been called into question by the low level of energy expenditure measured empirically during begging. This finding has prompted new theoretical work that shows that begging can be cost-free and yet still resolve parent-offspring conflict. Here, I report that begging is more costly than recent work suggests. My experimental evidence from captive canaries demonstrates a marginal cost of begging through impaired growth. Furthermore, I argue that previous studies of energy expenditure during solicitation do not measure the cost of begging, as defined theoretically. More generally, my results may account for the evolution of nestling growth rates, as well as the observation that begging is typically most flamboyant in older offspring.

Developmental changes and among-sibling variation of corticosterone levels in an altricial avian species.

The postnatal development of the activity of the brain-pituitary-adrenal axis was investigated in an altricial bird species by measurements of plasma corticosterone levels in nestling and fledgling canaries, Serinus canaria. Corticosterone was detectable (>2.6 ng/ml) in 30% of 5-day-old, 67% of 10-day-old, 72% of 15-day-old, and 88% of 23-day-old birds. When detectable, the corticosterone levels of 5-day-old nestlings were comparable to the baseline levels of adult birds. Levels were higher in 10- and 15- than in 5-day-old nestlings. The levels of 23-day-old fledglings (about 6 to 7 days after fledgling) were significantly higher than those of 15-day-old nestlings. They were intermediate between adult baseline and stress-induced levels. Sex did not influence this general profile, but levels varied with the order of hatching within broods. At the age of 15 and 23 days first hatched chicks had higher corticosterone levels than last hatched chicks, while second hatched chicks had intermediate levels. These differences were not correlated with body mass. The results suggest that (1) the brain-pituitary-adrenal axis of this altricial bird becomes fully functional after hatching and (2) birth order within broods influences corticosterone secretion during subsequent stages of development. It is unlikely that the brain-pituitary-adrenal axis matures at different rates in first and later hatched chicks or that the different levels of first and later hatched chicks were caused by capture and handling stress. Rather, they may result from such maternal effects as hatching asynchrony or differential concentrations of yolk steroids among the eggs in a clutch. Further studies will have to show whether this systematic variation of corticosterone levels among siblings during early life persists into adulthood and how it is related to behavior and fitness.

Fate of new neurons in adult canary high vocal center during the first 30 days after their formation.

Projection neurons are added to the high vocal center (HVC) of adult songbirds. Here we report on events associated with their initial arrival in HVC. Neurons formed in adult canaries were labeled with [(3)H]-thymidine and examined 8, 15, 22, and 31 days later. By 8 days, some [(3)H]-labeled cells with the nuclear profile of postmigratory neurons were already present in HVC but could not be retrogradely labeled by Fluoro-Gold injections in the robust nucleus of the archistriatum (RA); 7 days later, a few such cells could be backfilled from RA. Thus, new neurons may arrive in HVC as much as 1 week prior to establishing connections with RA. By 31 days, 43% of the [(3)H]-labeled neurons could be backfilled from RA. In no case were new neurons backfilled by tracer injections into Area X, suggesting that newly formed HVC cells do not establish a transient connection with this region. At all survival times, the somata of new neurons were often clustered tightly together with other HVC neurons that differed in age and projection. Between days 15 and 25 after their birth, half of the new HVC neurons disappeared. We conclude: (1) that neurons arrive in HVC earlier than previously thought, (2) that soon after their arrival they become part of cell clusters in HVC, and (3) that in addition to the previously described death of new neurons that occurs over a period of months, there is an early wave of death that occurs soon after new neurons adopt a postmigratory phenotype.