The regulation of methylation on the Z chromosome and the identification of multiple novel Male Hyper-Methylated regions in the chicken.

DNA methylation is a key regulator of eukaryote genomes, and is of particular relevance in the regulation of gene expression on the sex chromosomes, with a key role in dosage compensation in mammalian XY systems. In the case of birds, dosage compensation is largely absent, with it being restricted to two small Male Hyper-Methylated (MHM) regions on the Z chromosome. To investigate how variation in DNA methylation is regulated on the Z chromosome we utilised a wild x domestic advanced intercross in the chicken, with both hypothalamic methylomes and transcriptomes assayed in 124 individuals. The relatively large numbers of individuals allowed us to identify additional genomic MHM regions on the Z chromosome that were significantly differentially methylated between the sexes. These regions appear to down-regulate local gene expression in males, but not remove it entirely (unlike the lncRNAs identified in the initial MHM regions). These MHM regions were further tested and the most balanced genes appear to show decreased expression in males, whilst methylation appeared to be far more correlated with gene expression in the less balanced, as compared to the most balanced genes. In addition, quantitative trait loci (QTL) that regulate variation in methylation on the Z chromosome, and those loci that regulate methylation on the autosomes that derive from the Z chromosome were mapped. Trans-effect hotspots were also identified that were based on the autosomes but affected the Z, and also one that was based on the Z chromosome but that affected both autosomal and sex chromosome DNA methylation regulation. We show that both cis and trans loci that originate from the Z chromosome never exhibit an interaction with sex, whereas trans loci originating from the autosomes but affecting the Z chromosome always display such an interaction. Our results highlight how additional MHM regions are actually present on the Z chromosome, and they appear to have smaller-scale effects on gene expression in males. Quantitative variation in methylation is also regulated both from the autosomes to the Z chromosome, and from the Z chromosome to the autosomes.

Gene expression across mammalian organ development.

The evolution of gene expression in mammalian organ development remains largely uncharacterized. Here we report the transcriptomes of seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) across developmental time points from early organogenesis to adulthood for human, rhesus macaque, mouse, rat, rabbit, opossum and chicken. Comparisons of gene expression patterns identified correspondences of developmental stages across species, and differences in the timing of key events during the development of the gonads. We found that the breadth of gene expression and the extent of purifying selection gradually decrease during development, whereas the amount of positive selection and expression of new genes increase. We identified differences in the temporal trajectories of expression of individual genes across species, with brain tissues showing the smallest percentage of trajectory changes, and the liver and testis showing the largest. Our work provides a resource of developmental transcriptomes of seven organs across seven species, and comparative analyses that characterize the development and evolution of mammalian organs.

Fair concordance between Google Trends and Danish ornithologists in the assessment of temporal trends in Danish bird populations highlights the informational value of big data.

The ongoing depletion of natural systems and associated biodiversity decline is of growing international concern. Climate change is expected to exacerbate anthropogenic impacts on wild populations. The scale of impact on ecosystems and ecosystem services will be determined by the impact on a multitude of species and functional groups, which due to their biology and numbers are difficult to monitor. The IPCC has argued that surveillance or monitoring is critical and proposed that monitoring systems should be developed, which not only track developments but also function as "early warning systems." Human populations are already generating large continuous datasets on multiple taxonomic groups through internet searches. These time series could in principle add substantially to current monitoring if they reflect true changes in the natural world. We here examined whether information on internet search frequencies delivered by the Danish population and captured by Google Trends (GT) appropriately informs on population trends in 106 common Danish bird species. We compared the internet search activity with independent equivalent population trend assessments from the Danish Ornithological Society (BirdLife Denmark/DOF). We find a fair concordance between the GT trends and the assessments by DOF. A substantial agreement can be obtained by omitting species without clear temporal trends. Our findings suggest that population trend proxies from internet search frequencies can be used to supplement existing wildlife population monitoring and to ask questions about an array of ecological phenomena, which potentially can be integrated into an early warning system for biodiversity under climate change.

The mitoepigenome responds to stress, suggesting novel mito-nuclear interactions in vertebrates.

The mitochondria are central in the cellular response to changing environmental conditions resulting from disease states, environmental exposures or normal physiological processes. Although the influences of environmental stressors upon the nuclear epigenome are well characterized, the existence and role of the mitochondrial epigenome remains contentious. Here, by quantifying the mitochondrial epigenomic response of pineal gland cells to circadian stress, we confirm the presence of extensive cytosine methylation within the mitochondrial genome. Furthermore, we identify distinct epigenetically plastic regions (mtDMRs) which vary in cytosinic methylation, primarily in a non CpG context, in response to stress and in a sex-specific manner. Motifs enriched in mtDMRs contain recognition sites for nuclear-derived DNA-binding factors (ATF4, HNF4A) important in the cellular metabolic stress response, which we found to be conserved across diverse vertebrate taxa. Together, these findings suggest a new layer of mito-nuclear interaction in which the nuclear metabolic stress response could alter mitochondrial transcriptional dynamics through the binding of nuclear-derived transcription factors in a methylation-dependent context.

Practical application of a Bayesian network approach to poultry epigenetics and stress.

BACKGROUND: Relationships among genetic or epigenetic features can be explored by learning probabilistic networks and unravelling the dependencies among a set of given genetic/epigenetic features. Bayesian networks (BNs) consist of nodes that represent the variables and arcs that represent the probabilistic relationships between the variables. However, practical guidance on how to make choices among the wide array of possibilities in Bayesian network analysis is limited. Our study aimed to apply a BN approach, while clearly laying out our analysis choices as an example for future researchers, in order to provide further insights into the relationships among epigenetic features and a stressful condition in chickens (Gallus gallus). RESULTS: Chickens raised under control conditions (n = 22) and chickens exposed to a social isolation protocol (n = 24) were used to identify differentially methylated regions (DMRs). A total of 60 DMRs were selected by a threshold, after bioinformatic pre-processing and analysis. The treatment was included as a binary variable (control = 0; stress = 1). Thereafter, a BN approach was applied: initially, a pre-filtering test was used for identifying pairs of features that must not be included in the process of learning the structure of the network; then, the average probability values for each arc of being part of the network were calculated; and finally, the arcs that were part of the consensus network were selected. The structure of the BN consisted of 47 out of 61 features (60 DMRs and the stressful condition), displaying 43 functional relationships. The stress condition was connected to two DMRs, one of them playing a role in tight and adhesive intracellular junctions in organs such as ovary, intestine, and brain. CONCLUSIONS: We clearly explain our steps in making each analysis choice, from discrete BN models to final generation of a consensus network from multiple model averaging searches. The epigenetic BN unravelled functional relationships among the DMRs, as well as epigenetic features in close association with the stressful condition the chickens were exposed to. The DMRs interacting with the stress condition could be further explored in future studies as possible biomarkers of stress in poultry species.

Domestication effects on social information transfer in chickens.

Red junglefowl (RJF), ancestor of all domesticated chickens, is a highly social, omnivorous bird species, presumably with a capacity for social information sharing. During domestication, birds have been selected to live in large, dynamic groups, and this may have affected their social cognition. Here, we studied social information transfer in female RJF and domesticated White Leghorn (WL) chickens. Individuals were trained to open a puzzle-box feeder by pecking a lid and we then recorded the behaviour towards the same puzzle-box feeder for birds that had either observed the trained individual ("guided") or saw the puzzle-box feeder for the first time ("naïve"). WL were considerably faster in approaching the feeder regardless of prior demonstration and pecked more at it. Both breeds were significantly faster to approach the puzzle-box feeder and pecked more after prior demonstration, but the effects were significantly stronger in WL. We conclude that both RJF and WL can utilize social information to address a novel problem, but during domestication this ability appears to have increased. The effects can be an effect of either social learning or stimulus enhancement. Some caution in this conclusion is necessary since we tested relatively few WL. Furthermore, possible confounding explanations include higher fearfulness in RJF and different effects of dominance interactions between demonstrators and observers.

Breed group differences in the unsolvable problem task: herding dogs prefer their owner, while solitary hunting dogs seek stranger proximity.

The communicating skills of dogs are well documented and especially their contact-seeking behaviours towards humans. The aim of this study was to use the unsolvable problem paradigm to investigate differences between breed groups in their contact-seeking behaviours towards their owner and a stranger. Twenty-four dogs of ancient breeds, 58 herding dogs, and 17 solitary hunting dogs were included in the study, and their behaviour when presented with an unsolvable problem task (UPT) was recorded for 3 min. All breed groups interacted with the test apparatus the same amount of time, but the herding dogs showed a longer gaze duration towards their owner compared to the other groups and they also preferred to interact with their owner instead of a stranger. Interestingly, the solitary hunting dogs were more in stranger proximity than the other groups, and they also showed a preference to make contact with a stranger instead of their owner. Hence, we found differences in contact-seeking behaviours, reflecting the dog-human relationship, between breed groups that might not only be related to their genetic similarity to wolves, but also due to the specific breeding history of the dogs.

Shifts in growth, but not differentiation, foreshadow the formation of exaggerated forms under chicken domestication.

Domestication provides an outstanding opportunity for biologists to explore the underpinnings of organismal diversification. In domesticated animals, selective breeding for exaggerated traits is expected to override genetic correlations that normally modulate phenotypic variation in nature. Whether this strong directional selection affects the sequence of tightly synchronized events by which organisms arise (ontogeny) is often overlooked. To address this concern, we compared the ontogeny of the red junglefowl (RJF) (Gallus gallus) to four conspecific lineages that underwent selection for traits of economic or ornamental value to humans. Trait differentiation sequences in embryos of these chicken breeds generally resembled the representative ancestral condition in the RJF, thus revealing that early ontogeny remains highly canalized even during evolution under domestication. This key finding substantiates that the genetic cost of domestication does not necessarily compromise early ontogenetic steps that ensure the production of viable offspring. Instead, disproportionate beak and limb growth (allometry) towards the end of ontogeny better explained phenotypes linked to intense selection for industrial-scale production over the last 100 years. Illuminating the spatial and temporal specificity of development is foundational to the enhancement of chicken breeds, as well as to ongoing research on the origins of phenotypic variation in wild avian species.

The Concept, Practice, Application, and Results of Locally Based Monitoring of the Environment.

Locally based monitoring is typically undertaken in areas in which communities have a close attachment to their natural resource base. We present a summary of work to develop a theoretical and practical understanding of locally based monitoring and we outline tests of this approach in research and practice over the past 20 years. Our tests show that locally based monitoring delivers credible data at local scale independent of external experts and can be used to inform local and national decision making within a short timeframe. We believe that monitoring conducted by and anchored in communities will gain in importance where scientist-led monitoring is sparse or too expensive to sustain and for ecosystem attributes in cases in which remote sensing cannot provide credible data. The spread of smartphone technology and online portals will further enhance the importance and usefulness of this discipline.

Effect of contact incubation on stress, behavior and body composition in the precocial Red jungle fowl.

Birds use contact incubation to warm their eggs above ambient temperature required for embryonic development. In contrast, birds in the industry as well as many birds in breeding programs and scientific studies are incubated in conventional incubators that warm eggs via circulating warm air. This means that contact incubated eggs have different thermal properties than eggs incubated in a conventional incubator. In light of previous studies showing that small differences in incubation temperature can affect chicks post-hatching phenotype, we investigated the consequences of incubating Red jungle fowl eggs at the same temperature (37 °C) either via contact incubation or warm air incubation. We found that contact incubated chicks had a more robust body composition, were more explorative and had a higher temperature preference early in life, as well as a sex dependent difference in plasma Corticosterone levels pre-hatch (measured in down-feathers) and post-hatch (measured in plasma) compared to chicks incubated in a conventional warm air incubator. While previous studies have demonstrated that embryonic development and post-hatch phenotype is sensitive to small variations in temperature, our study demonstrates for the first time that the way heat is distributed to the egg has a similar magnitude of effect on post-hatch phenotype and highlights the sensitivity of the incubation period in shaping birds post-hatch phenotype.

Therapeutic concentration of ciprofloxacin and transfer across the human term placenta.

BACKGROUND: Pregnant women have an increased risk of infections, and early and decisive treatment is preferred to prevent complications. Although ciprofloxacin is very commonly used, safety aspects of maternal treatment during pregnancy are limited, and avoidance of its use during late pregnancy is recommended. OBJECTIVE: The aim is to estimate maternal-to-fetal transfer clearance of ciprofloxacin at a therapeutic concentration and to determine fetal exposure to maternally administered ciprofloxacin. STUDY DESIGN: Transplacental pharmacokinetics were determined with an ex vivo placental model, which is a reliable experimental model for estimating fetal drug exposure. Human placentas from uncomplicated term pregnancies were collected after delivery and a suitable cotyledon was cannulated. Ciprofloxacin was added at a therapeutic concentration (1.6 µg/mL) to the maternal compartment, and antipyrine was included as a reference drug (10.0 µg/mL). Samples were collected from the maternal and fetal compartment at 12 time points (-2 to 180 minutes), and the integrity and metabolic parameters were measured consecutively. Drug concentrations were determined using ultra-performance liquid chromatography-tandem mass spectrometry. RESULTS: A total of 5 human placentas from healthy term pregnancies were collected after delivery and cannulated with success. Ciprofloxacin crossed the placenta; its mean concentration in the fetal compartment was 0.3 µg/mL, accounting for 22% (0.29/1.30; range, 15%-31%) of the maternal concentration after 3 hours. The fetal/maternal ciprofloxacin concentration ratio increased gradually over time and reached 0.53. The transfer clearance for ciprofloxacin was 0.28 mL/min (range, 0.21-0.41 mL/min) during the first hour and 0.21 mL/min (range, 0.14-0.26 mL/min) during the following 2 hours. After end perfusion, the mean tissue concentration and proportion of ciprofloxacin were 0.7 µg/g and 11% (14/130; range, 7%-14%), respectively. CONCLUSION: Ciprofloxacin crossed the placenta at a slow, constant rate, indicating moderate fetal exposure. This study verifies an accumulation of ciprofloxacin in the placenta that may lengthen the duration of fetal exposure. These results are an essential element of fetal risk assessment, but further studies are needed to estimate fetal safety.

Cerebellum size is related to fear memory and domestication of chickens.

Red Junglefowl (Gallus gallus) were selected for divergent levels of fear of humans during eight generations, causing the selection lines to differ in fear levels as well as in the proportional brain and cerebellum masses. Birds from the two lines were then crossed to obtain an F3 intercross in order to study the correlations between brain mass and fear learning. We exposed 105 F3-animals individually to a fear habituation and memory test at 8 days of age, where the reactions to repeated light flashes were assessed on 2 consecutive days. After culling, the absolute and relative sizes of each of four brain regions were measured. Stepwise regression was used to analyse the effects of the size of each brain region on habituation and memory. There were no effects of any brain region on the habituation on day one. However, birds with a larger absolute size of cerebellum had significantly reduced reactions to the fearful stimuli on day two, indicating a better memory of the stimuli. No other regions had significant effects. We conclude that increased cerebellum size may have been important in facilitating chicken domestication, allowing them to adapt to a life with humans.

Human total fertility rate affected by ambient temperatures in both the present and previous generations.

Elevated temperatures negatively affect human reproduction through several processes that regulate nutrient uptake and resource allocation in pregnant women. These can interfere with foetal development, resulting in low birth weight neonates with altered development trajectories. Temperatures that affect the current generation could, therefore, also have an impact on the following generation. We asked whether heat stress affected offspring fertility by asking if current and past ambient temperatures influenced total fertility rates (TFR) in human populations distributed across the world. We analysed time series data in 65 countries using simple regression analyses based on maximum temperatures and temperature amplitudes over 55 years. Supplemental longer time series (up to 100 years) provided information on response patterns in Northern Europe and Greenland's colder climates. There were clear and strong effects of temperatures on the TFR in the concurrent and the previous generation. Our temperature-based models account for 71-95% of the variation in TRF in European countries and Greenland, and 56-99% of the variation in 65 countries worldwide. Our findings are consistent with studies of seasonal variation in fertility and suggest that increased temperatures will negatively influence populations subjected to monthly maximum temperatures above 15-20 °C, while fertility in colder climates benefits from elevated temperatures. Our results provide strong evidence that ambient temperatures have important effects on human fertility, and that these effects persist into the following generation.

Sex-biased microRNA expression in mammals and birds reveals underlying regulatory mechanisms and a role in dosage compensation.

Sexual dimorphism depends on sex-biased gene expression, but the contributions of microRNAs (miRNAs) have not been globally assessed. We therefore produced an extensive small RNA sequencing data set to analyze male and female miRNA expression profiles in mouse, opossum, and chicken. Our analyses uncovered numerous cases of somatic sex-biased miRNA expression, with the largest proportion found in the mouse heart and liver. Sex-biased expression is explained by miRNA-specific regulation, including sex-biased chromatin accessibility at promoters, rather than piggybacking of intronic miRNAs on sex-biased protein-coding genes. In mouse, but not opossum and chicken, sex bias is coordinated across tissues such that autosomal testis-biased miRNAs tend to be somatically male-biased, whereas autosomal ovary-biased miRNAs are female-biased, possibly due to broad hormonal control. In chicken, which has a Z/W sex chromosome system, expression output of genes on the Z Chromosome is expected to be male-biased, since there is no global dosage compensation mechanism that restores expression in ZW females after almost all genes on the W Chromosome decayed. Nevertheless, we found that the dominant liver miRNA, miR-122-5p, is Z-linked but expressed in an unbiased manner, due to the unusual retention of a W-linked copy. Another Z-linked miRNA, the male-biased miR-2954-3p, shows conserved preference for dosage-sensitive genes on the Z Chromosome, based on computational and experimental data from chicken and zebra finch, and acts to equalize male-to-female expression ratios of its targets. Unexpectedly, our findings thus establish miRNA regulation as a novel gene-specific dosage compensation mechanism.

Barriers and opportunities for prolonging working life across different occupational groups: the SeniorWorkingLife study.

BACKGROUND: Increasing retirement age is a pivotal issue in labour market reforms. This study analyses factors conditioning retirement intentions. METHODS: In SeniorWorkingLife, 11 444 employed workers ≥50 years replied to questions in random order about expected reasons for leaving and potential reasons for staying longer at the labour market. Respondents were stratified based on the Danish version of International Standard Classification of Occupations (ISCO). Representative estimates were produced using the SurveyFreq and SurveyLogistic procedures of SAS combined with model-assisted weights based on national registers. RESULTS: For ISCO groups 1-4 (seated work) main expected reasons for retiring were freedom to choose and desire for more leisure time, but many would consider staying longer if there were better possibilities for additional senior days, longer vacations and flexible working hours. For ISCO groups 5-9 (physical work), poor physical health and not being capable of doing the job were common expected reasons for retiring, but many would consider staying longer if the work were less physically demanding and there were more senior days. Possibility for pension was a general expected reason for retiring. Expected reasons differed to a less extent between genders than between ISCO groups, e.g. economic factors were more important for men and high work demands more important for women. CONCLUSION: Different barriers and opportunities for prolonging working life exist across different occupational groups of the labour market-with most consistent differences between those with seated and physical work. Targeting these specifically seems opportune for policy makers and future interventions.

Morphological diversity of integumentary traits in fowl domestication: Insights from disparity analysis and embryonic development.

The domestication of the fowl resulted in a large diversity of integumental structures in chicken breeds. Several integumental traits have been investigated from a developmental genetics perspective. However, their distribution among breeds and their developmental morphology remain unexplored. We constructed a discrete trait-breed matrix and conducted a disparity analysis to investigate the variation of these structures in chicken breeds; 20 integumental traits of 72 chicken breeds and the red junglefowl were assessed. The analyses resulted in slight groupings of breed types comparable to standard breed classification based on artificial selection and chicken type use. The red junglefowl groups together with bantams and European breeds. We provide new data on the red junglefowl and four chicken breeds, demonstrating where and when variation arises during embryonic development. We document variation in developmental timing of the egg tooth and feather formation, as well as other kinds of developmental patterning as in the anlagen of different type of combs. Changes in epithelial-mesenchymal signaling interactions may drive the highly diverse integument in chickens. Experimental and comparative work has revealed that the cranial neural crest mesenchyme mediates its interactions with the overlying epithelium and is the likely source of patterning that generates diversity in integumental structures.

Changes in pituitary gene expression may underlie multiple domesticated traits in chickens.

Domesticated animals share a unique set of morphological and behavioral traits, jointly referred to as the domesticated phenotype. Striking similarities amongst a range of unrelated domesticated species suggest that similar regulatory mechanisms may underlie the domesticated phenotype. These include color pattern, growth, reproduction, development and stress response. Although previous studies have focused on the brain to find mechanisms underlying domestication, the potential role of the pituitary gland as a target of domestication is highly overlooked. Here, we study gene expression in the pituitary gland of the domesticated White Leghorn chicken and its wild ancestor, the Red Junglefowl. By overlapping differentially expressed genes with a previously published list of functionally important genes in the pituitary gland, we narrowed down to 34 genes. Amongst them, expression levels of genes with inhibitory function on pigmentation (ASIP), main stimulators of metabolism and sexual maturity (TSHB and DIO2), and a potential inhibitor of broodiness (PRLR), were higher in the domesticated breed. Additionally, expression of 2 key inhibitors of the stress response (NR3C1, CRHR2) was higher in the domesticated breed. We suggest that changes in the transcription of important modulatory genes in the pituitary gland can account not only for domestication of the stress response in domestic chickens, but also for changes in pigmentation, development, and reproduction. Given the pivotal role of the pituitary gland in the regulation of multiple shared domesticated traits, we suggest that similar changes in pituitary transcriptome may contribute to the domesticated phenotype in other species as well.

Variationally Computed IR Line List for the Methyl Radical CH3.

We present the first variational calculation of a hot-temperature ab initio line list for the CH3 radical. It is based on a high-level ab initio potential energy surface and dipole moment surface of CH3 in the ground electronic state. The ro-vibrational energy levels and Einstein A coefficients were calculated using the general-molecule variational approach implemented in the computer program TROVE. Vibrational energies and vibrational intensities are found to be in very good agreement with the available experimental data. The line list comprises 9 127 123 ro-vibrational states ( J ≤ 40) and 2 058 655 166 transitions, covering the wavenumber range up to 10 000 cm-1 and should be suitable for temperatures up to T = 1500 K.

Genetical genomics of growth in a chicken model.

BACKGROUND: The genetics underlying body mass and growth are key to understanding a wide range of topics in biology, both evolutionary and developmental. Body mass and growth traits are affected by many genetic variants of small effect. This complicates genetic mapping of growth and body mass. Experimental intercrosses between individuals from divergent populations allows us to map naturally occurring genetic variants for selected traits, such as body mass by linkage mapping. By simultaneously measuring traits and intermediary molecular phenotypes, such as gene expression, one can use integrative genomics to search for potential causative genes. RESULTS: In this study, we use linkage mapping approach to map growth traits (N = 471) and liver gene expression (N = 130) in an advanced intercross of wild Red Junglefowl and domestic White Leghorn layer chickens. We find 16 loci for growth traits, and 1463 loci for liver gene expression, as measured by microarrays. Of these, the genes TRAK1, OSBPL8, YEATS4, CEP55, and PIP4K2B are identified as strong candidates for growth loci in the chicken. We also show a high degree of sex-specific gene-regulation, with almost every gene expression locus exhibiting sex-interactions. Finally, several trans-regulatory hotspots were found, one of which coincides with a major growth locus. CONCLUSIONS: These findings not only serve to identify several strong candidates affecting growth, but also show how sex-specificity and local gene-regulation affect growth regulation in the chicken.

Epigenetics and early domestication: differences in hypothalamic DNA methylation between red junglefowl divergently selected for high or low fear of humans.

BACKGROUND: Domestication of animals leads to large phenotypic alterations within a short evolutionary time-period. Such alterations are caused by genomic variations, yet the prevalence of modified traits is higher than expected if they were caused only by classical genetics and mutations. Epigenetic mechanisms may also be important in driving domesticated phenotypes such as behavior traits. Gene expression can be modulated epigenetically by mechanisms such as DNA methylation, resulting in modifications that are not only variable and susceptible to environmental stimuli, but also sometimes transgenerationally stable. To study such mechanisms in early domestication, we used as model two selected lines of red junglefowl (ancestors of modern chickens) that were bred for either high or low fear of humans over five generations, and investigated differences in hypothalamic DNA methylation between the two populations. RESULTS: Twenty-two 1-kb windows were differentially methylated between the two selected lines at p < 0.05 after false discovery rate correction. The annotated functions of the genes within these windows indicated epigenetic regulation of metabolic and signaling pathways, which agrees with the changes in gene expression that were previously reported for the same tissue and animals. CONCLUSIONS: Our results show that selection for an important domestication-related behavioral trait such as tameness can cause divergent epigenetic patterns within only five generations, and that these changes could have an important role in chicken domestication.