The mechanisms underlying seasonal timing of breeding : a multi-level approach using bi-directional genomic selection on timing of egg-laying

With climate change being one of the major threats to current biodiversity, it is essential for species to adapt sufficiently in order to survive. Some species adapt their phenology faster, in reaction to increasing temperatures, compared to others, resulting in mismatched timing. For many seasonal breeding avian species in temperate zones, such as the great tit, the reproductive period is short and coincides with warmer temperatures and increased food supplies required for successful rearing of offspring. Therefore, seasonal breeders time their reproductive cycle to the changing seasons in order to maximize reproductive success and offspring survival. With springs getting warmer earlier in the year, it is of importance for great tit females to start laying earlier to be able to raise their offspring in an optimal period (i.e. sufficient food abundance). However, females show large variation in timing of breeding, which lies in the underlying physiology: different cues are used and translated by a cascade of neuro-endocrine processes along the hypothalamic-pituitary-gonadal-liver (HPGL) axis into a laying date. Natural selection could act on this variation between females, but it is still unclear on which of the compartments (brain, ovary, liver) of the HPGL axis cues act and thus where the variation in timing between females arises. It is of importance to understand how the components of the physiological mechanism contribute to genetic variation in timing before one is able to understand how natural selection can act on timing of reproduction. In this thesis, the main aim was to explore the molecular basis of the physiological mechanisms underlying avian seasonal timing of breeding. A promising way to do this is by comparing (extremely) early and (extremely) late laying females. In Chapter 2, I describe a large-scale selection experiment, where we created selection lines for early and late egg-laying using genomic selection. In this chapter we show that genomic selection on a complex trait such as timing of breeding is possible, because we find that the early and late selection line birds differ genomically and that this difference increases over the generations. In addition, we find that F3 generation birds differ also phenotypically, with a significant average difference in egg-laying dates of ~10 days between selection lines. By housing pairs of the selection lines in climate-controlled aviaries and in outdoor aviaries for two consecutive years and in contrasting environments (either artificial or semi-natural), I was able to determine that temperature has a direct effect on timing of breeding instead of via food phenology and that females laid on average earlier in the warm environment (Chapter 3). Further, because we obtained two laying dates per female, we evaluated whether our selection on laying date also changed the birds’ phenotypic plasticity and found early selection line females to initiated egg laying consistently ~9 days earlier compared to late selection line females in outdoor aviaries, but no difference in the degree of plasticity. This suggests that while natural selection may lead to a change in phenotype in the average environment it is unlikely to result in a correlated response on the degree of plasticity in timing of breeding. I also aimed to determine whether individual differences in timing of breeding in females are reflected in differences in their molecular biology and if so where. In Chapter 4 we generated comprehensive RNA expression data from a set of three tissues important in the neuro-endocrine cascade (HPGL axis) underlying avian seasonal timing of breeding, from three different time points and from two temperature treatments and two selection lines for breeding time. Time was the strongest driver in this study, but we found an interesting interaction between time and temperature in hypothalamus, with several genes involved in circadian rhythms differentially expressed. Even though the hypothalamus has been considered the final integration point of environmental cues and guide top down hormonal regulation and in this way direct ovarian function to time breeding, we find evidence for downstream regulation of timing of breeding in Chapter 5. Differences in key reproductive candidate gene expression between phenotypically early and late laying females were found exclusively in the ovary and liver. This also suggests that adaptation in the HPGL axis to changing environments might be downstream. The effects of the environment need to be translated into gene transcription (Chapter 4 and 5), for which DNA methylation is a likely key regulator. Therefore, in Chapter 6, we investigated in great tits whether methylation changes were tissue-specific or tissue-general and whether such methylation changes were associated with expression changes within and between tissues. Overall, we found a positive correlation between changes in DNA methylation in red blood cells and liver, both genome-wide as well as for the sites within the promoter region or transcription start site (TSS) separately. Within the TSS of genes, hyper-methylation over time in red blood cells was highly correlated with a decrease in the expression of the associated gene in the ovary. Tissue-general changes in DNA methylation could potentially be informative for changes in gene expression in inaccessible tissues. I explored the molecular basis of the physiological mechanism underlying seasonal timing of breeding in an avian model species; the great tit. I looked at the phenotype, investigated candidate gene and genome-wide gene expression. In addition, we looked at DNA methylation (in relation to gene expression). The main conclusions are that (1) genomic selection is possible in wild populations, (2) temperature directly influences timing of breeding and (3) that timing of breeding is regulated downstream in the HPLG axis. However, we are only scratching the surface of this complex trait and further studies (also considering other ‘endo-phenotypes’ and their interactions, see Chapter 7) are necessary in order to make predictions about whether birds in general, and great tits specifically, will adapt to rapidly changing environments. </p

The role of social partners in the right of freedom of movement of workers

El material que se presenta responde al trabajo de investigación realizado por los representantes de los siete países participantes en el Seminario Internacional, consistente en analizar el papel que están llamados a desempeñar los agentes sociales en la implementación del derecho a la libertad de circulación de trabajadores en la Unión Europea. El citado análisis se ha realizado desde dos perspectivas: el nivel comunitario y el nacional, siendo este último el que ha permitido una mayor profundización respecto de la situación en que se halla la negociación colecitva y los interlocutores sociales respecto de la libertad de circulación de trabajadores.Asociación Italiana de Derecho del Trabajo y la Seguridad Social Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Rapid changes in DNA methylation associated with the initiation of reproduction in a small songbird

Species with a circannual life cycle need to match the timing of their life history events to the environment to maximize fitness. However, our understanding of how circannual traits such as timing of reproduction are regulated on a molecular level remains limited. Recent studies have implicated that epigenetic mechanisms can be an important part in the processes that regulate circannual traits. Here, we explore the role of DNA methylation in mediating reproductive timing in a seasonally breeding bird species, the great tit (Parus major), using genome-wide DNA methylation data from individual females that were blood sampled repeatedly throughout the breeding season. We demonstrate rapid and directional changes in DNA methylation within the promoter region of several genes, including a key transcription factor (NR5A1) known from earlier studies to be involved in the initiation of timing of reproduction. Interestingly, the observed changes in DNA methylation at NR5A1 identified here are in line with earlier gene expression studies of reproduction in chicken, indicating that the observed shifts in DNA methylation at this gene can have a regulatory role. Our findings provide an important step towards elucidating the genomic mechanism that mediates seasonal timing of a key life history traits and provide support for the idea that epigenetic mechanisms may play an important role in circannual traits.Peer reviewe

Seasonal Variation in Genome-Wide DNA Methylation Patterns and the Onset of Seasonal Timing of Reproduction in Great Tits

In seasonal environments, timing of reproduction is a trait with important fitness consequences, but we know little about the molecular mechanisms that underlie the variation in this trait. Recently, several studies put forward DNA methylation as a mechanism regulating seasonal timing of reproduction in both plants and animals. To understand the involvement of DNA methylation in seasonal timing of reproduction, it is necessary to examine within-individual temporal changes in DNA methylation, but such studies are very rare. Here, we use a temporal sampling approach to examine changes in DNA methylation throughout the breeding season in female great tits (Parus major) that were artificially selected for early timing of breeding. These females were housed in climate-controlled aviaries and subjected to two contrasting temperature treatments. Reduced representation bisulfite sequencing on red blood cell derived DNA showed genome-wide temporal changes in more than 40,000 out of the 522,643 CpG sites examined. Although most of these changes were relatively small (mean within-individual change of 6%), the sites that showed a temporal and treatment-specific response in DNA methylation are candidate sites of interest for future studies trying to understand the link between DNA methylation patterns and timing of reproduction.Peer reviewe

Temporal changes in DNA methylation and RNA expression in a small song bird: within- and between-tissue comparisons

BackgroundDNA methylation is likely a key mechanism regulating changes in gene transcription in traits that show temporal fluctuations in response to environmental conditions. To understand the transcriptional role of DNA methylation we need simultaneous within-individual assessment of methylation changes and gene expression changes over time. Within-individual repeated sampling of tissues, which are essential for trait expression is, however, unfeasible (e.g. specific brain regions, liver and ovary for reproductive timing). Here, we explore to what extend between-individual changes in DNA methylation in a tissue accessible for repeated sampling (red blood cells (RBCs)) reflect such patterns in a tissue unavailable for repeated sampling (liver) and how these DNA methylation patterns are associated with gene expression in such inaccessible tissues (hypothalamus, ovary and liver). For this, 18 great tit (Parus major) females were sacrificed at three time points (n=6 per time point) throughout the pre-laying and egg-laying period and their blood, hypothalamus, ovary and liver were sampled.ResultsWe simultaneously assessed DNA methylation changes (via reduced representation bisulfite sequencing) and changes in gene expression (via RNA-seq and qPCR) over time. In general, we found a positive correlation between changes in CpG site methylation in RBCs and liver across timepoints. For CpG sites in close proximity to the transcription start site, an increase in RBC methylation over time was associated with a decrease in the expression of the associated gene in the ovary. In contrast, no such association with gene expression was found for CpG site methylation within the gene body or the 10kb up- and downstream regions adjacent to the gene body.ConclusionTemporal changes in DNA methylation are largely tissue-general, indicating that changes in RBC methylation can reflect changes in DNA methylation in other, often less accessible, tissues such as the liver in our case. However, associations between temporal changes in DNA methylation with changes in gene expression are mostly tissue- and genomic location-dependent. The observation that temporal changes in DNA methylation within RBCs can relate to changes in gene expression in less accessible tissues is important for a better understanding of how environmental conditions shape traits that temporally change in expression in wild populations.</div

Fine-tuning of seasonal timing of breeding is regulated downstream in the underlying neuro-endocrine system in a small songbird

The timing of breeding is under selection in wild populations as a result of climate change, and understanding the underlying physiological processes mediating this timing provides insight into the potential rate of adaptation. Current knowledge on this variation in physiology is, however, mostly limited to males. We assessed whether individual differences in the timing of breeding in females are reflected in differences in candidate gene expression and, if so, whether these differences occur in the upstream (hypothalamus) or downstream (ovary and liver) parts of the neuroendocrine system. We used 72 female great tits from two generations of lines artificially selected for early and late egg laying, which were housed in climate-controlled aviaries and went through two breeding cycles within 1 year. In the first breeding season we obtained individual egg-laying dates, while in the second breeding season, using the same individuals, we sampled several tissues at three time points based on the timing of the first breeding attempt. For each tissue, mRNA expression levels were measured using qPCR for a set of candidate genes associated with the timing of reproduction and subsequently analysed for differences between generations, time points and individual timing of breeding. We found differences in gene expression between generations in all tissues, with the most pronounced differences in the hypothalamus. Differences between time points, and early- and late-laying females, were found exclusively in the ovary and liver. Altogether, we show that fine-tuning of the seasonal timing of breeding, and thereby the opportunity for adaptation in the neuroendocrine system, is regulated mostly downstream in the neuro-endocrine system.Peer reviewe

Managing Evidence in Food Safety and Nutrition

Evidence ('data') is at the heart of EFSA's 2020 Strategy and is addressed in three of its operational objectives: (1) adopt an open data approach, (2) improve data interoperability to facilitate data exchange, and (3) migrate towards structured scientific data. As the generation and availability of data have increased exponentially in the last decade, potentially providing a much larger evidence base for risk assessments, it is envisaged that the acquisition and management of evidence to support future food safety risk assessments will be a dominant feature of EFSA's future strategy. During the breakout session on 'Managing evidence' of EFSA's third Scientific Conference 'Science, Food, Society', current challenges and future developments were discussed in evidence management applied to food safety risk assessment, accounting for the increased volume of evidence available as well as the increased IT capabilities to access and analyse it. This paper reports on presentations given and discussions held during the session, which were centred around the following three main topics: (1) (big) data availability and (big) data connection, (2) problem formulation and (3) evidence integration. (C) 2019 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority

CGRP and migraine from a cardiovascular point of view: what do we expect from blocking CGRP?

Calcitonin gene-related peptide (CGRP) is a neuropeptide with a pivotal role in the pathophysiology of migraine. Blockade of CGRP is a new therapeutic target for patients with migraine. CGRP and its receptors are distributed not only in the central and peripheral nervous system but also in the cardiovascular system, both in blood vessels and in the heart. We reviewed the current evidence on the role of CGRP in the cardiovascular system in order to understand the possible short- and long-term effect of CGRP blockade with monoclonal antibodies in migraineurs. In physiological conditions, CGRP has important vasodilating effects and is thought to protect organs from ischemia. Despite the aforementioned cardiovascular implication, preventive treatment with CGRP antibodies has shown no relevant cardiovascular side effects. Results from long-term trials and from real life are now needed

Expanding the clinical and genetic spectrum of ALPK3 variants: Phenotypes identified in pediatric cardiomyopathy patients and adults with heterozygous variants

Introduction: Biallelic damaging variants in ALPK3, encoding alpha-protein kinase 3, cause pediatric-onset cardiomyopathy with manifestations that are incompletely defined. Methods and Results: We analyzed clinical manifestations of damaging biallelic ALPK3 variants in 19 pediatric patients, including nine previously published cases. Among these, 11 loss-of-function (LoF) variants, seven compound LoF and deleterious missense variants, and one homozygous deleterious missense variant were identified. Among 18 live-born patients, 8 exhibited neonatal dilated cardiomyopathy (44.4%; 95% CI: 21.5%-69.2%) that subsequently transitioned into ventricular hypertrophy. The majority of patients had extracardiac phenotypes, including contractures, scoliosis, cleft palate, and facial dysmorphisms. We observed no association between variant type or location, disease severity, and/or extracardiac manifestations. Myocardial histopathology showed focal cardiomyocyte hypertrophy, subendocardial fibroelastosis in patients under 4 years of age, and myofibrillar disarray in adults. Rare heterozygous ALPK3 variants were also assessed in adult-onset cardiomyopathy patients. Among 1548 Dutch patients referred for initial genetic analyses, we identified 39 individuals with rare heterozygous ALPK3 variants (2.5%; 95% CI: 1.8%-3.4%), including 26 missense and 10 LoF variants. Among 149 U.S. patients without pathogenic variants in 83 cardiomyopathy-related genes, we identified six missense and nine LoF ALPK3 variants (10.1%; 95% CI: 5.7%-16.1%). LoF ALPK3 variants were increased in comparison to matched controls (Dutch cohort, P = 1.6×10−5; U.S. cohort, P = 2.2×10−13). Conclusion: Biallelic damaging ALPK3 variants cause pediatric cardiomyopathy manifested by DCM transitioning to hypertrophy, often with poor contractile function. Additional extracardiac features occur in most patients, including musculoskeletal abnormalities and cleft palate. Heterozygous LoF ALPK3 variants are enriched in adults with cardiomyopathy and may contribute to their cardiomyopathy. Adults with ALPK3 LoF variants therefore warrant evaluations for cardiomyopathy

Erratum to: Methods for evaluating medical tests and biomarkers

[This corrects the article DOI: 10.1186/s41512-016-0001-y.]