Epigenetics and Early Life Stress : Experimental Brood Size Affects DNA Methylation in Great Tits (Parus major)

Early developmental conditions are known to have life-long effects on an individual's behavior, physiology and fitness. In altricial birds, a majority of these conditions, such as the number of siblings and the amount of food provisioned, are controlled by the parents. This opens up the potential for parents to adjust the behavior and physiology of their offspring according to local post-natal circumstances. However, the mechanisms underlying such intergenerational regulation remain largely unknown. A mechanism often proposed to possibly explain how parental effects mediate consistent phenotypic change is DNA methylation. To investigate whether early life effects on offspring phenotypes are mediated by DNA methylation, we cross-fostered great tit (Parus major) nestlings and manipulated their brood size in a natural study population. We assessed genome-wide DNA methylation levels of CpG sites in erythrocyte DNA, using Reduced Representation Bisulfite Sequencing (RRBS). By comparing DNA methylation levels between biological siblings raised in enlarged and reduced broods and between biological siblings of control broods, we assessed which CpG sites were differentially methylated due to brood size. We found 32 differentially methylated sites (DMS) between siblings from enlarged and reduced broods, a larger number than in the comparison between siblings from control broods. A considerable number of these DMS were located in or near genes involved in development, growth, metabolism, behavior and cognition. Since the biological functions of these genes line up with previously found effects of brood size and food availability, it is likely that the nestlings in the enlarged broods suffered from nutritional stress. We therefore conclude that early life stress might directly affect epigenetic regulation of genes related to early life conditions. Future studies should link such experimentally induced DNA methylation changes to expression of phenotypic traits and assess whether these effects affect parental fitness to determine if such changes are also adaptive.Peer reviewe

An ecologist's guide for studying DNA methylation variation in wild vertebrates

The field of molecular biology is advancing fast with new powerful technologies, sequencing methods and analysis software being developed constantly. Commonly used tools originally developed for research on humans and model species are now regularly used in ecological and evolutionary research. There is also a growing interest in the causes and consequences of epigenetic variation in natural populations. Studying ecological epigenetics is currently challenging, especially for vertebrate systems, because of the required technical expertise, complications with analyses and interpretation, and limitations in acquiring sufficiently high sample sizes. Importantly, neglecting the limitations of the experimental setup, technology and analyses may affect the reliability and reproducibility, and the extent to which unbiased conclusions can be drawn from these studies. Here, we provide a practical guide for researchers aiming to study DNA methylation variation in wild vertebrates. We review the technical aspects of epigenetic research, concentrating on DNA methylation using bisulfite sequencing, discuss the limitations and possible pitfalls, and how to overcome them through rigid and reproducible data analysis. This review provides a solid foundation for the proper design of epigenetic studies, a clear roadmap on the best practices for correct data analysis and a realistic view on the limitations for studying ecological epigenetics in vertebrates. This review will help researchers studying the ecological and evolutionary implications of epigenetic variation in wild populations

Avian ecological epigenetics : pitfalls and promises

Epigenetic mechanisms can alter gene expression without a change in the nucleotide sequence and are increasingly recognized as important mechanisms that can generate phenotypic diversity. Most of our current knowledge regarding the origin and role of epigenetic variation comes from research on plants or mammals, often in controlled rearing conditions. Epigenetic research on birds in their natural habitats is still in its infancy, but is needed to answer questions regarding the origin of epigenetic marks and their role in phenotypic variation and evolution. Here we review the potential for studying epigenetic variation in natural bird systems. We aim to provide insights into (1) the origin of epigenetic variation, (2) the relationship between epigenetic variation and trait variation, and (3) the possible role of epigenetic variation in adaptation to changing environments. As there is currently little research on epigenetics in wild birds, we examine how findings on other taxa such as plants and mammals relate to birds. We also examine some of the pros and cons of the most commonly used methods to study patterns of DNA methylation in birds, and suggest some topics we believe need to be addressed to develop the field of wild avian epigenetics further.Peer reviewe

Early developmental carry-over effects on exploratory behaviour and DNA methylation in wild great tits (Parus major)

Sepers B, Verhoeven KJF, van Oers K. Early developmental carry-over effects on exploratory behaviour and DNA methylation in wild great tits (Parus major). Evolutionary Applications . 2024;17(3): e13664.Adverse, postnatal conditions experienced during development are known to induce lingering effects on morphology, behaviour, reproduction and survival. Despite the importance of early developmental stress for shaping the adult phenotype, it is largely unknown which molecular mechanisms allow for the induction and maintenance of such phenotypic effects once the early environmental conditions are released. Here we aimed to investigate whether lasting early developmental phenotypic changes are associated with post-developmental DNA methylation changes. We used a cross-foster and brood size experiment in great tit (Parus major) nestlings, which induced post-fledging effects on biometric measures and exploratory behaviour, a validated personality trait. We investigated whether these post-fledging effects are associated with DNA methylation levels of CpG sites in erythrocyte DNA. Individuals raised in enlarged broods caught up on their developmental delay after reaching independence and became more explorative as days since fledging passed, while the exploratory scores of individuals that were raised in reduced broods remained stable. Although we previously found that brood enlargement hardly affected the pre-fledging methylation levels, we found 420 CpG sites that were differentially methylated between fledged individuals that were raised in small versus large sized broods. A considerable number of the affected CpG sites were located in or near genes involved in metabolism, growth, behaviour and cognition. Since the biological functions of these genes line up with the observed post-fledging phenotypic effects of brood size, our results suggest that DNA methylation provides organisms the opportunity to modulate their condition once the environmental conditions allow it. In conclusion, this study shows that nutritional stress imposed by enlarged brood size during early development associates with variation in DNA methylation later in life. We propose that treatment-associated DNA methylation differences may arise in relation to pre- or post-fledging phenotypic changes, rather than that they are directly induced by the environment during early development. © 2024 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd

The epigenetics of animal personality

van Oers K, van den Heuvel K, Sepers B. The epigenetics of animal personality. Neuroscience & Biobehavioral Reviews. 2023;150: 105194.Animal personality, consistent individual differences in behaviour, is an important concept for understanding how individuals vary in how they cope with environmental challenges. In order to understand the evolutionary significance of animal personality, it is crucial to understand the underlying regulatory mechanisms. Epigenetic marks such as DNA methylation are hypothesised to play a major role in explaining variation in phenotypic changes in response to environmental alterations. Several characteristics of DNA methylation also align well with the concept of animal personality. In this review paper, we summarise the current literature on the role that molecular epigenetic mechanisms may have in explaining personality variation. We elaborate on the potential for epigenetic mechanisms to explain behavioural variation, behavioural development and temporal consistency in behaviour. We then suggest future routes for this emerging field and point to potential pitfalls that may be encountered. We conclude that a more inclusive approach is needed for studying the epigenetics of animal personality and that epigenetic mechanisms cannot be studied without considering the genetic background

The epigenetics of animal personality

Animal personality, consistent individual differences in behaviour, is an important concept for understanding how individuals vary in how they cope with environmental challenges. In order to understand the evolutionary significance of animal personality, it is crucial to understand the underlying regulatory mechanisms. Epigenetic marks such as DNA methylation are hypothesised to play a major role in explaining variation in phenotypic changes in response to environmental alterations. Several characteristics of DNA methylation also align well with the concept of animal personality. In this review paper, we summarise the current literature on the role that molecular epigenetic mechanisms may have in explaining personality variation. We elaborate on the potential for epigenetic mechanisms to explain behavioural variation, behavioural development and temporal consistency in behaviour. We then suggest future routes for this emerging field and point to potential pitfalls that may be encountered. We conclude that a more inclusive approach is needed for studying the epigenetics of animal personality and that epigenetic mechanisms cannot be studied without considering the genetic background

Winter food selection and exploratory behavior vary with natal territory characteristics in wild great tits

Serrano-Davies E, Bircher N, Sepers B, van Oers K. Winter food selection and exploratory behavior vary with natal territory characteristics in wild great tits. Behavioral Ecology and Sociobiology. 2023;77(6): 69.Differences in habitat characteristics experienced during rearing associate with variation in a range of behavioral phenotypes such as exploratory behavior, foraging behavior and food selection. The habitat-dependent selection hypothesis predicts that animals develop behavioral characteristics fitted to their rearing environment. Yet, little is known about how habitat characteristics during rearing shape how animals face winter conditions and adjust their winter foraging behavior. The aim of this study was to explore how fine-scale rearing habitat characteristics associate with exploratory behavior, food selection, and foraging performance during winter. For this, we measured habitat characteristics during the breeding season in territories of wild great tits (Parus major) and tested first-year juvenile birds that fledged from these territories for exploratory and foraging behavior at feeders during winter. We found evidence that faster explorers were raised in territories with lower quality habitat characteristics. In addition, fast exploring fledglings visited the feeders significantly more (total visits). Moreover, the rearing environment, via caterpillar availability and tree species composition, determined diet selection during winter in first-year birds. These results show support for the habitat-dependent selection hypothesis, since exploratory behavior as well as food selection during winter associate with habitat features of the rearing territories during development. This pattern can be caused either by the kinds of natural foods prevalent during rearing at these sites or because of intrinsic individual differences. Further experiments are needed to disentangle these two. **Significance statement** Individuals vary in how they behaviorally adapt foraging and food selection strategies to the environmental conditions. A number of studies have shown that animals develop behavioral characteristics fitted to their rearing environment. However, how habitat characteristics during rearing shape the foraging strategy that animals use to face winter conditions is still unknown. We studied these links in yearling great tits using automated feeders that recorded their visits during winter. Fledglings with a higher exploratory score were born in territories with lower quality habitat characteristics and visited the feeders more. Furthermore, we found an association between caterpillar availability and tree species composition in the rearing territory of juveniles and their subsequent food selection in winter. Our study indicates that certain environmental conditions might favor the development of particular behaviors in birds and that early nutrition could shape food choice later in life

An ecologist’s guide for studying DNA methylation variation in wild vertebrates

The field of molecular biology is advancing fast with new powerful technologies, sequencing methods and analysis software being developed constantly. Commonly used tools originally developed for research on humans and model species are now regularly used in ecological and evolutionary research. There is also a growing interest in the causes and consequences of epigenetic variation in natural populations. Studying ecological epigenetics is currently challenging, especially for vertebrate systems, because of the required technical expertise, complications with analyses and interpretation, and limitations in acquiring sufficiently high sample sizes. Importantly, neglecting the limitations of the experimental setup, technology and analyses may affect the reliability and reproducibility, and the extent to which unbiased conclusions can be drawn from these studies. Here, we provide a practical guide for researchers aiming to study DNA methylation variation in wild vertebrates. We review the technical aspects of epigenetic research, concentrating on DNA methylation using bisulfite sequencing, discuss the limitations and possible pitfalls and how to overcome them through rigid and reproducible data analysis. This review provides a solid foundation for the proper design of epigenetic studies, a clear roadmap on the best practices for correct data analysis and a realistic view on the limitations for studying ecological epigenetics in vertebrates. This review will help researchers studying the ecological and evolutionary implications of epigenetic variation in wild populations.peerReviewe