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Experimental Competition Induces Immediate and Lasting Effects on the Neurogenome in Free-Living Female Birds

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Experimental Competition Induces Immediate and Lasting Effects on the Neurogenome in Free-Living Female Birds Experimental competition induces immediate and lasting effects on the neurogenome in free-living female birds Alexandra B. Bentza,b,1, Elizabeth M. Georgea,b, Sarah E. Wolfa,b, Douglas B. Ruschc, Ram Podichetic, Aaron Buechleinc, Kenneth P. Nephewd, and Kimberly A. Rosvalla,b aDepartment of Biology, Indiana University, Bloomington, IN 47405; bCenter for the Integrative Study of Animal Behavior, Indiana University, Bloomington, IN 47405; cCenter for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405; and dMedical Sciences Program, Indiana University School of Medicine, Bloomington, IN 47405 Edited by Gene E. Robinson, University of Illinois at Urbana–Champaign, Urbana, IL, and approved February 18, 2021 (received for review July 30, 2020) Periods of social instability can elicit adaptive phenotypic plasticity territorial encounters (9) and may not prompt a full neurogenomic to promote success in future competition. However, the underly- response, particularly in contexts that would otherwise culminate ing molecular mechanisms have primarily been studied in captive in direct physical contact (10). Furthermore, control treatments in and laboratory-reared animals, leaving uncertainty as to how nat- resident–intruder tests are often characterized by the absence of ural competition among free-living animals affects gene activity. interactions, making it unclear if effects are due to agonistic in- Here, we experimentally generated social competition among teractions or are merely a by-product of any social exchange. Thus, wild, cavity-nesting female birds (tree swallows, Tachycineta bi- despite the importance of competitive interactions in social sys- color). After territorial settlement, we reduced the availability of tems, there is uncertainty regarding the genomic response to key breeding resources (i.e., nest boxes), generating heightened competition because prior experimental work may not sufficiently competition; within 24 h we reversed the manipulation, causing ag- mimic natural social competition (11). gressive interactions to subside. We sampled females during the peak Competitive interactions may also have lasting effects if com- of competition and 48 h after it ended, along with date-matched petitive experiences are encoded in the genome. Animals involved controls. We measured transcriptomic and epigenomic responses to in recent territorial disputes tend to behave more aggressively and EVOLUTION competition in two socially relevant brain regions (hypothalamus and have a greater probability of winning future conflicts (i.e., winner ventromedial telencephalon). Gene network analyses suggest that effect) (12). However, little is known about how the accompanying processes related to energy mobilization and aggression (e.g., dopa- neurogenomic responses also change over time, with only a handful mine synthesis) were up-regulated during competition, the latter of of studies examining gene expression at multiple time points after a which persisted 2 d after competition had ended. Cellular mainte- competitive experience, all sampled within a couple of hours (13, nance processes were also down-regulated after competition. Com- 14). This has created a gap in our understanding of how social petition additionally altered methylation patterns, particularly in experiences generate long-lasting effects on behavioral and physi- pathways related to hormonal signaling, suggesting those genes ological processes (i.e., biological embedding) (15). Epigenetic were transcriptionally poised to respond to future competition. Thus, mechanisms, such as DNA methylation, are prime candidates to experimental competition among free-living animals shifts gene ex- mediate the lasting effects of the social environment on gene ac- pression in ways that may facilitate the demands of competition at tivity (16, 17). To date, methylation has been shown to respond to the expense of self-maintenance. Further, some of these effects per- sisted after competition ended, demonstrating the potential for epi- genetic biological embedding of the social environment in ways that Significance may prime individuals for success in future social instability. Social challenges are thought to be embedded in the genome, but gene network | MethylCap-seq | RNA-seq | dopamine | epigenetic little is known about how this adaptive modification of gene ac- tivity occurs in nature. We experimentally generated competition among free-living female tree swallows (Tachycineta bicolor)and ompetition is ubiquitous among social animals, and an indi- measured both transcriptomic and epigenomic changes in the vidual’s behavioral and physiological response to competition C brain. Social competition rapidly altered gene expression and can greatly impact fitness. For decades, research in this area was methylation, with effects that were enriched for hormone- and guided by hypotheses formed by studying males and was focused on neurotransmitter-related processes also being found two full days steroid hormones (i.e., challenge hypothesis) (1); however, recent later. These persistent effects on gene activity were correlated analyses indicate that androgen modulation is not necessarily a with epigenetic patterns as well as the degree of aggression ex- universal response to social competition (2–4). As more genome- perienced during the peak of competition. Thus, a single day of wide studies are performed, it has become clear that responses to competition in the wild is encoded in the brain, both during territorial aggression are mediated by changes in hundreds of genes competition and days later. in the brain (5–7), and yet, at least in vertebrates, this evidence largely comes from captive and laboratory-reared animals. Thus, Author contributions: A.B.B., E.M.G., S.E.W., K.P.N., and K.A.R. designed research; A.B.B., whether the key aggression-related pathways discovered in this E.M.G., S.E.W., and K.A.R. performed research; A.B.B., D.B.R., R.P., and A.B. analyzed data; past work are relevant to animals responding to more naturalistic A.B.B. and K.A.R. wrote the paper; and E.M.G., S.E.W., D.B.R., R.P., A.B., and K.P.N. pro- competition in the wild is unknown. This issue is especially im- vided manuscript feedback. portant because competition in nature unfolds as a complex series The authors declare no competing interest. of dynamic social interactions, often among multiple individuals in This article is a PNAS Direct Submission. nondyadic, brief interactions. To date, we are aware of only one Published under the PNAS license. experiment examining genome-wide neural responses to territorial 1To whom correspondence may be addressed. Email: [email protected]. intrusions in a free-living animal; however, they used simulated This article contains supporting information online at https://www.pnas.org/lookup/suppl/ intrusions involving an immobile intruder (8). There is evidence doi:10.1073/pnas.2016154118/-/DCSupplemental. that resident–intruder paradigms may insufficiently mimic natural Published March 22, 2021. PNAS 2021 Vol. 118 No. 13 e2016154118 https://doi.org/10.1073/pnas.2016154118 | 1of9 Downloaded by guest on September 29, 2021 territorial intrusions on the scale of hours (18); however, how Results competition-induced changes in methylation prime the genome Behavioral and Hormonal Responses to Competition. Fig. 1 summa- over longer time periods has yet to be tested, much less in a free- rizes the experimental design, which takes advantage of these living animal. This lends uncertainty as to whether and how natu- birds’ tendency to defend the area ∼20 m around their nest box, ralistic competition is biologically embedded to bring about lasting including additional boxes (23). Our goal was to induce compe- phenotypic changes. tition, concentrated near “focal” females, who were our primary Here, we experimentally generated a period of heightened ter- study subjects. To achieve this, we removed half of the occupied ritorial competition to explore the immediate and longer-lasting nest boxes along with all unoccupied boxes at multiple experi- effects on gene activity in the female brain. We used free-living mental sites, thereby guaranteeing a minimum number of com- tree swallows (Tachycineta bicolor), which are ideal for studying petitors (i.e., displaced females) in addition to any floater females ecologically relevant female–female competition. These songbirds also present. We erected new boxes to accommodate only half of the displaced females, placing them 20 m away from the un- are obligate secondary cavity nesters, meaning that a female’sre- touched boxes of focal territory holders. This positioning was close productive success is dependent on her ability to acquire a cavity enough to facilitate interactions between focal females and com- that she cannot excavate for herself (19). Nesting cavities are often petitors vying for the nearby new boxes. Less than 24 h after the limited, females must aggressively compete to obtain one, and fe- manipulation, we returned the removed boxes to their former male aggression is adaptive during territory establishment (20). positions, effectively reversing the experiment. Each experimental Furthermore, nonterritory-holding female “floaters” intrude at and unmanipulated control site was separated by >300 m, a dis- territories throughout the early breeding season, and they can evict tance that exceeds daily home ranges in this system (24). We or even kill other females (21), demonstrating that competition performed daily behavioral observations
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