bioRxiv preprint doi: https://doi.org/10.1101/2020.08.22.262493; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Age effects aggressive behavior: RNA-seq analysis in cattle with implications for studying 2 neoteny under domestication 3 Paulina G. Eusebi1, Natalia Sevane1, Thomas O´Rourke2,3, Manuel Pizarro1, Cedric Boeckx2,3,4, 4 Susana Dunner1 5 1Universidad Complutense de Madrid, Avenida Puerta de Hierro, s/n, 28040, Madrid, Spain. 6 2 Universitat de Barcelona, Gran Vía de les Corts Catalanes 585, 08007, Barcelona, Spain. 7 3UBICS, Carrer Martí Franqués 1, 08028 Barcelona, Spain. 8 4ICREA, Passeig Lluís Companys 23, 08010 Barcelona, Spain. 9 Corresponding author. [email protected] 10 Abstract 11 Aggressiveness is one of the most basic behaviors, characterized by targeted intentional actions 12 oriented to cause harm. The reactive type of aggression is regulated mostly by the brain’s 13 prefrontal cortex; however, the molecular changes underlying aggressiveness in adults have not 14 been fully characterized. Here we used an RNA-seq approach to investigate differential gene 15 expression in the prefrontal cortex of bovines from the aggressive Lidia breed at different age 16 stages: young three-year old and adult four-year-old bulls. A total of 50 up and 193 down- 17 regulated genes in the adult group were identified. Furthermore, a cross-species comparative 18 analysis retrieved 29 genes in common with previous studies on aggressive behaviors, 19 representing an above-chance overlap with the differentially expressed genes in adult bulls. 20 Particularly, we detected changes in the regulation of networks such as synaptogenesis, involved 21 in maintenance and refinement of synapses, and the glutamate receptor pathway, which acts as 22 excitatory driver in aggressive responses. Our results provide insights into candidate genes and 23 networks involved in the molecular mechanisms leading to the maturation of the brain. The bioRxiv preprint doi: https://doi.org/10.1101/2020.08.22.262493; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 reduced reactive aggression typical of domestication has been proposed to form part of a 25 retention of juvenile traits as adults (neoteny). The significant age-associated differential 26 expression of genes implicated in aggressive behaviors and concomitant increase in Lidia cattle 27 aggression validates this species as a novel model comparator to explore the impact of behavioral 28 neoteny under domestication. 29 Keywords gene expression, prefrontal cortex, cattle, aggressiveness, behavior genomics 30 31 Introduction 32 Aggression in animals, including humans, serves important purposes in securing mates, territory 33 and food and, therefore, is one of our most basic behaviors. We understand aggression as an 34 overt behavior directed at an object or a subject with an intention to cause harm or damage ( 35 Gannon et al. 2009). This definition encapsulates a heterogeneous and multifaceted construct, 36 including the important distinction between two different subtypes of aggression, reactive and 37 proactive (Smeets et al. 2017). Reactive aggression is the impulsive response to provocations, 38 frustrations or threats from the environment. Proactive aggression, is conscious and goal- 39 directed, planned with a specific outcome in mind (e.g. attainment of resources). While both 40 subtypes can co-occur, different neurobiological structures have been shown to underlie reactive 41 and proactive subtypes (Blair, 2013). 42 Animal studies have shown that reactive aggression is mediated by the limbic system, through a 43 circuit that runs from the amygdala to the hypothalamus, and from there to the periaqueductal 44 grey. This circuitry is regulated by frontal cortical regions, particularly by the prefrontal cortex 45 (PFC) and the anterior cingulate cortex (Blair, 2013). There is strong evidence that reactive 46 aggression often appears earlier in life than proactive aggression, and that heightened levels of bioRxiv preprint doi: https://doi.org/10.1101/2020.08.22.262493; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 47 reactive aggression at early stages are associated with increased levels of anxiety in adulthood. In 48 humans, childhood reactive aggression can be found in different neuropsychiatric that can lead to 49 conduct disorders during adulthood (Fite et al., 2010). Therefore, a comparison of gene 50 expression profiles in PFC tissues at different stages of development may be relevant to reveal 51 some of the molecular characteristics and mechanisms involved in aggressive-related 52 perturbations that characterize early stages of aggression and its progression into adulthood. 53 Research studies across different fields suggest that modern humans have undergone positive 54 selection for reduced reactive aggression relative to our archaic counterparts. Behavioral 55 comparisons with our closest extant ape relatives have shown that instances of reactive 56 aggression in our species are between two and three orders of magnitude lower (Wrangham, 57 2019). Modern humans also have marked reductions in hypothalamic-pituitary-adrenal (HPA) 58 axis activity (which regulates stress responses) compared to other primates (Chrousos et al ., 59 1982). Furthermore, evidence of convergent phenotypical changes in anatomically modern 60 humans and domesticated species strongly suggest that such reductions in aggressive behaviors 61 have continued in our recent evolutionary history, marking an important contributor to the 62 modern—archaic split: domesticated species ubiquitously display reductions in reactive 63 aggression when compared to their wild counterparts, often accompanied by broad phenotypical 64 changes known as the ``domestication syndrome” (reductions in cranial, brain and tooth size, 65 shortening of muzzles, depigmentation, the development of floppy ears, and attenuated HPA 66 activity) (Wilkins et al., 2014). Experimentally controlled selection for reduced reactive 67 aggression in farm-bred foxes brought about many of these broader changes (Dugatkin and Trut, 68 2017). Modern humans exhibit craniofacial changes relative to Neanderthals reminiscent of those 69 in domesticated species, including reductions in tooth size, contraction of the skull (in our case bioRxiv preprint doi: https://doi.org/10.1101/2020.08.22.262493; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 70 giving a more globularized shape), and flattening of the face (Sánchez-Villagra & van Schaik, 71 2019; Theofanopoulou et al., 2017). Genes identified within selective-sweep regions in modern 72 humans have been shown to have above-chance intersection with those implicated in domesticate 73 evolution, including disproportionate targeting glutamate receptor genes associated with 74 aggressive behaviors and implicated in regulation of the HPA axis (O’Rourke & Boeckx, 2020; 75 Theofanopoulou et al., 2017). This evidence for (self-) domestication in ours and domesticated 76 species has long been considered an instance of retained juvenile (neotenic) traits (see e.g. 5). 77 This includes behavioral traits such as diminished reactive aggression. A corollary of this 78 proposal is that Lidia cattle, for which reactive aggression increases with maturity, should have 79 non-juvenile gene expression patterns. 80 The Lidia cattle breed is a promising and natural animal model for studying reactive aggression, 81 given that the breed’s agonistic responses have been exacerbated through long-term selection of 82 aggressive behaviors. In relation to other domesticated cattle breeds, the selection regime that 83 Lidia cattle have undergone is analogous to that practiced in the farm-fox experiment, where 84 distinct fox lineages have been bred for tame versus aggressive behaviors. Although not perhaps 85 the most obvious models of human evolution, genes targeted by selective sweeps in tame cattle 86 (like dogs and unlike cats and horses) show above-chance intersection with genes falling within 87 selective sweep regions in modern humans (Theofanopolou et al., 2017). The Lidia is a primitive 88 breed, raised for centuries with the exclusive purpose of taking part in socio-cultural and 89 anachronistic events grouped under the term of “tauromaquias” (Eusebi et al., 2018). At these 90 festivities, male bovines are placed on a bullring in solitary with a human subject, resulting in a 91 series of aggressive reactions that measures the animal´s fighting capacities by means of reactive 92 responses, such as fighting, chasing and ramming. Different types of tauromaquia take place at bioRxiv preprint doi: https://doi.org/10.1101/2020.08.22.262493; this version posted November 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 93 the bullrings, perhaps the most popular are those known as major festivities: the “corrida” and 94 the “novillada”