Aggression is an adaptive behavior used to establish dominance hierarchies, compete for resources, protect offspring, and as a form of defense (Koolhaas and Bohus, 2003). It can be observed between conspecifics (as defined by Aronson and Tinbergen, 1953; and Lorenz, 1967), but also as a form of heterospecific interactions when competing for resources, especially when there is an overlap in the ecological niche (Grether et al., 2013). Studies usually focus on the ethological significance of aggressive behavior, its phylogenetic and ontogenetic development, or, in the context of human and veterinary medicine studies, understanding and controlling pathological aggression. However, a clear picture of the mechanisms underlying variation in aggressive behavior, within and across species, is still lacking (reviewed by Way et al., 2015). This is particularly true for non-mammalian species, including fish, despite the diversity of aggressive behaviors that can be found across the >30,000 described fish species (Froese and Pauly, 2024).

Aggressive behavior can be modulated by neurotransmitters, such as dopamine, 5-HT, histamine, nitric oxide, and by hormones from the hypothalamic-neurohypophysial system (HNS), hypothalamic-pituitary-interrenal axis (HPI) and hypothalamic-pituitary-gonadal axis (HPG) (Filby et al., 2010). The nonapeptides oxytocin (OXT) and vasotocin (VT) have also been implicated in the modulation of aggression (Kelly and Wilson, 2020). They are highly conserved across taxa and their evolution is tightly linked, with their genes located in the same chromosome, with evidence supporting the hypothesis that they are adjacent paralogous genes resulting from a local duplication (Theofanopoulou et al., 2021). The fish homologues of the mammalian OXT and VT have been referred to in the literature as isotocin (IT) and arginine vasotocin (AVT), respectively (Wircer et al., 2016). However, because their sequence differs only in two (OXT/IT) and one (VT/AVT) amino acid, a universal nomenclature for the ligands and receptors of this family has been proposed (Theofanopoulou et al., 2021) and here the terms oxytocin (OXT) and vasotocin (VT) are used. The two nonapeptides also share similar functions such as the regulation of reproductive behavior, stress response, metabolism, circadian and seasonal rhythms, cardiovascular system, and osmoregulation (Balment et al., 2006; Jurek and Neumann, 2018). They are also involved in the modulation of aggression, but their specific role and mechanisms of actions are still to be determined, with contradictory results reported in the literature. Generally, in mammals, VT is linked to an increase in the expression of aggression (Albers, 2012). A similar effect has been reported for some fish species, (e.g., non-territorial phase males of bluehead wrasse Thalassoma bifasciatum, Semsar et al., 2001; males of the beaugregory damselfish Stegastes leucostictus, Santangelo and Bass, 2006; zebrafish Danio rerio, Teles and Oliveira, 2016) although the opposite effect (e.g., territorial phase males of bluehead wrasse, Semsar et al., 2001; males of the brown ghost knifefish Apterootus leptorhynchus, Bastian et al., 2001; males and females of zebrafish, Filby et al., 2010) has also been described. VT has also been associated with social status and territorial behavior, for example: in the peacock blenny Salaria pavo, the cell size is higher in the parvocellular nuclei in parasitic sneaker males relative to nest-holder males and females (Grober et al., 2002); in the rock-pool blenny Parablennius parvicornis, the cell number is higher in smaller non-nesting male than in larger nesting males, if corrected for body mass (Miranda et al., 2003); in the masu salmon Oncorhynchus masou, the intensity of hybridization signal of VT in male breeders is lower in the parvocellular population and higher in the gigantocellular population, while no differences were detected in the magnocellular population (Ota et al., 1999); and, in zebrafish the VT cell number in dominant males is lower in the parvocellular population, and higher in the magnocellular and gigantocellular population (Larson et al., 2006). Thus, differences in the size and number of VT cells across brain areas appear to be linked to specific reproductive or dominance phenotypes. Hence, VT expression can correlate with alternative phenotypes (Greenwood et al., 2008) and have a role in shaping dominant-subordinate relationships (da Silva et al., 2021).

Less information is available on the role of OXT on aggression. In mammals, OXT seems to be associated with maternal aggression while in males its role is still ambiguous (reviewed in Lee et al., 2009). In fish, contradictory evidence on the relationship between OXT and aggression has also been reported. For example, OXT administration to the preoptic area/anterior hypothalamus (POA-AH) induces fictive aggressive vocalization in parasitic sneaker males, but not in territorial males, of the plainfin midshipman Porichthys notatus (Goodson and Bass, 2000); while in other species OXT administration does not produce any effects in aggressive displays (e.g., males of the beaugregory damselfish, Santangelo and Bass, 2006, and in males and females of the cichlid Neolamprologus pulcher, Reddon et al., 2012). Depending on the dominance and reproductive status, distinct OXT and VT expression patterns have been observed (e.g., in the Mozambique tilapia Oreochromis mossambicus, Almeida et al., 2012; in the three-spined stickleback Gasterosteus aculeatus, Kleszczyńska et al., 2012; in the zebrafish, Teles et al., 2016; and in the round goby Neogobius melanostomus, Sokołowska et al., 2020).

OXT and VT also have a reproductive role that is not only limited to the modulation of sexual and courtship behavior but can also act on steroidogenesis (VT) and in the regulation of the female reproductive cycle (OXT) (Gonçalves et al., 2024). Therefore, a connection between these two nonapeptides and sex steroids is plausible, especially because sex hormone receptors have been observed in different brain areas, including the preoptic area where OXT and VT neurons are located. Additionally, the presence of the VT receptor in Leydig cells in the testes, the primary site of androgen production, has been observed in two different fish species: the catfish Heteropneustes fossilis (Rawat et al., 2019) and bluehead wrasse (Lema et al., 2012). However, most studies aiming to understand the crosstalk between these two pathways have usually focused on how sex steroids modulate the nonapeptide systems (for a review see Mennigen et al., 2022) rather than exploring the reverse interaction.

The Siamese fighting fish Betta splendens has been emerging as a model species for the study of non-mammalian aggression because of its high levels of male aggression and stereotypical and conspicuous aggressive displays. Individuals of this species are solitary, with males establishing and vigorously defending a territory from other males and building a bubble nest from which they attract females for mating (Jaroensutasinee and Jaroensutansinee, 2001). The male-male aggression interactions in this species are well-defined, starting with threat displays that include the distension of fins, the opening of the opercula, and caudal swings toward the opponent, followed by attack behavior with bites and charges if the opponent is not deterred (Ramos et al., 2021; Simpson, 1968; Vu et al., 2020). The aggressiveness of this species, which is endemic to parts of Southeast Asia, has been recognized for centuries by locals, who began using males in staged fights as a national pastime (Smith, 1945). Winner lines are bred while loser lines are discarded, leading to the establishment of “fighter lines”, morphologically different and more aggressive than wild-type fish (Ramos and Gonçalves, 2019; Verbeek et al., 2007). The selection for winners also seems to have modulated the response of the endocrine system during aggression, with fighters showing a post-fight increase in plasma KT levels but not in cortisol (F), while wild-type fish show an increase in both hormones (Ramos and Gonçalves, 2022). The modulation of aggression in this species by VT and OXT is not yet clear, with only one study showing a negative effect of OXT administration on aggression (Oliveira et al., 2022), while no studies have been published on VT. On the other hand, a robust post-fight androgen response after an aggressive challenge has been found in this species independent of the type of stimulus – mirror, live conspecifics, or video playback – with androgen levels showing no correlation with the duration nor the frequency of aggressive behavior (Alex et al., 2023; Ramos et al., 2021; Ramos and Gonçalves, 2022).

Here, we tested the potential modulation of VT and OXT on the aggressive and endocrine responses elicited by the presence of a mirror in male B. splendens through intraperitoneal injection of VT and OXT antagonists. The aim was to contribute to the still poorly understood role of these neuropeptides in the modulation of aggressive behavior and their possible interaction with the described androgen response to an aggression challenge in B. splendens.