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This study tested the efficacy of mirror trials in studying aggressiveness in the matrinxã fish, Brycon amazonicus. The hypothesis was that a mirror would elicit an aggressive response in B. amazonicus juveniles, but show different behavioral and physiological profiles than those observed in trials with real opponents. Fish were tested using either a mirror trial (n = 7) or a real trial (n = 7), that is, placed alone with a mirror or paired with a same-sized opponent, respectively. All trials lasted for 20 min and took place in 96-L aquaria with water temperatures of 28°C. Fish in mirror trials exhibited less locomotion (mirror: 423.3 ± 39.1; real trial: 735.1 ± 31.9; p < .001), rushed less against the opponent (mirror: 3.1 ± 2.3; real trial: 39.6 ± 5.1; p < .001) and showed less escalation of aggressive behavior compared to the winner of a real trial. Regarding the physiological parameters, despite similar cortisol responses (mirror: 84.2 ± 13.8 ng/ml; real trial: 77.2 ± 16.9 ng/ml; p = .757), the fish that fought against a mirror had lower levels of plasma glucose than those of fish that fought against a real opponent (mirror: 76.8 ± 3.6 ng/dl; real trial: 103.8 ± 10.1 ng/ml; p = .037). This lower energy mobilization might be due to a lower cost of the fight in the absence of an opponent that flees from attacks, as the locomotion and number of rush totals were also lower in the mirror trials. This pattern of less locomotion is probably common in mirror trials because the opponent is always limited to one side of the arena. If the energetic cost is one of the factors that can determine the outcome of a fight, and if mirror trials do not evoke the physiological response observed in a real fight, the use of mirror trials must be reviewed in studies that consider the physiological responses.

Stress is an energy-demanding process, as well as the responses of the innate immune system, that impose a metabolic overload on cellular energy production, which can affect the cellular redox balance, causing oxidative damage. We evaluated the role of stress in the modulation of innate immune and oxidative/antioxidant mechanisms in juvenile pacu exposed to acute and chronic stressors. The experimental period lasted 30 days, and fish (113.7 ± 35.1 g) were fed commercial feed. During this period, half of the fish were not manipulated (Condition A), and the other half were chased with a dip net for 5 min twice a day (Condition C). After the 30-day period, fish from both groups were sampled (baseline sampling), and the remainders (not sampled) were air exposed for 3 min (acute stressor), returned to the tanks, and were sampled again 30 min, 3 h, 6 h, and 24 h after air exposure. We evaluated biomarkers of stress (circulating cortisol and glucose), the innate immune system (respiratory burst activity/RBA, hemolytic activity of the complement system (HA-AP) and serum concentration of lysozyme), oxidative damage (lipid peroxidation/LPO), and antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GSH-Px). Our results showed that stress, acutely or chronically, caused a transient reduction of RAL and activated the HA-AP. Acutely, stress increased the lysozyme concentration. Furthermore, both conditions caused oxidative stress in the liver, and differently they modulated the antioxidant system, enhancing SOD activity and impairing CAT and GSH-Px activity.