Repetitive aggressive encounters generate a long-lasting internal state in Drosophila melanogaster males

Abstract

Multiple studies have investigated the mechanisms of aggressive behavior in Drosophila; however, little is known about the effects of chronic fighting experience. Here, we investigated if repeated fighting encounters would induce an internal state that could affect the expression of subsequent behavior. We trained wild-type males to become winners or losers by repeatedly pairing them with hypoaggressive or hyperaggressive opponents, respectively. As described previously, we observed that chronic losers tend to lose subsequent fights, while chronic winners tend to win them. Olfactory conditioning experiments showed that winning is perceived as rewarding, while losing is perceived as aversive. Moreover, the effect of chronic fighting experience generalized to other behaviors, such as gap-crossing and courtship. We propose that in response to repeatedly winning or losing aggressive encounters, male flies form an internal state that displays persistence and generalization; fight outcomes can also have positive or negative valence. Furthermore, we show that the activities of the PPL1-γ1pedc dopaminergic neuron and the MBON-γ1pedc>α/β mushroom body output neuron are required for aversion to an olfactory cue associated with losing fights.

Keywords: Drosophila; aggression; generalization; persistence; valence.

Fig. 1.

Generation of a persistent winner and loser effect. (A) A single SH CS male was paired with a single opponent fly (hypoaggressive or hyperaggressive male) in three 30-min sessions, with a 1 h of rest in between sessions. Unfamiliar opponents were used in each fighting encounter. The winner or loser effect was tested 24, 48, or 72 h afterward. (B) Proportion of lunges received or executed during the last 5 min of training sessions of winners (n = 851) or losers (n = 897). A small proportion of fights in each group showed no dominance (tied). These males were used for all experiments described in this paper. (C) Trained flies were individually paired with naive SH CS males 24, 48, or 72 h after the last training session and the proportion of lunges executed or received quantified. The winner effect persisted for at least 72 h (one sample t test against 50%; n_winners = 19, 25, 35 at each respective time point; 24 h: **P < 0.01; 48 h: *P < 0.05; 72 h: **P < 0.01), while the loser effect persisted for 48 h (one sample t test against 50%; n_losers = 34, 37, 35 at each respective time point; 24 h: ***P < 0.001; 48 h: *P < 0.05; 72 h: P > 0.05). On average, the proportion of executed and received lunges for two naive SH CS males (left bar) was near 50% during the test period (one-sample t test, P > 0.05).

Fig. 2.

Valence of the winning and losing. (A) Naive SH CS males were trained to associate a particular fighting experience with an olfactory cue in three sessions, each consisting of a 30-min exposure to odor 1 (without opponent) followed by a 30-min exposure to odor 2 paired with the presence of an opponent fly: hypoaggressive male opponents were used to generate winners and hyperaggressive opponents were used to generate losers. New opponents were used in each training session. Flies were given a 1-h rest between sessions, and the memory tested 24 h after the end of training. Control flies were exposed to the two olfactory cues in the absence of opponents. (B) Compared with control flies (n = 17 experiments with groups of 24 males each), winners showed a conditioned preference (n = 17; ***P < 0.001), whereas losers showed a conditioned aversion (n = 16; ***P < 0.001) for the odor previously associated with the respective fighting experience. One-way ANOVA followed by Tukey’s multiple comparisons test.

Fig. 3.

Generalization of the loser and winner effect. Testing loser and winner flies in three different behavioral paradigms showed that the effect of persistently losing or wining fights extended to other behaviors. All behaviors were assayed 24 h after training. (A) Gap crossing. Winners and losers showed increased and decreased success, respectively, in crossing a 3-mm gap compared with naive CS males. Steel–Dwass multiple comparison test after nonparametric ANOVA, *P < 0.05, ****P < 0.0001; n_loser = 34, n_winner = 20, n_naive = 31. (B) Courtship. Losers exhibited significantly prolonged courtship latency compared with naive and winner flies, when paired with wild-type virgin females. Tukey’s multiple comparisons test after one-way ANOVA: *P < 0.05; **P < 0.01, n = 33 for all groups. (C) Object exploration. A greater number of flies with repeat winning experience (blue; n = 35) had moved toward and climbed the post by 76 s into the observations (green vertical line; t₅₀, based on concurrently run naive SH CS males, gray; n = 28), than those that had chronically lost (red; n = 43), χ², P = 0.0026. Prior fighting experience appeared to affect the exploration of males in comparison with naive males; however, the trends failed to be statistically significant: At t₅₀ (the time at which 50% of flies had reached the post), 63% of flies with repeat winning experience had found the post (winners vs. naive; χ², P = 0.2863) and yet only 29% of the chronic losers (losers vs. naive; χ², P = 0.0818). ns, nonsignificant.

Fig. 4.

The activities of PPL1- γ1pedc dopamine neuron and MBON-γ1pedc>α/β mushroom body output neurons are required for the memory of a cue associated with losing. (A) When trained at the restrictive temperature (30 °C), but not the permissive temperature (20 °C), MB438B-GAL4 > shi^ts (PPL1-γ1pedc blocked) showed a reduced conditioned aversion for an olfactory cue associated with the experience of losing. *P < 0.05, **P < 0.01; n = 12–16. (B) Similar results were obtained with MB112C-GAL4 > shi^ts1 (MBON-γ1pedc>α/β blocked). *P < 0.05; n = 20–21. One-way ANOVA was followed by the Tukey’s multiple comparisons test. Tests were carried out 24 h after training. ns, nonsignificant.