Does losing reduce the tendency to engage with rivals to reach mates? An experimental test

Male-male contests for access to females or breeding resources is critical in determining male reproductive success. Larger males and those with more effective weaponry are more likely to win fights. However, even after controlling for such predictors of fighting ability, studies have reported a winner-loser effect: previous winners are more likely to win subsequent contests, while losers often suffer repeated defeats. While the effect of winning-losing is well-documented for the outcome of future fights, its effect on other behaviors (e.g., mating) remains poorly investigated. Here, we test whether a winning versus losing experience influenced subsequent behaviors of male mosquitofish (Gambusia holbrooki) towards rivals and potential mates. We housed focal males with either a smaller or larger opponent for 24 hours to manipulate their fighting experience to become winners or losers, respectively. The focal males then underwent tests that required them to enter and swim through a narrow corridor to reach females, bypassing a cylinder that contained either a larger rival male (competitive scenario), a juvenile or was empty (non-competitive scenarios). The tests were repeated after one week. Winners were more likely to leave the start area and to reach the females, but only when a larger rival was presented, indicating higher levels of risk-taking behavior in aggressive interactions. This winner-loser effect persisted for at least one week. We suggest that male mosquitofish adjust their assessment of their own and/or their rival’s fighting ability following contests in ways whose detection by researchers depends on the social context.

Methods
Here, we examined whether winners/losers changed their behavior with the time that had elapsed since their previous contest experience. To do this, each male was tested twice in encounter tests: once immediately after the contest period ended (Test I), and then again one week later (Test II). To control for any potential effects of time and/or familiarity with the aquarium setup in Test II (Fig. 2), we ran an additional pair of encounter tests with another set of males who did not initially experience staged contests. After their first encounter test, focal males were isolated in individual 1L tanks for a week. Then they experienced contests for 24 hours (as above), after which we recorded these males’ behavior during their second encounter test (Test III).

The encounter test contained a start area at one end which was connected by a corridor to a reward area at the opposite end. The focal male was introduced into a mesh cylinder in the start area. Four large females were housed in the reward area. There was a transparent plastic cylinder at the center of the corridor that housed either a rival male, a juvenile, or was left empty. We recorded for each focal male: (i) initiation time: the time taken to enter the corridor; (ii) encounter time: the time spent < 3 cm (equivalent to 1 SL of the male) from the cylinder in the corridor; and (iii) total trial time: the time taken from release until reaching the reward area (see Supplementary Material for details). The trial concluded when the male entered the reward area, or when 10 minutes had elapsed.

A two-step analysis was conducted for each of the recorded behaviors. First, we examined the likelihood that the male: (1) left the start area, (2) entered the encounter area, and (3) entered the reward area. We ran separate generalized linear mixed models (GLMMs) with binomial error (glmmTMB package). Second, for those males who exhibited the above behaviors, we ran individual GLMMs (with Gaussian error) to analyze the time spent on each behavior. We log-transformed the time data to fulfil the model assumptions. Contest outcome (winner, loser), timing relative to contest/trial experience (Test I, Test II, Test III) and encounter type (male rival, juvenile, empty cylinder) were treated as fixed factors, and their three-way and two-way interactions were included in the initial model. Male identity and size-matched winner/loser pair’s identity was treated as a random factor to control for repeated measures from the same male. If there was a significant effect of contest outcome for a given response variable, we further compared data from Test II (second test, one week after contest experience) to Test III (second test, immediately after contest experience) using the same approach to test for the persistence of the observed winner/loser effect.