IntroductionOrganisms often use explicit signals in an attempt to compete for resources that are limiting, such as territory and mating opportunities, and to demonstrate dominance during a confrontation. Aggression elicited in both a conspecific and hetero-specific manner has the important adaptive function of increasing survival and reproduction prospects for the individual (Lynn et al., 2007). Intraspecific aggression is often ‘ritualised’ to prevent harm to either of the participants unless necessary (Price, 1969), an advantageous feature of agonistic behaviour in that it avoids unnecessary population reduction and the high energetic costs and risks associated with fighting.The ‘Siamese fighting fish’ Betta splendens (figure 1), provides an exemplary demonstration of aggressive and territorial behaviours. Male B. splendens exhibit several stereotyped displays when seemingly threatened by another male (Fantino, 1972) with the intent of communicating their level of fitness and their capacity to fight (Castro et al., 2006). B. splendens ward away those who are competing for territory and increase their reproductive success by protecting a ‘bubble nest’ containing female eggs for fertilisation (Forsatkar et al., 2016). The extension of the branchiostegal membrane whilst facing the opponent (figure 2) and the spread of the dorsal, caudal and ventral fins, the ‘broadside display’, are behaviours of interest with regards to aggression (Fantino et al., 1972). These signals are used to maximise and promote their size to the competitor and so are honest indicators of fitness as they cannot be replicated in the same way by inferior members of the species. The facing display was the emphasis in the following experiment as it appears to indicate aggression more significantly than the broadside display. Haplerin and Giri (1997) suggested that the broadside display is used in more than just aggressive social contexts, for instance it may be directed towards female B. splendens to advertise their fitness and reproductive capacity, whereas facing displays are mainly associated with aggression. The aim of the succeeding experiment was to determine whether rates and duration of facing displays towards an equally matched opponent are good predictors of the outcome of a contest between two male B. splendens. The null hypothesis to be tested was that there is no difference in the mean rates nor duration of facing displays towards an equally matched opponent between the winners and losers of a final contest. MethodA sample size of 93 pairs of male B. splendens was observed, sourced from pet shops. The experimental setup consisted of a fish tank with a capacity of around 14.5 litres divided equally into two sections, each section occupied by a male member of the B. splendens species, as shown in figure 3. Each half represented the perceived exclusive territory of the fish. Two different central dividers were used depending on the experimental stage, either a transparent divider or black opaque divider. A heater was used to reproduce the optimum living conditions for the tropical species (around 26°C), and the fish were acclimatized to the conditions of the tank and the laboratory setting over a period of two weeks preceding the experiment. The fish were fed equal quantities each day.First, the two fish were tested individually for their aggressive responses to an equally matched opponent. During this stage, the black divider was in place in order to prevent the two fish from seeing one another. To measure aggressive responses, a mirror was placed against the side of the tank, with the reflection mimicking an opponent of equal size, colour and other characteristics. Each fish was observed for 5 minutes. The cumulative duration of facing display towards the mirror was recorded using a stopwatch, and the number of facing displays that were carried out was counted using a tally counter. From the latter measurement, rate of display (displays per minute) was calculated. The subsequent stage of the experiment involved an acetate sheet that was fixed to the front of the tank, with a black line drawn 4cm from the central divider on either side, as shown in figure 3. The black divider was removed, leaving only the transparent barrier, and the fish were then allowed to view one another and defend their territory. The two fish were matched as closely as possible with regards to body size, colour and other characteristics. The total time (seconds) spent by each fish facing the opponent with half their body length in the central zone was measured using a stopwatch. The winner of the contest was defined as the individual who had the longest cumulative time.In order to test whether there was a significant difference in both the mean rates and duration of facing displays for the winners and losers of the two-membered contest, a t-test was used. For the purpose of the analysis, the samples were assumed to be independent. A parametric test was used as the sample size (n) for both groups was larger than 40, therefore according to the central limit theorem it can be presumed that sample means would exhibit a normal distribution.ResultsFigure 4 shows that the mean rate of display (displays per minute) towards their own reflection was greater for the winners of the final contest compared to the losers. The winners averaged at 8.1 displays per minute, whereas losers displayed at a rate of 7.2 times per minute. The standard error (SE±) bars have minor overlap, with an SE of 0.66 for winners and 0.52 for losers. An overlap in standard error bars could indicate a lack of a significant difference between the two mean rates of display.Figure 5 illustrates that the winners of the final contest between two members of the B. splendens species had a higher average duration of facing displays towards a reflection of themselves in the initial experiment. The winners showed a mean duration of 91.6 seconds compared to 73.1 seconds for the losers, a difference of 18.5 seconds. The standard error bars have no overlap, indicating that there could be a significant difference between the two mean durations of display. Mean duration of display for the winners showed a standard error of 6.33, compared to 4.80 for losers.Table 1 shows the summary statistics for an independent t-test used to test for a significant difference in mean rate of facing display for winners and losers of the contest. The test did not suggest a significant difference between winners and losers (two-tailed independent t-test, t175 = 1.05, p = 0.30). Conversely, the summary statistics in table 2 show that the test provided evidence to suggest that the mean duration of facing display for winners was significantly higher than that of the losers (two-tailed independent t-test, t171 = 2.32, p = 0.02). Overall, we can reject the null hypothesis that there is no significant difference in mean duration of facing displays for winners and losers, but cannot reach any conclusions about the effect of mean rate of display on the outcome of conflict. DiscussionOverall, a longer duration of facing display performed by B. splendens towards a reflection of themselves seems to be a significant predictor of victory in a subsequent dyadic conflict. On average, victors maintained facing displays for 18.5 seconds longer than losers. Displays carry high metabolic costs, therefore these are honest indicators of fitness as subordinate animals could not maintain as high a level of performance as those who are superior in resource gathering (Castro et al., 2006). Castro et al. found in their 2006 study that during a conflict, metabolic rates of B. splendens increase five-fold to “the maximum workload that organisms can sustain”. Therefore, a higher mean duration of facing display in the winners of the conflict may be a demonstration of higher fitness. This is supported by Haller and Wittenberger (1987) who found that winners were able to produce more energy per unit time than losers of the contest, in addition to a faster rate of metabolism of carbohydrates. Losers may therefore have a lower energy output and metabolism, accompanying a lower aptitude to maintain displays. Also, submissive fish may have spent less time in close proximity to the dominant individual with the intention of signalling their inferior social status and so the unwillingness to participate in the fight. The costs and risks to the submissive fish if it were to participate in a conflict with a superior individual are likely to be high, therefore avoided unless absolutely necessary.Rate of facing display towards a mirror image did not seem to predict the outcome of the final conflict. Although the overall mean rate was higher for winners, this difference was not found to be significant and likely occurred due to chance. It could be hypothesised that the rate of display does not signify dominance in the same way as duration of display. A longer duration may connote higher commitment and motivation to fight in that more time is spent in a stance that shows readiness. Directly facing the opponent leaves a lesser opportunity to flee before a fight incurs, placing the fish in a position for attack (Halperin and Giri, 1997). Simply performing the display intermittently may be considered less threatening as flexibility still remains for departure from the conflict. As aforementioned, independence of samples was assumed in the analysis. However, the experiment was conducted on 4 occasions using the same smaller sample of B. splendens. This may have created complications in that prior experience could have influenced the outcome of the contest. For instance, an individual who lost the contest in an earlier experiment may have then avoided confrontation in the following tests despite any predictions, skewing the results. The research of Karino and Someya (2007) suggests that a negative experience of loss in a previous fight may reduce aggressiveness thereafter. This could have had a confounding influence on the outcome of some fish to fish contests, yielding a result that may not reflect the impact of innate aggressiveness alone. The fish were obtained from pet shops, therefore their prior experience before the experiment is also unknown. Furthermore, the fish may have fatigued due to the high energy expenditure necessary to perform repeated aggressive displays, meaning that those that were seen to be less aggressive may have merely been in a state of recovery from their previous participation in the experiment. In order to improve future observations, fish should only be used on one occasion in order to remove the variable of previous experience with the other individual as well as exhaustion. A further issue in the experimental design arises when considering the temperature of the tank. The heater was only on the left side of the tank, and there was a palpable difference in water temperature in the right side of the tank. The barriers in the centre of the tank reduced water flow between the two sections, therefore heat may have not circulated completely around the tank. A water temperature that deviates from the optimum for the species may create unsuitable conditions in which behaviour may not be natural or the ability to display might be affected. To correct this issue, a water circulation device could be incorporated into the set up to ensure an equal temperature throughout the tank.In conclusion, the findings of the research indicated that assessing aggressive responses of B. splendens towards a mirror image may be useful when predicting the dominant individual and therefore the outcome of subsequent conflicts, especially with regards to duration of facing displays.