contest experiences
The latency to gill display and the latency to emerge from a shelter were positively correlated for individuals that received a winning, losing or no recent contest experience (P 0.061, Fig. 5), indicating a stable, positive correlation between aggressiveness and boldness in this fish. Although the latency to gill display also appeared to correlate positively with the latency to explore a novel object for individuals with different contest experiences, the trends never reached statistical significance (P 0.080, Fig. 6). In addition, there was no significant difference in aggression (latency to gill display) between the fish with on average better and those with on average worse learning performance (those that did or did not enter the target reservoir) for any of the contest experience treatments (P 0.331, Fig. 7. Five individuals that failed either training phase I or training phase II were excluded from this analysis).
These results indicated that, in K. marmoratus, there is a positive correlation between aggressiveness and boldness, but did not show a significant relationship between aggressiveness and exploratory tendency or between aggressiveness and learning performance.
3.2 Aggressiveness (latency to gill display): the effect of recent contest experience and its relationship with testosterone and cortisol levels
The Cox regression model indicated that contest experience had a significant effect on an individual‟s latency to gill display (P = 0.023, Table 1); a winning experience shortened while a losing experience prolonged the average time to display, although neither the effect of a winning (P = 0.058) nor of a losing experience (P = 0.386) was significant when compared individually with the control treatment of no recent contest experience. Strain type had a significant effect (P = 0.025) on this measurement; individuals of DAN2K strain took longer to display than individuals of HON9 strain (Fig. 8).
Baseline and post-experience levels of testosterone were negatively (P < 0.001) and positively (P = 0.001) related with the latency to gill display, respectively, indicating that individuals with higher levels of baseline testosterone tended to display more quickly (be more aggressive), while individuals with higher post-experience testosterone levels tended to display more slowly (were less aggressive). Neither baseline nor post-experience levels of testosterone were significantly correlated with the latency to gill display (P 0.123).
3.3 Boldness, exploratory tendency and learning performance: their
relationship with aggressiveness and levels of testosterone and cortisol
and how they are affected by contest experience
3.3.1 Boldness (latency to emerge from a shelter)
Cox regression (Table 2) showed a positive relationship between the latency to emerge from a shelter and the latency to gill display (P = 0.002), again confirming a positive relationship between aggressiveness and boldness. Contest experience, however, did not have a significant effect on the latency to emerge from a shelter (P = 0.571). There were no significant interaction effects between contest experience and the latency to gill display on the latency to emerge from a shelter (P = 0.520), indicating that the relationship between aggressiveness and boldness was not influenced by contest experience. Baseline testosterone level negatively related with the latency to emerge from shelter (P = 0.008) showing that individuals with higher baseline testosterone levels tended to emerge from the shelter sooner (be bolder).
Strain type had a significant effect (P = 0.011); individuals of RHL and VOL strain took longer to emerge from a shelter than individuals of HON9 strain (Fig. 9).
3.3.2 Tendency to explore (latency to explore a novel object)
Cox regression (Table 3) indicated that there was no significant relationship between the latency to gill display and the latency to explore a novel object (P = 0.133), confirming the lack of a significant relationship between aggressiveness and the tendency to explore. The tendency to explore was, however, significantly influenced by contest experience (P = 0.005): individuals with a losing experience approached the novel object more quickly (P = 0.022). There were no significant interaction effects between contest experience and the latency to gill display on the latency to explore (P = 0.444), indicating that the (lack of) relationship between aggressiveness and the tendency to explore was not influenced by contest experience.
Post-experience testosterone levels negatively related with the latency to explore (P =
0.029), i.e. individuals with higher post-experience testosterone levels had a higher tendency to explore.
3.3.3 Learning performance (probability of entering the target reservoir in the testing phase)
Logistic regression (Table 4) showed no relationship between the latency to gill display and the probability of entering the target reservoir (P = 0.729). Recent contest experience, however, had a significant effect on this probability (P = 0.004); more specifically, individuals with a losing experience were significantly less likely to enter the target reservoir than individuals with no recent contest experience (P = 0.045).
Individuals with winning experiences were more likely than those with no contest experience to enter the target reservoir, and although this difference did not reach significance level (P = 0.175), the contrast between the behavior of the winners and the losers contributed to the significance of contest experience overall. There were no significant interaction effects between contest experience and the latency to gill display on the probability (P = 0.8134), indicating that the (lack of) relationship between aggressiveness and learning ability was not influenced by contest experience.
Neither baseline nor post-experience levels of testosterone or cortisol had a significant relationship with this probability (P 0.500). In addition, last contest outcome had a significant effect on the probability; individuals that lost a contest more than one month previously had a higher probability of entering the target reservoir (P = 0.044).
3.4 Effect of contest experience on the levels of testosterone and cortisol
Post-experience testosterone levels, although positively correlated with baseline testosterone levels (P < 0.001), were not affected by the contest experience treatments
(P = 0.992) (Table 5). Similarly, post-experience cortisol levels were positively correlated with baseline cortisol levels (P < 0.001) but were also not influenced by contest experience treatments (P = 0.379) (Table 6).
Discussion
My study investigated aggression-related behaviors in K. marmoratus and how recent contest experiences affect both these behaviors and their relationship with hormones. The fish appeared to exhibit a behavioral syndrome including aspects of aggressiveness and boldness. Individuals that behaved aggressively were also bolder than others, and this relationship between aggressiveness and boldness was not influenced by recent contest experience. In addition, both the fishes‟ aggressiveness and their boldness positively correlated with their baseline testosterone levels, which lends support to the possibility that these two behaviors are positively correlated because they are modulated by common physiological mechanisms.
Neither the fishes‟ tendency to explore nor their learning performance was significantly correlated with aggressiveness, although as with aggressiveness, these two behaviors were influenced by recent contest experience. Individuals with a winning experience were more aggressive and had better learning performance but a lower tendency to explore than individuals with a losing experience. The fishes‟
tendency to explore was positively correlated with post-experience testosterone levels, whereas learning performance was not significantly related with levels of testosterone or cortisol.
4.1 Relationships between aggressiveness and boldness, tendency to explore and learning performance
There was a positive relationship between aggressiveness and boldness.
Individuals that had a shorter latency to gill display emerged from the shelter more quickly. This result is consistent with other studies in different species, suggesting a
relatively stable aggressiveness-boldness relationship across taxa (Kortet & Hedrick 2007; Moretz et al 2007; Pintor et al 2008).
There was no significant relationship between aggressiveness and either the tendency to explore or learning performance. This result differs from previous studies which found that, in general, more aggressive individuals were quicker to explore their environment than others (Benus et al 1987; Verbeek et al 2006), whereas less aggressive individuals took longer to explore their environment (van Oortmersse et al 1985; Benus et al 1987). It is therefore possible that the relationship between aggressiveness and both exploratory tendency and learning performance might vary with the study animal. There might also be a confounding effect between shelter-hiding tendency and exploratory tendency. Being in a novel environment is stressful, which might cause the individuals to flee or freeze in response. In my study, I provided a shelter in the area to be explored. Timid fish might therefore have approached the shelter to hide in it, rather than bold fish approaching the shelter to explore it.
4.2 The influence of recent contest experience on aggressiveness and boldness, the tendency to explore and learning performance, and on the relationships between them
4.2.1 Effect of contest experience on the different behaviors
Contest experience received one day previously significantly changed individuals‟ behavior in mirror tests (of aggressiveness). Individuals with a winning experience had a lower latency to gill display (were more aggressive) than those with
a losing experience. This result is consistent with previous studies of K. marmoratus (Hsu & Wolf 1999, 2001; Lee 2009; Lu 2010; Huang et al 2010; Lan 2010), which found that prior winning and losing experiences increased and decreased, respectively, individuals‟ aggressiveness (e.g. the probability of initiating gill display, attack) and the probability of winning a non-escalated contest.
In my study, contest experience received one day previously did not affect individuals‟ boldness. As well as the possibility that the effect of contest experience on boldness might vary with the study organism, it is possible that individuals with different personalities might react differently to contest experience. In a study of rainbow trout, the authors found that the boldness level of shy individuals increased no matter what contest experience they received, whereas bold individuals became shy after receiving a losing experience (Frost et al 2007). In my study, individuals were randomly assigned to receive a winning/losing/no experience regardless of their original personality, while the boldness levels of the individuals with different contest experience did not differ, suggesting that there were no significant experience effects on boldness rather than the dilution effect of different innate personalities.
Contest experience received one day previously significantly changed individuals‟ tendency to explore. Individuals which received a losing experience approached the novel object more quickly. These results might have occurred because the object we provided for the individuals to explore was a shelter. Individuals which had received a losing experience one day previously might have been inclined to hide in the shelter quickly rather than to explore the environment slowly.
Recent contest experience received one day previously had a significant effect on learning performance. Individuals which had received a losing experience were more likely to pick the wrong side of the reservoir than others, suggesting that a losing
experience reduced learning performance. This result might arise because losing increased anxiety levels, which subsequently impeded learning performance. Rats which were housed with cats to induce anxiety had lower locomotor activity in open field tests and worse performance in water maze tasks than the control group (Park et al 2001). Therefore, there appears to be a general trend for stress to reduce learning performance.
4.2.2 Effect of contest experience on the relationship between the behaviors
The different types of experience (W, L & N) had no significant effect on the relationships between aggressiveness and boldness, tendency to explore or learning performance. Aggressiveness and boldness were, however, highly correlated for all three experience types, suggesting a stable aggressiveness-boldness syndrome in K.
marmoratus. Recent studies have indicated that predation risk might be related to the
relationship between aggressiveness and boldness in sticklebacks (Bell 2005; Bell and Sih 2007): a positive relationship existed only in populations that were under high predation risk, suggesting that stressful conditions (e.g. high predation risk) might promote a tradeoff and cause correlations between aggressiveness and boldness. For instance, when predation risk is high, bolder individuals that tend to wander away from shelter are probably more likely to suffer higher mortality. This disadvantage of being bolder may be compensated by being aggressive toward conspecifics. When predation risk is high, selection against individuals which are both bold and not aggressive may therefore cause a positive relationship between boldness and aggressiveness.
Although contest experience had a significant effect on aggressiveness but not on boldness, the relationship between aggressiveness and boldness was not significantly affected by their contest experiences (W/N/L). This might be because the relationship between boldness and aggressiveness is stronger than the effect of contest experience on aggressiveness so that we could not detect a significant effect of contest experience on the relationship between them.
4.3 Relationships between hormones and behavioral traits
4.3.1 Effect of contest experience on individuals’ post-experience levels of testosterone and cortisol
Although winning and losing experiences caused the fish to become respectively more and less aggressive, they did not affect the fishes‟ post-experience levels of testosterone or cortisol. Previous studies of K. marmoratus reached similar conclusions on the effect of contest experiences on hormone levels (Lee 2009; Lu 2010). These results suggest that changes in the levels of these hormones are not the primary mechanisms for winner and loser effects in K. marmoratus. The physiological mechanisms that mediate winner and loser effects in the fish are yet to be uncovered.
Since losing experience did cause a deterioration in learning ability (see 4.2.1 above) as might be expected for animals under stress, but did not cause an increase in post-experience cortisol levels, it may be that cortisol does not mediate the stress response in this fish.
4.3.2 Relationships between hormones and each of the behaviors
There was a significant relationship between baseline testosterone levels and both aggressiveness and boldness. This result lends support to the possibility that the positive relationship between aggressiveness and boldness is caused by their being mediated by similar physiological mechanisms.
My finding of a positive relationship between aggressiveness and baseline testosterone levels is similar to that of a previous study of K. marmoratus. Li (2010) found a positive relationship between baseline testosterone and the fish‟s tendency to initiate aggressive acts and its persistence when fighting with a stronger opponent. I also discovered a negative relationship between aggressiveness and post-experience testosterone levels. However, two previous studies (Lee 2009, Lu 2010) of the fish showed that the aggressiveness (e.g. the likelihood of initiating gill displays, attacks and escalate contests) of individuals with a losing experience was positively correlated with post-experience testosterone and cortisol levels. One possible reason for the difference in findings between my and previous studies is different experimental designs. Lee (2009) and Lu (2010) provided their focal individuals with three losing contest experiences whereas the individuals in my study received only one contest experience. Three consecutive losing experiences might have caused a more significant relationship between post-experience hormone levels and contest behaviors than just one experience.
I did not find any significant relationship between cortisol levels and aggressiveness in the fish. Earley and Hsu (2008), however, reported a negative relationship between cortisol levels and aggressiveness. Earley and Hsu (2008) measured the hormone levels of both participants in a contest whereas I only measured the hormone levels of a fish displaying against its own reflection in a mirror,
which could have cause the difference in our findings about the relationship between aggressiveness and cortisol levels.
4.4 Conclusions
Overall, I found a stable aggressiveness-boldness relationship in K. marmoratus:
more aggressive individuals were also bolder. And, the relationship between aggressiveness and boldness remained stable regardless of whether the fish received a wining, a losing or no contest experience. The fish‟s tendency to explore and its learning performance, however, were not significantly correlated with its aggressiveness. In addition, baseline testosterone levels positively correlated with individuals‟ aggressiveness and boldness. The fish‟s tendency to explore correlated with post-experience testosterone levels, whereas learning performance did not have significant relationship with testosterone or cortisol levels. These results suggest that baseline testosterone levels might play an important role in mediating the relationship between aggression and boldness in the fish.
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