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. 2012 May 2;52(6):801–813. doi: 10.1093/icb/ics057

Aggression and Related Behavioral Traits: The Impact of Winning and Losing and the Role of Hormones

Ching Chang *, Cheng-Yu Li *, Ryan L Earley †, Yuying Hsu *,1
PMCID: PMC3501093  PMID: 22576819

Abstract

A suite of correlated behaviors reflecting between-individual consistency in behavior across multiple situations is termed a “behavioral syndrome.” Researchers have suggested that a cause for the correlation between different behaviors might lie in the neuroendocrine system. In this study, we examined the relationships between aggressiveness (a fish's readiness to perform gill display to its mirror image) and each of boldness (the readiness to emerge from a shelter), exploratory tendency (the readiness to approach a novel shelter), and learning performance (the probability of entering the correct reservoir in a T-maze test) in a mangrove rivulus, Kryptolebias marmoratus. We explored the possibility that the relationships between them arise because these behaviors are all modulated by cortisol and testosterone. We also tested the stability of the relationships between these behaviors shortly after using a winning or losing experience to alter individuals’ aggressiveness. The results were that aggressiveness correlated positively with boldness and the tendency to explore, and that these three behavioral traits were all positively correlated with pre-experience testosterone levels. Aggressiveness and boldness also positively correlated with pre-experience cortisol levels; exploratory tendency did not. The relationship between aggressiveness and boldness appeared to be stronger than that between either of them and exploratory tendency. These results suggest that testosterone and cortisol play important roles in mediating the correlations between these behavioral traits. Learning performance was not significantly correlated with the other behavioral traits or with levels of testosterone or cortisol. Recent experience in contests influenced individuals’ aggressiveness, tendency to explore, and learning performance but not their boldness; individuals that received a winning experience were quicker to display to their mirror image and performed better in the learning task but were slower to approach a novel object than were individuals that lost. Contest experience did not, however, significantly influence the relationships between aggressiveness and any of boldness, exploratory tendency, or learning performance. The results show that the individual components of a suite of correlated behaviors can preserve a flexibility to respond differently to environmental stimuli.

Introduction

In recent years, an increased interest in animals’ personality traits has led to a growing literature showing various behavioral characteristics to be linked to individual aggressiveness (Dingemanse and Réale 2005; Sih and Bell 2008; Stamps and Groothuis 2010). In particular, boldness (the tendency to take risks: e.g., willingness/readiness to encounter predators, or to stay outside shelter) is the characteristic most frequently investigated and reported to correlate with aggressiveness; the trend is for aggressive individuals to have a higher tendency to expose themselves to risky situations (Huntingford 1976; Dahlbom et al. 2011). The tendency to explore and manipulate the environment is another characteristic frequently found to correlate positively with aggressiveness (Verbeek et al. 1996; Koolhaas et al. 1999). In male rainbowfish (Melanotaenia duboulayi), dominant individuals were more aggressive, bold, and active than subordinates, despite the fact that neither the direct relationship between aggressiveness and boldness nor that between aggressiveness and activity levels were significant (Colléter and Brown 2011). Although not investigated as much, aggressive individuals of some species also perform better in avoidance-learning tasks than do nonaggressive ones (Benus et al. 1989; Zhuikov 1993).

The correlations between these behaviors could be a result of selection, because the combination of traits results in higher fitness, or they could arise because the traits share proximate mechanisms and are thus difficult to decouple (Sih et al. 2004; Bell and Sih 2007; Wolf et al. 2007; Budaev and Brown 2011). Hormones acting on multiple target tissues (Ketterson and Nolan 1999) and the pleiotropic effects of genes, for instance, could produce correlated behaviors. Selective breeding on the basis of the cortisol response to stress in rainbow trout (Oncorhynchus mykiss) yields individuals that differ not only in reactivity to stress (low-responsiveness [LR] and high-responsiveness [HR]), but also in several behavioral traits. LR individuals resume feeding sooner after stress, retain a learned fear response for longer, and show more aggression and social dominance than HR individuals (Pottinger and Carrick 1999, 2001; Moreira et al. 2004; Øverli et al. 2005, 2007). Moreover, risk-taking juvenile mulloway (Argyosomus japonicus) had significantly lower plasma cortisol concentrations in response to handling stress than did risk-avoiding individuals (Raoult et al. 2011), while risk-taking common carp (Cyprinus carpio) had lower expression of cortisol receptor genes in the head kidney and brain than did risk-avoiding individuals (Huntingford et al. 2010). Recently, a single gene, fgfr1a, was reported to modulate the expression of aggression, boldness, and exploratory behavior simultaneously in zebrafish, Danio rerio (Norton et al. 2011).

Interestingly, different, but correlated, behavioral traits could have different impacts on individual fitness. A meta-analysis of published studies showed that aggressiveness had a positive effect on reproductive success; boldness had positive and negative effects on reproductive success and survival respectively, while exploration had a positive effect on survival (Smith and Blumstein 2008). Moreover, in guppies (Poecilia reticulata), males that learned faster tended to score higher than did others in a female preference test (Shohet and Watt 2009), which may suggest a positive relationship between learning ability and reproductive success. The adaptive value of variations in aggressiveness therefore cannot be fully assessed without knowing what other behaviors are linked to aggression and considering the influence of these behaviors on individual fitness.

Aggression can be influenced by many extrinsic factors. In many animal species, for instance, the outcome of a recent fight has a substantial effect on individual aggression (Hsu et al. 2006; Rutte et al. 2006). After a recent victory, individuals are often more likely to initiate and escalate agonistic interactions and enjoy a higher chance of winning than are their naïve opponents (winner effect); after a defeat, individuals become less aggressive, retreat voluntarily, and suffer a higher chance of losing again (loser effect). If the correlations between aggression and the other behaviors result from them sharing common physiological mechanisms, a recent victory or defeat will probably alter behaviors correlated with aggression. This possibility has not been tested.

Using the mangrove rivulus, K. marmoratus, as the study organism, we examined the influence of winning and losing recent contests on the relationships between aggression and correlated behaviors. This fish is aggressive in the field and in the laboratory (Huehner et al. 1985; Taylor 1990). Two individuals confined in a 12 × 8 × 20 cm aquarium usually establish a dominant–subordinate relationship within an hour (Hsu et al. 2008). Aggressiveness (the likelihood of initiating gill display and attack, of escalating agonistic interactions, and of persisting in agonistic interactions) is modulated by recent wins and losses (Hsu and Wolf 1999, 2001; Huang et al. 2011; Lan and Hsu 2011). In addition, this fish's behavior in agonistic interactions is associated with its hormonal state: pre-fight cortisol related negatively, but pre-fight testosterone in the smaller opponent related positively to its initiation and winning of contests (Earley and Hsu 2008). Building on these findings, we examined (1) the relationship between the fish's aggression and boldness, exploratory tendency, and learning performance, (2) whether testosterone and cortisol mediated these relationships, and (3) whether winning or losing influences aggressiveness and its correlated behaviors in the same manner, such that, although the behavioral traits are altered, the relationships between them are preserved after the contests.

Methods

Study organism

Kryptolebias marmoratus is an internally self-fertilizing hermaphroditic fish (Taylor et al. 2001) living in mangrove swamps from coastal regions of Brazil and eastern Central America, throughout the Caribbean to central Florida (Harrington 1961). Natural populations consist mainly of isogenic homozygous hermaphrodites with <1% males, although an out-crossing heterozygous population with ∼20% males was discovered in Twin Cays, Belize (Mackiewicz et al. 2006).

The fish is often found in crab burrows and small ephemeral pools (Davis et al. 1990; Taylor et al. 2008), hiding under leaf litter (Huehner et al. 1985; Davis et al. 1990), or inside decaying mangrove logs (Taylor et al. 2008). A laboratory study showed that the dominant individual of a pair always enters and defends a shelter, when provided (Molloy et al. 2011). This tendency to explore and utilize shelters was used as the foundation for our tests of boldness, learning performance, and tendency to explore.

This study used F5–F10 generations of five strains of K. marmoratus originally collected by Dr D. Scott Taylor from various locations (DAN2K: Dangria, Belize; HON9: Utila, Honduras; RHL: San Salvador, Bahamas; SLC8E: St. Lucie County, FL, USA; VOL: Volusia County, Florida, USA). Fish were isolated within a week of hatching at the National Taiwan Normal University given a unique identification code and, except when used in experiments, kept alone in a 13 × 13 × 9 cm translucent polypropylene container filled with 550 ml 25 ppt synthetic sea water (Instant Ocean™ powder). Fish were kept at 25 ± 2°C on a 14:10-h photoperiod and fed newly hatched brine shrimp (Artemia) nauplii daily. Containers were cleaned and water replaced every 2 weeks. Experiments were conducted in accordance with National Taiwan Normal University Animal Care and Use Committee permit #96016.

Experimental design and procedures

Fish were isolated for more than a month after their previous experiment. Seven days before the start of this new experiment, two small pieces of pottery were placed in their containers to trigger their innate tendency to use shelters and hide. On Day 1, they were tested for baseline (pre-experience) hormone levels and then given a randomly allocated winning (W), losing (L), or no contest (N) experience. On Day 2, post-experience hormones were sampled and then boldness, aggressiveness, tendency to explore, and learning ability were measured as described below. After each hormone test, the fish was returned to its maintenance container, fed a small amount of brine shrimp and allowed to rest for 15 min before the next procedure. Each behavioral test was preceded by 1 h of acclimatization in the test aquarium. The first three tests were terminated after 30 min, whether or not the fish displayed the behavior concerned, and the fish moved on to acclimatize for the next test. The order of these first three tests was randomized. The test of learning ability took much longer and was carried out last. All behavioral tests were captured on digital video.

The sample size was 180, with 3 treatments (W, L, N) × 5 strains × 12 fish per strain, 6 of which won and 6 of which lost their most recent contest, more than a month previously.

Collection, extraction, and assay of hormones

Hormone samples were collected from 0900 to 1000 h on Days 1 and 2 by placing a fish for 1 h in a 400-ml glass beaker containing 200 ml 25 ppt clean synthetic seawater housed in a translucent plastic container. Although placing a fish in a beaker could cause it stress, this procedure is less invasive than bleeding, and water-born cortisol levels have been shown to be a good predictor of plasma cortisol levels (r = 0.6; Wong et al. 2008). Water was then removed from the beaker with a vacuum pump and passed through a C18 solid-phase column (Lichrolut RP-18, 500 mg, 3.0 ml; Merck) to extract hormones. Before use, the columns were washed twice each with 2 ml methanol and 2 ml pure water; after use, they were purged of sea salt with 2 × 2 ml pure water washes and stored at −80°C. Ellis et al. (2004) showed that freeze storage of water samples and columns does not influence steroid concentrations. Hormones were eluted from the columns by 2 × 2 ml ethyl acetate washes. The eluted solvent was evaporated at 37°C with nitrogen gas and the resulting hormone pellet was then re-suspended in 800 μl of enzyme-immunoassay (EIA) buffer supplied with the Cayman Chemicals Inc. EIA kits and the samples stored at −20°C until assay. Testosterone and cortisol were assayed using Cayman Chemicals Inc. EIA kits, following the manufacturer's recommended procedures. Plates were read at 405 nm on a BioMek microplate reader. Assay of cortisol and testosterone in mangrove rivulus using Cayman Chemicals Inc. EIA kits has been previously validated by Earley and Hsu (2008). All the data on hormone levels are presented as pg/ml. Intra-assay coefficients of variation were (assay plate 1–11) 6.3, 4.4, 10.3, 3.7, 8.8, 12.9, 4.2, 2.5, 20.0, 7.7, and 9.1% for testosterone and 4.1, 5.3, 9.6, 11.8, 4.8, 10.0, 6.5, 3.8, 10.2, 6.3, and 4.4% for cortisol. The inter-assay coefficient of variation was 11.8% for testosterone and 11.0% for cortisol.

Provision of winning, losing, or no-contest experiences

Focal individuals assigned to receive W or L experiences were placed at random into one of two symmetrical compartments of a 12 × 8 × 20 cm aquarium containing 2 cm gravel and 12 cm water divided by an opaque partition. A much larger/smaller fish (standard winner/loser) which had won/lost several fights with similarly sized opponents was placed in the other compartment and the partition removed after 15 min acclimatization. A winning experience was completed when the standard loser persistently retreated from the focal individual's display/attack for 1 min. A losing experience was completed when the focal individual persistently retreated from the standard winner's display/attack for 1 min. The pre-designated experience was acquired quickly and the partition re-inserted. Fish assigned N experience were treated as above except with no opponent.

Test of aggressiveness

Aggressiveness was quantified by a mirror test in a 12 × 16 × 20 cm aquarium, containing 2 cm gravel and 12 cm water. The fish acclimatized behind an opaque partition, 10 cm from a mirror on one of the narrow sides of the aquarium. The partition was removed and the elapsed time until the fish first displayed its gills to its mirror image (erecting its operculum during display) was used as the index of aggressiveness, with more aggressive fish displaying more quickly.

Test of boldness

Latency to emerge from a shelter was used to measure boldness (modified from Brown et al. 2005), with bolder individuals emerging more quickly. A 12 × 16 × 20 cm aquarium was fitted with a 6 × 6 × 8 cm dark black plastic shelter. The focal fish was placed in the shelter for acclimatization with the shelter's removable door closed. When the door was opened, the time until the fish first emerged was recorded.

Test of tendency to explore

The time to arrive within 4 cm of a novel shelter placed in a new environment was used as the index of tendency to explore, with fish showing a greater tendency to explore arriving sooner (Verbeek et al. 1994). The fish acclimatized behind a black partition 6 cm from one narrow end of a 12 × 34 × 26 cm aquarium containing 16 cm water and 2 cm gravel. A novel pottery shelter was placed at the opposite end. When the partition was lifted, the elapsed time until the fish went within 4 cm of the shelter was recorded.

Test of spatial-learning ability

The fish were classified as having better, or worse, spatial-learning ability with a pass/fail T-maze test, which built on the fish's known tendency to seek out shelter. Fish that found their way back to the shelter-rich target reservoir at one, randomly chosen, end of the T-maze to which they had been directed in two previously administered and successive training sessions passed; others failed. The T-maze (Fig. 1) was a modified version of that used by Darland and Dowling (2001) for testing cognitive ability in zebrafish, and consisted of a down piece (35 cm long) joining the mid-point of a cross piece (70 cm long), both 10-cm wide and 12-cm deep with 5 cm of water. Symmetrically arranged at the two ends of the cross piece, behind removable opaque partitions, were 22.5 × 22.5 × 22.5 cm reservoirs, with 1 cm gravel, 3 shelters, and 14.5 cm water. After acclimatization behind a further removable partition 10 cm from the end of the down piece, the test fish was given two training sessions with the partition to the randomly chosen target reservoir open.

Fig. 1.

Fig. 1

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The setting for the spatial learning ability test (T-maze test): the T-maze consisted a cross piece (70 cm), with a down piece (35 cm) joining the mid-point of cross piece. Both sections were 10-cm wide, 12-cm deep, and contained water 5-cm deep. There was a 22.5 × 22.5 × 22.5 cm reservoir at each end of the cross piece, with 1 cm of gravel, 3 shelters, and water 14.5-cm deep. There was an opaque partition at the entrance to each reservoir and 10 cm away from the end of the down piece of the T-maze.

Training periods started when the start partition was opened and ended after the fish entered the target reservoir and had been confined there for 5 min. If the fish did not move from the down piece in 30 min, it was forced to the maze junction with a 10-cm-wide plastic ruler and left to make its own turning decision there. If it had still not arrived at the target reservoir after a further 30 min in the first training session it was forced there; one animal which failed to arrive at the target reservoir within 30 min in the second training session was deemed to have failed the training and the test, which were then terminated. After each training period, the fish was placed back at the starting point for 10 min of acclimatization.

All three partitions were lifted for the testing phase. Fish were allowed 60 min to find their way to the correct reservoir. Fish that did not move from the starting point in 30 min were forced out as above. Fish that did not enter the correct reservoir in 60 min or entered the wrong reservoir first were deemed to have failed.

Analysis of data

Spearman's rank correlations were used to measure the overall relationships between aggressiveness, boldness, exploratory behavior, and pre-contest and post-contest levels of testosterone and cortisol. Individuals that did not display the expected behavior in 30 min (aggression 18; boldness 2; exploration 2) were assigned a ceiling score of 180 s. The relationships between learning performance and the other behavioral traits and the levels of testosterone and cortisol were determined with Wilcoxon tests to examine whether individuals that entered/did not enter the correct reservoir in the maze test differed in these attributes.

We used the Wilcoxon paired-sample test to analyze the difference between pre-experience and post-experience hormone levels for individuals that were assigned different contest experiences. We used Cox regression to analyze the relationship between the three experience treatments (W, N, and L) and aggressiveness—18 individuals that did not perform gill display in the mirror test were treated as censored data. Cox regression models also analyzed whether the W/L/N contest experience given to the fish in their training (hereafter “contest experience”) affected the relationships between aggressiveness (rank score for latency to gill display) and both boldness and exploratory behavior. Eighteen fishes that did not display were assigned a rank of 163; the two individuals that did not leave the shelter in the boldness test and the two individuals that did not approach the novel shelter in the exploration test were treated as censored data in the respective models. (Cox regression models the hazard or instantaneous risk of an event occurring—a higher hazard indicating a faster performance or a shorter latency—and allows for censored data in which the event was not completed before a cut-off time.) A multiple logistic regression model examined whether contest experience affected the relationship between aggressiveness (rank score) and learning performance. Testosterone and cortisol levels (ln transformed) were included in the models to examine the relationship between hormones and the various behaviors. All regression models controlled for strain, standard length, and the outcome of the fish's previous contest. JMP (v. 5.0.1 SAS Institute Inc., Cary, NC, USA) was used for the statistical analyses.

Results

Correlations between different types of behavior and between behaviors and hormones

As summarized in Table 1, the latency to display to a mirror image, to emerge from a shelter, and to approach a novel object were all positively correlated, but the relationship between the latencies to display and to emerge were stronger than were the relationships between either of these and the latency to approach the novel object. There was no significant relationship between the likelihood that the fish entered the correct reservoir and any of the latency to display, to emerge, or to approach the novel shelter.

Table 1.

Relationships between behaviors and hormones

Latency to display Latency to emerge Latency to approach Pre-exp-T Pre-exp-Cort Post-exp-T Post-exp-Cort
Latency to display (Aggressiveness) rs = −0.16 (P = 0.033) rs = −0.19 (P = 0.009) rs = 0.05 (P = 0.430) rs = −0.11 (P = 0.157)
Latency to emerge (Boldness) rs = 0.31 (P < 0.001) rs = −0.27 (P < 0.001) rs = −0.33 (P < 0.001) rs = −0.102 (P = 0.174) rs = −0.17 (P = 0.021)
Latency to approach (Exploratory tendency) rs = 0.15 (P = 0.044) rs = 0.15 (P = 0.042) rs = −0.15 (P = 0.041) rs = −0.06 (P = 0.421) rs = −0.14 (P = 0.068) rs = 0.00 (P = 0.982)
Correct reservoir/not (Learning) Z = −0.12 (P = 0.902) Z = 0.22 (P = 0.827) Z = 0.62 (P = 0.540) Z = −0.55 (P = 0.580) Z = −0.432 (P = 0.666) Z = 0.47 (P = 0.639) Z = −0.91 (P = 0.360)
Pre-exp-T rs = 0.29 (P < 0.001) rs = 0.63 (P < 0.001) rs = 0.19 (P = 0.009)
Pre-exp-Cort rs = 0.19 (P = 0.009) rs = 0.52 (P < 0.001)
Post-exp-T rs = 0.24 (P = 0.001)
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Spearman’s rank correlation was used to test the relationship between behavioral traits (except for whether or not the fish entered the correct reservoir), between hormones (Pre-exp-T: pre-experience testosterone level; Post-exp-Cort: post-experience cortisol level, etc.), and between behavioral traits and hormones. Wilcoxon rank test (normal approximation) was used to examine the relationships between whether or not the fish entered the correct reservoir and the other behavioral traits and hormones.

The correlations between the behavioral measurements and the hormones are also provided in Table 1. Hormonal levels were positively correlated with each other. All three latencies were negatively correlated with pre-experience testosterone levels, indicating that individuals with higher pre-experience testosterone levels tended to be faster to display to their mirror image, to emerge from a shelter and to approach a novel object. Individuals with higher pre-experience cortisol levels were also faster to display to their mirror image and to emerge from a shelter; however, pre-experience cortisol levels did not have a significant relationship with the latency to approach a novel object. The only significant relationship between behavioral measurements and post-experience hormones was between latency to emerge from a shelter and post-experience cortisol; all other correlations between behavioral measurements and post-experience hormones were nonsignificant. The likelihood that an individual would enter the correct reservoir was not significantly correlated with pre-experience or post-experience levels of either testosterone or cortisol.

The effect of winning/losing

There was no significant difference between pre-experience and post-experience hormone levels (for either cortisol or testosterone) for any of the three experience treatments (Fig. 2). Since pre-experience levels of testosterone and cortisol were significantly correlated with the behavioral traits, they were included in the regression models as control factors when testing the relationships between aggression and the other behavioral traits.

Fig. 2.

Fig. 2

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Pre-experience (clear bars) and post-experience (shaded bars) levels (ln transformed, mean ± SE) of (A) testosterone and (B) cortisol for individuals that were assigned a winning, no contest or a losing experience. Wilcoxon paired-sample tests (normal approximation) were used to compare the pre- and post-experience levels of hormones.

We first examined the effect of contest experience on the latency to perform gill display to a mirror. Cox regression indicated that contest experience had a significant effect on the hazard (instantaneous risk) of gill display (Table 2). When analyzed separately, winning and losing had nonsignificant positive and negative effects, respectively. Thus, although winning and losing a contest tended, respectively, to shorten and prolong the time to gill display, it was only the combined effect of the two that reached significance. The model also confirmed that individuals with higher pre-experience levels of either testosterone or cortisol had higher hazards, i.e., were quicker to display.

Table 2.

Cox regression modeling the effect of contest experience on the latency to gill display, controlling for strain, body size, outcome of the fish’s previous contest, and pre-experience levels of testosterone (Pre-exp-T), and cortisol (Pre-exp-Cort)

Latency to display
Variable df b ± SE χ2 P
Contest experience 2 6.43 0.040*
    Wa 1 0.312 ± 0.194 2.58 0.108
    La 1 −0.191 ± 0.197 0.94 0.331
Strain 4 10.04 0.040*
Body size 1 −0.100 ± 0.054 3.39 0.066
Previous outcome-Lb 1 −0.110 ± 0.160 0.47 0.493
Pre-exp-T 1 0.298 ± 0.149 4.03 0.045*
Pre-exp-Cort 1 0.152 ± 0.069 4.88 0.027*
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Note: Cox regression models the hazard (instantaneous risk) of the event (display); a higher hazard of display (positive coefficient) indicates a shorter latency to gill display (i.e., more aggressive). *P < 0.05.

aAn indicator variable for the focal individuals that received a pre-designated winning (W) or losing (L) experience; the baseline group comprised the individuals that received no experience.

bAn indicator variable for the focal individuals that lost more than 1 month previously; the baseline group comprised the individuals that won a contest more than 1 month previously.

We next analyzed the influence of contest experience on the other behavioral traits and on their relationships with aggression (summarized in Table 3). Previous contest experience did not significantly influence the latency to emerge from the shelter. However, individuals that took longer to display had a lower hazard of emerging from the shelter (i.e., were slower or took longer to do so), which confirmed the positive correlation found between the two latencies in the previous section. The interaction effect between previous contest experience and the latency to gill display was not significant, indicating that such experience did not significantly alter the relationship between the two latencies (Fig. 3A). The model also confirmed that individuals with higher pre-experience levels of testosterone or cortisol had higher hazards, i.e., were quicker to emerge from the shelter.

Table 3.

Regression models examining the effect of contest experience on the latency to emerge from a shelter (Cox regression), the latency to approach a novel object (Cox regression) and the probability of entering the correct reservoir (logistic regression) and their relationships with the latency to gill display (Experience × Display)

Latency to emerge from the shelter
 Latency to approach a novel object
 Probability of entering the correct reservoir
Variable df b ± SE χ2 P b ± SE χ2 P b ± SE χ2 P
Contest experience 2 1.14 0.566 8.69 0.013* 13.21 0.001*
    Wa 1 −0.158 ± 0.191 0.68 0.409 −0.223 ± 0.194 1.32 0.250 0.648 ± 0.398 2.70 0.101
    La 1 0.033 ± 0.187 0.03 0.860 0.354 ± 0.199 3.17 0.075 −0.798 ± 0.394 4.22 0.040*
Latency to display-rank 1 −0.006 ± 0.002 12.53 <0.001* −0.004 ± 0.002 4.97 0.026* −0.000 ± 0.003 0.00 0.973
Experience × Display 2 0.38 0.826 1.33 0.513 1.79 0.409
Strain 4 12.43 0.014* 9.24 0.055 2.47 0.649
Body size 1 −0.005 ± 0.047 0.01 0.913 0.005 ± 0.049 0.01 0.910 −0.073 ± 0.098 0.55 0.459
Previous outcome-Lb 1 −0.074 ± 0.157 0.22 0.636 0.098 ± 0.160 0.37 0.540 0.623 ± 0.326 3.72 0.054
Pre-exp-T 1 0.430 ± 0.146 8.93 0.003* 0.345 ± 0.133 6.95 0.008* 0.122 ± 0.284 0.19 0.667
Pre-exp-Cort 1 0.157 ± 0.067 5.39 0.020* 0.033 ± 0.064 0.27 0.602 −0.079 ± 0.135 0.33 0.563
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All models controlled for strain, body size, previous fighting outcome, and pre-experience testosterone (Pre-exp-T) and cortisol (Pre-exp-Cort) levels.

Note: Cox regression models: the hazard (instantaneous risk) of an event (emerging from the shelter or approaching the novel object); a higher hazard (positive coefficient) indicates a shorter latency to emerge from the shelter or approach the novel object (i.e., bolder or greater exploratory tendency). * P < 0.05.

aAn indicator variable for the focal individuals that received a pre-designated winning (W) or losing (L) experience; the baseline group comprised the individuals that received no experience.

bAn indicator variable for the focal individuals that lost more than 1 month previously; the baseline group comprised the individuals that won a contest more than 1 month previously.

Fig. 3.

Fig. 3

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The relationship between the latency to display (rank score) and (A) the latency to emerge from the shelter (rank score), (B) the latency to approach a novel object (rank score), and (C) whether the test individual entered the correct reservoir for different experience treatments (red: winning experience, black: no experience, blue: losing experience). The bars in (C) represent the mean rank scores (±SE); and the sample size for each bar is indicated in the parentheses.

Previous contest experience significantly influenced the latency to approach a novel shelter. When analyzed separately, a winning experience nonsignificantly lowered while a losing experience nonsignificantly raised the hazard of approaching the shelter. These trends, for individuals with a winning experience to be slower and those with a losing experience to be quicker to approach the novel object, were the opposite of what might be expected. The model also showed that less aggressive individuals (those with a longer latency to display) had a lower hazard of approaching the novel shelter (i.e., were slower or took longer to approach), which confirmed the positive correlation found between the two latencies in the previous section. The interaction effect between previous contest experience and the latency to gill display was not significant, indicating that such experience did not significantly alter the relationship between the two latencies (Fig. 3B). The model also confirmed that individuals with higher pre-experience levels of testosterone had higher hazards, i.e., were quicker to approach the novel shelter.

Logistic regression showed that previous contest experience significantly influenced the probability that the fish would enter the correct reservoir; individuals with a winning experience had a nonsignificantly higher probability of entering the correct reservoir than those with no contest experience, while those with a losing experience had a significantly lower probability. The model also confirmed that latency to gill display, pre-experience testosterone, and pre-experience cortisol were not significantly correlated with the probability of entering the correct reservoir. The interaction effect between previous contest experience and the latency to gill display was not significant, indicating that such experience did not significantly alter the relationship between the probability of entering the correct reservoir and the latency to gill display (Fig. 3C).

Discussion

This study showed that individual mangrove rivulus, like some other species of fish (see Budaev and Brown 2011 for a review), behaved consistently in different situations (context generality) (Groothuis and Trillmich 2011): individuals that were quicker to display to their own mirror image were also quicker to emerge from a shelter and quicker to approach a novel shelter, which suggests an aggression–boldness–exploration syndrome. All three behaviors correlated positively with pre-experience testosterone levels. How fast the fish displayed to its mirror image and emerged from a shelter also correlated positively with pre-experience levels of cortisol. The two behaviors that correlated positively with levels of both testosterone and cortisol were more strongly correlated with each other than with the tendency to explore, which correlated positively with testosterone but not with cortisol. These results indicate that testosterone and cortisol play important roles in mediating the correlations among these behaviors. Testosterone has long been linked to aggression although this relationship can be complex and modulated by season, sex, and mating system (Wingfield 2005). There are fewer studies relating boldness and exploratory tendency with testosterone, and studies that do explore such relationships do not always reach the same conclusions. In house sparrows (Passer domesticus), no relationship was found between exploratory behavior (the total number of objects visited in 30 min in a novel environment) and testosterone levels (Mutzel et al. 2011). In great tits (Parus major), fast explorers (based on scores from novel-environment and novel-object tests) were more aggressive and had better competitive ability than slow explorers (Verbeek et al. 1996), but, surprisingly, the fast explorers had lower baseline testosterone levels than did the slow explorers (van Oers et al. 2011). To complicate matters further, Japanese quail chicks (Coturnix japonica) that hatched from testosterone-injected eggs approached novel objects sooner and took longer to start distress vocalizing in open-field tests than did those hatched from ordinary eggs (Daisley et al. 2005).

Glucocorticoids are often associated with stress, but the relationships between glucocorticoids and aggression, boldness, and exploration are far from clear. Aggression can be stressful for both the initiator and the receiver (Summers et al. 2005). Acute elevation of glucocorticoids intensifies aggression (rats; Mikics et al. 2004) but chronically elevated glucocorticoids tend to reduce aggression (rainbow trout; Øverli et al. 2002). In rainbow trout (Oncorhynchus mykiss), individuals that display a low cortisol response to stress resume feeding sooner after the stressor than those displaying higher cortisol responses, show enhanced aggression and social dominance, and retain a learned fear response for longer (Pottinger and Carrick 1999, 2001; Moreira et al. 2004; Øverli et al. 2005, 2007). Similarly, in great tits, fast and slow explorers differed in their aggressiveness (Verbeek et al. 1996), but although their baseline levels of corticosteroid did not differ, the slow explorers’ corticosteroid levels were elevated after being exposed to a resident while the fast explorers’ levels remained relatively unchanged (Carere et al. 2003). In contrast to these trends, cortisol levels were positively correlated with androgen levels in bison bulls (Bison bison) and were higher in the more highly ranked dominant bulls, which also copulated more frequently (Mooring et al. 2006). To summarize, the relationships between behaviors and testosterone and glucocorticoids appear to differ among species and contexts. Our study reveals that, in K. marmoratus, the readiness to display, to emerge from a shelter, and to approach a novel shelter is positively correlated with the level of testosterone and/or cortisol. Interestingly, these behavioral traits are more strongly related to hormone levels occurring before the experience (hereafter called “pre-experience hormones levels”) than to those occurring after the experience (hereafter called “post-experience hormone levels”), despite the fact that post-experience hormones were measured on the same day as, and immediately before, the behavioral tests, whereas pre-experience hormones were measured 1 day previously. It therefore appears that these behavioral traits are better predicted by baseline (i.e., pre-experience) hormone levels. As the levels of neither testosterone nor cortisol were systematically altered by contest experience but the relationships between pre-experience and post-experience hormones were not perfect (rs for testosterone = 0.63, for cortisol = 0.52), it may be that contest experience acted as a disturbance to the hormones, causing them temporarily and unsystematically to deviate from baseline levels such that post-experience hormones displayed weaker relationships with the behavioral traits than did pre-experience hormones. In juvenile mulloway, the measurements of cortisol concentrations of the blood taken 1 month previously were still predictive of the fish's boldness measured 1 month later (Raoult et al. 2011), indicating that the relationship between baseline hormones and behavioral traits can remain stable for a long time. Another possible explanation of why the behavioral traits display stronger relationships with pre-experience hormones is that they represented the fish's behavioral and hormonal responses when it encountered a novel test procedure. When a fish was put through a behavioral test, it was always the first time that the fish encountered the tank set-up and experimental procedures for the test. When collecting the water samples for pre-experience hormones, it was also the first time the fish went through the procedures, but for post-experience hormones, it was the second time that the fish went through the procedures. It is, however, worth noting that post-experience hormones were highly correlated with pre-experience levels, indicating that individual variation in nonmanipulated testosterone and cortisol levels in this fish are useful in predicting the individual variation in the levels of the same hormones after exposure to different contest experiences (Williams 2008).

The result that the fish's aggressiveness (readiness to display to mirror image) was positively correlated with testosterone levels is comparable with the findings by Earley and Hsu (2008), who staged contests between a larger and a smaller fish and examined whether and how their baseline hormones might influence their interactions during contests. The study found that the larger opponents’ probability of initiating attacks was associated positively with the larger opponent's testosterone level but negatively with the smaller opponent's. When the larger opponents won, their frequency of delivering post-retreat attacks to the losers was positively correlated with their testosterone levels although no significant relationship was observed when the smaller opponents won. Overall, these findings and the results in this study reveal a robust positive relationship between testosterone levels and aggressiveness in the fish. The relationship between cortisol levels and aggressiveness in this fish, however, was not nearly as straightforward. In the study of Earley and Hsu (2008), the larger contestant's probability of initiating attacks had no significant relationship with its own cortisol level but was strongly and positively correlated with the smaller contestant's cortisol level. When the larger contestants won, their post-retreat attack frequency was negatively associated with their cortisol level but, when the smaller contestants won, their post-retreat attack frequency was positively associated with their cortisol level. Considering these findings together with those of this study, the relationship between cortisol levels and aggressiveness in this fish can depend on the role of the individual vis-à-vis its opponent (the larger or the smaller contestant), the type of interaction (dyadic contest or observing mirror image), and the potential cost of the behavior being measured (attacks being more dangerous and thus more costly than displays). Further studies will be necessary to understand the role of cortisol in aggression in this fish better.

The fish's levels of testosterone or cortisol were not significantly altered after a winning, losing or no contest experience. A recent victory, however, influenced the behavioral traits of K. marmoratus in different ways than did a recent defeat. A winning experience tended to have a positive effect on the readiness to perform gill displays to its mirror image, had no effect on the readiness to emerge from a shelter, a negative effect on readiness to approach a novel shelter, and a positive effect on learning performance, while a defeat influenced these behaviors in opposite directions. Interestingly, although winning and losing had different effects on aggression, boldness, and tendency to explore, it did not have any significant effect on the correlations between them. Conversely, although victories and defeats affected aggression and learning performance in the same way, this did not bring about a positive relationship between them. Thus, it appears that, in this fish, the relationships between different behaviors are relatively robust: even though individual behaviors are affected in different ways by winning/losing experiences, the relationships between them remain relatively stable across the different experience treatments. These results may indicate that the physiological mechanisms underlying behavioral correlations differ from the mechanisms mediating the effects of experience on these behaviors—an indication that deserves further investigation. These results also show that individual behavioral components of a suite of correlated behaviors can be flexible to respond differently to environmental stimuli while maintaining the relationships between them. That different behavioral components of a suite of correlated behaviors may respond differently to the same environmental factors is also reported for zebrafish (Norton et al. 2011). The zebrafish study showed that a single gene, fgfr1a, simultaneously modulates aggressiveness, boldness, and exploratory activity. The authors went on to compare aggressiveness and boldness (but not exploratory activity) between fish from different locations, and found significant differences in between-location aggressiveness but none in boldness, which led them to conclude that aggressiveness is more likely to respond to environmental factors than is boldness. Thus, our study and that of Norton et al. (2011) both showed that behaviors that are linked by common proximate mechanisms could maintain differential flexibility in response to environmental factors.

Our study showed that the learning performance of K. marmoratus (whether or not a fish entered the reservoir that it was trained to enter) was not significantly correlated with the readiness to display to a mirror image, to emerge from a shelter or approach a novel object; nor did it correlate with levels of testosterone or cortisol. Previous studies of learning performance in some other species have shown it to correlate positively with aggressiveness, dominance status, and/or boldness and negatively with glucocorticoids. Aggressive individuals performed better in avoidance-learning tasks in male mice (Benus et al. 1989) and guppies (Zhuikov 1993). In rainbow trout, bolder individuals learned faster in conditional-feeding tasks (Sneddon 2003) and individuals that had low post-stress levels of cortisol tended to retain a learned fear response for longer than did those with high post-stress cortisol levels (Moreira et al. 2004). In male CD-1 mice, individuals with a greater drop in corticosterone levels between two sampling events (roughly 6 weeks apart) performed better in T-maze tests with a food reward (Fitchett et al. 2009). In mountain chickadees (Poecile gambeli), subordinates and dominants did not differ in corticosteroid levels but the subordinates cached less food, were less efficient at retrieving caches, and performed worse in the spatial memory task than did dominants (Pravosudov et al. 2003). Although the probability of K. marmoratus entering the correct reservoir was not related to its aggressiveness or to hormone levels, it was positively influenced by winning and negatively influenced by losing. This accords with the findings that dominant individuals perform better in spatial learning than do subordinates. In male African cichlid fish (Astatotilapia burtoni), individuals that learned in a spatial learning test had higher immediate early gene mRNA levels in the dorsolateral telencephalon than those that did not learn (Wood et al. 2011). Future exploration of the influence of winning or losing on the immediate early gene activity within the dorsolateral telencephalon might provide useful information on how winning/losing modifies fish's learning performance.

In this study, a winning experience negatively influenced and a losing experience positively influenced the readiness to approach a novel object; this was the opposite of what we expected. One potential explanation for these trends is that the object used was a shelter and that individuals of K. marmoratus, being more eager to hide after losing, approached the shelter more quickly. However, although winning and losing did significantly influence how quickly individuals approached the shelter, they did not influence the probabilities that the fish would enter it (W = 30/60, N = 29/60, L = 34/60). As defeated individuals tend to behave more submissively and lose subsequent contests with other individuals, being more willing to explore new resources could perhaps increase their chances of finding alternative, unoccupied resources, which could be an adaptive trait for losers.

Overall, our study showed that aggression is positively correlated with boldness and exploratory tendency and that testosterone and cortisol play important roles in the correlations between these behavioral traits. Interestingly, the three correlated behavioral traits responded differently to winning and losing but the correlations among the behaviors were preserved. Although learning performance was not significantly correlated with aggression, it too was modulated by winning and losing. This study shows that individuals change multiple behavioral traits in response to one single environmental stimulus, and that although the behavioral traits are correlated with each other, the responses to such stimuli can differ. A more comprehensive knowledge of these types of changes could perhaps enable us to have a more integrative understanding of how animals adapt to variable environments.

Funding

Supported by Taiwan National Science Council (NSC 97-2621-B-003-005-MY3).

Acknowledgments

We thank Alan Watson for help with the manuscript. We thank Hal Heatwole and two anonymous reviewers for thorough and helpful comments that improved the quality of the article. This work was presented at the symposium “Mangrove ‘Killifish’: An Exemplar of Integrative Biology” thanks to support by an NIH Conference Grant R13HD070622 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development; SICB through the DCE, DCPB, DAB, and the C. Ladd Prosser Fund; and the College of Agriculture and Natural Resources, University of Maryland.

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