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Steroid hormones and agonistic behavior in a cichlid teleost, Aequidens pulcher
HORMONES AND BEHAVIOR
19, 353-371 (1985)
Steroid Hormones and Agonistic Behavior in a Cichlid Teleost, Aequidens p&her A. D. MUNRO’
AND
T. J.
PITCHER
School of Animal Biology, University College of North Wales, Bangor, Gwynedd, LL.57 2UW, United Kingdom The effects of three steroid hormones on the agonistic behavior of female Aequidens pulcher have been evaluated. Testosterone, estradiol, and cortisol were tested using an immersion technique to minimize trauma, and we also examined metyrapone, a blocker of cortisol biosynthesis. Two different experimental protocols were employed, the first investigating agonistic interactions within groups of fish, and the second examining the responses of isolated fish to models and mirrors. Differences between replicates were small, and both protocols supported similar conclusions. Each of the three hormones produced a characteristically different spectrum of behaviors when compared to the controls. Testosterone increased agonistic behavior in all experimental situations, while estradiol had a generally opposite effect; this may reflect the natural modulation of behavior by hormones during the reproductive cycle of A. pulcher. Cortisol also had distinct behavioral effects; available evidence suggests that this steroid increases submissive components of agonistic behavior, and that observed increases in some aggressive components are an indirect consequence, dependent upon the feedback of social information received by each fish. Metyrapone treatment greatly reduced all agonistic behaviors, groups of fish forming shoals typical of juveniles. This was not reversed by replacement therapy with cortisol, which suggests that metyrapone affects behavior by an alternative, possibly toxic, mechanism. 0 1985 Academic Press. Inc.
INTRODUCTION Agonistic behaviors have been observed in a wide variety of teleost fish, particularly in the context of reproductive behavior. This has lead to the hypothesis that reproductive hormones are important modulators of aggressive behavior, although attempts to test this hypothesis have often yielded negative or conflicting results (Liley, 1969; Liley and Stacey, 1983; Munro and Pitcher, 1983). There is evidence that testosterone does stimulate aggression in some cichlids (Rixner, cited by Reinboth, 1972; Wapler-Leong and Reinboth, ’ To whom correspondence should be addressed: Department of Zoology, The National University of Singapore, Kent Ridge, Singapore 0511. 353 0018-506X/85 $1.50 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
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1974; Fernald, 1976),although work published to date is not well quantified. In this respect, it is surprising that cichlids have not received more attention, in view of the numerous studies of agonism and its underlying motivation in these fish (e.g., Baerends and Baerends-van Roon, 1950; Greenberg, Zylstra, and Baerends, 1965; Heiligenberg, 1974; Barlow and Ballin, 1976; Vodegel, 1978). The first aim of this report is to confirm quantitatively that testosterone does affect aggressive and other components of agonistic behavior in a cichlid fish, Aequidens pulcher (the blue acara). Following Barlow and Ballin (1976), we have also considered submissive components of agonist interactions, in order to gain a broader perspective of the behavioral effects of this steroid. In mammals, testosterone must first be aromatized to estradiol in order to affect some behavioral activities (Leshner, 1978); therefore, we have also examined the effects of estradiol treatment on agonism in A. p&her. Like sex steroids, corticosteroids may also affect agonistic behavior in some mammals (Leshner, 1978, 1981; Brain, 1980). Unlike testosterone, which increases aggressive components in some mammals, corticosteroids act by increasing mainly the submissive components of agonistic interactions. Surprisingly, there are no studies reported for the behavioral effects of corticosteroids in nonmammals. We have therefore also examined the effects of the principal teleost corticosteroid, cortisol, on agonistic behavior in A. p&her. In addition we have also tested the effects of metyrapone, a drug which blocks the biosynthesis of corticosteroids. Previous work on the endocrine control of agonistic behavior in teleosts is subject to various criticisms (Liley, 1969; Liley and Stacey, 1983; Munro and Pitcher, 1983). All lack a thorough analysis of the data, which generally relate only to the aggressive components of agonistic behavior. Only one test situation was used in most cases, which makes it difficult to be sure whether any observed effects are relevant to the fishes’ natural life history, or are merely a product of the artificial test environment. We have attempted to avoid these criticisms by comparing results for two quite different experimental protocols: isolated fish presented with mirrors and models (Munro, 1985), and hierarchical groups. In l&ra&icaJ groups, we have examined the effects of steroid treatment on submissive as well as aggressive behaviors, and we have also assessed the effects on hierarchical structure. We have used hierarchical groups to avoid the mortalities often associated with territorial situations. If sex steroids do affect aggression, then using fish of both sexes or at widely different stagesof reproductive development might be expected to give wide variation between individuals; we have used only immature females to avoid these sources of variability, and we have also made observations only at a particular time of day to avoid any variability due to diurnal fluctuations in behavioral activity.
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Previous workers have administered testosterone by manual injection. We have used an immersion technique, as this has been shown to produce more stable elevated levels of circulating hormone (Eales, 1974; TerkatinShimony and Yaron, 1978), besides being less traumatic. The dosages used here are based on patallel physiological studies on A. p&her (Munro, in preparation). MATERIALS AND METHODS Animals. A. p&her (A. latifrons), mean weight 7.7 -+ 0.9 g and mean standard length 59.0 + 5.8 mm, were purchased from Aquatic Nurseries Ltd. (Hampton, Middlesex). Only maturing females, likely to be in the final stages of vitellogenesis (Munro, 1982), were used: males (identified by their conical genital papilla) were discarded. The fish were maintained in a stock tank prior to use. All stock and experimental fish were maintained under a 12L: 12D photoperiod, at 27 + 1°C. They were fed daily, early in the light cycle. Hormone treatments. The steroids used were testosterone propionate, estradiol-17P benzoate, and 17o-hydroxycorticosterone (cortisol) (Sigma, U.K.). The dosage used was 500 pg.liter-’ of aquarium water, each 500 (ug of the steroid having initially been dissolved in 50 ~1 of dimethyl formamide (DMF; Sigma, U.K.). Control fish received an equivalent dose of DMF alone. Other fish were treated with 30 mg.liter-’ of metyrapone (SU 4885; Ciba-Geigy Ltd., U.K.); at this dosage, the pituitaryadrenocortical axis is activated due to inhibition of corticosteroid synthesis (Munro, 1984b). Also, other fish were treated with 30 mg.liter-’ metyrapone in combination with 500 pg.liter-’ cortisol. Experimental procedures. (1) Groups of fish. A suitable population density for experiments on dominance hierarchies proved to be six fish in a 60 x 24 x 24-cm observation tank. Each experimental fish of such a group was individually isolated for 4 days prior to the start of the behavioral experiments. The appropriate hormone treatment (dosages as above) was added to each isolation tank on the first day of this acclimation period. The water received no filtration, aeration, or further treatment. On the fourth day of the acclimation period, each of the fish was identified by a subdermal injection of red or blue rubber latex (Riley, 1966), and the members of the group were placed together in a freshly treated, bare observation tank screened on three sides. Behavioral observations were recorded on an audiotape from behind a curtain; they commenced the day after the members of the group had been introduced to each other, and continued over 4 days. Each group was observed for 10 min daily, recordings being made late in the photophase (7-8 hr after lights on, and 5-6 hr after feeding), to avoid any interference of feeding on hierarchical structure (Barlow and Ballin, 1976). Eight
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behaviors were scored: Frontal and Lateral Displays (after Baerends and Baerends-van Roon, 1950), Charge, Approach, Flee, Avoid, Bite/Bite At, and Forage (all after Barlow and Ballin, 1976). Courtship behaviors (Baerends and Baerends-van Roon, 1950) were not observed. The transcript provided details of each agonistic encounter between identified fish. On the basis of these various encounters, a dominance hierarchy (Myrberg, 1972; Barlow and Ballin, 1976) was constructed. Activity was measured from the number of times that individual fish crossed two vertical lines on the tank front; this was recorded for 2-min periods immediately before and after the main observation period, and incidentally confirmed the absence of territoriality in all cases. The testosterone, estradiol, cortisol, metyrapone, and control treatments were repeated on five different groups of fish. Three groups of fish were treated with metyrapone in combination with cortisol. 2. Isolated fish. After a 4-day acclimation period in isolation similar to the groups above, test fish were transferred to separate 18 x 24 x 24-cm observation tanks containing freshly treated water. Again no filtration or aeration was supplied. Next day, and for the 3 days thereafter, each fish was presented with a 87 x 64-mm mirror, lowered into the tank from behind a screen. Observations were made from behind the screen, and recorded onto audiotape. Recording commenced with the first aggressive response of the fish to the mirror, and continued for the next 2 min, after which the mirror was removed. Three aggressive behaviors were scored: Frontal and Lateral Displays, and Bite. These behaviors are identical to those seen in groups of fish. Thirty minutes later, a model of a dominantly colored (barred) conspecific, standard length 46 mm, was lowered into the tank, and the fish’s behavior recorded for the next 2 min. The data of Heiligenberg (1974) indicate that 30 min was sufficient time for aggression to return to near baseline levels in another cichlid, Haplochromis burtoni. Control fish received DMF alone. In the first series of experiments, there were nine fish for each of the treatments control, 500 pg.liter-’ testosterone propionate and 500 pg.liter-’ estradiol-17/3 benzoate. In the second series, there were three fish for each of the treatments: control, 500 pg.liter-’ cortisol, and 30 mg.liter-’ metyrapone. RESULTS
1. Groups of Fish Members of each group interacted as a relatively stable nip-dominant hierarchy (Myrberg, 1972), with the exception of metyrapone-treated groups which schooled and had significantly higher activity scores (Fig. 1). As each group member was identifiable from its latex marking, its social ranking in relation to other group members could be determined using the “combined index of attack” of Barlow and Ballin (1976). The
STEROIDS AND CICHLID AGONISM
40.
”
CORTISOL
P-
OESTRADIOL
40
100
*
ttlllllflnnn 40.
-
*
rt A
357
-
In
TESTOSTERONE *
* *
FIG. I. Bar graphs of the mean frequencies of each of the behaviors scored in groups of treated fish for 10 min. together with activity scores; those significantly different from control values at the 5% level are indicated by an asterisk (LSD test with planned comparisons).
three most dominant fish ((Y,p, and y), and the most submissive (5) could be easily recognized with all treatments except metyrapone. Defining an agonistic encounter as an unbroken sequence of agonistic behavioral interactions between two identified fish, the numbers of agonistic encounters per IO-min observation period for each treatment were compared using two-way analysis of variance (Table la). There were no significant differences among the 4 days of each treatment, nor was there any significant interaction; there was a significant difference between hormone treatments, however. Testosterone significantly increased the mean frequency of agonistic encounters, while both estradiol and metyrapone significantly reduced their frequency (Table lb). The mean frequency seen in cortisol-treated groups was intermediate between those for controls and testosterone, and not significantly different from either. Groups treated simultaneously with cortisol and metyrapone were comparable with those treated with metyrapone alone (three replicate groups of fish, mean frequency of agonistic encounters 4.2/10 min). For the initiator of an agonistic encounter, the outcome may be either
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TABLE 1A Analysis of the Total Frequencies of Agonistic Interactions Scored in Groups of Six A. p&her for the Five Treatments Two-way analysis of variance
Hormones Days Interaction Residual Total
d!
Sum of squares
4 3 12 80 99
92,467 191 1,307 32,840 126,805
Variance
F value
23,117 64 108 411
56.3 0.2 0.3
P
successful (the initiator wins the encounter, with the other fish either Avoiding or Fleeing), unsuccessful (it loses), or inconclusive (there is no obvious winner). The steroid treatments each affect the balance between these various possible outcomes. In control groups, a mean proportion of 17.4% of all successful attacks for the initiator are directed against a higher ranking fish; the mean value for cortisol-treated groups, 10.7%, is significantly lower (F = 14.4, $1 and 15; P < O.Ol),while the values for both testosterone (12.5%) and estradiol (12.7%) are intermediate, and significantly different from neither. For all four treatments, the great majority of both unsuccessful and inconclusive attacks were initiated against fish of a higher rank. There are no significant differences among treatments for the relative frequencies of either unsuccessful or inconclusive encounters. However, there is a significant positive correlation between the frequencies of total and inconclusive agonistic encounters in both testosterone- and estradiol-treated groups; and a significant negative correlation in those treated with cortisol; the trend for control groups is not significant (Table 2). No such correlations were detectable for the frequencies of unsuccessful attacks. TABLE 1B Tukey (LSD) Test of the Means of Each of the Five Treatments Mean frequency of agonistic interactions Testosterone Cortisol Control Estradiol Metyrapone
92.4 81.0 ,I 71.4 55.4 5.3
Note. Lines join pairs of means which are not significantly different at the 95% confidence level.
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STEROIDS AND CICHLID AGONISM TABLE 2 Correlations for Treated Groups for the Total Numbers of Agonistic Encounters with the Frequencies of Inconclusive Interactions (in which neither participant clearly wins): 20 Replicates in Each Case Pearson coefficient
Treatment Testosterone Cortisol Control Estradiol
+ 0.592 - 0.605 + 0.218 +0.515
F
P
11.34 7.52 1.25 6.15
co.01 co.01 NS co.05
It is thus apparent that hormone treatments may alter both the frequency and the pattern of interactions within a hierarchy. A broadly similar pattern is seen if the frequencies of the eight behaviors scored (the seven agonistic behaviors together with Forage) are summated and compared by three-way analysis of variance for the five treatments over the 4 days (Table 3a). Again there are no significant differences between the four replicate days. The hormone treatments produced significant differences in behavior and in addition this analysis clearly demonstrated that the behaviors responded differently to the five treatments (Table 3a). The mean frequency for total behavior is significantly lowered in metyrapone-treated groups, and significantly raised in those treated with testosterone, compared to controls (Table 3b). Groups treated with estradiol are not significantly different from controls. The cortisol-treated groups show a significantly greater mean value for total numbers of TABLE 3A Three-Way Analysis of Variance of the Eight Behaviors Scored for Groups of Six A. pulcher for the Five Treatments” Sum of squares
Variance
F
P
4 7 3 4 28 12 21
121 70,931 244 33,381 19,299 442 2,013
30.3 10,132.9 81.4 8,345.5 689.3 36.8 95.9
0.2 14.7 0.6 12.1 4.6 0.3 0.6
CO.8 <0.001 CO.6
84 636 799
3,111 94,836 224,380
37.0 149.1
0.2
df Replicates Behaviors Days Hormones Behaviors x Hormones Days x Hormones Behaviors x Days Behaviors x Days x Hormones Residual Total
a Fifty-seven percent of the total variation is accounted for.
CO.8
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MUNRO AND PITCHER TABLE 3B Tukey (LSD) Test for the Mean Number of Behaviors for Each of the Five Treatments Mean number of behaviors Testosterone Cortisol Control Estradiol Metyrapone
25.71** 23.12* 19.19 16.16 1.36**
* Significantly different from control (P < 0.05). ** Significantly different from control (P < 0.01).
behaviors (Table 3b), but this result for cortisol is not seen if Forage is excluded from the analysis. The mean frequencies for each of the eight behaviors for each hormone treatment were compared with those for controls using a least significant difference (LSD) test (planned comparisons, Sokal and Rohlf, 1%9), as indicated in Fig. 1. Metyrapone significantly reduced the frequencies of all eight behaviors. Testosterone significantly elevated the frequencies of five of the seven agonistic behaviors: Approach, Charge, Avoid, and both Displays. Cortisol only caused a significant increase in Charge and Lateral Display; it also significantly elevated the frequency of Forage, despite the absence of food or detritus on the tank bottom. Overall, estradiol-treated groups were most similar to controls, but each agonistic behavior (except for Lateral Display) tended to be less frequent, this reduction being significant for Approach (Fig. 1). The frequencies of the seven agonistic behaviors scored, together with Forage, were compared using one-way analysis of variance both (i) within each treatment, among the six ranks, and (ii) between each treatment for each of the six ranks (Table 4). This was in order to determine whether hormone treatments which alter the frequencies of specific behaviors (Fig. 1) do so for all group members equally, or whether these effects are restricted primarily to one or more ranks. Attack behaviors showed a positive correlation of frequency with rank, although the only significant difference is for Charge in a-fish compared to all other ranks for all treatments (Table 4a). Comparison of particular ranks between treatments indicates that mean Charge frequencies are significantly raised in the a-fish of cortisol-treated groups, and significantly reduced in the p-fish of groups treated with estradiol; all other changes are nonsignificant (Table 4b). The other attack behavior, Approach, is not significantly different for comparisons between ranks, but it is with comparisons between treatments for a-fish: frequencies for estradiol-
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TABLE 4 The Effects of Hormone Treatments on the Organization of Hierarchies, Using OneWay Analysis of Variance Tests (T, Groups Treated with Testosterone; E2, Those Treated with Estradiol; F, Those Treated with Cortisol; and C, Controls) Behavior
F
C
22.6’ 5.6 1.0” 1.8 4.9c,D 0.5 4.3 6.7 1.3” 2.2A.c 0.7’
11.3A 8.4 0.9d 3.P 3.3O 0.5 4.2 4.5 1.9 1.2 1.2
b. Comparisons within individual ranks between treatments 17.4 10.6” 22.6‘+’ 4.0”’ 9.2’ 5.6 ; Approach 12.6’ 8 3”.b 12.4A Avoid 4.18 3:6.” 1.8’++ ; Frontal display 2.7B 2.1 1.3b a 2.2,Q 1.2b o.7b Y 5 5A.B.C Lateral display 4.0” 2.9’ 6: lA,C 2.2 2.9”
11.3b 8.4* 10.3 3.1 1.9 1.2” 1.9 2.0
Charge Avoid Flee Frontal display
Charge
Rank
T
E2
a. Comparisons between ranks for each treatment 17.4= 10.6’ 9.2’ 4.0 ; O.jd 0.6“ 4.P 3.6* ; 4.jD 3.6D Y 0.2 o.2b,c ; 3.6’.” 4.4B Y 7.0” 4.6’ 2.7 2.1 ; 2.0 1.4 Y 2.2 1.2
Note. The values given are the means for 20 replicates. Key to superscripts: A signi8cantly greater than n (P < 0.05); B significantly greater than b (P < 0.01); and C significantly greater than c (P < 0.001).
treated groups are reduced compared to those treated with either testosterone or cortisol, with controls occupying an intermediate position (Table 4b). Both of the flight behaviors, Avoid and Flee, show a negative correlation with rank for each treatment, but this is generally only significant for (Yfish compared to other ranks (Table 4a). However, the frequency of Avoid in the p-fish of groups treated with cortisol is also significantly lower than the frequency for more subordinate fish. Comparison of the same rank between treatments (Table 4b) indicates that Avoid is significantly less frequent in the P-fish of cortisol-treated groups than in groups treated with either testosterone or estradiol. Cortisol-treated groups are unusual in that the frequency of Frontal Display in p-fish is significantly higher than that for either a-fish or yfish (Table 4a). Treatment with testosterone increases the frequencies of Frontal Display mainly for the top three ranks, compared to other groups, but this difference is significant only for y-fish (Table 4b).
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Lateral Display is significantly increased for the top two ranks of testosterone-treated groups compared to other treatments. For all treatments, there was no correlation of the frequency of Forage with rank, with each fish showing a wide variation over the 4-day observation period. To conclude, it is apparent that each hormone treatment has different effects on particular ranks in affecting the overall frequencies of particular behaviors (Fig. 1). Thus the significant increase in frequency of Charge in cortisol-treated groups, and the two Displays in testosterone-treated groups can be attributed to significant changes in one or more ranks, while other significant changes in overall behavioral frequencies are apparently more evenly spread over several ranks (e.g., Charge and Approach in groups treated with testosterone, and Lateral Display in those treated with cortisol). Contrariwise, although some behaviors may show no significant overall change as a result of hormone treatment, there are significant effects when individual ranks are compared: for example, the effects of cortisol on Frontal Display. Principal Components analysis (Maxwell, 1977) is a way of clearly summarizing differences between the overall spectrum of behaviors shown by an individual: it is free of the bias introduced when separate behaviors are selected for measurement and analysis (Huntingford, 1984). Such an analysis, based on a correlation matrix of the eight scored behaviors together with the measure for Activity across the five treatment groups, confirmed that each treatment had a different effect. No rotation was performed. Since analysis of variance had shown that there were no significant differences, we were justified in using the means over all 20 replicates (days x replicate groups) to simplify the analysis. The first two Principal Components account for 95% of the total variance; as the third and subsequent Components contribute only 5% or so, they can be safely be omitted from consideration. Each of the behaviors has a high and roughly equal loading on the first Component (Fig. 2), demonstrating that the distinguishing features of the acara’s behavior are genuinely multivariate. On the second Component, the most important behaviors are the two Displays, Charge, and Forage, confirming that no single behavior best discriminates the effects of the hormone treatments. Figure 3 illustrates the location of the five treatments on the first two Principal Components (i.e., the five treatments plotted in “hormone space”). The figure shows a clear separation of behavior for testosterone, cortisol, and metyrapone. Behaviors for these three treatments show a clear separation from estradiol and controls, whose positions in the center of the plot reflect behavior closest to the overall average for the experiment. On this basis, estradiol and control groups have the most similar behaviors. Cortisol is the next most similar to this group; testosterone is similar to cortisol on Component 1, but very dissimilar on Component 2. Component 1 mainly serves to separate metyrapone-treated groups from the others.
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P.C.2
P.C.l +2
0
-2
El-’
0
+2
i
’
FIG. 2. The distribution of the eight scored behaviors and activity within the first two principal components of the analysis of groups of fish. Principal component 1 (PCl) accounts for 85% of the explained variability, and PC2 for 10% of this, over the five treatments.
The results of an “inverse” Principal Components analysis (i.e., the eight behaviors plotted in hormone space) are shown in Fig. 4. This indicates that the eight component behaviors fall roughly into three clusters: Approach with Avoid, Charge with Flee, and the two Displays together with Bite and Forage. Each of the behaviors in the first two clusters is mutually complementary: Approach by one fish is frequently accompanied by the other fish Avoiding, for example. The feature shared by the third cluster is an expansion of the buccal cavity in the absence of any major forward movement; if Forage on the bare tank bottom represents a t3
P.C.2 t
CORTISOL
I
OEFRADIOL
I
0 METYRAPONE
TESTOSTERONE l
-3 1 FIG. 3. Principal components of controls and the four hormone treatments in “behavior space.” Both control and estradiol-treated groups lie close to the origin, while the other three treatments each occupy an isolated position within the plot.
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t3 AVOID l
t
0 APPROACH FORAGE BITE .t FRONTALtZ .DISPUY
P.C.1 -2
LATERAL
DISPLAY
FLEE .
0 CHARGE
FIG. 4. Principal components analysis of the eight behaviors scored for groups of fish plotted in “treatment space.” Approach and avoid are clearly separated as a unit from charge and flee; forage, bite/bite at, and frontal display together with lateral display form a third cluster.
displacement activity (see Discussion), then this also shares a basis of conflict with the two Displays (Baerends, 1974). 2. Individually
Isolated Fish
Fish consistently show more aggression to mirrors than to models of dominantly colored conspecifics. Testosterone increases the frequency of aggressive incidents for both model and mirror presentations (Table 5a), this increase being also seen for each of the three component behaviors scored (Lateral and Frontal Display, Bite/Bite At) apart from Bites against mirrors (Table 5b). Estradiol reduces the frequency of aggressive responses directed to models and, especially, to mirrors (Table 5a), but, of the three behaviors, there is only a significant difference for Frontal Displays (Table 5b). Cortisol also increased the frequency of aggressive incidents, but this is significant only for model presentations. Conversely, metyrapone reduced aggressiveness overall, but this reduction is only significant for mirrors (Table 5a). DISCUSSION
Our results confirm that sex steroids have a direct effect on aggressive behavior in cichlids. There was no difference between the four replicate
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TABLE SA Mean Frequencies of Aggressive Interactions for Mirror and Model Presentations to Isolated Fish” Mean frequency per 2-min response Treatment
Mirrors
Testosterone Cortisol
15.6
Estradiol Metyrapone Control
2.4 7.6
Models 5.6
7.8
6.3 0.4 2.3 I
1.8 5.3
0.3 2.3I
’ Lines join means which are not significantly different at the 95% confidence level (Tukey test); although the cortisohmetyrapone experiment (12 replicates) had to be run separately from that with testosterone/estradiol (36 replicates), their respective means are not significantly different.
days throughout the experiments, which suggests that stable, elevated levels of circulating hormones resulted from our immersion technique with the three steroids; and that the effects of metyrapone were also maintained over a prolonged period. In contrast, hormones have been administered by injection in most previous behavioral studies on teleosts, with the disadvantage that, where aggression is affected (e.g., Fernald, 1976),the subsequent distinct peak is difficult to compare across treatments. In our work, we have been able to show that each of the three steroids, and also metyrapone, had a characteristic effect on the pattern of agonistic behavior, which was encouragingly consistent across replicates and across both experimental protocols. TABLE 5B Mean Frequencies for the Three Aggressive Behaviors Shown by Isolated Fish to Mirror and Model Stimuli Mean frequency per 2-min response Behavior Lateral display
Frontal display Bite
Treatment
Mirrors
Models
Testosterone Control Estradiol Testosterone Control Estradiol Testosterone Control Estradiol
21.1 8.1 3.2 34.6 18.2 5.3 7.6 4.1 0.9
8.9 3.4 1.2 9.7 4.6 0.6 3.8 1.0 0.0 I
L?Lines join means not significantly different from control values at the 95% confidence level (Tukey test, 12 replicates for each).
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Testosterone Treatment Testosterone stimulated overall agonistic behavior in both experimental protocols. Lateral Display, Frontal Display, and Bites directed against mirrors or models increased in isolated fish treated with this hormone. Similarly, in groups treated with testosterone the two displays were significantly elevated, together with three of the five other agonistic behaviors scored: except for Lateral Display, these increases were uniform across all ranks in the group hierarchy. At present, we do not know if testosterone itself alters behavior, or whether a metabolite such as Sa-dihydrotestosterone or 1I-ketotestosterone is involved. 1I-Ketotestosterone is the principal androgen for the control of male secondary sex characters in teleosts (e.g., Hackman, 1974), but preliminary experiments suggest that it is not behaviorally active (Munro, unpublished). Stimulation of aggression by testosterone has been reported previously for both sexes of two mouth-brooding cichlids, Hemihaplochromis multicolor (Rixner, cited by Reinboth, 1972)and Haplochromis burtoni (WaplerLeong and Reinboth, 1974; Fernald, 1976) but changes in behavior following their single injections of hormone are difficult to interpret in detail. Estradiol Treatment Estradiol inhibited aggression by reducing the overall frequency of agonistic encounters. There was also a tendency to affect individual encounters through an increase in the number of agonistic acts per encounter, which is apparent in groups of fish with the failure of estradiol to reduce significantly the frequencies of six out of the seven scored agonistic behaviors below control values, despite the significant decrease in the frequency of agonistic encounters. A similar trend is seen in the mirror/model experiment. Principal Components analysis locates the effects of estradiol close to those of control, which raises the question of whether this hormone does in fact have a distinct affect on aggression; however, the distinct effects of estradiol on the responsiveness of isolated fish suggests that there is a definite effect. The effects of estradiol are different from those of testosterone, so we may safely conclude that testosterone does not have to be aromatized in order to affect agonistic behavior. It is not clear whether estradiol has a direct inhibitory effect on agonistic behavior (in which case, the ratio testosterone:estradiol may be more important than absolute levels of the individual hormones in the regulation of behavior); or whether it affects aggression indirectly, by inhibiting the secretion of pituitary gonadotropin, and hence gonadal testosterone, through negative feedback (Munro and Pitcher, 1983).
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Cortisol Treatment In affecting agonistic behavior, cortisol has a distinct action: its effects appear to be selective by behavior, by rank, and by stimulus. In isolated fish, aggressive encounters are increased for model presentations, whereas this is not the case for mirror experiments, nor for groups of fish. Nevertheless, there are differential effects in groups of fish compared to other treatments: some behaviors are generally increased (e.g., Lateral Display), some are elevated selectively across the hierarchy (e.g., Charge for a-fish), while others differ more among ranks than in control groups although there may be no overall significant change (e.g., Frontal Display). Leshner (1981) found similar, seemingly equivocal evidence for the effects of corticosteroids on agonistic behavior in mammals, due in part to differences between experimental protocols. Leshner concluded that the main effect of corticosteroids was to enhance submissiveness, on the basis of experiments with pairs of rodents. While directly parallel experiments remain to be performed with cichlids, an increase in submissiveness would explain the lack of an increase in aggression for mirror presentations, where fish would experience an abnormally immediate feedback (Pitcher, 1979) for any aggressive act. Dummies on the other hand provide no such feedback, so that with these cortisol would appear to increase aggression. Such an enhancement of submissiveness would also account for certain details of the behavior of groups of fish. Thus, fewer successful attacks were initiated by low rankers against high-ranking fish. Furthermore, in contrast to all other treatments, the greater the frequency of agonistic encounters in cortisol-treated groups, the fewer the number of inconclusive encounters, and hence the more defined the hierarchical structure. Despite the absence of food, cortisol treatment elevated the frequency of the behavior “Forage” in groups of fish (this behavior was not often seen in isolated fish). This behavior may have been “displacement foraging,” which occurs just before or after territorial boundary fights in other cichlids (Baerends and Baerends-van Roon, 1950; Heiligenberg, 1974; Fernald and Hirata, 1977). If our interpretation of Forage as a displacement activity is correct, then this complements our general conclusion that the principal effect of cortisol on agonism is to increase the submissive components of behavior. In this way, cortisol may increase aggression in high-ranking fish indirectly, by increasing submissiveness in lower ranks. As far as we are aware, this is the first experiment to describe the effects of cortisol on social groups of any vertebrate. Metyrapone Treatment This drug virtually abolishes agonistic encounters. In groups of fish, schooling behavior (Pitcher, 1983)occurs, associated with a general increase
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in activity. Aggression is also greatly reduced for mirror presentations to isolated fish: the smaller reduction for models may again reflect the differential feedback of these two stimulus situations for isolated fish. The effects of metyrapone could be due to three causes: (1), inhibition of corticosteroid synthesis through blocking 1lfi-hydroxylase; (2), inhibition of 11-ketotestosterone synthesis by the same mechanism; or (3), a nonspecific toxic effect. Simultaneous administration of cortisol with metyrapone should lead to elevated, rather than reduced, blood cortisol levels; the fact that groups of fish treated with this combination are behaviorally comparable with those which were treated with metyrapone therefore eliminates alternative (1) above. A derivative of testosterone, 1I-ketotestosterone is the main androgen regulating the development of secondary morphological sexual characters in teleosts (Hackman, 1974). Metyrapone blocks the conversion of testosterone to 11-ketotestosterone in the testes, leading to a elevated plasma testosterone levels (Duggan and Bolton, 1981). Preliminary experiments (Munro, unpublished) suggest that 11-ketotestosterone does not affect aggression in isolated blue acaras, unlike testosterone; this suggests that alternative (2) can also be ruled out. Metyrapone impairs learning in birds by a toxic action (Martin, 1978), and we suggest that this is the basis for our results with this drugalternative (3) above. Similarly, environmental stressors (including low temperatures) have been reported to induce schooling in other cichlids (Baerends and Baerends-van Roon, 1950; Greenberg et al., 1965). Such an effect was seen in the metyrapone-treated fish although levels of testosterone can be presumed to be rising in the plasma (Duggan and Bolton, 1981); this suggests that the behavioral effects of the androgen were being overridden, even after cortisol replacement therapy. The mechanism by which metyrapone overcomes the expected behavioral effects of these steroids is unknown. Conclusions
As in various other teleosts, aggressive interactions are not restricted to a particular context (for example, during reproduction) in cichlids: this is particularly true for aquarium populations, where aggression is generally increased (Liley and Stacey, 1983; Munro and Pitcher, 1983). Stress, and continued exposure to the same individuals, may contribute to the increased aggression seen in the aquarium (Barlow, 1974). This raises problems when trying to evaluate the significance of hormonal effects; nevertheless, the consistency of our results between the two different protocols-hierarchical groups and isolated fish-strongly suggests that the steroids tested may have behavioral roles in the natural life history of A. pulcher.
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In the wild, aggression in substrate-spawning cichlids like the blue acara apparently is mainly restricted to guarding of a spawning site (reviewed by Munro and Pitcher, 1983); indirect evidence suggests that plasma testosterone levels may be highest in both sexes at this time (Bogomolnaya, Rothbard, and Yaron, 1983), which suggests a causal relationship between circulating hormone levels and behavior. In common with mammals, cortisol secretion is elevated by a variety of stressors in teleosts, including social inferiority (Terkatin-Shimony, Ilan, Yaron, and Johnston, 1980; Schreck, 1981). In affecting submissiveness, elevated cortisol levels may be behaviorally important in the context of social inferiority, as has also been suggested for mammals (Leshner, 1981). As some, but not all, other stressors are reported to induce schooling behavior (which is the behavior normally seen in juvenile fish: Baerends and Baerends-van Roon, 1950), such stressors presumably override the behavioral effects of elevated cortisol levels (Munro and Pitcher, 1983). As with all previous work on teleosts, and seemingly also that on mammals and birds, we have used only one dosage of hormone. The absence of significant differences between individual isolated fish tends to rule out the possibility of large differences in sensitivity between fish. The possibility that there may be dose-dependent effects of these hormones on agonistic behavior remains to be tested: the experiments reported here suggest that tests using isolated fish would give valid results. In conclusion, although mammals have been used for most previous experimental work on steroids and agonism, teleost fish can provide useful general insights, especially in elucidating the subtleties of effects in relatively simple social groups which show fairly stylized behaviors. ACKNOWLEDGMENTS A.D.M. would like to thank the Science and Engineering Research Council for their support.
REFERENCES Baerends, G. P. (1975). An evaluation of the conflict hypothesis as an explanatory principle for the evolution of displays. In G. P. Baerends, C. Beer, and A. Manning (Eds.), Function and Evolution in Behavior, pp. 225-273. Oxford Univ. Press (Clarendon) London/New York. Baerends, G. P., and Baerends-van Roon, J. M. (1950). An introduction to the study of the ethology of cichlid fishes. Behaviour, Suppl. 1, 1-243. Barlow, G. W. (1974). Contrasts in social behaviour between Central American cichlid fishes and coral reef surgeon fishes. Amer. Zool. 14, 9-34. Barlow, G. W., and Ballin, P. J. (1976). Predicting and assessing dominance from size and colouration in the polychromatic Midas cichlid. Anim. Behav. 24, 793-813. Bogomolnaya, A., Rothbard, S., and Yaron, Z. (1983). Sex steroids in tilapia at various phases of the breeding cycle: Preliminary in vivo and in vitro studies. Absrruc?, tenth meeting of the European August. 1983.
Society for Comparative
Endocrinology,
Shejjield,
U.K.,
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Brain, P. F. (1980). Adaptive aspects of hormonal correlates of attack and defence in laboratory mice: A study in ethobiology. Progr. Bruin Res. 53, 391-413. Duggan, R. T., and Bolton, J. P. (1981). Androgen production by the plaice, PIeuronectes platessa: Effects of gonadotrophic preparations and metyrapone. Abstract, ninth meeting of the European
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Endocrinology,
Jerusalem,
August,
1981.
Eales, J. G. (1974). Creation of chronic physiological elevations of plasma thyroxine in brook trout, Salvelinus fontinalis (Mitchill) and other teleosts. Gen. Comp. Endocrinol. 22, 209-217. Fernald, R. D. (1976). The effect of testosterone on the behaviour and colouration of adult male cichlid fish (Haplochromis burtoni Gunther). Hormone Res. 7, 172-178. Femald, R. D., and Hirata, N. R. (1977). Field study of Haplochromis burtoni: Quantitative behavioural observations. Anim. Behav. 25, 964-975. Greenberg, B., Zylstra, J. J., and Baerends, G. P. (I%$ A quantitative description of the behaviour changes during the reproductive cycle of the cichlid fish Aeyuidens portalegrensis (Hensel). Proc. K. Ned. Akad. Wet. Ser. C 68, 135-149. Hackman, E. (1974). Einfluss von Androgenen auf die Geschlechtsdifferenzierung verschiedener Cichliden (Teleostei). Gen. Camp. Endocrinol. 24, 44-52. Heiligenberg, W. (1974). Processes governing behavioural states of readiness. Advances in the Study of Behaviour, Vol. 5, Academic Press, New York. Huntingford, F. (1984). The Study of Animal Behaviour, 411 pp. Chapman and Hall, London. Leshner, A. I. (1978). An Introduction to Behavioural Endocrinology. Oxford Univ. Press, London/New York. Leshner, A. I. (1980). The role of hormones in the control of submissiveness. In P. F. Brain and D. Benton (Eds.), Multidisciplinary Approaches to Aggression Research, pp. 309-321, Elsevier/North Holland, Amsterdam. Liley, N. R. (1969). Hormones and behaviour in fishes. In W. S. Hoar and D. J. Randall (Eds.), Fish Physiology, vol. 3, pp. 73-116, Academic Press, New York. Liley, N. R., and Stacey, N. E. (1983). Hormones, pheromones, and reproductive behaviour in fish. In W. S. Hoar, D. J. Randall, and E. M. Donaldson (Eds.), Fish Physiology, vol. 9B, pp. l-63, Academic Press, New York. Martin, J. T. (1978). The influence of metyrapone and pituitary-adrenal hormones on approach behaviour and imprinting in newly-hatched ducklings, Anas platyrhynchus. In P. J. Gaillard and H. H. Boer (Eds.), Comparative Endocrinology, p. 190, Elsevieri North Holland, Amsterdam. Maxwell, A. E. (1977). Multivariate Analysis in Behavioural Research, pp. 164, Chapman and Hall, London. Munro, A. D. (1982). Comparative Studies of the Forebrain of Aequidens p&her (Teleosrei; Cichlidae), with Particular Reference to the Control of the Pituitary Gland, 2 volumes. Ph.D. thesis, University of Wales. Munro, A. D. (1985). The effects of D-amphetamine, apomorphine, and chlorpromazine on the aggressiveness of isolated Aequidens p&her (Teleostei; Cichlidae). Psychoin press. pharmacology, Munro, A. D., and Pitcher, T. J. (1983). Hormones and agonistic behaviour in teleosts. In J. C. Rankin, T. J. Pitcher, and R. T. Duggan (Eds.), Control Processes in Fish Physiology, pp. 155-175, Groom Helm, London. Myrberg, A. A. (1972). Social dominance and territoriality in the bicolor damselfish, Eupomacentrus partitus (Poey) (Pisces, Pomacentridae). Behavior 41, 207-231. Pitcher, T. J. (1979). Sensory information and the organisation of behaviour in a shoaling cyprinid fish. Anim. Behav. 27, 126-149. Pitcher, T. J. (1983). Heuristic definitions of shoaling behaviour. Anim. Behav. 31, 611613.
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Reinboth, R. (1972). Some remarks on secondary sex characters, sex and sexual behaviour in teleosts. Gen. Comp. Endocrinol. Suppl. 3, 565-570. Riley, J. (1966). Liquid latex marking technique for young fish. J. Cons. Inr. Explor. Mer 30, 354-357. Schreck, C. B. (1981). Stress and compensation in teleostean fishes: response to social and physical factors. In A. D. Pickering (Ed.), Stress and Fish, pp. 295-321, Academic Press, New York/London. Sokal, R. R., and Rohlf, F. J. (1969). Biometry. Freeman, San Francisco. Terkatin-Shimony, A., and Yaron, 2. (1978). Oestrogens and oestrogenic effects in Tilapia aurea (Cichlidae, Teleostei). Ann. Biol. Anim. Biochem. Biophys. 18, 1007-1012. Terkatin-Shimony, A., Ran, Z., Yaron, Z., and Johnston, D. W. (1980). Relationship between temperature, ovarian recrudescence, and plasma cortisol levels in Tilapia aurea (Cichlidae, Teleostei). Gen. Comp. Endocrinol. 40, 143-148. Vodegel, N. (1978). A study of the motivation underlying some communicative behaviours of Pseudofropheus zebra (Pisces, Cichlidae): A mathematical model. Proc. K. Ned. Akad. Wef. Ser. C 81, 211-240. Wapler-Leong, D. C. Y., and Reinboth, R. (1974). The influence of androgenic hormones on the behaviour of Haplochromis burtoni (Cichlidae). Fortschr. Zool. 22, 334-339.
19, 353-371 (1985)
Steroid Hormones and Agonistic Behavior in a Cichlid Teleost, Aequidens p&her A. D. MUNRO’
AND
T. J.
PITCHER
School of Animal Biology, University College of North Wales, Bangor, Gwynedd, LL.57 2UW, United Kingdom The effects of three steroid hormones on the agonistic behavior of female Aequidens pulcher have been evaluated. Testosterone, estradiol, and cortisol were tested using an immersion technique to minimize trauma, and we also examined metyrapone, a blocker of cortisol biosynthesis. Two different experimental protocols were employed, the first investigating agonistic interactions within groups of fish, and the second examining the responses of isolated fish to models and mirrors. Differences between replicates were small, and both protocols supported similar conclusions. Each of the three hormones produced a characteristically different spectrum of behaviors when compared to the controls. Testosterone increased agonistic behavior in all experimental situations, while estradiol had a generally opposite effect; this may reflect the natural modulation of behavior by hormones during the reproductive cycle of A. pulcher. Cortisol also had distinct behavioral effects; available evidence suggests that this steroid increases submissive components of agonistic behavior, and that observed increases in some aggressive components are an indirect consequence, dependent upon the feedback of social information received by each fish. Metyrapone treatment greatly reduced all agonistic behaviors, groups of fish forming shoals typical of juveniles. This was not reversed by replacement therapy with cortisol, which suggests that metyrapone affects behavior by an alternative, possibly toxic, mechanism. 0 1985 Academic Press. Inc.
INTRODUCTION Agonistic behaviors have been observed in a wide variety of teleost fish, particularly in the context of reproductive behavior. This has lead to the hypothesis that reproductive hormones are important modulators of aggressive behavior, although attempts to test this hypothesis have often yielded negative or conflicting results (Liley, 1969; Liley and Stacey, 1983; Munro and Pitcher, 1983). There is evidence that testosterone does stimulate aggression in some cichlids (Rixner, cited by Reinboth, 1972; Wapler-Leong and Reinboth, ’ To whom correspondence should be addressed: Department of Zoology, The National University of Singapore, Kent Ridge, Singapore 0511. 353 0018-506X/85 $1.50 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
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1974; Fernald, 1976),although work published to date is not well quantified. In this respect, it is surprising that cichlids have not received more attention, in view of the numerous studies of agonism and its underlying motivation in these fish (e.g., Baerends and Baerends-van Roon, 1950; Greenberg, Zylstra, and Baerends, 1965; Heiligenberg, 1974; Barlow and Ballin, 1976; Vodegel, 1978). The first aim of this report is to confirm quantitatively that testosterone does affect aggressive and other components of agonistic behavior in a cichlid fish, Aequidens pulcher (the blue acara). Following Barlow and Ballin (1976), we have also considered submissive components of agonist interactions, in order to gain a broader perspective of the behavioral effects of this steroid. In mammals, testosterone must first be aromatized to estradiol in order to affect some behavioral activities (Leshner, 1978); therefore, we have also examined the effects of estradiol treatment on agonism in A. p&her. Like sex steroids, corticosteroids may also affect agonistic behavior in some mammals (Leshner, 1978, 1981; Brain, 1980). Unlike testosterone, which increases aggressive components in some mammals, corticosteroids act by increasing mainly the submissive components of agonistic interactions. Surprisingly, there are no studies reported for the behavioral effects of corticosteroids in nonmammals. We have therefore also examined the effects of the principal teleost corticosteroid, cortisol, on agonistic behavior in A. p&her. In addition we have also tested the effects of metyrapone, a drug which blocks the biosynthesis of corticosteroids. Previous work on the endocrine control of agonistic behavior in teleosts is subject to various criticisms (Liley, 1969; Liley and Stacey, 1983; Munro and Pitcher, 1983). All lack a thorough analysis of the data, which generally relate only to the aggressive components of agonistic behavior. Only one test situation was used in most cases, which makes it difficult to be sure whether any observed effects are relevant to the fishes’ natural life history, or are merely a product of the artificial test environment. We have attempted to avoid these criticisms by comparing results for two quite different experimental protocols: isolated fish presented with mirrors and models (Munro, 1985), and hierarchical groups. In l&ra&icaJ groups, we have examined the effects of steroid treatment on submissive as well as aggressive behaviors, and we have also assessed the effects on hierarchical structure. We have used hierarchical groups to avoid the mortalities often associated with territorial situations. If sex steroids do affect aggression, then using fish of both sexes or at widely different stagesof reproductive development might be expected to give wide variation between individuals; we have used only immature females to avoid these sources of variability, and we have also made observations only at a particular time of day to avoid any variability due to diurnal fluctuations in behavioral activity.
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Previous workers have administered testosterone by manual injection. We have used an immersion technique, as this has been shown to produce more stable elevated levels of circulating hormone (Eales, 1974; TerkatinShimony and Yaron, 1978), besides being less traumatic. The dosages used here are based on patallel physiological studies on A. p&her (Munro, in preparation). MATERIALS AND METHODS Animals. A. p&her (A. latifrons), mean weight 7.7 -+ 0.9 g and mean standard length 59.0 + 5.8 mm, were purchased from Aquatic Nurseries Ltd. (Hampton, Middlesex). Only maturing females, likely to be in the final stages of vitellogenesis (Munro, 1982), were used: males (identified by their conical genital papilla) were discarded. The fish were maintained in a stock tank prior to use. All stock and experimental fish were maintained under a 12L: 12D photoperiod, at 27 + 1°C. They were fed daily, early in the light cycle. Hormone treatments. The steroids used were testosterone propionate, estradiol-17P benzoate, and 17o-hydroxycorticosterone (cortisol) (Sigma, U.K.). The dosage used was 500 pg.liter-’ of aquarium water, each 500 (ug of the steroid having initially been dissolved in 50 ~1 of dimethyl formamide (DMF; Sigma, U.K.). Control fish received an equivalent dose of DMF alone. Other fish were treated with 30 mg.liter-’ of metyrapone (SU 4885; Ciba-Geigy Ltd., U.K.); at this dosage, the pituitaryadrenocortical axis is activated due to inhibition of corticosteroid synthesis (Munro, 1984b). Also, other fish were treated with 30 mg.liter-’ metyrapone in combination with 500 pg.liter-’ cortisol. Experimental procedures. (1) Groups of fish. A suitable population density for experiments on dominance hierarchies proved to be six fish in a 60 x 24 x 24-cm observation tank. Each experimental fish of such a group was individually isolated for 4 days prior to the start of the behavioral experiments. The appropriate hormone treatment (dosages as above) was added to each isolation tank on the first day of this acclimation period. The water received no filtration, aeration, or further treatment. On the fourth day of the acclimation period, each of the fish was identified by a subdermal injection of red or blue rubber latex (Riley, 1966), and the members of the group were placed together in a freshly treated, bare observation tank screened on three sides. Behavioral observations were recorded on an audiotape from behind a curtain; they commenced the day after the members of the group had been introduced to each other, and continued over 4 days. Each group was observed for 10 min daily, recordings being made late in the photophase (7-8 hr after lights on, and 5-6 hr after feeding), to avoid any interference of feeding on hierarchical structure (Barlow and Ballin, 1976). Eight
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behaviors were scored: Frontal and Lateral Displays (after Baerends and Baerends-van Roon, 1950), Charge, Approach, Flee, Avoid, Bite/Bite At, and Forage (all after Barlow and Ballin, 1976). Courtship behaviors (Baerends and Baerends-van Roon, 1950) were not observed. The transcript provided details of each agonistic encounter between identified fish. On the basis of these various encounters, a dominance hierarchy (Myrberg, 1972; Barlow and Ballin, 1976) was constructed. Activity was measured from the number of times that individual fish crossed two vertical lines on the tank front; this was recorded for 2-min periods immediately before and after the main observation period, and incidentally confirmed the absence of territoriality in all cases. The testosterone, estradiol, cortisol, metyrapone, and control treatments were repeated on five different groups of fish. Three groups of fish were treated with metyrapone in combination with cortisol. 2. Isolated fish. After a 4-day acclimation period in isolation similar to the groups above, test fish were transferred to separate 18 x 24 x 24-cm observation tanks containing freshly treated water. Again no filtration or aeration was supplied. Next day, and for the 3 days thereafter, each fish was presented with a 87 x 64-mm mirror, lowered into the tank from behind a screen. Observations were made from behind the screen, and recorded onto audiotape. Recording commenced with the first aggressive response of the fish to the mirror, and continued for the next 2 min, after which the mirror was removed. Three aggressive behaviors were scored: Frontal and Lateral Displays, and Bite. These behaviors are identical to those seen in groups of fish. Thirty minutes later, a model of a dominantly colored (barred) conspecific, standard length 46 mm, was lowered into the tank, and the fish’s behavior recorded for the next 2 min. The data of Heiligenberg (1974) indicate that 30 min was sufficient time for aggression to return to near baseline levels in another cichlid, Haplochromis burtoni. Control fish received DMF alone. In the first series of experiments, there were nine fish for each of the treatments control, 500 pg.liter-’ testosterone propionate and 500 pg.liter-’ estradiol-17/3 benzoate. In the second series, there were three fish for each of the treatments: control, 500 pg.liter-’ cortisol, and 30 mg.liter-’ metyrapone. RESULTS
1. Groups of Fish Members of each group interacted as a relatively stable nip-dominant hierarchy (Myrberg, 1972), with the exception of metyrapone-treated groups which schooled and had significantly higher activity scores (Fig. 1). As each group member was identifiable from its latex marking, its social ranking in relation to other group members could be determined using the “combined index of attack” of Barlow and Ballin (1976). The
STEROIDS AND CICHLID AGONISM
40.
”
CORTISOL
P-
OESTRADIOL
40
100
*
ttlllllflnnn 40.
-
*
rt A
357
-
In
TESTOSTERONE *
* *
FIG. I. Bar graphs of the mean frequencies of each of the behaviors scored in groups of treated fish for 10 min. together with activity scores; those significantly different from control values at the 5% level are indicated by an asterisk (LSD test with planned comparisons).
three most dominant fish ((Y,p, and y), and the most submissive (5) could be easily recognized with all treatments except metyrapone. Defining an agonistic encounter as an unbroken sequence of agonistic behavioral interactions between two identified fish, the numbers of agonistic encounters per IO-min observation period for each treatment were compared using two-way analysis of variance (Table la). There were no significant differences among the 4 days of each treatment, nor was there any significant interaction; there was a significant difference between hormone treatments, however. Testosterone significantly increased the mean frequency of agonistic encounters, while both estradiol and metyrapone significantly reduced their frequency (Table lb). The mean frequency seen in cortisol-treated groups was intermediate between those for controls and testosterone, and not significantly different from either. Groups treated simultaneously with cortisol and metyrapone were comparable with those treated with metyrapone alone (three replicate groups of fish, mean frequency of agonistic encounters 4.2/10 min). For the initiator of an agonistic encounter, the outcome may be either
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TABLE 1A Analysis of the Total Frequencies of Agonistic Interactions Scored in Groups of Six A. p&her for the Five Treatments Two-way analysis of variance
Hormones Days Interaction Residual Total
d!
Sum of squares
4 3 12 80 99
92,467 191 1,307 32,840 126,805
Variance
F value
23,117 64 108 411
56.3 0.2 0.3
P
successful (the initiator wins the encounter, with the other fish either Avoiding or Fleeing), unsuccessful (it loses), or inconclusive (there is no obvious winner). The steroid treatments each affect the balance between these various possible outcomes. In control groups, a mean proportion of 17.4% of all successful attacks for the initiator are directed against a higher ranking fish; the mean value for cortisol-treated groups, 10.7%, is significantly lower (F = 14.4, $1 and 15; P < O.Ol),while the values for both testosterone (12.5%) and estradiol (12.7%) are intermediate, and significantly different from neither. For all four treatments, the great majority of both unsuccessful and inconclusive attacks were initiated against fish of a higher rank. There are no significant differences among treatments for the relative frequencies of either unsuccessful or inconclusive encounters. However, there is a significant positive correlation between the frequencies of total and inconclusive agonistic encounters in both testosterone- and estradiol-treated groups; and a significant negative correlation in those treated with cortisol; the trend for control groups is not significant (Table 2). No such correlations were detectable for the frequencies of unsuccessful attacks. TABLE 1B Tukey (LSD) Test of the Means of Each of the Five Treatments Mean frequency of agonistic interactions Testosterone Cortisol Control Estradiol Metyrapone
92.4 81.0 ,I 71.4 55.4 5.3
Note. Lines join pairs of means which are not significantly different at the 95% confidence level.
359
STEROIDS AND CICHLID AGONISM TABLE 2 Correlations for Treated Groups for the Total Numbers of Agonistic Encounters with the Frequencies of Inconclusive Interactions (in which neither participant clearly wins): 20 Replicates in Each Case Pearson coefficient
Treatment Testosterone Cortisol Control Estradiol
+ 0.592 - 0.605 + 0.218 +0.515
F
P
11.34 7.52 1.25 6.15
co.01 co.01 NS co.05
It is thus apparent that hormone treatments may alter both the frequency and the pattern of interactions within a hierarchy. A broadly similar pattern is seen if the frequencies of the eight behaviors scored (the seven agonistic behaviors together with Forage) are summated and compared by three-way analysis of variance for the five treatments over the 4 days (Table 3a). Again there are no significant differences between the four replicate days. The hormone treatments produced significant differences in behavior and in addition this analysis clearly demonstrated that the behaviors responded differently to the five treatments (Table 3a). The mean frequency for total behavior is significantly lowered in metyrapone-treated groups, and significantly raised in those treated with testosterone, compared to controls (Table 3b). Groups treated with estradiol are not significantly different from controls. The cortisol-treated groups show a significantly greater mean value for total numbers of TABLE 3A Three-Way Analysis of Variance of the Eight Behaviors Scored for Groups of Six A. pulcher for the Five Treatments” Sum of squares
Variance
F
P
4 7 3 4 28 12 21
121 70,931 244 33,381 19,299 442 2,013
30.3 10,132.9 81.4 8,345.5 689.3 36.8 95.9
0.2 14.7 0.6 12.1 4.6 0.3 0.6
CO.8 <0.001 CO.6
84 636 799
3,111 94,836 224,380
37.0 149.1
0.2
df Replicates Behaviors Days Hormones Behaviors x Hormones Days x Hormones Behaviors x Days Behaviors x Days x Hormones Residual Total
a Fifty-seven percent of the total variation is accounted for.
CO.8
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MUNRO AND PITCHER TABLE 3B Tukey (LSD) Test for the Mean Number of Behaviors for Each of the Five Treatments Mean number of behaviors Testosterone Cortisol Control Estradiol Metyrapone
25.71** 23.12* 19.19 16.16 1.36**
* Significantly different from control (P < 0.05). ** Significantly different from control (P < 0.01).
behaviors (Table 3b), but this result for cortisol is not seen if Forage is excluded from the analysis. The mean frequencies for each of the eight behaviors for each hormone treatment were compared with those for controls using a least significant difference (LSD) test (planned comparisons, Sokal and Rohlf, 1%9), as indicated in Fig. 1. Metyrapone significantly reduced the frequencies of all eight behaviors. Testosterone significantly elevated the frequencies of five of the seven agonistic behaviors: Approach, Charge, Avoid, and both Displays. Cortisol only caused a significant increase in Charge and Lateral Display; it also significantly elevated the frequency of Forage, despite the absence of food or detritus on the tank bottom. Overall, estradiol-treated groups were most similar to controls, but each agonistic behavior (except for Lateral Display) tended to be less frequent, this reduction being significant for Approach (Fig. 1). The frequencies of the seven agonistic behaviors scored, together with Forage, were compared using one-way analysis of variance both (i) within each treatment, among the six ranks, and (ii) between each treatment for each of the six ranks (Table 4). This was in order to determine whether hormone treatments which alter the frequencies of specific behaviors (Fig. 1) do so for all group members equally, or whether these effects are restricted primarily to one or more ranks. Attack behaviors showed a positive correlation of frequency with rank, although the only significant difference is for Charge in a-fish compared to all other ranks for all treatments (Table 4a). Comparison of particular ranks between treatments indicates that mean Charge frequencies are significantly raised in the a-fish of cortisol-treated groups, and significantly reduced in the p-fish of groups treated with estradiol; all other changes are nonsignificant (Table 4b). The other attack behavior, Approach, is not significantly different for comparisons between ranks, but it is with comparisons between treatments for a-fish: frequencies for estradiol-
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TABLE 4 The Effects of Hormone Treatments on the Organization of Hierarchies, Using OneWay Analysis of Variance Tests (T, Groups Treated with Testosterone; E2, Those Treated with Estradiol; F, Those Treated with Cortisol; and C, Controls) Behavior
F
C
22.6’ 5.6 1.0” 1.8 4.9c,D 0.5 4.3 6.7 1.3” 2.2A.c 0.7’
11.3A 8.4 0.9d 3.P 3.3O 0.5 4.2 4.5 1.9 1.2 1.2
b. Comparisons within individual ranks between treatments 17.4 10.6” 22.6‘+’ 4.0”’ 9.2’ 5.6 ; Approach 12.6’ 8 3”.b 12.4A Avoid 4.18 3:6.” 1.8’++ ; Frontal display 2.7B 2.1 1.3b a 2.2,Q 1.2b o.7b Y 5 5A.B.C Lateral display 4.0” 2.9’ 6: lA,C 2.2 2.9”
11.3b 8.4* 10.3 3.1 1.9 1.2” 1.9 2.0
Charge Avoid Flee Frontal display
Charge
Rank
T
E2
a. Comparisons between ranks for each treatment 17.4= 10.6’ 9.2’ 4.0 ; O.jd 0.6“ 4.P 3.6* ; 4.jD 3.6D Y 0.2 o.2b,c ; 3.6’.” 4.4B Y 7.0” 4.6’ 2.7 2.1 ; 2.0 1.4 Y 2.2 1.2
Note. The values given are the means for 20 replicates. Key to superscripts: A signi8cantly greater than n (P < 0.05); B significantly greater than b (P < 0.01); and C significantly greater than c (P < 0.001).
treated groups are reduced compared to those treated with either testosterone or cortisol, with controls occupying an intermediate position (Table 4b). Both of the flight behaviors, Avoid and Flee, show a negative correlation with rank for each treatment, but this is generally only significant for (Yfish compared to other ranks (Table 4a). However, the frequency of Avoid in the p-fish of groups treated with cortisol is also significantly lower than the frequency for more subordinate fish. Comparison of the same rank between treatments (Table 4b) indicates that Avoid is significantly less frequent in the P-fish of cortisol-treated groups than in groups treated with either testosterone or estradiol. Cortisol-treated groups are unusual in that the frequency of Frontal Display in p-fish is significantly higher than that for either a-fish or yfish (Table 4a). Treatment with testosterone increases the frequencies of Frontal Display mainly for the top three ranks, compared to other groups, but this difference is significant only for y-fish (Table 4b).
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Lateral Display is significantly increased for the top two ranks of testosterone-treated groups compared to other treatments. For all treatments, there was no correlation of the frequency of Forage with rank, with each fish showing a wide variation over the 4-day observation period. To conclude, it is apparent that each hormone treatment has different effects on particular ranks in affecting the overall frequencies of particular behaviors (Fig. 1). Thus the significant increase in frequency of Charge in cortisol-treated groups, and the two Displays in testosterone-treated groups can be attributed to significant changes in one or more ranks, while other significant changes in overall behavioral frequencies are apparently more evenly spread over several ranks (e.g., Charge and Approach in groups treated with testosterone, and Lateral Display in those treated with cortisol). Contrariwise, although some behaviors may show no significant overall change as a result of hormone treatment, there are significant effects when individual ranks are compared: for example, the effects of cortisol on Frontal Display. Principal Components analysis (Maxwell, 1977) is a way of clearly summarizing differences between the overall spectrum of behaviors shown by an individual: it is free of the bias introduced when separate behaviors are selected for measurement and analysis (Huntingford, 1984). Such an analysis, based on a correlation matrix of the eight scored behaviors together with the measure for Activity across the five treatment groups, confirmed that each treatment had a different effect. No rotation was performed. Since analysis of variance had shown that there were no significant differences, we were justified in using the means over all 20 replicates (days x replicate groups) to simplify the analysis. The first two Principal Components account for 95% of the total variance; as the third and subsequent Components contribute only 5% or so, they can be safely be omitted from consideration. Each of the behaviors has a high and roughly equal loading on the first Component (Fig. 2), demonstrating that the distinguishing features of the acara’s behavior are genuinely multivariate. On the second Component, the most important behaviors are the two Displays, Charge, and Forage, confirming that no single behavior best discriminates the effects of the hormone treatments. Figure 3 illustrates the location of the five treatments on the first two Principal Components (i.e., the five treatments plotted in “hormone space”). The figure shows a clear separation of behavior for testosterone, cortisol, and metyrapone. Behaviors for these three treatments show a clear separation from estradiol and controls, whose positions in the center of the plot reflect behavior closest to the overall average for the experiment. On this basis, estradiol and control groups have the most similar behaviors. Cortisol is the next most similar to this group; testosterone is similar to cortisol on Component 1, but very dissimilar on Component 2. Component 1 mainly serves to separate metyrapone-treated groups from the others.
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P.C.2
P.C.l +2
0
-2
El-’
0
+2
i
’
FIG. 2. The distribution of the eight scored behaviors and activity within the first two principal components of the analysis of groups of fish. Principal component 1 (PCl) accounts for 85% of the explained variability, and PC2 for 10% of this, over the five treatments.
The results of an “inverse” Principal Components analysis (i.e., the eight behaviors plotted in hormone space) are shown in Fig. 4. This indicates that the eight component behaviors fall roughly into three clusters: Approach with Avoid, Charge with Flee, and the two Displays together with Bite and Forage. Each of the behaviors in the first two clusters is mutually complementary: Approach by one fish is frequently accompanied by the other fish Avoiding, for example. The feature shared by the third cluster is an expansion of the buccal cavity in the absence of any major forward movement; if Forage on the bare tank bottom represents a t3
P.C.2 t
CORTISOL
I
OEFRADIOL
I
0 METYRAPONE
TESTOSTERONE l
-3 1 FIG. 3. Principal components of controls and the four hormone treatments in “behavior space.” Both control and estradiol-treated groups lie close to the origin, while the other three treatments each occupy an isolated position within the plot.
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t3 AVOID l
t
0 APPROACH FORAGE BITE .t FRONTALtZ .DISPUY
P.C.1 -2
LATERAL
DISPLAY
FLEE .
0 CHARGE
FIG. 4. Principal components analysis of the eight behaviors scored for groups of fish plotted in “treatment space.” Approach and avoid are clearly separated as a unit from charge and flee; forage, bite/bite at, and frontal display together with lateral display form a third cluster.
displacement activity (see Discussion), then this also shares a basis of conflict with the two Displays (Baerends, 1974). 2. Individually
Isolated Fish
Fish consistently show more aggression to mirrors than to models of dominantly colored conspecifics. Testosterone increases the frequency of aggressive incidents for both model and mirror presentations (Table 5a), this increase being also seen for each of the three component behaviors scored (Lateral and Frontal Display, Bite/Bite At) apart from Bites against mirrors (Table 5b). Estradiol reduces the frequency of aggressive responses directed to models and, especially, to mirrors (Table 5a), but, of the three behaviors, there is only a significant difference for Frontal Displays (Table 5b). Cortisol also increased the frequency of aggressive incidents, but this is significant only for model presentations. Conversely, metyrapone reduced aggressiveness overall, but this reduction is only significant for mirrors (Table 5a). DISCUSSION
Our results confirm that sex steroids have a direct effect on aggressive behavior in cichlids. There was no difference between the four replicate
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TABLE SA Mean Frequencies of Aggressive Interactions for Mirror and Model Presentations to Isolated Fish” Mean frequency per 2-min response Treatment
Mirrors
Testosterone Cortisol
15.6
Estradiol Metyrapone Control
2.4 7.6
Models 5.6
7.8
6.3 0.4 2.3 I
1.8 5.3
0.3 2.3I
’ Lines join means which are not significantly different at the 95% confidence level (Tukey test); although the cortisohmetyrapone experiment (12 replicates) had to be run separately from that with testosterone/estradiol (36 replicates), their respective means are not significantly different.
days throughout the experiments, which suggests that stable, elevated levels of circulating hormones resulted from our immersion technique with the three steroids; and that the effects of metyrapone were also maintained over a prolonged period. In contrast, hormones have been administered by injection in most previous behavioral studies on teleosts, with the disadvantage that, where aggression is affected (e.g., Fernald, 1976),the subsequent distinct peak is difficult to compare across treatments. In our work, we have been able to show that each of the three steroids, and also metyrapone, had a characteristic effect on the pattern of agonistic behavior, which was encouragingly consistent across replicates and across both experimental protocols. TABLE 5B Mean Frequencies for the Three Aggressive Behaviors Shown by Isolated Fish to Mirror and Model Stimuli Mean frequency per 2-min response Behavior Lateral display
Frontal display Bite
Treatment
Mirrors
Models
Testosterone Control Estradiol Testosterone Control Estradiol Testosterone Control Estradiol
21.1 8.1 3.2 34.6 18.2 5.3 7.6 4.1 0.9
8.9 3.4 1.2 9.7 4.6 0.6 3.8 1.0 0.0 I
L?Lines join means not significantly different from control values at the 95% confidence level (Tukey test, 12 replicates for each).
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Testosterone Treatment Testosterone stimulated overall agonistic behavior in both experimental protocols. Lateral Display, Frontal Display, and Bites directed against mirrors or models increased in isolated fish treated with this hormone. Similarly, in groups treated with testosterone the two displays were significantly elevated, together with three of the five other agonistic behaviors scored: except for Lateral Display, these increases were uniform across all ranks in the group hierarchy. At present, we do not know if testosterone itself alters behavior, or whether a metabolite such as Sa-dihydrotestosterone or 1I-ketotestosterone is involved. 1I-Ketotestosterone is the principal androgen for the control of male secondary sex characters in teleosts (e.g., Hackman, 1974), but preliminary experiments suggest that it is not behaviorally active (Munro, unpublished). Stimulation of aggression by testosterone has been reported previously for both sexes of two mouth-brooding cichlids, Hemihaplochromis multicolor (Rixner, cited by Reinboth, 1972)and Haplochromis burtoni (WaplerLeong and Reinboth, 1974; Fernald, 1976) but changes in behavior following their single injections of hormone are difficult to interpret in detail. Estradiol Treatment Estradiol inhibited aggression by reducing the overall frequency of agonistic encounters. There was also a tendency to affect individual encounters through an increase in the number of agonistic acts per encounter, which is apparent in groups of fish with the failure of estradiol to reduce significantly the frequencies of six out of the seven scored agonistic behaviors below control values, despite the significant decrease in the frequency of agonistic encounters. A similar trend is seen in the mirror/model experiment. Principal Components analysis locates the effects of estradiol close to those of control, which raises the question of whether this hormone does in fact have a distinct affect on aggression; however, the distinct effects of estradiol on the responsiveness of isolated fish suggests that there is a definite effect. The effects of estradiol are different from those of testosterone, so we may safely conclude that testosterone does not have to be aromatized in order to affect agonistic behavior. It is not clear whether estradiol has a direct inhibitory effect on agonistic behavior (in which case, the ratio testosterone:estradiol may be more important than absolute levels of the individual hormones in the regulation of behavior); or whether it affects aggression indirectly, by inhibiting the secretion of pituitary gonadotropin, and hence gonadal testosterone, through negative feedback (Munro and Pitcher, 1983).
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Cortisol Treatment In affecting agonistic behavior, cortisol has a distinct action: its effects appear to be selective by behavior, by rank, and by stimulus. In isolated fish, aggressive encounters are increased for model presentations, whereas this is not the case for mirror experiments, nor for groups of fish. Nevertheless, there are differential effects in groups of fish compared to other treatments: some behaviors are generally increased (e.g., Lateral Display), some are elevated selectively across the hierarchy (e.g., Charge for a-fish), while others differ more among ranks than in control groups although there may be no overall significant change (e.g., Frontal Display). Leshner (1981) found similar, seemingly equivocal evidence for the effects of corticosteroids on agonistic behavior in mammals, due in part to differences between experimental protocols. Leshner concluded that the main effect of corticosteroids was to enhance submissiveness, on the basis of experiments with pairs of rodents. While directly parallel experiments remain to be performed with cichlids, an increase in submissiveness would explain the lack of an increase in aggression for mirror presentations, where fish would experience an abnormally immediate feedback (Pitcher, 1979) for any aggressive act. Dummies on the other hand provide no such feedback, so that with these cortisol would appear to increase aggression. Such an enhancement of submissiveness would also account for certain details of the behavior of groups of fish. Thus, fewer successful attacks were initiated by low rankers against high-ranking fish. Furthermore, in contrast to all other treatments, the greater the frequency of agonistic encounters in cortisol-treated groups, the fewer the number of inconclusive encounters, and hence the more defined the hierarchical structure. Despite the absence of food, cortisol treatment elevated the frequency of the behavior “Forage” in groups of fish (this behavior was not often seen in isolated fish). This behavior may have been “displacement foraging,” which occurs just before or after territorial boundary fights in other cichlids (Baerends and Baerends-van Roon, 1950; Heiligenberg, 1974; Fernald and Hirata, 1977). If our interpretation of Forage as a displacement activity is correct, then this complements our general conclusion that the principal effect of cortisol on agonism is to increase the submissive components of behavior. In this way, cortisol may increase aggression in high-ranking fish indirectly, by increasing submissiveness in lower ranks. As far as we are aware, this is the first experiment to describe the effects of cortisol on social groups of any vertebrate. Metyrapone Treatment This drug virtually abolishes agonistic encounters. In groups of fish, schooling behavior (Pitcher, 1983)occurs, associated with a general increase
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in activity. Aggression is also greatly reduced for mirror presentations to isolated fish: the smaller reduction for models may again reflect the differential feedback of these two stimulus situations for isolated fish. The effects of metyrapone could be due to three causes: (1), inhibition of corticosteroid synthesis through blocking 1lfi-hydroxylase; (2), inhibition of 11-ketotestosterone synthesis by the same mechanism; or (3), a nonspecific toxic effect. Simultaneous administration of cortisol with metyrapone should lead to elevated, rather than reduced, blood cortisol levels; the fact that groups of fish treated with this combination are behaviorally comparable with those which were treated with metyrapone therefore eliminates alternative (1) above. A derivative of testosterone, 1I-ketotestosterone is the main androgen regulating the development of secondary morphological sexual characters in teleosts (Hackman, 1974). Metyrapone blocks the conversion of testosterone to 11-ketotestosterone in the testes, leading to a elevated plasma testosterone levels (Duggan and Bolton, 1981). Preliminary experiments (Munro, unpublished) suggest that 11-ketotestosterone does not affect aggression in isolated blue acaras, unlike testosterone; this suggests that alternative (2) can also be ruled out. Metyrapone impairs learning in birds by a toxic action (Martin, 1978), and we suggest that this is the basis for our results with this drugalternative (3) above. Similarly, environmental stressors (including low temperatures) have been reported to induce schooling in other cichlids (Baerends and Baerends-van Roon, 1950; Greenberg et al., 1965). Such an effect was seen in the metyrapone-treated fish although levels of testosterone can be presumed to be rising in the plasma (Duggan and Bolton, 1981); this suggests that the behavioral effects of the androgen were being overridden, even after cortisol replacement therapy. The mechanism by which metyrapone overcomes the expected behavioral effects of these steroids is unknown. Conclusions
As in various other teleosts, aggressive interactions are not restricted to a particular context (for example, during reproduction) in cichlids: this is particularly true for aquarium populations, where aggression is generally increased (Liley and Stacey, 1983; Munro and Pitcher, 1983). Stress, and continued exposure to the same individuals, may contribute to the increased aggression seen in the aquarium (Barlow, 1974). This raises problems when trying to evaluate the significance of hormonal effects; nevertheless, the consistency of our results between the two different protocols-hierarchical groups and isolated fish-strongly suggests that the steroids tested may have behavioral roles in the natural life history of A. pulcher.
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In the wild, aggression in substrate-spawning cichlids like the blue acara apparently is mainly restricted to guarding of a spawning site (reviewed by Munro and Pitcher, 1983); indirect evidence suggests that plasma testosterone levels may be highest in both sexes at this time (Bogomolnaya, Rothbard, and Yaron, 1983), which suggests a causal relationship between circulating hormone levels and behavior. In common with mammals, cortisol secretion is elevated by a variety of stressors in teleosts, including social inferiority (Terkatin-Shimony, Ilan, Yaron, and Johnston, 1980; Schreck, 1981). In affecting submissiveness, elevated cortisol levels may be behaviorally important in the context of social inferiority, as has also been suggested for mammals (Leshner, 1981). As some, but not all, other stressors are reported to induce schooling behavior (which is the behavior normally seen in juvenile fish: Baerends and Baerends-van Roon, 1950), such stressors presumably override the behavioral effects of elevated cortisol levels (Munro and Pitcher, 1983). As with all previous work on teleosts, and seemingly also that on mammals and birds, we have used only one dosage of hormone. The absence of significant differences between individual isolated fish tends to rule out the possibility of large differences in sensitivity between fish. The possibility that there may be dose-dependent effects of these hormones on agonistic behavior remains to be tested: the experiments reported here suggest that tests using isolated fish would give valid results. In conclusion, although mammals have been used for most previous experimental work on steroids and agonism, teleost fish can provide useful general insights, especially in elucidating the subtleties of effects in relatively simple social groups which show fairly stylized behaviors. ACKNOWLEDGMENTS A.D.M. would like to thank the Science and Engineering Research Council for their support.
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Reinboth, R. (1972). Some remarks on secondary sex characters, sex and sexual behaviour in teleosts. Gen. Comp. Endocrinol. Suppl. 3, 565-570. Riley, J. (1966). Liquid latex marking technique for young fish. J. Cons. Inr. Explor. Mer 30, 354-357. Schreck, C. B. (1981). Stress and compensation in teleostean fishes: response to social and physical factors. In A. D. Pickering (Ed.), Stress and Fish, pp. 295-321, Academic Press, New York/London. Sokal, R. R., and Rohlf, F. J. (1969). Biometry. Freeman, San Francisco. Terkatin-Shimony, A., and Yaron, 2. (1978). Oestrogens and oestrogenic effects in Tilapia aurea (Cichlidae, Teleostei). Ann. Biol. Anim. Biochem. Biophys. 18, 1007-1012. Terkatin-Shimony, A., Ran, Z., Yaron, Z., and Johnston, D. W. (1980). Relationship between temperature, ovarian recrudescence, and plasma cortisol levels in Tilapia aurea (Cichlidae, Teleostei). Gen. Comp. Endocrinol. 40, 143-148. Vodegel, N. (1978). A study of the motivation underlying some communicative behaviours of Pseudofropheus zebra (Pisces, Cichlidae): A mathematical model. Proc. K. Ned. Akad. Wef. Ser. C 81, 211-240. Wapler-Leong, D. C. Y., and Reinboth, R. (1974). The influence of androgenic hormones on the behaviour of Haplochromis burtoni (Cichlidae). Fortschr. Zool. 22, 334-339.