General and Comparative Endocrinology 147 (2006) 184–189
www.elsevier.com/locate/ygcen
Male 11-ketotestosterone levels change as a result of being watched
in Siamese Wghting Wsh, Betta splendens
Teresa L. Dzieweczynski a,¤, Amy C. Eklund b, William J. Rowland b,9
a
b
Department of Psychology, University of New England, Biddeford, ME 04005, USA
Department of Biology, Jordan Hall 142, 1001 E, 3rd Street, Indiana University, Bloomington, IN 47405, USA
Received 8 November 2005; revised 19 December 2005; accepted 30 December 2005
Available online 13 February 2006
Abstract
This study investigated the eVects of nesting status and the presence of an audience on 11-ketotestosterone (11KT) levels in male
Siamese Wghting Wsh, Betta splendens. Prior studies have demonstrated that both nesting status, an indicator of territory-holding power
and reproductive state, and the sex of a conspeciWc audience lead to diVerences in male behavior during aggressive encounters. Since
behavioral changes have already been demonstrated, we chose to investigate whether 11KT levels were also inXuenced by nesting status
and audience presence as 11KT both stimulates, and is stimulated by, reproductive and aggressive behaviors in male teleosts. Male 11KT
levels were measured from water samples taken from containers holding Wsh both before and after interaction. Males interacted under
three treatment conditions: no audience, female audience, and male audience. Within these treatments were two nest paradigms: both
males had nests or neither male had a nest. 11KT levels varied depending on nesting status and audience type. In general, 11KT levels
were lower in interacting males when a female audience was present or when males had nests. Overall, 11KT showed increases or
decreases as aggression increased or decreased, as shown by already established behavioral Wndings [see Dzieweczynski T.L., Green T.M.,
Earley R.L., Rowland W.J., 2005. Audience eVect is context dependent in Siamese Wghting Wsh, Betta splendens. Behav. Ecol. 16, 1025–
1030; Doutrelant, C., McGregor, P.K., Oliveira, R.F., 2001. EVect of an audience on intrasexual communication in male Siamese Wghting
Wsh (Betta splendens). Behav. Ecol. 12, 283–286.]. Our results suggest that 11KT levels are inXuenced by reproductive status, as indicated
by nest ownership, and audience presence and are most likely modulated by territorial behavior and social environment.
© 2006 Elsevier Inc. All rights reserved.
Keywords: 11-Ketotestosterone; Social behavior; Aggression; Siamese Wghting Wsh
1. Introduction
While androgens are typically thought of as controlling
seasonal changes in male reproductive behavior, studies in
recent years have demonstrated that androgen levels can be
inXuenced by interactions with conspeciWcs. Social interactions have been found to inXuence reproduction through a
number of ways including regulating ovulation (Cheng,
1986; Stern and McClintock, 1998) and producing dramatic
transformations such as sex changes (wrasse (Thalassoma
*
9
Corresponding author.
E-mail address: [email protected] (T.L. Dzieweczynski).
Deceased.
0016-6480/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygcen.2005.12.023
dupperrey) Larson et al., 2003; clownWsh Buston, 2003).
Sexual maturation may be suppressed in the presence of
dominant individuals in a number of social species including an African cichlid Wsh, Haplochromis burtoni (e.g., Davis
and Fernald, 1990; Sapolsky, 1993). As a consequence,
dominant members of a hierarchy experience greater reproductive success than subordinate animals (e.g., primates,
Ellis, 1995; dwarf mongeese, Creel et al., 2002; African cichlid Wsh (Haplochromis burtoni), Hofmann et al., 1999). In all
vertebrates, Wnal regulation of androgen levels occurs in the
hypothalamic–pituitary–gonadal (HPG) axis, beginning
with the GnRH neurons, which are directly inXuenced by
aggressive interactions (e.g., Francis et al., 1993). This indicates that some form of reciprocal relationship likely exists
T.L. Dzieweczynski et al. / General and Comparative Endocrinology 147 (2006) 184–189
between androgens and behavior and may provide a way
for individuals to adjust to changes in their social environment (see Oliveira et al., 2002 for review in teleosts).
According to the challenge hypothesis (WingWeld et al.,
1990), interactions between males should stimulate the production of androgens within these males; in turn, the
amount of androgen stimulation should be a product of the
stability of a male’s social environment. Under this hypothesis, we would expect males of higher social status to have
greater androgen levels because dominant/territorial males
must be more aggressive in order to maintain their territories. We would also expect these levels to be highest during
periods of social instability, such as initial territory formation or the establishment of a dominance hierarchy (e.g.
Cardwell and Liley, 1991; Pankhurst and Barnett, 1993). In
contrast, when the social environment is relatively stable,
levels of aggression will drop and androgen levels will no
longer be closely associated with aggression levels. Finally,
androgen levels should be negatively correlated with the
degree of paternal care; this is supported by the Wnding that
males in the parental phase of a breeding cycle tend to
decrease their androgen levels (e.g. Townsend and Moger,
1987). These predictions would suggest that manipulating
the reproductive context (i.e., presence or absence of a nest)
during an interaction between males may impact the androgen levels of male Siamese Wghting Wsh.
In addition, manipulating the audience type should also
aVect androgen levels. Audience, or bystander eVects, on
behavior are well-documented (e.g., Cheney and Seyfarth,
1985; Evans and Marler, 1992; Mennill and RatcliVe, 2004).
They are often sex-speciWc and can cause both individuals
that are being watched and those watching the interaction,
known as eavesdroppers, to modify their behavior (e.g.,
chickens, Evans and Marler, 1994; lions, Grinnell and
McComb, 2001; stickleback, Dzieweczynski and Rowland,
2004; Bettas, Doutrelant et al., 2001; Oliveira et al., 1998).
Many of the studies on bystander eVects have demonstrated that male–male interactions are aVected by the presence of an audience in Siamese Wghting Wsh, Betta splendens
(e.g., Doutrelant et al., 2001; Matos and McGregor, 2002;
McGregor et al., 2001). Whether or not a male has a nest
also inXuences the costs and beneWts of being aggressive in
the presence of an audience. For example, males that have
invested energy into producing a nest are less likely to Wght
in the presence of a male audience, perhaps because the risk
of losing a territory to a potential usurper is too great
(Dzieweczynski et al., 2005). It would seem logical that hormone levels would change as a result of these behavioral
changes. While it has been demonstrated that watching
Wghts leads to elevated hormone levels in a male eavesdropper cichlid Wsh (Oliveira et al., 2001), we are unaware of any
studies in Siamese Wghting Wsh that investigate how hormonal levels amongst interacting males might be aVected
by being watched.
Siamese Wghting Wsh males are known for their coloration, long Wns, and highly conspicuous and stereotypical
displays. Males direct aggressive behaviors such as bites,
185
gill Xares, and tail beats at other males when Wghting (Simpson, 1968) and use many of these same behaviors when they
are interacting with females. However, the frequency and
intensity of the display elements may diVer (Simpson, 1968).
Given these Wndings, one would expect that the type of
individuals (i.e., bystanders) that are near a given male
would aVect his behavior. Males compete for territories
and, once they have obtained a territory, build a nest of
mucous-coated air bubbles on the water surface. This nest
will serve to entice females as well as provide a home for
eggs and newly hatched fry (Simpson, 1968). Building a nest
and establishing a territory is presumably energetically
costly and nesting status inXuences male behavior in
aggressive interactions (Dzieweczynski et al., 2005).
Because we previously demonstrated (Dzieweczynski
et al., 2005) behavioral diVerences when audience and reproductive context were varied in Siamese Wghting Wsh, we were
interested in investigating whether hormonal changes
accompanied these behavioral changes. We chose to assay
11-ketotestosterone (11KT), a principle Wsh androgen,
because other studies have found that 11KT is an inXuencer
of, and is inXuenced by, aggressive interactions in Wsh (e.g.,
Fostier et al., 1983; Hay and Pankhurst, 2005; Liley and Stacey, 1983; Oliveira et al., 1996). Circulating plasma levels of
11KT are also higher in males while testosterone levels are
generally similar between males and females, indicating that
11KT is likely important in modifying male behavior (Borg,
1994). If androgens are produced in response to social interactions, we would expect that hormone levels would change
after an interaction. In addition, changing the context of an
interaction by altering the sex of an audience and male nesting status should inXuence male androgen levels after an
interaction. We predicted that hormone levels would be
higher in males after interacting than before. We also predicted that males with nests would have lower 11KT levels
than those without nests, and that males interacting in the
presence of an audience would have elevated 11KT levels
compared to males interacting without an audience.
2. Materials and methods
2.1. Animals
Siamese Wghting Wsh subjects were obtained from a commercial distributor in Indianapolis, Indiana in May 2003. Males were measured for standard length (distance between the mouth and the base of the tail) and
weighed (g) before being placed into 500 ml opaque containers to prevent
exposure to other males prior to testing. Subjects were maintained at
24.4 °C and a 16L/8D photoperiod throughout the testing period. Subjects
were fed Tetra BettaMin once a day.
2.2. Experimental design
For a behavioral trial, males were allowed to interact for 20 min under
varying nesting conditions (neither, one or both males had nests) and audience paradigms (female, male, or no audience) for a total of nine combined
treatments (Dzieweczynski et al., 2005). All Wsh were tested in separate
tanks that abutted one another (see Fig. 1). These tanks were separated by
opaque partitions prior to testing. None of the individuals used in this
study, regardless of whether they served as a subject or audience Wsh, were
186
T.L. Dzieweczynski et al. / General and Comparative Endocrinology 147 (2006) 184–189
Fig. 2. Plot of concentration of 11KT in blood plasma samples as compared to water samples for 8 non-interacting control males.
Fig. 1. Diagram of experimental set-up as described in detail in
Dzieweczynski et al. (2005). The box represents a small clear plastic box
the audience Wsh was placed into to restrict its movement. All Wsh, both
interactants and the audience, were house in individual, abutting tanks
and separated by opaque partitions prior to testing.
used more than once. The males used for the hormonal study were a subset
of those used for the behavioral work. Since the “one nest” condition was
comprised of pairs of males in which one male had a nest while the other
did not, we eliminated these treatments from the current investigation, to
give a total of seven treatments (see Table 1). For a more detailed description of the behavioral methodology see Dzieweczynski et al. (2005).
Before males were placed in testing tanks, we collected hormone samples to obtain a measure of baseline 11KT levels. To collect hormone samples, males were placed individually into new, opaque containers with
250 ml water. After 2 h, Wsh were removed from these holding containers
and placed into the testing tanks. The eVect of placing Wsh in these containers for 2 h was not tested here, however, Mayer et al. (1990) conducted
a test of container conWnement on hormone levels and found little diVerence between conWned and aquarium-housed males. Water from the container was stored at ¡20 °C until steroid extraction. Males were kept in the
testing tanks for 24 h and then allowed to interact under the various treatment conditions. After the 20 min of interaction time, males were again
placed into opaque containers with 250 ml of water for 2 h. Again, water
samples were stored at ¡20 °C until steroid extraction. We analyzed hormone samples from 10 males for each of the following conditions: 2 nests,
male audience; 2 nests, female audience; 2 nests, no audience; no nests,
male audience; no nests, female audience; no nests, no audience (Table 1).
We also analyzed 40 water samples from males prior to interacting in a
trial. Thus, a total of 100 water samples, all from diVerent males, were
analyzed.
It is important to note that we chose to collect androgen samples from
water rather than plasma. Steroids are released into the water via the gills,
urine, and feces and can be extracted from water in which a Wsh is kept.
This provides a non-invasive method of analysis that does not cause additional stress for the subjects. In addition, hormone extraction from water
provides a means of measuring both before- and after-interaction hormone levels, which could not be achieved by traditional methods that
require the subject be sacriWced in order to obtain a suYcient volume of
plasma. While we did not have adequate data here to assess the correlation
between plasma and water 11KT levels (but see Fig. 2), others have tested
this and found a positive correlation; urinary androgen levels have been
shown to reliably correlate with plasma androgen levels in rainbow trout
(Scott and Liley, 1994), and water steroid levels are known to correlate
with plasma levels (Greenwood et al., 2001), indicating that this is a suitable method of analysis.
2.3. Hormone measurements
Water samples were Wltered to remove Wne particulates (glass microWber Wlters, VWR). One hundred milliliters of the Wltered sample was then
extracted using solid-phase C18 matrix cartridges (Maxi-Clean High
Capacity—300 mg, Alltech), activated with 4 ml of 100% ethanol and then
4 ml of distilled water. Steroids were eluted from the columns using 4 ml of
100% ethanol and evaporated under nitrogen at 40 °C. The free steroid
fraction was collected by extracting this eluent with diethyl ether and
reconstituting the dried organic phase in 200 l enzyme immunoassay
(EIA) buVer (Cayman Chemical). To obtain the sulfated fraction, the
polar phase remaining after ether extraction was evaporated under nitrogen at 40 °C. One ml of triXuoroacetic acid/ethylacetate (1:100 v/v) was
added and the samples were placed in a 40 °C water bath overnight. After
the incubation period, the samples were evaporated under nitrogen and
0.5 ml of 0.1 M sodium acetate buVer (pH 4.5) was added. Samples were
then extracted with diethyl ether and resuspended in buVer, as previously
described. To obtain the glucuronidated fraction, the remaining polar
phase was evaporated and then 15 l -glucuronidase (from Helix pomatia, ICN) was added to the samples. After overnight incubation at 37 °C,
this fraction was extracted with diethyl ether and resuspended in buVer, as
described above. All samples were stored at ¡20 °C until analysis. Free,
sulfated, and glucuronidated fractions were then diluted 1:5 in EIA buVer
and assayed for 11KT by EIA (Cayman Chemical). The assay speciWcity
was: 100% for 11KT, 0.01% for 4-androsten-11,17-diol-3-one, <0.01%
for testosterone, 5-androstan-17-ol-3-one, and 5-androsten-3,17diol. The limit of detection for the assay was 1.3 pg/ml and the extraction
eYciency was 79%.
2.4. Statistical analysis
Because of the non-normality of the data and standard transformations
did not normalize our data, we conducted a two-way analysis of variance
(ANOVA) to examine the eVects of audience and nest on 11KT levels.
Before interaction samples were not included in this ANOVA. Post hoc tests
Table 1
Summary of experimental treatments included in hormonal study
Before interaction:
After interaction:
Before
Interacting male had:
Nest
No nest
No audience
No-2
No-0
Female audience
Female-2
Female-0
Each of 7 treatments for which male 11KT levels were measured, and, in bold, the abbreviation used for each treatment.
Male audience
Male-2
Male-0
T.L. Dzieweczynski et al. / General and Comparative Endocrinology 147 (2006) 184–189
were then performed using Holm–Sidak tests. All statistical tests were twotailed and were analyzed using the SigmaStat statistical program.
3. Results
Whether or not males had nests inXuenced 11KT levels
(ANOVA: F1,64 D 10.887, P D 0.002), with males without
nests having higher 11KT levels than males with nests
(Holm–Sidak: t D 3.30, P D 0.002). The type of audience
present also inXuenced 11KT levels (ANOVA: F2,64 D 4.314,
P D 0.017); 11KT levels were higher in males with a male or
no audience present than in males with a female audience
present (Holm–Sidak: t 7 2.187, P 6 0.032). However, since
a strong nest £ audience interaction was found (ANOVA:
F2,64 D 3.786, P D 0.028; Table 2), these two factors cannot
be analyzed independently. Using a Kruskal–Wallis
ANOVA, we found that 11KT levels were higher in our
before interaction group of males than in males that interacted in the presence of a female audience, regardless of
whether or not males had nests (Holm–Sidak: t D 2.406,
N D 100, P D 0.018), and males with nests that interacted in
Table 2
Comparison of behavioral and hormonal eVects of presence of audience
(F, M, or No) and nest (0, 1, or 2)
Female, 2
Female, 0
No, 2
No, 0
Male, 2
Male, 0
None
Higher
None
None
None
None
None
Lower
Lower
Lower
Higher
None
None
None
Higher
None
Lower
Lower
None
None
Lower
Before
Female, 2
Female, 0
No, 2
No, 0
Male, 2
Values in the table reXect the treatment on the right compared to the
treatment on the top and are the results of the ANOVA. Comparisons
with no signiWcant diVerence are indicated by “none.” Treatments with
greater 11KT levels are indicated by “higher” and comparisons with
decreased 11KT levels are indicated by “lower.”
Fig. 3. Mean 11-ketotestosterone concentrations in nanograms for before,
female audience, 2 nests (F,2), female audience, no nests (F,0), no audience, 2 nests (0,2), no audience, no nests (0,0), male audience, 2 nests (M,2),
male audience, no nests (M,0) § 1 SEM in nanograms.
187
the presence of a male audience (Holm–Sidak: t D 2.345,
N D 100, P D 0.021; Fig. 3).
Males without nests that interacted with a male or no audience present had higher 11KT levels than those that interacted with a female audience present (Holm–Sidak: t 7 3.226,
it P 6 0.002; Fig. 3). When no audience was present, males
without nests had higher 11KT levels than those with nests
(Holm–Sidak: t D 2.510, P D 0.015). Males that did not have
nests and interacted in the presence of a male or no audience
had higher 11KT levels than males that had nests and interacted in the presence of a female (Holm–Sidak: t 7 2.571,
N D 100, P 6 0.12; Fig. 3). Finally, males without nests that
interacted with a male audience present had higher 11KT levels than those with nests (Holm–Sidak: t D 3.494, P < 0.001).
4. Discussion
Our results demonstrate that both the sex of the audience and nesting status inXuence 11KT levels in interacting
males (Table 2). Our hypothesis that males with nests
would have lower 11KT levels was supported. The highest
11KT levels were generally found in males that did not
have nests. The exception to this was for males without
nests that interacted with a female audience. Our hypothesis that 11KT levels would be higher with an audience present was partly supported; type of audience clearly mattered.
Males interacting with females present had lower 11KT levels than males with no audience. However, males interacting with a male audience present tended to have higher
levels than those with no audience or a female audience.
Our hypothesis that 11KT levels would rise after interacting was also not supported; intermediate 11KT levels were
found for males before interaction, and either rose or fell
after interaction, depending on audience type.
The change in 11KT levels in male interactants is not surprising given the changes that occur in aggressive behavior as
a result of nesting condition and audience diVerences (Doutrelant et al., 2001; Dzieweczynski et al., 2005). In general,
males with nests and males that interacted with a female
present had the lowest 11KT levels. Males that did not have
nests and interacted in the presence of a male audience had
elevated 11KT levels in comparison to those of males that
had nests and interacted in the presence of a male. This echoed the Wnding that males are more aggressive in the presence of a male audience when they do not have nests
compared to when they do (Dzieweczynski et al., 2005). It is
possible that a reduction in aggression and 11KT beneWts
males in this situation, as building a nest is likely to be energetically costly to a male; therefore, losing a Wght in the presence of two potential usurpers could be disadvantageous.
Alternatively, the increased 11KT levels males show
after interacting in the presence of a male audience when
they do not have nests could reXect elevated aggression levels as a result of males attempting to compete with both
their opponents and the audience males. A male that
quickly escalates a Wght may be seen as a more diYcult
opponent and may deter both the rival and the audience.
188
T.L. Dzieweczynski et al. / General and Comparative Endocrinology 147 (2006) 184–189
Overall, males tended to have higher 11KT levels after
interactions in which they had a lot invested. For example,
males that are attempting to establish a territory (i.e., males
without nests) in the presence of a male or no audience are
highly aggressive and have elevated 11KT levels that mirror
this increased aggression.
In addition, we saw higher male 11KT levels after males
without nests had interacted in the presence of a male or no
audience compared to males that had nests and interacted
with a female present. These diVerences are not reXected in
male aggression during these interactions (Dzieweczynski
et al., 2005). The males with nests may have had decreased
11KT levels for one of two reasons. First, it is possible that
males have fought to completion (i.e., a winner has been
determined) and this is accompanied by a decrease in available 11KT after the interaction in the presence of the
female. This is a possibility since these interactions tended
to be the most aggressive out of the various treatment
groups (Dzieweczynski et al., 2005). Alternatively, males
with nests may be more in a courtship phase (i.e., ready to
mate) than males that are not nest holders. If this is the
case, males that are more motivated to court may have
reduced 11KT levels so that they do not act too aggressively towards potential mates. Males that act aggressively
towards females risk discouraging females from mating.
Territorial intrusions have been demonstrated to inXuence androgen levels in teleosts. Established male stoplight
parrotWsh experienced peaks in androgen levels when
another male intruded into the territory (Cardwell and
Liley, 1991). Hirschenhauser et al. (2004) found that 11KT
levels were long-lasting and elevated in males of a polygynous cichlid species and were relatively unaVected by a simulated intruder. A rise in 11KT levels was found after male
cichlids had interacted with females (Borges et al., 1998)
and a number of studies have demonstrated that territorial
males have higher androgen levels than non-territorial
males in teleost Wsh (e.g., Cardwell and Liley, 1991; Elofsson et al., 2000; Francis et al., 1993; Oliveira et al., 1996).
Our study adds an additional layer to these Wndings: androgen levels in male Siamese Wghting Wsh are indeed inXuenced by territorial status but are inXuenced by the
presence of observers as well.
This experiment complements prior Wndings that males
modify their behavior when an audience is present, by demonstrating that there is a subsequent alteration in 11KT levels after these observed interactions. As androgens likely
mediate changes within a male that are required for attentiveness and readiness to Wght, and behavior, in turn, can
inXuence androgen levels, it is not surprising that we Wnd
diVerences in 11KT levels before and after Wghting as well
as audience- and nest-dependent changes.
Acknowledgments
The authors thank Rich Granquist, Rick Alvey, Heather
Bleakley, Troy Smith, and Emilia Martins, and two anonymous reviewers for their assistance during the preparation
of this manuscript. We also thank Matthew Grober’s laboratory for assistance with the steroid extraction protocol.
This study used the facilities of the Animal Behavior lab
within the Center of the Integrative Study of Animal
Behavior (CISAB) and was supported by grants from
CISAB and the Indiana Academy of Sciences.
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