Socially induced and rapid increases in aggression are inversely r.docx

Socially induced and rapid increases in aggression are inversely
related to brain aromatase activity in a sex-changing fish,
Lythrypnus dalli
This paper investigates a social trigger for rapid changes in
sexual phenotype and its effect on AA and behavior. The paper
analyses the effect of a changing social environment on
aggressive behavior and brain aromatase activity (bAA) in a
sex-changing fish, Lythrypnus dalli. Aromatase is responsible
for the conversion of androgen or testosterone into estradiol. It
can modulate behavior in adult birds and mammals.
When males were removed from the group, aggression in
females was increased. These females had lower brain
aromatase activity and similar gonadal aromatase activity. This
aggressive behavior was inversely proportional to bAA. In this
fish when males were removed, some behavioral and
morphological modifications were produced which changed
sexual phenotype from female to male.
The paper compared AA and behavior of females in the early
stage of sex change to control females, established males, and
recently sex changed fishes.
Materials
Fishes were collected from Catalina Island California and the
experiment was carried out with 19 social groups. The groups
were selected such that each group had one large male, one
large female which was smaller than the male and two females
smaller than the large female. They were kept for five days so
that they adjust to new social condition. The observation on
female behavior was observed on forth day for 10 minutes. This
was done both in the morning as well as in the afternoon. The
behavior was recorded in the form of jerks, approaches and
displacements. Displacements are ritualized aggression defined
as moving within 5 cm of another fish and resulting in that fish
moving away and jerks are male-typical courtship swims.
The groups were divided into four types mainly dominance

phase, sex changed, control females and males. In dominance
phase males were removed in the morning on the fifth day and a
female’s behavior was observed for 10 min, 10 min in the
afternoon and 10 min in the morning the next day. They
sacrificed large female groups after any of these three
observation periods.
All dominance phase fishes had been sacrificed after the third
observation period. The time was recorded and female tissue
was frozen. The dominance phase fishes are at an early stage in
the sex change process.
In sex-changed groups, the male was also removed on the fifth
day and the large female was allowed to fully change sex. Once
the sex changer fertilized eggs as a male, it was sacrificed.
In control female groups, the male remained in the group and
the large female was sacrificed at the same time as in the sex
changer groups. The sex changer and control groups were paired
two by two before experiments began and the large females in
these groups were sacrificed in parallel on the same days.
Six males that had remained in control groups were also
sampled at the same time as control females and their brains and
gonads were collected for analysis.
The fishes were sacrificed via decapitation and the brain and
then the gonad were removed and frozen on dry ice. The tissues
were kept for aromatase activity assay.
Aromatase Assay
The frozen brain and gonad samples were weighed,
homogenized and assayed for AA. This was done by measuring
tritiated water production from androstenedione. The
homogenized tissue was incubated with 25 nM androstenedione
for 1 hr for brain and 15 minutes for gonadal tissue. They
determined the protein content using micro modification of the
Bradford method.
Displacement behavior was compared before male removal and
before sacrifice. Linear regressions were used to analyses
relationships between AA levels, genitalia, behavior and latency
between male removal and sacrifice.

This graph shows the observation of aggressive displacement
behavior and as is clear from the graph, on day 4 prior to
removal, there is not much difference. On day 5 the female
dominance increased.
In this graph we can see that Brain AA was significantly lower
in dominance phase and sex-changed individuals compared to
control females.
In this graph we can clearly see that established males had
lower brain AA than all other groups and lower gAA than all
groups except dominance phase females (second and third
panels).
This graph depicts the regression of bAA against socially
induced increases in aggression in Lythrypnus dalli during the
dominance phase of sex change.
In this model we can clearly see that estradiol production
decreases, while testosterone levels increase. Higher levels of
testosterone (T) substrate could then increase conversion to 11-
ketotestosterone.
From the results obtained it is quite clear that the removal of
the male from stable social groups results in a rapid (within
hours) increase in aggression in the largest female, correlated
with lower bAA but not gAA.
The results are novel as –
(i) demonstrate socially induced decreases in bAA levels that
correspond with increases in aggressive behaviour,
(ii) identify this process as a possible neurochemical mechanism
regulating the induction of behavioural, and subsequently
gonadal, sex change and
(iii) show differential regulation of bAA versus gAA resulting
from social manipulations

Proc. R. Soc. B (2005) 272, 2435–2440
doi:10.1098/rspb.2005.3210
on September 9,
2015http://rspb.royalsocietypublishing.org/Downloaded from
Socially induced and rapid increases in aggression
are inversely related to brain aromatase activity in
a sex-changing fish, Lythrypnus dalli
Michael P. Black
1,*, Jacques Balthazart
2
, Michelle Baillien
2
and Matthew S. Grober
1
1
Center for Behavioural Neuroscience, Georgia State University,
PO Box 3966, Atlanta, GA 30302-3966, USA
2
Center for Cellular and Molecular Neurobiology, University of
Liège, 4020 Liège, Belgium
Published online 14 September 2005

* Autho
Received
Accepted
Social interactions can generate rapid and dramatic changes in
behaviour and neuroendocrine activity. We
investigated the effects of a changing social environment on
aggressive behaviour and brain aromatase
activity (bAA) in a sex-changing fish, Lythrypnus dalli.
Aromatase is responsible for the conversion of
androgen into oestradiol. Male removal from a socially stable
group resulted in rapid and dramatic
(R200%) increases in aggression in the dominant female, which
will become male usually 7–10 days later.
These dominant females and recently sex-changed individuals
had lower bAA but similar gonadal
aromatase activity (gAA) compared to control females, while
established males had lower bAA than all
groups and lower gAA than all groups except dominant females.
Within hours of male removal, dominant
females’ aggressive behaviour was inversely related to bAA but
not gAA. These results are novel because
they are the first to: (i) demonstrate socially induced decreases
in bAA levels corresponding with increased
aggression, (ii) identify this process as a possible
neurochemical mechanism regulating the induction of

behavioural, and subsequently gonadal, sex change and (iii)
show differential regulation of bAA versus gAA
resulting from social manipulations. Combined with other
studies, this suggests that aromatase activity
may modulate fast changes in vertebrate social behaviour.
Keywords: vertebrate; plasticity; oestrogen; teleost; 11-
ketotestosterone; testosterone
1. INTRODUCTION
Aromatase, the enzyme that converts testosterone to
oestradiol, has been implicated in the early development
and later adult expression of sexual behaviour in a wide
range of vertebrate species. Early treatments with
aromatase inhibitors that generated rats with a bisexual
phenotype (Bakker et al. 1993) or reversed gonadal sex in
chickens (Elbrecht & Smith 1992) exemplify the import-
ant role of aromatase during sexual development.
Aromatase in the brain also modulates behaviour in
adult birds and mammals (Lephart 1996; Pinckard et al.
2000). Most effects of oestrogens derived from testoster-
one aromatization are thought to reflect specific changes in

the transcription of oestrogen-dependent genes (McEwen
& Alves 1999) but oestrogen can have rapid actions
through non-genomic mechanisms in a variety of biologi-
cal systems (Kelly & Ronnekleiv 2002) and its production
can be rapidly regulated via changes in brain aromatase
activity (AA) in birds (Balthazart et al. 2003). Considering
this fast regulation of brain AA and rapid oestrogen effects
on brain and behaviour, we investigated a social trigger for
rapid changes in sexual phenotype and its effect on AA and
behaviour. In a socially stable group of bluebanded gobies,
Lythrypnus dalli, removal of the dominant male produces
dramatic behavioural and morphological modifications in
the dominant female, which then changes sexual pheno-
type from female to male. Within minutes to hours of male
r for correspondence ([email protected]).
10 December 2004
8 June 2005
2435
removal, the dominant female increases her aggressive
behaviour, an accurate early indicator that the female will

morphologically change sex to male (Reavis & Grober
1999). This system therefore exhibits socially mediated
transitions between sexual phenotypes and an early and
unambiguous behavioural change that robustly predicts
the individual that will change sexual phenotype.
In preliminary studies of L. dalli, males had lower AA in
both brain and gonad than females, suggesting that a
decrease in AA occurs during sex change. Changing social
interactions in L. dalli could downregulate AA (Grober
1997) and cause a dramatic change in behaviour (Reavis &
Grober 1999). To test this hypothesis, we compared AA
and behaviour of females in the early stage of sex change to
control females, established males, and recently sex-
changed fishes.
2. MATERIAL AND METHODS
(a) Subjects and in vivo manipulations
Fishes were collected off the coast of Catalina Island,
California (permit no. SC-003083), and then maintained in
a fish facility in Atlanta, Georgia. Our experiment ran from

February to March of 2003 with 19 social groups. Each group
had one large male (standard length (SL)Z37.27G0.53 mm,
meansGs.e.m.), one large female at least 3 mm smaller than
the male (SLZ30.85G0.61 mm), and two females at least
3 mm smaller than the large female (SLZ22.82G0.35 mm).
These sizes assured male dominance over all fishes and the
largest female’s dominance over all females in the group. Each
q 2005 The Royal Society
http://rspb.royalsocietypublishing.org/
2436 M. P. Black and others Socially induced changes in brain
aromatase
on September 9,
group was placed in a 40 l aquarium with a PVC nesting tube
and given 5 days’ adjustment to new social conditions. On the
fourth day, the largest female’s behaviour was observed for
10 min in both the morning and afternoon. Recorded
behaviour included approaches, displacements and jerks
(Reavis & Grober 1999). Briefly, displacements are ritualized
aggression defined as moving within 5 cm of another fish and
resulting in that fish moving away, and jerks are male-typical

courtship swims. Each behaviour was averaged for the day as
baseline frequency.
There were four group types: dominance phase, sex-
changed, control females and males. In dominance phase
groups (nZ8), males were removed in the morning on the
fifth day and a female’s behaviour was observed for 10 min
after male removal, 10 min in the afternoon and 10 min in the
morning the next day. The large female in these groups was
sacrificed after any of these three observation periods once
she met behavioural criteria for dominance, as assessed by
exclusive access to the nest tube and/or a doubling of her
baseline frequency of aggressive displacement behaviour. All
dominance phase fishes had been sacrificed after the third
observation period. In each case, we recorded the time from
when the male was removed from the social group to when
the female’s tissue was frozen (see below). These latencies are
conservative time estimates because if a male had been in the
nest tube, it may have taken some time before the female

discovered that the male was missing. The dominance phase
fishes are at an early stage in the sex change process.
In sex-changed groups (nZ4), the male was also removed
on the fifth day and the large female was allowed to fully
change sex. Once the sex changer fertilized eggs as a male, it
was sacrificed.
In control female groups (nZ4), the male remained in the
group and the large female was sacrificed at the same time as
in the sex changer groups. The sex changer and control
groups were paired two by two before experiments began and
the large females in these groups were sacrificed in parallel on
the same days (8.5G2.53 days) after male removal in the sex-
changed groups.
To provide additional reference values, six males that had
remained in control groups were also sampled at the same
time as control females and their brains and gonads were
collected for analysis. Four males came from the same groups
as the four control females used in this study. The other two
males came from control groups where ‘females’ were

excluded because gonad structure in females did not
correspond with their genitalia (see below). No difference
was detected between these two subgroups of males in brain
or gonadal AA or last recorded displacements toward the
dominant female (unpaired t-test, tZ1.28, pZ0.27 and
tZ1.40, pZ0.24, tZ0.27, pZ0.80, respectively).
All fishes above were rapidly sacrificed via decapitation
and the brain and then the gonad were removed and frozen on
dry ice. Tissues were kept at K78 8C until shipping, on dry
ice, to Belgium for AA assays.
The genital papilla (external genitalia) of each fish was
photographed before and after the experiment and length:
width ratios were measured (Carlisle et al. 2000). The ratios
were used to assess the subjects’ sex at the experiment’s start.
However, the genital papilla of L. dalli is not a perfect
predictor of functional sex (St. Mary 1993). Before freezing,
gonad inspection verified initial genital-based sex assignment
and five fishes coming from three different groups were found
to have gonads that were not consistent with the initial sex

Proc. R. Soc. B (2005)
assignment. These groups were removed from the experiment
before we were aware of their brain or gonadal AA, their
papillae data were not included, and numbers presented
above correspond to the final sample sizes.
(b) Aromatase assay
All frozen brain and gonad samples were weighed, homogen-
ized, and assayed for AA by measuring the tritiated water
production from [1b-3H]-androstenedione, as described by
Roselli & Resko (1991), with minor modifications (Baillien &
Balthazart 1997). Homogenates containing about 1 mg of
fresh weight tissue per assay were incubated with 25 nM
androstenedione at 37 8C for 1 h for brain and 15 min for
gonadal tissue. The incubation durations were selected based
on preliminary experiments to limit the amount of substrate
metabolized so that the enzymatic reactions could proceed
linearly during the entire incubation period (data not shown).
Preliminary assays had confirmed that the substrate concen-
tration used here is saturating (at least five times Km) in

L. dalli as it is in goldfish (Zhao et al. 2001).
Within each experiment, controls using boiled brain or
brain samples with an excess (final concentration 40 mM) of
the potent and specific aromatase inhibitor, R76713
(Racemic vorozole, Janssen Pharmaceutica, Beerse, Belgium)
never exceeded 300–600 dpm while active control samples
had radioactivities ranging between 2000 and 150 000 dpm.
Assays were performed so that each run had controls and
samples from each of the experimental groups. A recovery of
93G2% was usually obtained from samples of 10 000 dpm
tritiated water conducted throughout the entire purification
procedure (incubation, centrifugation and Dowex column).
Protein content of all homogenates was determined
in triplicate by a micromodification of the Bradford
method (Bradford 1976). Enzyme activity was expressed in
pmol h
K1
mg
K1

protein after correction of the counts for
quenching, recovery, blank values and percentage of tritium
in b-position in the substrate.
(c) Data analysis
Statistics were performed using JMP 5.0.1, STATVIEW 5.0 and
SAS 8.02 (SAS Institute, Cary, NC). A repeated measures
ANOVA was used to compare displacement behaviour before
male removal (day 4) and before sacrifice across groups,
followed by paired t-tests with Bonferroni correction to
compare changes in displacement behaviour within each
experimental group. A MANOVA followed by one-way
ANOVAs was used to compare differences between groups
and was followed when appropriate by Fisher protected least
significant difference tests to compare groups two by two.
Linear regressions were used to analyse relationships between
AA levels, genitalia, behaviour and latency between male
removal and sacrifice. All data in the text are presented as
meansGs.e.m.

3. RESULTS
(a) Behaviour
There was an overall difference in displacement behaviour
before male removal (day 4) and displacements before
sacrifice, but no overall difference between treatment
groups (F1,13Z12.09, pZ0.004 and F2,13Z0.645,
pZ0.54, respectively; figure 1a; repeated measures
ANOVA). There was also a significant interaction between
these factors (F2,13Z4.21, pZ0.039). Further post hoc
average for day 4
(male present)
average for day 5
observation before sacrifice
control
female
dominance
phase female
sex-changed
individual
control
female

dominance
phase female
sex-changed
individual
male
0
20
30
ag
gr
es
si
ve
b
eh
av
io
r
(d
is
pl
ac
em

en
ts
/1
0
m
in
ut
es
)
0
4
8
12
16
20
0
40
80
120
160

br
ai
n
ar
om
at
as
e
ac
ti
vi
ty
(p
m
ol
/h
/m
g
pr
ot
ei
n)
go
na

da
l
ar
om
at
as
e
ac
ti
vi
ty
(p
m
ol
/h
/m
g
pr
ot
ei
n)
a
a

a, b
a
b b
b
c
NA
10
(a)
(b)
(c)
Figure 1. Aggressive displacement behaviour and aromatase
activity (AA) in the brain and gonad of Lythrypnus dalli. For
behaviour (a), on day 4 (prior to male removal), there was no
statistical difference in average daily displacements (baseline
frequency) between the largest females. On day 5 the male
was removed from dominance phase and sex-changed groups
and dominant females increased their aggressive behaviour.
Dominance phase fishes have no average (NA) day 5 data
because they were sacrificed during day 5 or just after. Brain
(b) but not gonadal (c) AA was significantly lower in
dominance phase and sex-changed individuals compared to
control females, and established males had lower brain AA
than all other groups and lower gAA than all groups except
dominance phase females (second and third panels). Bars
with a different letter are significantly different ( p!0.05)

based on post hoc Fisher PLSD tests following a significant
overall ANOVA.
2
4
6
8
10
12
14
16
br
ai
n
ar
om
at
as
e
ac
ti
vi
ty

(p
m
ol
/h
/m
g
pr
ot
ei
n)
7 8 9 10 11 12 13 14
increases in aggressive acts / 10 minutes
(displacements following male removal
minus displacements with the male present)
y = 2 2543.91–1332.67x; r 2 = 0.62
(3 : 00)
(26 : 15)
(3 : 20)
(2 : 50)
(3 : 00)(29 : 40)
(29 : 51)
Figure 2. Regression of brain aromatase activity against

socially induced increases in aggression in Lythrypnus dalli
during the dominance phase of sex change. Increased
aggression is scored as the number of aggressive acts
(displacements) performed after male removal during the
last test period before sacrifice minus the number of acts
performed in the presence of the male (prior to male removal).
The number next to each data point represents the time from
male removal (the social cue) to when the brain was frozen on
dry ice (e.g. 3 : 20 is 3 h and 20 min).
Socially induced changes in brain aromatase M. P. Black and
others 2437
on September 9,
analysis revealed that dominance females had statistically
higher displacement behaviour at sacrifice compared to
levels prior to male removal ( pZ0.003) and sex changers
showed a trend in the same direction ( pZ0.057), while
control females did not show a significant change in their
levels of displacement behaviour ( pZ0.767).
(b) Brain and gonadal aromatase
Dominance phase females had their brains frozen on dry
ice an average of 12.76G4.65 h after male removal. This
relatively large average latency is due to the fact that some
subjects went through two observation periods without
meeting criteria, and so had to be kept overnight for a third

observation period the following day. Thus, although most
groups reached criterion in the first 10 min observation
period (62.5%), our median time to collect brain tissue
was about 3.79 h after male removal. All groups that
reached criterion in the first observation period were
frozen less than 4.25 h after male removal.
Both brain and gonadal AA differed across groups
(MANOVA, Wilks’ Lambda, F6,34Z6.39, p!0.001).
Brain AA (bAA) and gonadal AA (gAA) were significantly
different between experimental groups (bAA: F3,18Z10.71,
p!0.001; gAA: F3,18Z4.72, pZ0.01; figure 1b,c). Post hoc
analysis showed that bAA was significantly higher in control
females than in the early dominance phase females (pZ0.01)
and sex changers ( p!0.01). The early dominance females
were not different from the sex changers ( pO0.05). In
addition, established males had lower bAA than all other
groups ( p!0.05). Post hoc analysis showed lower gAA in
established males than in all other groups ( p!0.05), except
dominance phase females ( pO0.05). Among all groups
other than established males, including those groups that
were in the process of changing sex and those that had just
changed sex and fertilized eggs as a male, there was no
significant difference in gAA ( pO0.05; figure 1c).

(c) Regressions
Multiple regression for those dominance phase females
that increased their rates of aggression in response to male
removal (nZ7) showed that there was no significant
relationship between bAA and both time after male
removal and increases in aggressive behaviour (F2,4Z
4.193, R
2Z0.677, pZ0.104) and there was no relation-
ship between bAA and time after male removal ( p-value
for individual regression coefficientZ0.455), but there
XO
HO
OH
OH
aromatase
testosterone
estradiol
11b
-hyd

roxy
lase
,
11b
- hy
drox
yste
roid
deh
ydro
gen
ase
O
OH
11-ketotestosterone
O
?
Figure 3. Model for the potential neurosteroidal consequences
of decreased aromatase activity (note grey X). First, estradiol
production decreases, while testosterone levels increase
(indicated by grey arrows). Higher levels of testosterone (T)
substrate
could (see question mark) then increase conversion to 11-
ketotestosterone (11-KT; grey arrow). The increased T and/or
greater

conversion to 11-KT, reduced oestrogen, or the greater
androgen : oestrogen ratio could be affecting the brain,
behaviour and
morphology of sex changing individuals.
aromatase
on September 9,
might be for bAA and increases in aggression ( p-value for
individual regression coefficientZ0.044). Simple linear
regression on the individual variables demonstrated that
bAA levels were not associated with the amount of time
after male removal (F1,5!0.001, R
2!0.001, pZ0.99),
but rather with the increased aggressive behaviour of these
dominance phase fishes (figure 2; F1,5Z8.22, R
2Z0.62,
pZ0.04). Moreover, the level of aggression in these
females, either prior to removal of the male or just before
sacrifice, was not significantly associated with bAA
(F1,5Z0.006, R
2Z0.001, pZ0.94 and F1,5Z0.881,
R
2Z0.15, pZ0.39, respectively). Thus, it is the increase

in aggressive behaviour following male removal that
appears to linked to lower bAA.
Regression showed no relationship between final
genitalia length : width ratio and bAA or gAA of all fishes
sacrificed (R
2!0.03, pO0.05). Further, there was no
relationship between gAA and bAA or the increase in
aggression of dominance phase fishes (R
2!0.03, pO0.05).
4. DISCUSSION
We show here higher bAA in females than in male L. dalli,
contrary to what is observed in birds and mammals
(Schumacher & Balthazart 1986; Roselli 1991), but
consistent with some fishes (e.g. Callard et al. 1978;
Contractor et al. 2004). More importantly, removal of the
male from stable social groups results in a rapid (within
hours) increase in aggression in the largest female,
correlated with lower bAA but not gAA. The female that
establishes dominance through this increased aggression
will fertilize eggs as a male, but the sex-changed individual

resulting from this process still has similar bAA and gAA
levels as dominance phase females. In contrast, in
established males, bAA and gAA are significantly lower
than in individuals that have recently changed sex from
female to male. These results are novel in that they are the
first to: (i) demonstrate socially induced decreases in bAA
levels that correspond with increases in aggressive
behaviour, (ii) identify this process as a possible neuro-
chemical mechanism regulating the induction of beha-
vioural, and subsequently gonadal, sex change and
(iii) show differential regulation of bAA versus gAA
resulting from social manipulations.
As noted above, established males showed much lower
bAA and gAA relative to recently sex-changed fishes.
Because testicular tissue is built up faster than ovarian
tissue is broken down, ovarian tissue remains in recently
sex-changed L. dalli (Black et al. 2005). Visual inspection
of gonads confirmed that sex-changed individuals in our

study still had ovarian tissue while males did not. The
ovarian tissue remaining in sex-changed fishes may both
generate high levels of gAA and regulate bAA through
gonadal oestrogen production. Since oestrogen upregu-
lates bAA in other fishes (Pasmanik et al. 1988; Kishida &
Callard 2001), ovarian oestrogen may prevent a drop in
bAA to levels observed in established males that have
completely degraded their ovarian tissue.
We demonstrated dramatic differences in AA between
the brain and gonad, but the differential regulation of bAA
and gAA is not unexpected. In goldfish and zebrafish, the
aromatase CYP19B gene is expressed more in the brain,
while CYP19A predominates in the gonad (Callard &
Tchoudakova 1997; Tchoudakova & Callard 1998).
Moreover, tissue-specific promoters can differentially
regulate aromatase expression in mice and humans
(Simpson et al. 2000). On a shorter-term basis, it has
also been shown in quail that brain and ovarian aromatase

react differentially to calcium and various phosphorylating
conditions (M. Baillien & J. Balthazart, unpublished
data). Several mechanisms are therefore available to
differentially regulate AA in the brain and gonad.
One important question that arises from the rapid
change in both behaviour and bAA, but not gAA is, does
Socially induced changes in brain aromatase M. P. Black and
others 2439
on September 9,
the decrease in bAA cause behavioural sex change? Our
data suggest that early in the process of brain reorganiz-
ation from female to male, bAA drops dramatically.
Decreased bAA in L. dalli should limit oestrogen
synthesis, leave more testosterone available for conversion
into 11-ketotestosterone (11-KT), a potent fish androgen
(Borg 1994; figure 3), and thus increase the brain
androgen : oestrogen (A : E) ratio. This increased A : E

ratio (or an increase in 11-KT production alone) may be
responsible for the increased aggression that negatively
correlates with bAA and is the earliest predictor of sex
change (Reavis & Grober 1999). In fishes, behaviour can
change rapidly in response to 11-KT (Remage-Healey &
Bass 2004), and several studies support the idea that
increased production of androgen, such as 11-KT,
increases aggression (e.g. Brantley et al. 1993; Borg
1994; Oliveira et al. 2001). Moreover, similar negative
correlations between bAA and aggression have been
observed in mammals. For example, in Peromyscus mice,
increases in aggressive behaviour correlate with reduced
bAA in the bed nucleus of the stria terminalis, and
experimentally reduced aromatase levels resulted in
shorter attack latencies (Trainor et al. 2004).
A second important question that arises from our data
is, does the decrease in bAA cause morphological sex
change? Androgens promote a variety of male-typical traits

in L. dalli, including testicular growth, secondary sex
characters like the accessory gonadal structure, and
increases in genitalia length : width ratios (Carlisle
2001). As changes in bAA can affect peripheral levels of
steroids in the zebra finch (male oestrogen levels;
Schlinger & Arnold 1991), the lower bAA in dominance
phase and sex-changed fishes may have been sufficient to
affect peripheral levels of oestrogen and androgens. The
results of the present study suggest the intriguing
possibility that a change in the social environment causes
early downregulation of bAA, which can act in two
possible ways to affect morphological sex: (i) decreased
bAA triggers a cascade of events resulting in altered serum
androgen levels and morphological sex change, or (ii)
decreased bAA changes morphological sex via direct
affects on serum hormone levels. In either case, the
brain leads the gonad in this process of sexual rediffer-
entiation (e.g. Grober & Bass 1991; Francis 1992). This

mechanism is consistent with studies showing that blue-
head wrasse behaviourally change sex in the absence of
their gonads and gonadally derived steroids (Godwin et al.
1996). This is also consistent with our model of down-
regulation of AA (Grober 1997) driving behavioural
changes that independently precede gonadal changes.
Finally, changes in aromatase function may signifi-
cantly alter brain and serum steroid levels and steroids are
known to have potent effects on sex-changing fishes
(Devlin & Nagahama 2002). Variation in AA among
different sexual phenotypes has been found in several
fishes (e.g. Schlinger et al. 1999) including sex changers
(e.g. Kincl et al. 1987; Lee et al. 2002). Consistent with
our results, treatment with aromatase inhibitor induces
female to male sex change in blackeye and coral gobies
(Kroon & Liley 2000; Kroon et al. 2005), but blocks
protandrous (male to female) sex reversal in the black
porgies (Lee et al. 2002). These studies implicate the role

of aromatase in the sex change process, but do not identify
the exact nature or timing of that role. Future studies in
L. dalli will focus on how quickly bAA changes following
male removal, whether a causal relationship between bAA,
aggression and sex change exists, and what mechanisms
decrease bAA.
We thank C. Mizell, E. Stokes, E. Rodgers, J. Netherton and
K. Felton for help with behavioural observations,
J. Pylkkanen and C. Drilling for the help in catching fishes,
E. Broadwater for papilla measurements, R. Earley and
C. Derby for statistical consulting, and R. Earley and
E. Rodgers for helpful comments on the manuscript. This
material is based upon work supported in part by the STC
Program of the National Science Foundation under
Agreement no. IBN-9876754, the Georgia Research Alliance
and GSU-RPE program, NSF-IBN 9723817 to M.S.G.
and NIMH (MH50388) and the Belgian FRFC (2.4562.05)
to J.B.
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http://rspb.royalsocietypublishing.org/Socially induced and
rapid increases in aggression are inversely related to brain
aromatase activity in a sex-changing fish, Lythrypnus
dalliIntroductionMaterial and methodsSubjects and in vivo
manipulationsAromatase assayData
analysisResultsBehaviourBrain and gonadal
aromataseRegressionsDiscussionWe thank C. Mizell, E. Stokes,
E. Rodgers, J. Netherton and K. Felton for help with
behavioural observations, J. Pylkkanen and C. Drilling for the
help in catching fishes, E. Broadwater for papilla
measurements, R. Earley and C. Derby for statistical
c...References

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