2015. Published by The Company of Biologists Ltd | Biology Open (2015) 4, 6978 doi:10.1242/bio.

20149597

RESEARCH ARTICLE

Brain nonapeptide and gonadal steroid responses to deprivation of

heterosexual contact in the black molly

ABSTRACT
Fish may respond to different social situations with changes in both
physiology and behaviour. A unique feature of fish is that social
interactions between males and females strongly affect the sexual
characteristics of individuals. Here we provide the first insight into the
endocrine background of two phenomena that occur in mono-sex
groups of the black molly (Poecilia sphenops): masculinization in
females and same-sex sexual behaviour, manifested by gonopodial
displays towards same-sex tank mates and copulation attempts in
males. In socially controlled situations, brain neurohormones impact
phenotypic sex determination and sexual behaviour. Among these
hormones are the nonapeptides arginine vasotocin (AVT) and
isotocin (IT), counterparts of the well-known mammalian arginine
vasopressin and oxytocin, respectively. To reveal potential hormone
interactions, we measured the concentrations of bioactive AVT and IT
in the brain, along with those of the sex steroids 17b-estradiol and 11ketotestosterone in the gonads, of females, masculinized females,
males displaying same-sex sexual behaviour and those who did
not. These data were supplemented by morphological and
histological analyses of the gonads. Correlations between brain
nonapeptides and gonadal steroids strongly suggest a cross talk
between hormonal systems. In the black molly, the masculinization
process was associated with the production of brain AVTand gonadal
steroids, whereas same-sex sexual behaviour involves both brain
nonapeptides, but neither of the sex steroids. This study extends
current knowledge of endocrine control of phenotypic sex and sexual
behaviour in fish and for the first time links brain nonapeptides with
the occurrence of male-male sexual behaviour in lower vertebrates.
KEY WORDS: Arginine vasotocin, Isotocin, Sex steroids,
Masculinization, Same-sex sexual behaviour, Black molly

INTRODUCTION

Fish may respond to different social situations with changes in

both physiology and behaviour. A unique feature of fish is
that social interactions between males and females strongly
affect the sexual characteristics of individuals (Devlin and
Nagahama, 2002). Moreover, some teleosts, even in adulthood,
spontaneously undergo phenotypic sex reversal in response to
Department of Genetics and Marine Biotechnology, Institute of Oceanology of
Polish Academy of Sciences, Powstancow Warszawy 55, 81-712 Sopot, Poland.
*These authors contributed equally and are jointly considered the first author
`

Author for correspondence (ekulczykowska@iopan.gda.pl)

This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution
and reproduction in any medium provided that the original work is properly attributed.

Received 22 July 2014; Accepted 29 October 2014

signals from the external environment (Godwin, 2010). A

diversity of sexual patterns in fishes has been reviewed
excellently by Sadovy de Mitcheson and Liu (Sadovy de
Mitcheson and Liu, 2008). This lability in sex expression
makes fish useful in studies of endocrine mechanisms
underlying socially related changes of sexual phenotype and
behaviour. The historically dominant view that sexual behaviour
is under the control of sex steroids has recently shifted towards a
paradigm in which brain neurotransmitters and neuromodulators
play a decisive role in the determination of sexual phenotype and
behaviour in the many fish species where sex change is under
social control (Semsar and Godwin, 2003; Godwin, 2010). In
socially controlled changes, brain neurohormones integrate
and interpret social cues (Perry and Grober, 2003). There are
several categories of signalling molecules in the brain that may be
related to the sexual plasticity of fish, namely gonadotropinreleasing hormone (GnRH) peptides, monoamines (serotonin,
noradrenaline, dopamine), and the nonapeptides arginine
vasotocin (AVT) and isotocin (IT) (for review: Foran and Bass,
1999; Bass and Forlano, 2008; Godwin, 2010; Godwin and
Thompson, 2012). AVT and IT, the teleostean equivalents of
mammalian vasopressin and oxytocin, respectively (Acher and
Chauvet, 1995), have attracted much attention due to their role
in the regulation of social and reproductive behaviours (for
review: Bass and Forlano, 2008; Godwin, 2010; Godwin and
Thompson, 2012). More than 60 years ago Pickford (Pickford,
1952) demonstrated that neurohypophysial extracts induced a
spawning reflex in hypophysectomized killifish (Fundulus
heteroclitus) and later proposed AVT as an active component
(Pickford and Strecker, 1977). Subsequent studies on AVT
mRNA expression, AVT-immunoreactivity, the size of AVTproducing cells (for review: Godwin and Thompson, 2012) and
AVT receptors in the brain (Semsar et al., 2001; Semsar and
Godwin, 2004; Huffman et al., 2012; Lema et al., 2012; Soares
et al., 2012; Oldfield et al., 2013) have revealed the contribution
of AVT to social and reproductive behaviours in various fish
species. In contrast, much less research has been dedicated to IT,
which regulates paternal behaviour in a monogamous cichlid fish
(OConnell et al., 2012), courtship in the three-spined stickleback
(Gasterosteus aculeatus) (Kleszczynska et. al., 2012), social
approach of males in goldfish (Carassius auratus) (Thompson
and Walton, 2004) and vocal communication in the plainfin
midshipman (Porichthys notatus) (Goodson and Bass, 2000).
Recently, analyses of AVT and IT concentrations in the brains of
several fish species have shown that these nonapeptides are
related to specific phases of breeding or the social status of
individuals (Ripley and Foran, 2010; Kleszczynska et al., 2012;
Sokoowska et al., 2013).
While the role of AVT in reproductive behaviour is well
established in many fish species, its involvement in process of
69

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Ewa Kulczykowska*,`, Hanna Kalamarz-Kubiak*, Marta Nietrzeba and Magdalena Gozdowska

RESEARCH ARTICLE

70

towards tank mates and copulation attempts, and those that did
not. Any relationships between nonapeptides and steroids were
verified statistically. We also performed morphological and
histological analyses of gonads. Finally, we considered the nature
of the signal triggering masculinization and same-sex sexual
behaviour in the black molly. Our findings extend current
knowledge of the endocrine background of phenotypic sex
and sexual behaviour in fish, and for the first time link
brain nonapeptides with the occurrence of same-sex sexual
behaviour.
RESULTS

The brain AVT (Fig. 1A) and IT (Fig. 1B) concentrations were
determined in different groups of the model fish black molly:
younger and older females and masculinized females, males that
displayed same-sex mating (called nave males) and those that did
not. Brain AVT levels were lowest in masculinized females and
nave males. Brain IT levels were similar in all females and
younger masculinized females, but significantly lower in older
masculinized females and males. Nave males had significantly
lower brain levels of both nonapeptides than males. In
females and masculinized females, the concentrations of these
neurohormones were age dependent: brain levels of AVT and IT
were significantly lower in older females and older masculinized
females, respectively.
Calculation of the AVT:IT ratios for the different groups gave
the following values: masculinized females 1:7 (12 months old)
and 1:9 (18 months old), females 1:1 (12 months old) and 1:2
(18 months old), nave males 1:3 and males 1:2. The
proportions of AVT and IT in masculinized females were
markedly different from those in females and males. The
AVT:IT ratio also differed between nave males and males. In
females and masculinized females, this ratio changed with age.
Concentrations of gonadal 11-KT (Fig. 2A) and E2 (Fig. 2B)
were measured in younger and older masculinized females,
females, nave males and males. In females, 11-KT levels were
low and E2 levels were high, whereas the opposite was found in
all males. In all masculinized females, 11-KT levels were
significantly higher than those in females, but lower than in all
males. E2 levels were lowest in masculinized females. In nave
males, the 11-KT and E2 levels were similar to those of males.
There were no significant differences in the GPI (gonopodial
index) between males (18.47 6 0.50), nave males (18.97 6 0.81)
and younger (16.75 6 0.4) or older (17.56 6 0.6) masculinized
females.
Pearsons correlation analysis (Table 1) showed no
relationship between brain AVT and IT concentrations in
females, masculinized females or males. However, a strong
positive correlation (r50.71; P,0.001) was demonstrated in
nave males. Positive correlations were also identified between IT
and E2 (r50.52; P,0.001) in older masculinized females,
and between AVT and E2 in males (r50.43; P,0.01).
Negative correlations were demonstrated between the AVT and
11-KT levels in nave males (r520.50; P,0.01), older females
(r520.54; P,0.001) and older masculinized females (r520.62;
P,0.001).
Images of the gonads of females, males, nave males and
masculinized females were compared. The gonads of
masculinized females were bigger and heavier than male testis
(0.030 6 0.004 vs 0.013 6 0.002 g respectively, P,0.01). In
contrast to testes, the masculinized individuals gonads contained
disorganized spermatocysts (spermatogenic cysts) and irregularly

Biology Open

masculinization or sex change is less well characterized (Grober

and Sunobe, 1996; Reavis and Grober, 1999; Godwin et al.,
2000). Furthermore, in comparison with AVT, very little is
known about the action of IT. Lastly, despite the fact that samesex sexual behaviour has been described in many fish species
(Morris, 1951; Bagemihl, 1999; Field and Waite, 2004; Tobler
et al., 2005; Cureton et al., 2010, Bierbach et al., 2013), the
endocrine background of this phenomenon has never been studied
in fish or in other lower vertebrates. The present study is an
attempt to fill these knowledge gaps. Previous studies have
identified relationships between AVT and androgen levels, and
sexual phenotypes in fish (see for example: Goodson and Bass,
2001; Semsar and Godwin, 2004; Oliveira et al., 2005; AubinHorth et al., 2007). The aim of the present study was to examine
the response of female and male fish to deprivation of sexual
partners by measuring its effect on the levels of nonapeptides in
the brain and steroid levels in the gonads. We used a procedure to
determine the concentration of free nonapeptides AVT and IT
after their dissociation from non-covalent complexes. This was
important because only this nonapeptide fraction binds to specific
receptors to act as neurotransmitters and/or neuromodulators
in the brain. This analytical procedure, which permits the
measurement of bio-active nonapeptides AVT and IT at their
site of action, has been used successfully, with slight
modifications, in several fish species (Gozdowska et al., 2006;
Kleszczynska et al., 2012; Almeida et al., 2012; Kleszczynska
and Kulczykowska, 2013; Sokoowska et al., 2013; Martos-Sitcha
et al., 2013). However, in small fishes such as black molly, a
limitation of the procedure is that AVT and IT levels can only be
analyzed in whole brains, and not in separate populations of
neurones, and AVT mRNA expression in magnocellular and
parvocellular neurones in the POA (preoptic area) appears to be
regulated independently during sex change (Perry and Grober,
2003).
Several years of research on the black molly (Poecilia
sphenops) conducted in our laboratory indicate that this wellknown aquarium fish is a good model candidate for the present
study. This species has several useful distinctive characteristics. It
displays conspicuous sexual dimorphism: the males are smaller
than the females and have a larger dorsal fin and an anal fin
modified into an intromittent organ, the gonopodium. When kept
in a mono-sex group, females undergo masculinization and males
exhibit same-sex sexual behaviour, manifested by gonopodial
displays towards same-sex tank mates and copulation attempts.
These phenomena are triggered off in a reproducible manner
under laboratory conditions. The masculinization process
proceeds only in one direction, is irreversible and generates
individuals with distinct features: they are the size of females,
have well formed gonopodia and changed morphology of gonads.
Moreover, they manifest male-like sexual behaviour with
courting (gonopodium swinging, see later) and attempted
copulation with females. On the other hand, males in male-only
groups display male-to-male sex attraction, with behaviour
equivalent to attempted copulation. In mixed sex groups, all
males mate only with females. Conveniently, there is no social
hierarchy within a group of black mollies, so social rank did not
influence the results of this study (Backstrom and Winberg, 2009;
Maruska et al., 2013).
Using the black molly model, we have compared the brain
concentrations of AVT and IT, and the gonadal concentrations of
17b-estradiol (E2) and 11-ketotestosterone (11-KT) in females,
masculinized females, males that exhibited gonopodial displays

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RESEARCH ARTICLE

Biology Open (2015) 4, 6978 doi:10.1242/bio.20149597

distributed large bundles of spermatozeugmata (Fig. 3A,B). The

results of Experiment 4 showed that masculinized individuals
were infertile. In nave males, the microscopic structure of the
gonads were similar to those of males (Fig. 4AD).
DISCUSSION

Responses of black molly females and males to the lack of mates

in the group were different: masculinization and same-sex sexual
behaviour, respectively. Our findings revealed that
masculinization involves brain AVT and gonadal steroids,
whereas same-sex sexual behaviour involves both brain
nonapeptides, AVT and IT, but not sex steroids.
There is evident sexual dimorphism in brain AVT and IT
concentrations in the black molly, just like that described
previously in the three-spined stickleback (Kleszczynska et al.,
2012; Kleszczynska and Kulczykowska, 2013). In female brains,
irrespective of age, we measured significantly higher AVT and IT
concentrations than in the brains of males. As was confirmed in
younger and older masculinized females, the process of
masculinization is apparently linked to a decrease in whole
brain AVT levels towards values typical for males. A reduction in
brain IT was also noted, but only in older masculinized females.
The masculinization process appears to be associated not only
with alterations in the concentrations of single nonapeptides, but
also with the AVT:IT ratio in the brain. The activity of cells
producing AVT and the abundance of its receptor transcript in the

brain are sex-dependent and coupled with changes in sex

phenotype in some fish species (Grober and Sunobe, 1996;
Reavis and Grober, 1999; Godwin et al., 2000; Semsar and
Godwin, 2003; Maruska et al., 2007; Lema et al., 2012). In the
present study, we observed masculinization in younger or older
individuals, but the factors responsible for the timing of the
occurrence of this phenomenon remain unknown. We also
demonstrated the influence of age on brain nonapeptide levels
(Fig. 1A,B) which should be taken into consideration when
planning future studies.
Sexually nave males exhibiting gonopodial displays towards
tank mates and copulation attempts had significantly lower
levels of both AVT and IT than males not displaying such
behaviour (Fig. 1A,B). The AVT:IT ratios were also different.
This finding is the first indication of a relationship between brain
nonapeptides and the occurrence of same-sex sexual behaviour
in fish. There have been no other similar studies in lower
vertebrates, but in humans, Swaab and Hofman (Swaab and
Hofman, 1990) found an enlarged vasopressinergic subnucleus
in the SCN (suprachiasmatic nuclei) of the hypothalamus of
homosexual men, suggesting a link between the nonapeptide
vasopressin, the human counterpart of vasotocin, and types of
sexual attraction in humans. In general, little is known about
neurohormonal mechanisms controlling mammalian sexual
attraction, not to mention lower vertebrates. Besides indicating
the involvement of nonapeptides AVT and IT in same-sex
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Fig. 1. Arginine vasotocin (A) and isotocin

(B) in brains of males, nave males, younger
and older females and masculinized
females of Poecilia sphenops. Values are
presented as means 6 SEM. Statistical
differences are indicated as: (A) a: P,0.001 vs
the others, b: P,0.001 vs nave males,
younger and older masculinized females, c:
P,0.05 vs nave males and older
masculinized females. (B) a: P,0.05 vs nave
males, b: P,0.001 vs males and nave males,
c: P,0.001 vs males, nave males and older
masculinized females, d:
P,0.01 vs nave males, younger and
older females.

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Fig. 2. 11-ketotestosterone (A) and 17-b estradiol

(B) concentrations in gonads of males, nave males,
older females and younger and older masculinized
females of Poecilia sphenops. Values are presented
as means 6 SEM. Statistical differences are indicated
as: (A) a: P,0.001 vs older females, b: P,0.05 vs
younger masculinized females, c: P,0.001 vs older
females and younger masculinized females, d: P,0.01
vs older females and older masculinized females, e:
P,0.01 vs older females, f: P,0.001 vs older females.
(B) a: P,0.001 vs the others, b: P,0.05 vs younger
masculinized females, c: P,0.001 vs older
masculinized females, d: P,0.05 vs younger
masculinized females, e: P,0.01 vs older
masculinized females.

mating in fish, our data also suggest that the production

of nonapeptides in the male brain is stimulated by sexual
contact with females, because AVT and IT concentrations were
significantly higher in female-experienced individuals than in
sexually nave ones (Fig. 1A,B).

Correlation analysis showed no relationship between brain

levels of AVT and IT in females, masculinized females or males
that do not display same-sex mating behaviour. This is not
surprising because it is known that AVT and IT are synthesized
independently in separate neurones in the POA (Holmqvist and

AVT/IT
11-KT/E2
AVT/11-KT
AVT/E2
IT/11-KT
IT/E2

Males
n511

Nave males
n56

Younger females
n55

Older females
n59

Younger masculinized females

n58

Older masculinized females

n58

20,17
0,09
20,29
0,43**
0,25
20,11

0,71***
20,72***
20,50**
20,03
0,11
20,31

0,07
_
_
_
_
_

0,14
20,25
20,54***
0,25
20,13
20,04

20,35
_
_
_
_
_

20,10
20,09
20,62***
20,10
0,04
0,52***

***P,0.001, **P,0.01.

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Table 1. Pearsons correlation analysis

Fig. 3. Transverse section of gonad of male (A) and masculinized

female (B) of Poecilia sphenops. In male and masculinized female, germ
cells are in all spermatogenic stages. Connective tissue constituted about
5% of both gonads. (A) Progressing stages of germ cells are distributed from
the distal to proximal part. Spermatogonia (1) are restricted to the distal
termini of the lobules. Spermatozeugmata (2) are regularly distributed within
central part of the lobules. (B) Spermatocysts (spermatogenic cysts) (3) are
irregularly organised. Large bundles of spermatozeugmata (2) are irregularly
distributed.

Ekstrom, 1995; Saito et al., 2004). However, the finding of a

strong positive correlation between brain AVT and IT levels in
nave males displaying same-sex mating was unexpected. Perhaps
this suggests a direct functional connection between AVT- and
IT-producing neurones (or a common regulatory mechanism) that
is linked to specific behaviour or the lack of sexual experience of
sexually nave males. Although this observation is currently
difficult to explain, it certainly warrants further study.
In this study, we also examined the involvement of gonadal
steroids in the determination of phenotypic sex and sexual
behaviour, and the relationship between these sex hormones and
brain nonapeptides. Gonads are the main sources of circulating
sex hormones (Kime, 1993) therefore the analysis of 11-KT and
E2 in gonads is applicable when plasma collection is not possible
or the plasma volume is low (Becker et al., 1992). This approach
is also useful when a link between a local production of sex
hormones and gonads morphology is discussed as it is in this
case. In black molly masculinized females, the levels of 11-KT
were significantly higher and E2 significantly lower than in

Biology Open (2015) 4, 6978 doi:10.1242/bio.20149597

females. This result was not unexpected because a similar

observation was made in the Hawaiian saddleback wrasse
(Thalassoma duperrey), a protogynous reef species, (Nakamura
et al., 1989) where sex hormones were measured in plasma. In
addition, Cardwell and Liley (Cardwell and Liley, 1991a;
Cardwell and Liley, 1991b) found an elevation of 11-KT and a
decline in E2 in the plasma of stoplight parrotfish (Sparisoma
viride) during a female-to-male sex change. It is also known that
gonopodium formation depends on androgens (Ogino et al.,
2004), and the stage of development of this organ should reflect
changes in the androgen level. However, in black molly, 11-KT
levels were considerably lower in masculinized females than in
males, despite the fact that both have fully developed gonopodia.
It should be noted that the macroscopic and microscopic
structures of the gonads of males and masculinized females
were markedly different. The masculinized individuals are steril
(Experiment 4), cannot reproduce and cannot regress back into
females. This maladaptive phenotype probably resulted from
generations of domestic inbreeding.
On the other hand, it should be stressed that the microscopic
structure of the gonads and sex steroid levels were identical in all
males, irrespective of whether or not they displayed same-sex
mating. Thus the occurrence of same-sex mating seems to be
independent of the sex steroid status. A strong negative
correlation between 11-KT and E2 levels was only observed in
sexually nave males, which suggests that an intrinsic negative
feedback mechanism may operate in the gonads that is linked to a
specific behavioural phenotype and/or the lack of sexual contact
with females. In addition, the timing of the masculinization
seemed to be related to the steroid concentration: the later this
process occurred, the higher the 11-KT level and the lower the E2
level. However, the number of individuals examined is too small
to draw any firm conclusions.
Given that the only way that social cue (the lack of a mate in
this study) can affect the gonads and behaviour is via the brain,
we suppose that neural mechanisms initiate socially-dependent
phenotypic sex and behavioural changes in the black molly.
Among several neuroendocrine pathways that could be engaged
in the transduction of social signals, a likely candidate is the
gonadotropic signalling pathway. A recent study by Kim et al.
(Kim et al., 2012) showed that GnRH peptides play important
roles in the regulation of the hypothalamic-pituitary-gonadal axis
and probably control gonad development and sex change in the
cinnamon clownfish (Amphiprion melanopus). It is probable that
a GnRH-related cue is responsible for the increased 11-KT and
decreased E2 levels detected in the gonads of black molly
masculinized individuals. However, gonadotropic signalling is
not the only neurohormonal mechanism that can stimulate
phenotypic sex change and control the behavioural phenotype
in fish. In the present study, we have shown that brain AVT is
linked to socially dependent masculinization and changes in
sexual behaviour of females. The male-like behaviour of
masculinized individuals, i.e. movements of the gonopodium
(full swing), courting and copulation attempts with females, is
associated with a lower male brain AVT level. Because the
brain nonapeptide levels were measured when the process of
masculinization was complete (confirmed by the fully developed
gonopodium and analysis of gonads), we can only speculate that
the decrease in brain AVT concentration acts to trigger the
phenotypic and behavioural changes. On the other hand, the
male-like behaviour of masculinized females does not seem to be
linked to the brain IT, because levels of this nonapeptide were
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similar in all females and younger masculinized individuals, and

were lower only in older masculinized individuals that behave
just the same as younger ones.
Previous studies have demonstrated relationships between
AVT, androgen levels and sexual phenotypes in various fish
species (see for example: Goodson and Bass, 2001; Semsar and
Godwin, 2004; Oliveira et al., 2005; Aubin-Horth et al., 2007).
The nature of the relationship between the GnRH- and AVT/ITrelated signalling systems in black molly remains an open
question. There is evidence that the GnRH and AVT pathways
overlap in Teleostei. GnRH-synthesizing neurones are widely
distributed in the POA hypothalamus region adjacent to the
AVT-containing neurons in the brain of the green molly Poecilia
latipinna (Batten et al., 1990). In the ventral POA of dwarf
gourami (Colisa lalia), IT-ir neurons are also immunoreactive to
GnRH (Maejima et al., 1994). The significant correlation
between brain nonapeptides and gonadal steroids that we have
identified in black molly is a strong indication of cross talk
between these hormonal systems. Interestingly, the character of
this interaction, be it positive or negative, depended on the
sexual phenotype of individuals. Because a correlation refers to
any relationship between two variables and does not identify the
cause or the effect, two different scenarios may be considered:
(1) GnRH stimulates AVT/IT neurones directly via neural
pathways and/or via sex steroids, and (2) AVT/IT triggers
GnRH-related cues, i.e. sex steroid production. Both of these
scenarios receive support in the literature (Saito et al., 2003;
Rodrguez and Specker, 1991).
The biological context of the occurrence of masculinization
and same-sex mating in black molly deserves some comment. In
the present study, only females from an overcrowded group
underwent masculinization. This suggests that high-density stress
plays some role in promoting the process of masculinization. In
some fish species, cortisol or its metabolites produced and
secreted during stress cause masculinization of females or induce
sex change (Hattori et al., 2009; Grillitsch et al., 2010), probably
74

because the molecular structure of glucocorticoids and 11-KT are

similar. As demonstrated during high temperature-induced
masculinization in several fish species, cortisol can also
promote 11-KT production in gonads (Fernandino et al., 2012).
However, in the case of the black molly, none of the females
showed any of the typical signs of stress or discomfort such as
aggressiveness, going into hiding or avoidance behaviour.
Therefore, we propose that other cues, such as pheromones or
visual stimulation, may be engaged in the masculinization
process. Recently, Mangiamele et al. (Mangiamele et al., 2013)
showed that exposure of male goldfish to androstenedione, a
pheromone released by males and females, inhibits social
approaches towards other males and increases AVT gene
expression in the POA. There is growing evidence on female
responses to pheromones (Corkum et al., 2008) and it seems
likely that these chemical cues play a pivotal role in causing black
molly females to change sexual phenotype. An abundance of E2
and its derivatives released by many females and/or a lack of
male chemical markers (11-KT and/or its derivatives) may
influence the proportion of sex steroids in the gonads, which
results in masculinization. The very low E2 levels measured in the
gonads of masculinized individuals, that were even below male
values, seems to confirm this. Vision plays a pivotal role in the
reproductive behaviour of many fish species, especially in the
Poeciliidae (Kodric-Brown and Nicoletto, 2001; Plath et al.,
2005; Tobler et al., 2006), so a second candidate for the signal
triggering masculinization in the black molly is visual
stimulation. In the present case, the visual stimulus is the
perception of many females in close proximity. Recently,
Amorim et al. (Amorim et al., 2013) demonstrated that visual
courtship signal is relevant in mate choice in the painted goby
(Pomatoschistus pictus). In a similar way, a lack of sensory cues
from females and a presence of visual or chemical signals from
males, may promote the same-sex sexual behaviour in nave
males. Both of the above scenarios, pheromones- and visionrelated, are possible and further research is required to establish

Biology Open

Fig. 4. Transverse section of testis of male

(A,B) and nave male (C,D) of Poecilia
sphenops. (A,C) spermatozeugmata, bundles
of spermatozoa with heads pointing outwards
and tails in the center; (B,D) regularly
organised cysts with different stages of
spermatogenesis: spermatocytes (1),
spermatids (2).

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which cue is critical in initiating masculinization and same-sex

sexual behaviour in black molly females and males.
In summary, our findings show that brain AVT and gonadal
steroids are engaged in masculinization of the black molly
females. On the other hand, the same-sex sexual behaviour of
sexually nave males is linked to brain AVT and IT levels, but
does not involve gonadal steroids. The occurrence of
masculinized individuals that display typical male behaviour
allows females to gain sexual experience in situations where there
is a dearth of natural sexual partners in the group. Same-sex
mating appears in mono-sex groups of sexually nave males or
sexually experienced males, with only the latter showing
aggressive behaviour. In lower vertebrates, male-male sexual
attraction is usually considered in the context of dominance rank,
but in our model species, there is no dominance hierarchy. It
should be noted that males have only a passing predisposition to
same-sex mating; when introduced into a mixed-sex group,
sexually experienced or nave males court and copulate only with
females. In non-human animals, most males that engage in samesex encounters also mate with females (Bailey and Zuk, 2009). In
conclusion, we have demonstrated, for the first time in lower
vertebrates, that brain AVT and IT may be involved in socially
controlled sexual acts that are not directly linked with procreation
and reproductive success.
MATERIALS AND METHODS
Animals and experimental design

The viviparous black molly (Poecilia sphenops), a well-known aquarium

fish, is a short-finned hybrid of Poecilia latipinna, the sailfin molly that is
not present in nature. In this year-round breeder, fertilization is internal.
In both P. latipinna and P. sphenops, male mate choice copying has been
found (Gabor and Aspbury, 2008). A laboratory stock of black mollies
(males and females) was bred and maintained at a temperature of
27 6 1 C in water of 0.2 ppt salinity with a 12L:12D photoperiod in
aerated aquaria at the Institute of Oceanology PAN (IO PAN Sopot,
Poland), where all experiments were carried out. The fish were fed
commercial Ichtio-vit flake food, and frozen mosquito and brine shrimp
larvae. In each experiment, the fish were sampled 3 h after the onset of
the light period. The following experiments were performed using fish
from the laboratory stock:
Experiment 1 females that do not undergo masculinization and
males that do not display same-sex sexual behaviour
Adult sexually mature females of 910 months of age were randomly
selected from the mixed sex laboratory stock of black mollies. At the
beginning of the experiment all females were of equivalent reproductive

state. When kept at low density (5 fish in an aquarium of 50 L) none of

these females underwent masculinization. The fish behaved indifferently
towards each other. The experiment was repeated 6 times and provided
30 females for further analyses. The fish were sampled at the ages of 12
and 18 months to be comparable to females which underwent
masculization at the ages of 12 and 18 months, respectively (Table 2).
Two adult males of 1218 months of age selected from the stock were
kept with two adult females of the same age in a 50-L aquarium. The
males directed their sexual interest only towards the females and showed
a mating preference for large individuals. They courted females,
performing a full swing (gonopodium swung forward 180 from its
resting position), and attempted to copulate with them. The experiment
was repeated 11 times and provided 22 males for further analyses. The
males were aged between 12 and 18 months when sampled (Table 2).
Experiment 2 masculinized females
Adult females of 910 months of age were randomly selected from the
mixed sex laboratory stock of black mollies to constitute female-only
groups. In all groups of females kept at high density (15 females in an
aquarium of 50 L) only one individual underwent masculinization. The
masculinization was completed in individuals at the age of 12 or 18
months. Therefore masculinized females were divided up into two groups
(younger and older) and compared with females from Experiment 1
sampled at the ages of 12 and 18 months, respectively. At the start of the
masculinization process an individual with a barely visible gonopodium
behaved indifferently towards conspecifics, but later on when its
gonopodium had developed in size, it performed a half swing
(gonopodium swung forward 90 from its resting position) towards
females. Individuals with a fully developed gonopodium displayed a
full swing and attempted to copulate with females. Full development
of the gonopodium occurred over about 3 months. The masculinization
process was irreversible and produced individuals with distinct features.
The masculinized individuals were as big as females, had a larger dorsal
fin than females and a gonopodium of the same size as that of males and
changed morphology of gonads. In this study, only individuals with a
fully developed gonopodium were sampled. The experiment was
repeated 24 times and provided 24 masculinized females for further
analyses (Table 2).
Experiment 3 triggering same-sex sexual behaviour
Since just after hatching, juveniles were kept in a 40-L aquarium for six
months. The males were removed as soon as their gonopodium became
visible. Males of about the same age (1218 months) and body size
(44.7 6 1.4 mm and 0.96 6 0.13 g) were put into two 50-L aquaria of 12
individuals per aquarium. All of these female-inexperienced males (nave
males) exhibited same-sex sexual behaviour manifested by gonopodial
displays towards same-sex tank mates and copulation attempts. When the
group was divided between six 50-L aquaria of 4 individuals per

Table 2. Animals and sampling protocols in Experiments 13

Older n516 (18 months old)

Males n522 (between 12
and 18 months old)
Nave males n524 (between 12
and 18 months old)

Experiment 2

Experiment 3

8 brains for AVT and IT; 4 gonads for

11KT and E2; 4 gonads for histology
8 brains for AVT and IT; 8 gonads for
11KT and E2; 8 gonads for histology

11 brains for AVT and IT; 11gonads for

11KT and E2; 11 gonads for histology
8 brains for AVT and IT; 6 gonads
for 11KT and E2; 12 gonads for
histology

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Biology Open

Experiment 1
Females n530
Younger n512 (12 months old) 6 brains for AVT and IT; 6 gonads
for histology
Older n518 (18 months old)
11 brains for AVT and IT; 9 gonads for
11KT and E2; 9 gonads for histology
Masculinized females n524
Younger n58 (12 months old)

aquarium gonopodial displays and copulation attempts had become even

more pronounced. It should be noted that female-experienced males in
male-only groups also displayed same-sex sexual behaviour. However, in
this situation, the female-experienced males were always aggressive
towards each other, whereas the sexually nave males were not. In this
study, we sampled and analyzed only the nave males to avoid any impact
of aggressive behaviour on the results (Backstrom and Winberg, 2009;
Lema and Nevitt, 2004; Iwata et al., 2010). The experiment provided 24
sexually nave males displaying same-sex sexual behaviour. The males
aged between 12 and 18 months were sampled from six 50-L aquaria for
further analyses (Table 2).
Experiment 4 determining the fertility of masculinized individuals
One masculinized female at the age of 12 or 18 months and two
unfertilized mature females of between 12 and 18 months old from the
low-density female-only group (Experiment 1) were put together in a 50L aquarium for 3 months. The masculinized female was then removed
and the females were observed for the next 3 months. This experiment
was repeated many times, but offspring were never produced. In
comparison, when one adult male from the stock (aged between 12 and
18 months with a fully developed gonopodium) and two unfertilized
mature females from the low-density female-only group were put
together in a 50-L aquarium for 3 months, this usually resulted in the
appearance of progeny.

Biology Open (2015) 4, 6978 doi:10.1242/bio.20149597

Technologies, USA) with a diode array detector. Chromatographic

separation of peptides was carried out on an Ultrasphere ODS column
(250 mm 6 4.6 mm; 5 mm) following a pre-column (45 6 4.6 mm I.D.)
comprised of the same material (both columns obtained from Beckman
Coulter, USA). Separation of peptides was achieved with a linear
gradient system consisting of 2040% solvent B (0.1% trifluoroacetic
acid (TFA) in acetonitrile:water, 3:1) in solvent A (0.1% TFA in water)
over 20 min. The column temperature was 20 C and flow rate 1 mL
min21. UV detection was performed at 215 nm. The equation of the AVT
calibration curve was y50.795x (R250.999), and that for IT, y50.864x
(R250.997). The limits of detection (LOD) of AVT and IT were
evaluated at a signal-to-noise ratio of 3:1 as 15 and 18 pmol mL21,
respectively. The intra-day precision of the method, expressed as relative
standard deviation (RSD), was within the ranges 3.66.1% and 4.45.9%
for AVT and IT, respectively. The concentrations of these brain
neurohormones were expressed as ng per g wet weight of tissue.
Gonadal steroids
Gonads were individually sonicated in 0.5 mL of phosphate buffer
(0.05 M, pH 7.4) supplemented with sodium azide (NaN3) using a
MicrosonTM XL 2000 (USA). The sonicates were centrifuged at 20,000 g
for 20 min at 4 C and the supernatants stored at 270 C prior to the
analysis of E2 and 11-KT levels.

Chemical analyses

11-ketotestosterone
The 11-KT concentration in gonad organic extracts was determined using
a Caymans competitive enzyme immunoassay (EIA) kit (Ann Arbor,
MI, USA). Gonad supernatants (250 mL) were extracted with 1 mL of
ethyl acetate/hexane (50:50 v/v) according to the method recommended
in the EIA kit protocol. The samples were then held at 220 C for 15 min
to separate the layers. The ethyl/hexane layer was decanted into a glass
tube and evaporated under a stream of nitrogen. This procedure was
repeated three times. Dried extracts were stored at 220 C prior to
analysis. The recovery rate of the extraction was between 98115%.
Extracts were dissolved in 0.5 mL of EIA buffer and samples of 50 mL
were taken for analysis. 11-KT-acetylocholinesterase (AChE) conjugate
was used as a tracer. A standard curve was prepared using eight standard
dilutions of 11-KT: 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 pg mL21.
The assay was conducted in microplates according to the EIA kit
manufacturers instructions with slight modifications. Microplates were
gently shaken for 15 min, incubated for 18 hours at 4 C and washed with
buffer using a HydroFlex strip-washer (Tecan, Austria). The plates were
then developed by shaking with Ellmans reagent in the dark for 90 min
and absorbance at 412 nm was read using a Sunrise Absorbance Reader
(Tecan, Austria). All samples were assayed in duplicate. The detection
limit of the assay was 13 pg mL21. The intra-assay coefficient of
variation was 0.8%. The inter-assay coefficient of variation was 7.8%.
The concentration of 11-KT was expressed as ng per g wet weight of
tissue.

Arginine vasotocin and isotocin

The AVT and IT concentrations in fish brains were measured using the
procedure described by Kulczykowska (Kulczykowska, 1995). Briefly,
the brains were individually sonicated in 1 mL of 0.25% acetic acid using
a Microson XL 2000 (Misonix Inc, NY, USA) and the sonicates were
placed in a boiling water bath for 3.5 min. The extracts were cooled on
ice and then centrifuged (20,000 g, 20 min, 4 C). The supernatants were
removed and loaded onto solid phase extraction (SPE) columns
(Bakerbond C18, 100 mg mL21, JT Baker, USA) that had been preconditioned with methanol followed by water. Impurities were washed
from the loaded columns with 1 mL of 5% acetic acid followed by 1 mL
of water and 1 mL of 5% methanol. Peptides were eluted with 2 mL of an
ethanol:HCl mixture (2000:1) and this elution was repeated once. The
pooled eluates were evaporated to dryness in a TurboVap LVTM (Caliper
Life Science, USA). Using the procedure described by Kulczykowska
(Kulczykowska, 1995), the efficiency of AVT and IT recovery following
SPE extraction was estimated as 8994% and 9295%, respectively.
High performance liquid chromatographic (HPLC) analyses were
performed using a 1200 series Quaternary HPLC system (Agilent

17b-estradiol
The E2 concentration in gonad organic extracts was determined using a
Spectria Estradiol radioimmunoassay (RIA) kit (Orion Diagnostica,
Finland). Gonad supernatants (200 mL) were extracted with 1.6 mL of
ethyl ether according to a modification of the method of Mori and Kano
(Mori and Kano, 1984). Samples were vortexed for 1 min at 350 g, for
30 min at 35 g and then held at 220 C for 30 min to separate the layers.
The ethyl ether layer was decanted into a glass tube and evaporated under
a stream of nitrogen. Dried extracts were stored at 220 C prior to
analysis. The recovery rate of the extraction was between 86109%.
Extracts were dissolved in phosphate buffer (0.05 M, pH 7.4)
supplemented with NaN3 and samples of 100 mL were taken for RIA
analysis. Iodinated E2 with 125I was used as a tracer. A standard curve
was prepared using six standard dilutions of 50, 150, 500, 1500, 5000 and
15,000 pmol L21. The assay was conducted in RIA tubes according to
the kit manufacturers instructions with slight modifications. The samples
were added to tubes that had been pre-coated with polyclonal anti-rabbit
antiserum. After vortexing for 10 seconds, the tubes were incubated for

Tissue sampling

Fish selected for further analyses were anaesthetized by immersion in MS

222 (tricaine methanesulphonate; Sigma-Aldrich, USA) solution in water
(50 mg L21), their spinal cords were sectioned and whole brain together
with pituitary and gonads removed (Table 2). The brains were weighed
and stored at 270 C prior to AVT and IT analyses. The gonads were
weighed, subjected to morphological examination and then either stored
at 270 C prior to E2 and 11-KT measurement or immediately fixed in
4% formalin solution for histology. The gonopodial index (GPI) was
calculated by expressing the gonopodial length as a percentage of the
total body length.
The ranges of total length and weight of individuals were as follows:
males 44.7 6 1.4 mm and 0.96 6 0.13 g; younger females 40.7 6
0.5 mm and 1.00 6 0.07 g; older females 54.9 6 0.5 mm and 3.44 6
0.61 g; younger masculinized females 47.8 6 1.2 mm and 1.62 6 0.17 g;
older masculinized females 51.4 6 2.4 mm and 2.35 6 0.39 g. The older
females and masculinized females were significantly bigger than younger
ones (p,0.01) and younger masculinized females were significantly bigger
than younger females (p,0.01).
All experiments complied with EC Directive 2010/63/EU for animal
experiments and with the guidelines of the Local Ethics Committee on
Animal Experimentation.

76

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RESEARCH ARTICLE

2 hours at 37 C, decanted, washed with 1 mL of 66 concentrated Tween

20 solution and decanted again. Radioactivity in each tube was measured
for 1 min using a Wallac Wizard 1470 gamma counter (Perkin Elmer
Life Science, USA). All samples were assayed in duplicate. The detection
limit of the assay was 37 pmol L21. The intra-assay coefficient of
variation was 6.5%. The inter-assay variation was not determined
because all samples were measured in the same assay. The concentration
of E2 was expressed as ng per g wet weight of tissue.
Histological analysis of gonads

Whole gonads were fixed in 4% formalin solution, dehydrated in

ethanol and embedded in paraffin. Sections of 5 mm were cut using a
RM2245 microtome (Leica Microsystems GmbH, Germany) and stained
with Mayers haematoxylin and eosin. Slides prepared from each gonad
were examined with a Leica HI1210 light microscope (Leica
Microsystems GmbH, Germany). The developmental stage of testes
was determined according to the classification worked out for oviparous
teleosts by Grier (Grier, 1981) and modified by Kinnberg et al.
(Kinnberg et al., 2003), while the developmental stage of oocytes was
determined according to the classification worked out for the guppy,
Poecilia reticulate, by Takano (Takano, 1964) and modified by Koya
(Koya et al., 1998).
Statistical analyses

Statistical analyses of data were carried out using Statistica 5.1 software.
Values are presented as means 6 SEM. For multiple comparisons of
hormone levels, analysis of variance (one-way ANOVA) was performed
followed by post hoc tests (Tukeys test for equal numbers of cases or
Spjotvoll and Stolines test for unequal numbers of cases). Pearsons
correlation coefficient was used to measure the strength and direction of
the linear relationship between hormones (all combinations). Students ttest was used to detect differences in GPI. Significance was accepted at
P,0.05.
Acknowledgements
Authors wish to thank Dr Joanna Szulc (Department of Fish Anatomy, West
Pomeranian University of Technology, Szczecin, Poland) for providing helpful
advice in histological analysis.

Competing interests
The authors have no competing interests to declare.

Author contributions
E.K. developed the concept of this paper, H.K.K., M.N. and M.G. carried out all
experiments and data analyses. E.K. and H.K.K. prepared the manuscript.

Funding
This study was supported by National Science Center [grant 2011/01/N/NZ8/
02333 to M.N.] and by Institute of Oceanology of Polish Academy of Sciences
statutory research task IV.2.2. to E.K. and M.G.

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