Ecotoxicology

https://doi.org/10.1007/s10646-018-02012-y

Mating under the influence: male Siamese fighting fish prefer EE2-
exposed females
Rebecca A. Cram1 Jaslynn M. Lawrence1 Teresa L. Dzieweczynski
● ●
1

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Countless pharmaceuticals and endocrine disrupting chemicals (EDCs) exist on the market with more added each day. Many
of these compounds are not removed during the wastewater treatment process and enter bodies of water in their active form.
EDCs are known to have physiological and behavioral effects in exposed organisms. Exposure to the synthetic estrogen 17α-
ethinylestradiol (EE2), a common EDC found in birth control pills, has been found to lead to population collapse after only a
few generations in some fish species. Mechanisms identified as potential driving forces for collapse include feminization of
males and altered fecundity in both sexes. However, an additional way in which EE2 could lead to population collapse is by
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altering courtship behavior, which could then change mating preferences and decrease mating opportunities. The current
study had the following objectives: determine if exposing female Siamese fighting fish, Betta splendens, to EE2 changes
mate choice in males; assess if the dose and duration of female exposure matters; and examine if exposing males to EE2
influences their mating preferences. Both unexposed and exposed males were presented with pairs of females that differed in
EE2 dose and exposure duration. The results indicate that males were more responsive to EE2-exposed females than
unexposed females, with males being most responsive to females exposed to the low versus high dose. Furthermore, exposed
males responded less overall than unexposed males. If EE2 concentration increases in the environment, the likelihood of
successful mating could decrease and, therefore, potentially lead to adverse population impacts.
Keywords Endocrine disrupting chemicals Mate choice 17α-ethinylestradiol Courtship
● ● ●

Introduction treatment facilities lack the ability to properly detect, dilute,

Humans use hundreds, if not thousands, of pharmaceuticals
and personal care products (PPCPs) each year worldwide.
As populations grow and age, the use of PPCPs is only
likely to increase (La Farre et al. 2008; Blair et al. 2013;
Gaw et al. 2014). While PPCPs are used by humans, non-
target organisms face effects of exposure as many of these
PPCPs are designed to have an effect at small doses and
remain in their active state even after they are excreted (Frye
et al. 2012; Arnold et al. 2013). Estradiol is excreted in a
conjugated, inactive form, but is then returned to a biolo-
gically active form during the sewage treatment process
(Ternes et al. 1999). Additionally, most wastewater
* Teresa L. Dzieweczynski
tdzieweczynski@une.edu
1
Department of psychology, University of New England, 11 Hills
Beach Road, Biddeford, ME 04005, USA
or degrade PPCPs before they are released in effluent into
surrounding bodies of water (Baronti et al. 2000; Arnold
et al. 2013). This means that PPCPs often enter the envir-
onment in active form where they adversely affect exposed
wildlife. Perhaps, the most well-studied and prevalent class
of PPCPs are endocrine disrupting chemicals (EDCs),
which act by mimicking or inhibiting the normal
functioning of steroid hormones (Gleason and Nacci 2001;
Sumpter 2005). Through this interference, EDCs can impact
any organism within an exposed environment, although the
severity and specific mechanism of the effects require
greater study.
Exposure to EDCs has produced morphological, phy-
siological, behavioral, and even genetic changes in aquatic
organisms (Kime 1998; Tyler et al. 1998). Because many of
the EDCs found in the environment have estrogen
mimicking properties (Kime 1998), the bulk of the studies
done on the effects of exposure to these compounds have
focused on reproductive changes. Negative consequences of
exposure include year-round production of vitellogenin

(Purdom et al. 1994), reduced fertilization and hatching
success (Länge et al. 2001), and occurrence of intersex in
male fish and reptiles (Guillette et al. 1995; Jobling et al.
1998) as well as decreases in courtship and aggression in
exposed males (Bell 2001). Changes observed in the
behavior of males, such as altered social hierarchies (Coe
et al. 2008), impaired ability to acquire mates (Saaristo et al.
2009), delayed nest building (Saaristo et al. 2010), and
reduced social status (Colman et al. 2009), are likely to
produce population-level consequences. Indeed, exposure
to one of the most prevalent EDCs, the estrogen mimic 17α-
ethinylestradiol (EE2), has led to population collapse in
multiple fish species (Nash et al. 2004; Kidd et al. 2007;
Palace et al. 2009). Given that EE2 reduces courtship
behaviors in males, changes in mate preference that result
from these courtship reductions could be one potential
mechanism contributing to these collapses.
EE2 is a synthetic estrogen that is the active component
in oral contraceptives and is commonly used in many types
of hormone replacement therapies. Concentrations of EE2
in aquatic ecosystems are usually between 1 and 10 ng/L
but concentrations as high as 50–60 ng/L have been found
in highly populated areas of the world (Larsson et al. 1999;
Kolpin et al. 2002; Muller et al. 2008; Ciocan et al. 2010).
EE2 binds with higher affinity to estrogen receptors and
degrades at slower rates than natural steroids, making
exposed organisms highly susceptible to its effects (Shore
et al. 1993). Because of these properties, EE2 may bioac-
cumulate and has been found in exposed fish at levels up to
300 times greater than those found in the environment
(Länge et al. 2001; Lai et al. 2002). Therefore, given EE2’s
slow degradation, high binding affinity, and potential to
bioaccumulate, it poses a serious threat to organisms living
in aquatic ecosystems where EE2 is present.
It is well established that females across taxa choose
males based on traits that are related to their fitness
(Andersson 1994; Stapley 2008; Hunt et al. 2009). There-
fore, if EE2 exposure influences courtship and aggression in
males, it may alter female mating decisions and social
dynamics. While females tend to be the choosier sex, males
may also prefer females with some trait variants over those
with different variants. Degree of male preference may
relate to degree of male investment such as if males are
building nests or are involved in caring for offspring. Male
preferences may change if physical attributes of either sex
are affected by EE2 exposure. This, in turn, could reduce
the number of matings within a population or alter mate
choice. For example, male threespine stickleback court
females with distended abdomens more vigorously than
females with less distended abdomens (Rowland 1982) and
male desert gobies adjust their courtship efforts based on
female size (Lehtonen et al. 2011). Studying how mate
choice changes following EE2 exposure could be especially
R. A. Cram et al.
important as it would provide a behavioral cause for
population collapse even if exposed individuals appear to be
unchanged morphologically.
Siamese fighting fish, Betta splendens, is a model species
that is widely used in laboratory settings in part due to the
ritualized aggressive and mating displays used to commu-
nicate with members of both sexes (Simpson 1968). Recent
work in this species has focused on these displays (e.g.,
Jaroensutasinee and Jaroensutasinee 2001; Clotfelter et al.
2006; Verbeek et al. 2008), how they might vary within-
and between-individuals (e.g., Dzieweczynski et al. 2010;
Dzieweczynski and Hebert 2013), and if they are impacted
by social network (e.g., Doutrelant and McGregor 2000;
Matos and Schlupp 2005). Additionally, over the past
decade, exposure to various EDCs has been found to
influence this species behaviorally, reproductively, and
neurologically (Clotfelter et al. 2010; Stevenson et al. 2011;
Dzieweczynski et al. 2014). Much work has been done on
how EE2 alters social behavior in this species. For example,
males suppress female-directed behaviors following EE2
exposure on either an acute or chronic scale (Dzieweczynski
and Hebert 2013; Dzieweczynski et al. 2014), females avoid
males exposed to EE2 (Dzieweczynski and Kane 2017), and
males are less attentive to males that have been exposed to
EE2, especially when they themselves have been exposed
(Dzieweczynski and LaMonica 2016). These studies pro-
vide further support for the utility of this species as a model
for effects of EDCs on reproductive behaviors that may
have individual or population-level consequences in the
wild.
In order to examine if changes in mate choice may be a
factor in population collapse following EE2 exposure, we
allowed both unexposed and exposed male Siamese fighting
fish to view females that differed in their level of EE2
exposure (0, 5, and 10 ng/L). We presented males with
females that had been exposed for 1 week and 2 weeks to
determine if duration of exposure influenced male choice.
Both of the doses utilized in this study fall within the range
of EE2 levels found in the wild. Based on previous studies
that have found a decrease in male courtship behavior,
including gill flaring, fin spreading, and tail beating, fol-
lowing exposure to EE2 (Dzieweczynski et al. 2014), we
predicted that exposed males would perform less female-
directed courtship behavior overall than unexposed males.
We also predicted that these exposed males would express a
weaker preference than unexposed males due to a lack of
courtship motivation as exposure to EE2 should reduce
testosterone levels (Adeel et al. 2017), thereby decreasing
male-typical behaviors. EE2 exposure has led to reductions
in a wide range of male-typical behaviors including
aggression in fathead minnows (Salierno and Kane 2009),
nest building in sand gobies (Saaristo et al. 2009) and
threespine stickleback (Brian et al. 2006), and courtship and
Mating under the influence: male Siamese fighting fish prefer EE2-exposed females
reproductive success in guppies and sand gobies (Kris-
tensen et al. 2005; Saaristo et al. 2010). In terms of male
preference towards females experiencing the three different
EE2 exposure levels, we hypothesized that males would
prefer exposed over unexposed females. This was hypo-
thesized because exposed female rats have heightened
receptivity following an influx of estrogen (Asarian and
Geary 2002). Additionally, increased estrogen may lead to
the production of more eggs, a trait males prefer and assess
via female size (Doyle et al. 2013). There was no specific
prediction as to male response to females exposed to a
higher or lower dose of EE2 or if duration of exposure
would have an effect as these aspects have not been studied
previously.
Methods
Subjects
Male and female Siamese fighting fish (males: 66.43 ±
7.97 mm, 1.84 ± 0.56 g; females: 52.67 ± 5.27 mm, 1.44 ±
0.27 g), B. splendens, that were sexually mature but had
never mated, were purchased from a commercial distributor
and then allowed to acclimate to laboratory conditions
(26.7 °C; 16:8 h light:dark cycle) for a minimum of two
weeks before testing. Fish were purchased at two different
time points (January 2016: 60 males; 100 females; Sep-
tember 2016: 20 males; 60 females). A total of 80 male
subjects (40 control; 40 EE2-exposed) and 160 females (50
control; 110 EE2-exposed) were used. Males were housed
in opaque 475 mL containers to prevent them from obser-
ving conspecifics that could elicit aggressive behavior.
However, because female Siamese fighting fish typically
form shoals, they were housed in 475 mL transparent con-
tainers so that they could see other females. Males and
females were housed on separate shelving units that pre-
vented them from seeing one another outside of the
experimental testing. All fish were fed Hikari Bio-Pure©
freeze-dried bloodworms ad libitum daily and received
100% water changes daily during testing.
17α-Ethinylestradiol exposure
Male subjects were randomly assigned to one of the two
groups: those that received exposure to EE2 during testing
and those that remained unexposed. To administer EE2, a
stock solution of EE2 was first created by dissolving EE2
(98% purity; Cayman Chemical Company; CAS 57-63-6) in
95% ethanol (Bell 2001 for methodology). Exposed males
then received 5 µL of this solution in their home containers
after the daily water change to reach a total concentration of
5 ng/L. Males in the unexposed group had daily water
changes but no EE2, only ethanol, added to their home
containers. This exposure was done daily over a 2-week
testing period. Females were assigned to one of the three
exposure groups: control (0 ng/L), low dose (5 ng/L), or
high dose (10 ng/L), and exposure for the females was done
in an identical manner to that for the male subjects
throughout the 2-week experimental period. Both low- and
high-dose exposures were used to determine whether
varying degrees of exposure had an effect on male pre-
ference. Both concentrations used fall within the spectrum
of levels in the wild. The low dose is well within the range
found in the aquatic environment (e.g., Baronti et al. 2000)
and the high dose, while higher than the amount found in
most areas, is a dose commonly used in ecotoxicology
studies (Frye et al. 2012). Additionally, the high dose is a
pharmacologically relevant dose and could be seen more
frequently in bodies of freshwater in the future due to
increased use of EE2 and population growth. Concentra-
tions of EE2 in the storage containers were verified by
taking water samples collected right before the daily water
changes on day 1, day 6, and day 13 from a subset of
randomly selected control, low-dose females, high-dose
females, and exposed males (n = 5 each group). EE2 was
extracted from the water samples via C18 solid-phase car-
tridges (Sep-pack Plus C18 cartridges) and then GC-MS
was used to analyze the samples to determine EE2 con-
centrations (control: below detectable limits; low dose:
4.8 ± 0.6 ng/L; high dose: 10.2 ± 1.1 ng/L; exposed males:
4.7 ± 0.8 ng/L).
Experimental setup and procedure
The experimental setup consisted of one 37.9-liter tank
(51 × 25 × 30 cm) centered between two 20.5-liter tanks
(43 × 23 × 28 cm) on either side of it (Fig. 1). The male was
allowed to swim freely about the experimental tank while
the females were confined in containers within their tanks
so that these females were always at a set distance from the
focal tank. This design allowed the male to easily observe
and interact with the females while preventing the females
from viewing one another and keeping both females in
standardized locations due to the distance between the tanks
and the placement of the female containers. The experi-
mental tank was visually divided into different areas: a
neutral zone (middle third of the tank) and two preference
zones at the end of the tank (two fish lengths from the
compartment containing the female in the adjacent tank).
To start a trial, opaque partitions were first placed
between the experimental tank and the two adjacent female
tanks. Then a male and two females were placed into the
appropriate tanks. After a 5-min acclimation period and
after the male had swam into the neutral zone of the
experimental tank, the opaque partitions separating the

Female Tank
Preference
Zone
Fig. 1 Diagram of experimental tank setup. The test tank in the middle contained the male subject, while the two tanks on the sides were where the
females were presented. Opaque dividers between the tanks were removed at the start of a trial
tanks were removed and the 5-min trial started. All trials
were recorded with a Sony HandyCam digital camcorder
positioned in front of the experimental setup so that all three
tanks were in view. When the trial was completed, the
opaque partitions were replaced and the male started the
next acclimation period.
Five different viewing combinations of females (HL =
high + low exposure; HH = high + high exposure; LC =
low + control; CC = control + control; and HC = high +
control) were used to examine if male behavior changed as
a byproduct of female exposure to EE2. To reduce risk of
habituation due to repeated testing, the video exposures
were divided so that a male had three combinations of
females 1 day and the other two combinations on the second
testing day. Each week, a group of 10 males was selected
for testing. Measures were taken to control for side bias and
order effects, such as randomizing combination order and
the side on which a female of a given exposure type was
presented. Additionally, males never saw the same female
more than once. Testing lasted 2 weeks with males seeing
females that had been exposed to EE2 for 1 week during the
first week of testing and then females exposed for two
weeks the second week. This was done to determine if the
length of time females were exposed affected male
preferences.
Behaviors measured
Behaviors were scored using the event recording program,
Event-PC. Five behaviors were measured toward a given
female: time spent within a preference zone, time orienting
to a female, time tracking, amount of time spent gill flaring,
R. A. Cram et al.
Preference Female Tank
Neutral Zone
Zone
and number of tail beats to a female. Since there were two
females within each viewing combination, these five
behaviors were coded for each female, for a total of 10
measures in each trial. Time in a preference zone was
defined by a male being within two body lengths of a
female. Time spent orienting to a female was defined as a
male being within a preference zone facing the female.
Time spent tracking (following the female while orienting
to and within a body length of that female), time spent gill
flaring (extension of the opercular flaps), and number of tail
beats (deliberate slaps of the tail directed at a conspecific)
were only counted when a male was in a preference zone.
These behaviors have been found to be suitable proxy
measures of mate choice in this species (Jaroensutasinee
and Jaroensutasinee 2001; Clotfelter et al. 2006; Dzie-
weczynski et al. 2013). To minimize observer bias, blinded
methods were used when all behavioral data were scored by
RAC and JML.
Statistical analyses
First, a series of three-way repeated-measures analyses of
variance (RMANOVAs) on ranks with treatment (stimulus
presentation types), male exposure (control and exposed),
and duration of female exposure (week 1 and week 2) as
factors was run. This was done to examine if side bias
affected total amount of response or difference in response
to one female versus the other (e.g., time by female on
left–time by female on right). Next, we examined if the
strength of preference to one female versus the other varied
across the five treatments. In other words, whether male
response to one female differ based on the type of female he
Mating under the influence: male Siamese fighting fish prefer EE2-exposed females

was presented with. To do this, two different series of

Table 1 Results of the three-way RMANOVAs run on the difference scores of the five different behaviors measured towards each of the two females within a presentation type (time by the

exposure. When females of the same level of exposure were presented simultaneously, the behavior towards the female on the right was subtracted from the amount of that behavior towards the
female on the left. Male exposure (Exposure), female presentation type (Treatment), and duration of female exposure (Duration) were factors in the RMANOVAs, and all possible interaction
Difference scores were obtained by subtracting the behavior towards the female with the least amount of EE2 exposure in a treatment from that behavior towards the female with the greater
three-way ANOVAs on ranks with treatment, male expo-
sure, and duration of female exposure as factors and all

F4,799 = 0.33, p = 0.86

F4,799 = 0.31, p = 0.87

F4,799 = 0.14, p = 0.97

F4,799 = 0.61, p = 0.66

F4,799 = 0.51, p = 0.73

Treatment × duration ×
possible interaction effects were run. One series was run on
the total amount of behavior within a trial and the other was
run on the difference scores. The total amount of behavior,
exposure

regardless of which female the behavior was directed at

(e.g., time orienting to female A + time orienting to female
female, time spent orienting to the female, time spent tracking the female, time spent gill flaring towards the female, and number of tail beats directed at a female)

B), was calculated for each individual in each treatment and

then ANOVAs on ranks with post hoc Wilcoxon tests and
Duration × exposure

F1,799 = 0.93, p =

F1,799 = 1.79, p =

F1,799 = 2.23, p =

F1,799 = 0.06, p =

F1,799 = 0.22, p =
Bonferonni corrections were conducted on these scores.
Additionally, difference scores were calculated for each
male in each treatment (e.g., time by high-exposure
female–time by low-exposure female) and the same sta-
0.34

0.18

0.14

0.81

0.64

tistical tests were run on these scores. For these difference

scores, female A was always the female with the greatest
F4,799 = 2.60, p = 0.03

F1,799 = 0.16, p = 0.69 F4,799 = 0.35, p = 0.85 F4,799 = 2.35, p = 0.06

F1,799 = 3.67, p = 0.06 F1,799 = 1.08, p = 0.30 F4,799 = 0.57, p = 0.68 F4,799 = 0.16, p = 0.14

F1,799 = 0.09, p = 0.77 F1,799 = 6.74, p = 0.01 F4,799 = 1.26, p = 0.28 F4,799 = 1.18, p = 0.32
Treatment × exposure

exposure concentration (e.g., high exposure–control). In the

F1,799 = 0.98, p = 0.32 F1,799 = 2.79, p = 0.10 F4,799 = 0.20, p = 0.94 F4,799 = 12.29, p <

treatments where both females had the same level of

exposure (e.g., high exposure and high exposure), female A
was always the female on the left of the testing tank and
0.0001

female B was on the right side of the tank.

Treatment × duration

Results
F4,799 = 0.67, p =

Side bias
0.612

Side bias did not significantly influence any of the depen-

dent measures (treatment: F4,799 ≤ 1.18, p ≥ 0.32; duration:
F1,799 ≤ 3.22, p ≥ 0.07; male exposure: F1,789 ≤ 3.89, p ≥
F1,799 = 0.0004, p =

0.06; treatment × duration: F4,799 ≤ 2.47, p ≥ 0.12; treat-

ment × male exposure: F4,799 ≤ 1.95, p ≥ 0.10; treatment ×
duration × male exposure: F4,799 ≤ 1.92, p ≥ 0.11). This
Exposure

means that whether a particular female was presented on

0.98

the left or right side of the tank did not affect any of the
measured behaviors.
F1,799 = 0.0001, p =

F1,799 = 0.0002, p =

Difference scores
effects were included. Significant effects are bolded

A positive difference score indicates that more behavior

Duration

was directed to the higher exposure female, a negative

0.99

0.99

difference score shows that males are more responsive to

females of lower exposure, and a difference score close to
zero demonstrates that there is little difference in behavior
F4,799 = 121.65, p <

Orienting F4,799 = 108.26, p <

towards the two females in a viewing condition. Treatment

F4,799 = 54.75, p <

F4,799 = 56.13, p <

Tail beats F4,799 = 30.97, p <

had a significant effect on the difference scores (Beha-

viorFemaleA − BehaviorFemaleB) for time spent within the
Treatment

preference zone (F4,799 = 121.65, p < 0.0001); however,

0.0001

0.0001

0.0001

0.0001

0.0001

because a treatment × male exposure interaction effect was

found (F4,799 = 2.60, p = 0.03), this factor cannot be ana-
lyzed separately (Table 1). Difference scores for unexposed
Tracking
Time by

males in the high-exposure + low-exposure treatment were

flaring
Gill

more negative than all other treatments by female exposure


a
a a a exposed female to the control female (Z ≥ 6.08, p < 0.0001).
b
b male exposure (treatment: F4,799 ≥ 56.13, p < 0.0001; treat-
b b
c sure female treatment (Z ≥ 4.22, p < 0.0001; Fig. 3a). The
d exposed males in this same treatment (Z ≥ 2.00, p ≤ 0.046).
Fig. 2 Difference scores (BehaviorFemaleA − BehaviorFemale B) for time
spent by the females in the five different female pairings males
received: control + control (CC), high exposure + high exposure
(HH), low exposure + control (LC), high exposure + control (HC),
and high exposure + low exposure (HL) for both control and exposed
males. Significant differences are denoted by differing letters. Error
bars represent ± 1 SEM and α = 0.05
dose combinations for time spent in the preference zone
(Z ≥ 2.61, p ≤ 0.001; Fig. 2). This means that control males
spent more time near the female exposed to the lower
concentration of EE2 than to the higher concentration when
encountering both simultaneously. Exposed males had more
negative difference scores in this same treatment than all
other treatments by exposure combinations with the
exception of unexposed males in the high-exposure + low-
exposure treatment (Z ≥ 5.14, p ≤ 0.0001). Difference scores
for time spent in the preference zone were the highest in the
low-exposure + control and high-exposure + control treat-
ments, regardless of whether or not the males were exposed
to EE2, meaning that males preferred exposed females (Z ≥
5.01, p ≤ 0.0001; Fig. 2). Difference scores in treatments in
which males received females of the same exposure level
(e.g., control + control) were close to zero, indicating that
males directed similar amounts of behavior to both females.
Treatment affected the difference scores for time spent
orienting as well as the amount of time spent tracking
(F4,799 ≥ 54.75, p < 0.0001; Table 1). The difference scores
within the high-exposure + low-exposure treatment were
more negative than those found in the other four treatments
for both of these behaviors suggesting a low-exposure
female was preferred over a high-exposure one (Z ≥ 6.04, p
< 0.0001). Additionally, the difference scores for time spent
orienting and time spent tracking were the most highly
positive in the low-exposure + control and high-exposure
R. A. Cram et al.
+ control treatments, meaning that males preferred the
For the difference between the amount of time males
spent gill flaring to one female versus the other female, both
treatment and an interaction between treatment type and
ment × exposure: F4,799 ≥ 12.29, p < 0.0001; Table 1) had an
influence. Difference scores were the most negative for
unexposed males in the high-exposure female + low-expo-
second most negative difference score was found for
Difference scores for gill flaring were the most positive for
unexposed males in the low-exposure + control and high-
exposure + control treatments meaning both exposed and
unexposed males preferred low-exposure females over
those from the high exposure (Z ≥ 3.63, p ≤ 0.003; Fig. 3a)
and the next highest difference scores occurred when
exposed males were in these same two treatments showing
gill flaring was increased when females were exposed to
either EE2 treatment (Z ≥ 3.35, p < 0.001). Again, difference
scores were close to zero for both control and exposed
males in the high-exposure + high-exposure and control +
control treatments.
Difference scores for tail beats were influenced by male
exposure, treatment, and a male exposure × treatment
interaction (exposure: F1,799 ≥ 6.74, p = 0.01; treatment:
F4,799 ≥ 30.97, p < 0.0001; exposure × treatment: F4,799 ≥
2.78, p = 0.03; Table 1). Control males in the high-expo-
sure + control and low-exposure + control treatments had
the highest difference scores in the number of tail beats they
delivered to the two females (Z ≥ 2.52, p < 0.01; Fig. 3b).
Exposed males in these same two treatments had the next
highest difference scores indicating EE2 treated females
were preferred by both exposed and unexposed males (Z ≥
2.04, p ≤ 0.04). Finally, the most negative difference scores
were found in the high-exposure + low-exposure treatment
meaning low-exposure females elicited more tail beats than
high-exposure females (Z ≥ 2.76, p ≤ 0.006; Fig. 3b).
Total overall responses
Male exposure type (control vs. EE2-exposed) and female
viewing combination influenced the total amount of time
males spent in the preference zones (exposure: F1,799 =
99.80, p < 0.0001; treatment: F4,799 = 9.23, p < 0.0001;
Table 2). Exposed males spent less time overall in the
preference zones than unexposed males (Z = 9.99, p <
0.0001). Males spent less time in the preference zones when
females of the same level of exposure were present (i.e.,
control + control) compared to when females differing in
EE2 exposure levels were present (Z ≥ 2.52, p ≤ 0.01). Male
exposure, treatment, and an interaction effect between these
Mating under the influence: male Siamese fighting fish prefer EE2-exposed females
a)
e
e
d d
c
c c c
b
a
Fig. 3 Difference scores for a the amount of time males spent gill
flaring to and b number of tail beats directed they directed at the
females in the five treatments: control + control (CC), high exposure
+ high exposure (HH), low exposure + control (LC), high exposure +
control (HC), and high exposure + low exposure (HL). A male’s
response to the female with the lower level of exposure was always
two factors, affected the total amount of time males spent
orienting to the females (exposure: F1,799 = 133.36, p <
0.0001; treatment: F4,799 = 9.47, p < 0.0001; exposure ×
treatment: F4,799 = 3.22, p = 0.01; Table 2). Overall,
exposed males spent less time orienting than unexposed
males, regardless of treatment type (Z ≥ 5.27, p ≤ 0.0001;
Fig. 4). Additionally, exposed males spent less time
orienting to the females when the females had the same
level of EE2 exposure versus differing levels (e.g., control
+ control versus high exposure + control; Z ≥ 3.02, p ≤
0.003).
The total amount of time males spent tracking was
influenced by duration of female exposure to EE2 and
treatment type (Table 2). Males spent less time tracking
when females had been exposed to EE2 for 2 weeks com-
pared to 1 week (Z = 3.77, p = 0.0002). This suggests that
females may behave differently the longer they are exposed
to EE2. Males also spent more time tracking when females
of different levels of EE2 exposure were present than when
females of the same amount of EE2 exposure were present
(Z ≥ 2.10, p ≤ 0.04). The time males spent gill flaring was
affected by duration of female exposure and subject expo-
sure but no other factors (duration: F1,799 = 5.14, p = 0.02;
exposure: F1,799 = 153.62, p < 0.0001; Table 2). Males
spent less time gill flaring to females that were exposed to
EE2 for 2 weeks than those exposed for 1 week (Z = 2.27,
p = 0.02). Exposed males also spent less time gill flaring
than control males (Z = 12.39, p < 0.0001). Finally, the total
b)
a a
b b
c c c c
d d
subtracted from the response to the female with the higher level of
exposure. When the pair experienced equivalent amounts, the behavior
to the female on the right was subtracted from the female on the left.
Significant differences are denoted by differing letters. Error bars
represent ± 1 SEM and α = 0.05
number of tail beats was affected by male exposure (F1,799
= 50.45, p < 0.0001; Table 2). As with the majority of the
measured behaviors, exposed males performed fewer tail
beats than unexposed males (Z = 7.10, p < 0.0001).
Discussion
The presence of the estrogen mimic EE2 in aquatic eco-
systems has known physiological and behavioral effects on
organisms living in these waters (Frye et al. 2012). Most of
the research in this area has focused on changes in males
with females receiving little attention. In this study, we
examined whether exposure to EE2 affected females in a
manner that might influence male preference. To address
this, changes in male behavior towards females were
examined as proxy measures of altered female behavior
and/or reproductive physiology. It has recently been found
that females avoid males that have been exposed to EE2
(Dzieweczynski and Kane 2017). Therefore, by examining
how exposure to EE2 produces changes in male and female
reproductive behavior, we can gain a more complete picture
of how this compound impacts mating systems. The results
suggest that male Siamese fighting fish actually prefer
females that have been exposed to EE2, perhaps because the
addition of estrogen increases female receptivity. Increased
male preference could be beneficial for females if it
decreases aggression during courtship (Persaud and Galef
 R. A. Cram et al.

a a a

Male exposure (Exposure), female presentation type (Treatment), and duration of female exposure (Duration) were factors in the RMANOVAs, and all possible interaction effects were included.
a
a

b b

F4,799 = 0.34, p = 0.86

F4,799 = 0.31, p = 0.87

F4,799 = 1.22, p = 0.30

F4,799 = 0.75, p = 0.56

F4,799 = 1.26, p = 0.28

Treatment × duration ×

c
c
exposure
Treatment × duration Treatment × exposure Duration × exposure

F1,799 = 1.66, p =

F1,799 = 1.99, p =

F1,799 = 2.40, p =

F1,799 = 0.01, p =

F1,799 = 0.03, p =
0.20

0.16

0.12

0.94

0.86
F4,799 = 3.22, p =
F4,799 = 2.36, p =

F4,799 = 2.04, p =

F4,799 = 0.39, p =

F4,799 = 0.60, p =

Fig. 4 The total amount of time males spent orienting to the females
(i.e., BehaviorFemaleA − BehaviorFemale B) was influenced by an inter-
action effect between the combination of females they viewed: (con-
trol + control (CC), high exposure + high exposure (HH), low
0.06

0.01

0.09

0.82

0.66
Table 2 Results of the three-way RMANOVAs run on the total behavior directed to both females within a treatment

exposure + control (LC), high exposure + control (HC), and high

exposure + low exposure (HL)) and their own exposure level. Sig-
nificant differences are denoted by differing letters. Error bars repre-
F1,799 = 99.80, p < 0.0001 F4,799 = 1.07, p =

F4,799 = 0.85, p =

F4,799 = 1.33, p =

F4,799 = 0.69, p =

F4,799 = 0.96, p =

sent ± 1 SEM and α = 0.05

2005; Dzieweczynski et al. 2013) or if it provides oppor-

tunities for females to gain access to higher quality mates
0.37

0.50

0.26

0.60

0.43

(Edward and Chapman 2011). Males preferred females

exposed to a lower dose of EE2 over those exposed to a
F1,799 = 50.45, p = 0.01
F1,799 = 0.54, p = 0.46

higher concentration, indicating that, is all females in a

F1,799 = 133.36, p <

F1,799 = 153.62, p <

population are exposed, there may be a point where

exposed females are no longer more attractive. Levels of
EE2 in aquatic ecosystems may reach as high as 50 ng/L
Exposure

(Ciocan et al. 2010), a concentration at which it is unlikely

0.0001

0.0001

males would exhibit a preference for females as they would

be completely feminized. Additionally, EE2 exposure has
negative reproductive consequences on males including
F1,799 = 5.14, p = 0.02
F1,799 = 0.07, p = 0.79

F1,799 = 0.08, p = 0.78

F1,799 = 2.71, p = 0.10

reduced courtship rates and lower sperm viability (e.g.,

F4,799 = 4.38, p = 0.002 F1,799 = 14.21, p =

Kristensen et al. 2005; Dzieweczynski and Kane 2017).

Since both males and females would simultaneously
Duration

experience an EE2 exposure in the wild, the overall effects

0.0002

of exposure on reproduction are likely to be negative such

as decreased courtship attempts from males and altered
receptivity in females.
F4,799 = 1.45, p = 0.22

Tail beats F4,799 = 0.26, p = 0.90

Difference scores and total responsiveness both

F4,799 = 9.23, p <

F4,799 = 9.47, p <

Significant effects are bolded

demonstrate that males alter their behavior towards females

based on whether or not females had experienced EE2
Treatment

exposure. Males performed more courtship behaviors to the

0.0001

0.0001

exposed than the unexposed female in a pair, reflected by

greater positive difference scores. Additionally, males
Orienting

preferred females exposed to a lower dose of EE2 more

Tracking
Time by

flaring

than females exposed to a higher dose when the two were

Gill

presented simultaneously. Male preference when a low-

Mating under the influence: male Siamese fighting fish prefer EE2-exposed females
exposure female and a high-exposure female were present
was greater than when an unexposed female and an exposed
female were presented together. This indicates that males
have stronger preferences when they can simultaneously
assess females exposed to different concentrations of EE2.
When females of identical levels of exposure were present,
males responded similarly towards both females, indicating
that the observed preferences were due to the difference in
EE2 concentration rather than some inherent differences
between females in the study.
Whether or not males experienced EE2 exposure them-
selves also had an influence on reproductive behavior.
While both unexposed and exposed males exhibited similar
preferences, these preferences were weaker in exposed
males. Exposed males may have a more difficult time
assessing differences between females because EE2 expo-
sure has been found to affect recognition abilities and
attention in other exposed species (Zala and Penn 2004;
Mills and Chichester 2005; Peterson et al. 2017). Most
importantly, and not surprisingly, exposed males responded
less than males that were not exposed to EE2, further
supporting the existing body of literature on reductions in
male-typical behaviors following exposure.
It is possible that EE2 exposure in females increased
female receptivity, causing behavioral or morphological
changes that were apparent to males but not detected by the
human observers. Female Siamese fighting fish display
receptive bars along their sides when they are preparing for
copulation (Simpson 1968) and increased estrogen may
have caused subtle changes in this barring pattern to appear.
There was no difference in the number of females that
displayed these bars with the three exposure levels, but the
intensity of the bar coloration or duration of expression may
have differed in a manner that was noticeable to the male
subjects but not noticeable to the observers. Female body
size and mass are often positively correlated with estrogen
levels as well as male preference (Doyle et al. 2013),
however, these factors were controlled for in this study so
they cannot be responsible for differences in male pre-
ference across treatments. Disappointingly, few studies
have assessed the effects of EE2 on female receptivity (but
see Dzieweczynski and Kane 2017). EE2 at low doses sti-
mulates egg production but higher amounts decrease ferti-
lity in female zebrafish (Silva et al. 2012; Söffker et al.
2012). Early exposure to EE2 increases bottom-dwelling
behavior but has no effect on social or reproductive beha-
viors in adulthood in female guppies (Volkova et al. 2012).
Thus, determining the precise morphological and/or beha-
vioral changes that occur in females following exposure to
EDCs that then, in turn, alter male choices is an important
area for further study.
In closing, this study demonstrates that males prefer
females exposed to EE2, as reflected by both overall
responses and difference scores. However, there is a limit to
this preference as, when high- and low-dose females were
both present, males directed more behavior to the female
with the lower exposure. These results suggest that altera-
tions in mate choice may be a potential mechanism by
which EE2 exposure could lead to changes in population
structure and/or population collapse. Chronic exposure to
a low dose of EE2 quickly led to population
collapse in fathead minnows (Kidd et al. 2007) and zebra-
fish (Nash et al. 2004) by altering oogenesis in females and
producing intersex in males. Both males and females
influence reproduction and likely experience exposure in
the wild simultaneously, although they may differ in how
much exposure they encounter. As human population sizes
grow, levels of EDCs worldwide will continue to persist
and likely increase (Adeel et al. 2017; Gaw et al. 2014).
Therefore, even if exposure to EE2 is not directly detri-
mental to exposed females, it could still have negative
implications because EE2 decreases male-typical behaviors
necessary for successful reproduction (Peterson et al. 2017).
In other words, even if unexposed males prefer exposed
females in our system, any benefit of this is expected to be
is minimal for two reasons; first, EE2 decreases male-
typical behaviors (Peterson et al. 2017) and, second,
because females have been shown to avoid males exposed
to EE2 (Dzieweczynski and Kane 2017). The current results
further demonstrate the negative effects EE2 has on males
because exposed males, while exhibiting similar preferences
to unexposed males, had weaker preferences and were less
responsive overall to females. Thus, EE2 could contribute
to population collapse even if exposed individuals are still
physiologically capable of mating. Future studies should
examine mating behavior and examine offspring viability
following exposure. In the current study design, males and
females were separated from one another and, therefore,
could only interact through visual means. The role of
pheromonal information in mating interactions within this
species should be examined as it might be influenced by
EE2 exposure as well. By understanding how reproductive
behaviors such as mate choice are impacted by exposure to
EDCs, we can obtain a more accurate picture of the long-
term consequences of exposure.
Data availability
All data analyzed during this study are available via the
Open Science Framework (osf.io/e3qj9).
Acknowledgements The authors would like to thank Haley LaMonica
for her assistance in running behavioral trials. They also thank Jessica
Kane, Kelley Portrais, Megan Stevens, and two anonymous reviewers
for their feedback on a previous version of this manuscript. Amy
Keirstead and Molly Wright were instrumental in creating the
EE2 solution and analyzing EE2 in water samples.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval All applicable international, national, and/or institu-
tional guidelines for the care and use of animals were followed.
Animal care and experimental protocols were approved by the UNE
IACUC and covered under protocol UNE-20130910DZIET.
Informed consent All authors have approved this version of the work.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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