Caitlin Grant10/18/2015

Capstone Project

The Effects of Arsenic on Eavesdropping Behavior in Female Betta splendens

Introduction

Arsenic (As) is found as a widely distributed environmental pollutant in both

organic and inorganic form. It is distributed into the environment via anthropogenic

activities through industrial processes and farming (Nandi et al., 2005), but is also

naturally present in bedrock, with high levels often found in New England wells. Maine

drinking water standard allows up to 10 ug/L of arsenic to be safely present for public

consumption (Maine CDC, 2012), and is also the maximum concentration considered

safe for aquatic fauna (CONAMA, 2005). Alarmingly, areas of Maine and other New

England states have areas where concentrations exceed 100 ug/L. Even sublethal levels

may have subtle impacts on species performance that can in turn lead to important

indirect effecArsenic exposure has been seen to lead to neuronal loss in three

neurotransmission systems: cholinergic, GABAergic, and glutamatergic, impairing their

involvement in the creation and retrieval of memory (Barros et al., 2005). Arsenic acts on

the cholinergic system, affecting the mechanisms of high-affinity choline uptake and the

disulfide group of acetylcholinesterases, a neurotransmitter of the central nervous

system that regulates autonomic, cognitive and motor functions (Trevor et al., 1978).

Additionally, an increase in glutamic acid decarboxylase (GAD), part of the GABAergic

inhibitory neurotransmission system and an indicator of neuronal activity, showed a

decrease in certain regions of the brain, paired with an increase in the excitatory

neurotransmitter, glutamate. When in excess, glutamate can become excitotoxic, a

process by which neurons are damaged by over-activations of receptors, leading to

neuronal death (Tekkok et al., 2007). The aim of this work is to evaluate the impacts of

arsenic on communication signaling networks and how the ability of individuals to utilize

and respond to these signals when exposed.

Arsenic is considered an endocrine disrupting chemical (EDC) that is widely

present in the marine environment. EDC’s are chemicals that can affect steroid levels in

organisms by altering rates of synthesis or targeting receptors and can have significant

impacts even at low concentrations (Baccarelli et al., 2000). Along with arsenic,

considerable work has been done on phytoestrogens, another EDC present in the

aquatic environment. These chemicals accumulate via effluent from wood pulp mills,

sewage treatment plants and agricultural runoff. Exposure to endocrine disrupting

chemicals has been shown to suppress and alter male-typical aggressive behaviors in

multiple species of fish. These effects have been observed in Betta splendens by a

reduction in latency to respond to a mirror stimulus, along with a decrease in frequency

and duration of opercular displays utilized during courtship and aggressive male-male

interactions (Clotfelter, 2006). Additionally, development of secondary sexual

characteristics and delayed sexual maturity were observed in Gulf pipefish, with males

developing iridescent bars, a secondary sexual trait normally restricted to females when

exposed to the phytoestrogen 17α -ethinylestradiol. The suppression of aggressive

behaviors in betta fish, and the reproductive dysfunction and developmental

abnormalities in pipefish may be influenced by the down-regulation of androgens,

namely testosterone. As well as potentially acting as a serotonin reuptake inhibitor that

could directly affect the reduction of aggression in fish, (Clotfelter, 2006), androgen also

plays a direct part of trait expression in both male and female pipefish (Partridge, 2010).

This disrupted sexual differentiation may deter females from mating with males with

similar physical similarities, decreasing mating opportunities and ultimately impacting

reproductive fitness (Partridge, 2010).

Betta splendens show very distinctive male-male aggressive behaviors, as well as

characteristic inter-species observations amongst one another, making them useful in a

multitude of behavioral studies. These behaviors are often witnessed by third parties who

observe these interactions and gain important information about the individuals, known

as eavesdropping. This phenomenon is beneficial to the fitness of an individual by the

ability to gather information about the competitors with little to no cost to themselves, and

provides this access prior to any communication with the interacting individuals. There

are multiple trait qualities that can be advertised and received by an audience while

eavesdropping occurs, including aggression level, territory quality, social status, physical

condition, and/or effective courtship displays and parental ability (Dugatkin & Fitzgerald,

1997). Male display behavior has also been found as an accurate predictor of bubble

nest size, and thus eavesdropping on two males interacting serves as an available

indicator to females as well, as to whom a successful prospective mate may be

(Clotfelter et al., 2006). This behavior of monitoring aggressive interactions should

therefore be a useful source of information to other individuals.

Little work has been done directly relating to the effects arsenic has on

eavesdropping behavior. The behavioral and neurotoxic impacts of arsenic have

however been explored in Zebrafish, Danio rerio. One-trial inhibitory avoidance tests

were carried out, where fish were conditioned to avoid a dark section of the tank by

being administered a shock. Arsenic was shown to impair long-term memory, even at

concentrations considered safe for aquatic fauna, indicating the fish’s ability to archive

and utilize learned experiences were adversely affected when exposed to arsenic (de

Castro et al., 2009). As previously noted mentioned in this paper, arsenic acts on the

central and peripheral nervous system, inducing neuronal loss and may be the cause of

the observed cognition defects.

In an eavesdropping scenario, there are two individuals interacting (the signalers),

with a third party or audience (the receivers) of information gained from observing the

interaction. The effects of the EDC 17α -ethinylestradiol on how the presence of

eavesdroppers influence the nature of interactions, the “audience effect” was explored by

Dzieweczynski & Buckman (2013). In unexposed fish, Doutrelant et al. (2001) found that

the presence of females or males changed the intrasexual interaction between two

fighting males. With a female audience, males performed fewer aggressive displays

(bites) used only in male-male interactions, and used more conspicuous displays (tail

beats), used in the presence of both sexes. With a male audience, interacting males

increased all aggressive displays. When exposed with a phytoestrogen, male betta fish

were less responsive in male-male interactions, and adjusted their behavior less when

an audience was present (Dzieweczynski & Buckman, 2013). Exposure to EDC’s

interfere with communication between both members of the same sex and those of the

opposite, detrimental to their ability to simultaneously communicate to multiple

individuals. If selection acts on individuals who can readily alter their behavior when

interacting with different individuals, the elimination of this behavior may lead to

population crisis over time if left unchecked.

Previous studies have shown that females who monitor aggressive interactions

between two males do in fact use the information gained by eavesdropping in initial

stages of subsequent mate choice (Doutrelant & McGregor, 2000). Females were

observed to spend significantly more time near and displaying towards the “winner”,

whereas females who had not seen the interaction showed no significant behavioral

differences. That leads us to the question: will a female Betta splendens who has been

exposed to higher than ecologically safe levels of arsenic retain the information of who

was the victor of the aggressive male-male interaction?

Fish behavior is linked closely with its ability to survive and its inherent and

derived strategies for adaptation, including predator avoidance, mate selection and

social interactions. Pollutant exposure has a direct effect on the success and survival of

a species, even with doses below mortality levels. Behavioral indicators of toxicity are

extremely useful in assessing sub-lethal impacts of contaminants in animal species (de

Castro et al., 2009), and are an important factor in assuring the process of sexual

selection maintains behavioral variation within and between populations.

The null hypothesis for this study is that eavesdropping behavior in female Betta

splendens will be unaffected by arsenic exposure. Our hypothesis asks if females

exposed to arsenic will be able to retain and utilize information gathered while

eavesdropping and behave significantly differently towards the “winner” and the “loser” of

a male-male interaction.

Materials and Methods

The popular anabantid aquarium fish Betta splendens are native to slow moving

streams and rice patties in the southern portion of Asia. For this experiment, we

purchased subjects (red morph) from an Aquarium Wholesaler Seacrest Farms (Florida,

USA). Females and males were housed in 1 L and 5 L aquaria respectively, at the

Aquaculture Research Center at the University of Maine campus in Orono, Maine. Males

were housed in opaque tanks to prevent aggressive behaviors between individuals.

Females were housed in clear tanks permitting interactions between subjects. The

difference in aquaria setup is due to the implication of isolating females having

detrimental effects on female behaviors.

Water changes were conducted weekly using well water filtered through a reverse

osmosis filtration system. KENT R/O Right salts were added to mimic subject’s native

environment. All fish were fed daily (Aqueon Betta Pellets, Betta food), and kept at a

constant temperature between 24-30℃ with a 12:12 h light/dark photoperiod regime.

Arsenic Dosage

Adult female Betta splendens were dosed with either 10 ppb, or 100 ppb of

standard grade sodium arsenate combined with purified water, using 0 ppb as a control

group. Solutions of arsenic water were renewed daily and females were returned to

clean water post-experimentation.

Behavioral Observations

Females Used

Nineteen females were used for this experiment (0 ppb, n = 6; 10 ppb, n =

7; 100 ppb, n = 6). Females were exposed to arsenic solution for 96 hours prior to

experimentation. Females were observed for the presence of an ovipositor as an

indicator of sexual maturation (Doutrelant & McGregor 2000).

Males Used

Thirty-eight males were used for this experiment (0 ppb, n = 12; 10 ppb, n

= 7; 100 ppb, n = 6). Males were paired together by similar body length to reduce

the likelihood of size being a strong influence on female preference (Clotfelter et

al. 2006).

Tank Setup

Female Betta splendens trials were carried out in 208.2 liter rectangular

aquariums divided into 3 sections set with silicone caulk. The right quarter of the

tank was divided into two sections, housing each male where the female can

eavesdrop on both simultaneously. An “interaction zone” was marked on the

aquarium glass to record when the females were within eavesdropping distance of

the males. A one-way mirror film was adhered on female/male partition so males

were unaware of being observed by female. In the interim before and between

trials, an opaque partition was placed between males divider to ensure no

interactions. Subjects were allowed 1 hour to acclimate within testing tanks to

ensure normal behavior, with the recorder placed in view for 10 minutes prior to

trial. Water in tanks was replaced in between each trial to mitigate effects of

previous subjects olfactory cues.

Eavesdropping Phase

Female’s interaction time within 5 cm of male’s section of the tank was recorded

while female is eavesdropping on the two males interacting to confirm the phenomenon

is occurring. This was carried out by recording the amount of time the female spends

within the marked section closest to the interacting males for a 15-minute period (Figure

1). Recording was stopped once female returned to “neutral” zone and resumed once

she crossed back into the interaction zone. The same tank set up was used for the male

gill flare trials, conducted simultaneously as the female interaction time trial for fifteen

minutes. Gill flaring in betta fish is defined as the “sudden increase in distance between

the distal edge of the operculum and the body” (Braddock & Braddock, 1955). Frequency

and duration of gill flares ultimately tell us which male was the “winner” or “loser” of the

interaction from the female’s point of view (Clotfelter et al., 2006; Bronstein, 1984).

Interaction Phase

For the second part of the experiment, a tube was placed within the female’s

section of the tank, to house each stimulus male in turn, with each trial was recorded for

10 minutes. The time it took for the female to enter within a body’s length of the male

(latency time) was recorded, along with the duration in which she remained inside the

body length area around the tube (association time) (Figure 2). The partitions were

placed between the female section and the non-stimulus male not currently being utilized

to ensure singular attention on the stimulus male.

After the trials, females were placed in clean, non-arsenic R/O water in 1 L tanks

and males were placed back into original 5 L tanks. All research conformed to the

protocols approved by the Institutional Animal Care and Use Committee. Once all trials

were conducted, subjects were sacrificed using MS222 and tissues were extracted for

processing in the University of Maine histology lab.

Video Playback

A high definition video camera was used to record the trials to allow for a more

detailed scoring of female/male behaviors to gain a clearer visual of preference patterns.

Each video trial was transferred to a hard drive to then be analyzed.

During playback, association time was recorded for 15 minutes with a stopwatch

whenever the female entered the “association zone” and was stopped and restarted

when she exited and then reentered the zone. The same video was then replayed to

record the frequency and duration of each male, recording time starting when gill flare is

initiated and stopped when operculum closes, with each gill flare display adding up to a

final duration at the end of the 15 minute trial for individual males.

For the evaluation of latency time to interact with stimulus males, a stopwatch was

again utilized to record the length of time from when the female was placed in the tank to

when the first behavior towards stimulus male was observed. Recording time began

when female came within one body length of the males holding tube.

Winners and losers of the male-male interactions were determined by which fish

had a higher display rate (duration and frequency).

Figure 1. Female mate preference tank set up #1 in 208.2 liter aquariums for interaction

time trials and gill flare duration trials.

Figure 2. Female mate preference tank set up #2 in 208.2 liter aquarium for female association and latency time towards each stimulus male individually.

Results

Eavesdropping Phase

Females of all treatment groups spent significantly more time in the interaction zone

than the rest of the tank, indicating use of eavesdropping behavior (mean ±SE time

spent in interaction zone: control group = 770.8± 28.7; 10 ppb treatment = 693.3 ±23.6;

100 ppb treatment = 628.8 ± 67.8; paired t-test for control: t = 11.168, df = 5, p< 0.01)

Males scored as “winners” did significantly more gill flaring than males scored as

“losers”, which is an indicator of fight outcome. (mean ± SE difference between W/L:

control group = 109.2 ± 29.6; 10 ppb treatment = 101.1 ±28.3; 100 ppb treatment = 63.8

± 17.4; one-sample t-test: t = 3.687, df = 5, p< 0.01; t = 3.578, df = 6, p< 0.01, t = 3.66, df

= 5, p< 0.01)

Interaction Phase

Females did not behave differently towards winners or loser in association time

trials (mean ± SE: control = -32.3 ± 60.5; 10 ppb treatment = 29 ± 50; 100 ppb treatment

= 10.8 ± 56) or latency time trials (mean ± SE: control = -31.2 ± 61.5; 10 ppb treatment =

12.6 ± 19.5, 100 ppb treatment = -62 ± 27), regardless of arsenic dosage. The difference

in behavior was calculated by: time towards winner – time towards loser. Positive

numbers = more time towards winner, negative numbers = more time towards loser.

These results are likely due to small sample sizes per treatment group. ANOVA tests

were performed between groups for association time analysis (F = 0.326, p = 0.727) and

latency time analysis (F = 0.970, p = 0.400)

0ppb 10ppb 100ppb0

100

200

300

400

500

600

700

800

900

In Interaction ZoneOut of Interaction zone

96 Hour Arsenic Dosage

Inte

ract

inon

Tim

e

Figure 3. Exposed female time in interaction zone in seconds. Paired t-test was used and superscripts indicate statistical significance.

0ppb 10ppb 100ppb0

20

40

60

80

100

120

96 Hour Arsenic Dosage

Dif

fere

nce

in G

ill F

lare

: Win

ner

-Lo

ser

(s)

Figure 4. Difference in gill flare time between winners and losers in seconds for 0, 10 and 100ppb concentrations. T-tests were used and asterisk (*) indicates significance from zero.

0ppb 10ppb 100ppb-40

-30

-20

-10

0

10

20

30

40

96 hour arsenic dosage

Ass

ocia

tion

tim

e (s

)

Figure 5. Comparison of association time female spent among each treatment group. ANOVAs were used to compare means among treatment groups. All means ± SE.

0ppb 10ppb 100ppb-70

-60

-50

-40

-30

-20

-10

0

10

20

Series1

96 hour arsenic dosage

Dif

fere

nce

in la

ten

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s)

Figure 6. Comparison in difference of latency time female showed in each treatment group. ANOVAs were used to compare means among treatment groups. All means ± SE.

Discussion

Eavesdropping Phase

Our results showed that regardless of treatment group, females spent significantly

more time within the interaction zone than the rest of the tank. This indicates that the

female is actively observing the two males interacting and receiving any signals being

portrayed (Figure 3). It also validates out tank setup as a way to observe if

eavesdropping is occurring among the subjects.

The duration of male-male interaction (15 minutes) was chosen because it is long

enough for differences in displays to become apparent, but short enough to prevent

submissive coloration (horizontal stripes) from being displayed, strongly suggesting

females receive information from the exchange of signals between interacting males

(Doutrelant & McGregor, 2000).

When scoring the males as an indicator of fight outcome, males scored as

“winners” did significantly more gill flaring than males scored as “losers” (Figure 4).

These results have shown that using male displays as a measure of fight outcome is a

reliable way to establish the winner of an aggressive interaction, a method also utilized

by Doutrelant & McGregor (2000) and Oliveira et al. (1998).

Interaction Phase

There was no significant difference found in female behavior towards winners or

losers in association time or latency time, regardless of arsenic dosage (Figure 5 & 6).

We expected to see a distinct difference between behavior towards fish scored as

winners or losers in our control group, following results found by Doutrelant & McGregor

(2000) in a study done similar to ours. However, we did not receive these results, in

either the amount of time the female spent within one body length of the stimulus male,

or in the time it took her to interact with the stimulus male.

If we had seen the females behaving significantly differently towards the winner or

the loser in the control groups, as we had expected to, we would have been able to say

that the results we saw in the exposed treatment groups supported our hypothesis that

the female would behave similarly to both males, regardless of fight outcome due to the

effects of the arsenic. Unfortunately we were unable to do so, given our control group

showed the results it did, therefore we accepted our null hypothesis that eavesdropping

behavior would not be affected by exposure to arsenic concentrations. The wide range of

means between the error bars seen in Figures 5 and 6, our contradictory control data

showing that unexposed females don’t differentiate between winners and losers, and

most importantly, the small samples sizes could all play a role in why we didn’t see the

outcomes we expected to in this study.

Conclusion

Endocrine disrupting chemical inputs into the environment show no sign of being

mitigated anytime soon, and with increases in the human population, it would be safe to

assume that there will be an increase in these anthropogenic factors from where EDCs

derive. Further research should be conducted to evaluate how EDC’s such as arsenic

have a short-term and more importantly, long-term effects on populations of wild fish.

The implications of their negative impact on individual fitness, reproductive and

developmental stability, and population dynamics have been well established, and

therefore should serve as a motivator to continue research on how arsenic may impair

communication networks that serve as critical intersexual and intrasexual information

exchange between individuals.

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