Volume 6, Spring 2008 - Saddleback College

Fall 2007 Biology 3A Abstracts
There have been very few studies that have
tested the relationship between aggression in aquatic
animals and estrogen. A study performed by Lai et al.
(2002) looked at the effects of natural and synthetic
steroid estrogens in relation to their environmental
occurrence. It was found that fatal effects have only
been observed in juvenile fish exposed to a high
concentration of estrogens. The development of sexual
and reproductive characteristics in vertebrates is
sensitive to exogenous estrogens. If the alterations of
sexual characteristics affect the reproductive
performance of the organisms, either temporarily or
permanently, the exposure to steroid estrogens may
have an effect at the population level. Exposure to
estrogens may cause other physiological effects in
animals. It is apparent that exposure to steroid
estrogens results in a diverse range of effects on a large
number of species.
Betta splendens, commonly referred to as
Betta fish or the Siamese fighting fish, are a breed of
fish that live in the waters around Southeast Asia and
are from the family Anabantidae. Betta splendens are a
labyrinth fish that gulp air to get oxygen. They have a
special auxiliary breathing mechanism called the
labyrinth that has a pair of irregular passages to provide
supplemental oxygen to the blood. Hence, they can live
in low oxygen level waters. The male Betta fish are
renowned for their long tails and fins that they use to
display aggression toward perceived threats. They
exhibit a behavior of fixed action patterns that are
sequences of movements determined genetically (Allen
and Nicoletto, 1997). Researchers found that
environmental stimuli promote these patterns. In the
case of the Betta, the stimulus is most likely another
male Betta.
The male fighting fish is so aggressive, or
agonistic, that in a community tank where other fish
are present there can only be one male Betta. When
fighting, males will nip at the other’s fins until one of
them is exhausted. Betta fish will display their fins to a
mirror since they do not recognize their reflections and
think another male is infringing on their territory. The
stretching of the fins and opening of the gills to display
the membrane enables the male to look twice his
resting size and is called flaring, or displaying.
Betta splendens have historically been used in
experiments because of their easily recognized and
well documented social displays (Oiverira et al., 1998).
This experiment exposed male Betta fish to high levels
of ethinyl estradiol, and it was expected that they
would behave less aggressively to stimuli.
Methods and Materials
Subjects and setup
Six male Betta fish were purchased from Pet
Palace (Glendora, CA) on April 2, 2008 and stored
separately in 1 L Pyrex beakers: each containing 1 L of
water. Azusa municipal tap water was dechlorinated
by allowing it to sit for 24 hours in an open mouthed
container. For the duration of the experiment, the
beakers housing the Betta fish were wrapped in white
paper in order to minimize external stimulation. All
the Betta fishes were approximately the same size,
appeared to be in good health, and maintained in
similar conditions. They were fed four Aqua Culture
Betta Pellet (HBH Pet Products, Springville, UT) twice
daily.
Testing procedure
After a week of living under “normal”
conditions, basal levels of aggression were measured.
One at a time, the Betta fish were transferred into a
paper wrapped 2.5 L Aqueon Mini-tank (Central
Garden & Pet Company, Walnut Creek, CA) filled
with dechlorinated water and allowed to acclimate to
the new environment for an hour. A portion of the
white paper was then removed and replaced with a 3 x
4.5-inch reflective mirror. The time it took the fish to
respond to the mirror was measured in seconds using a
stop watch. Aggression was determined by the time it
took to begin fin flaring which includes the pelvic fin,
the ventral fin, the caudal fin, the dorsal fin, the gill
operculum, and the 90° twist of the body.
In order to simulate estrogen pollution, thirty
Yasmin 28 (Berlex, Nomtville, NJ) birth control pills
each containing 0.03 mg ethinyl estradiol were ground
using a ceramic mortar and pestle. Since ethinyl
estradiol has low solubility in water, the resultant
powder was first dissolved in 4 mL of ethanol to make
a stock solution of 7.6 mM. Now able to dissolve in
water, 220 μL of the stock solution was then added to
10 mL of dechlorinated water. Each fish received 1 ml
of this diluted solution in their 1 L beakers for a final
concentration of approximately 60 μg/L. The dosed
Betta fish were allowed to acclimate to the new
environmental conditions for a week. Once again, the
Betta fish were transferred to the 2.5 L aquarium now
containing estradiol treated water, and their agonistic
behavior was recorded in manner described above.
Data analysis was completed using Microsoft
Excel (Microsoft Corporation, Sylmar, CA). A paired
t-test was performed to assess whether there was a
significant difference in the time to start the fixed
action pattern of agonistic aggressive pre and post
ethinyl estradiol exposure. Differences were considered
significant at P