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2017-04-03
The Effect of the Herbicide Roundup on
Female Mate Choice in Guppies (Poecilia
reticulata)
Yemc, Madison
http://hdl.handle.net/10456/42746
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of the U.S. Code.
The Effect of the Herbicide Roundup on Female Mate
Choice in Guppies (Poecilia reticulata)
By
Madison Yemc
April 3, 2017
Allegheny College
Department of Biology
Yemc | 1
The Effect of the Herbicide Roundup on Female
Choice in Guppies (Poecilia reticulata)
By
Madison Yemc
April 3, 2017
A Senior Comprehensive Project in Partial Fulfillment of the Requirements for a
Bachelor of Science Degree from Allegheny College
I hereby recognize and pledge to fulfill my responsibilities as defined in the
Honor Code and to maintain the integrity of both myself and the College
community as a whole.
___________________________________________
Pledge
Approval by the Senior Thesis Committee:
___________________________________________________
Dr. Mumme
___________________________________________________
Dr. Webb
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Abstract
Roundup, which contains non-ionic, polyethoxylated tallowamine (POEA) surfactant and
the active ingredient glyphosate, is one of the most commonly used glyphosate-based herbicides
globally. According to the World Health Organization, Roundup does not pose a threat to
animals; however, concerns remain about how this herbicide may negatively affect aquatic
ecosystems due to the longevity and persistence of chemicals in these environments. Presently,
not much is known about how fish behave under the presence of Roundup in their environment.
Because guppies (Poecilia reticulata) are a model organism for sexual selection and toxicology
studies, I studied the effect of an environmentally relevant, sublethal concentration of Roundup
on female choice in guppies. Over a period of 16 days, female guppies were exposed to 700 μg/L
of Roundup. On days 1, 2, 4, 7, 11, and 14, female fish underwent preference trials to evaluate
their inclination for bright males. On days 15 and 16, I evaluated female guppies’ sexual
receptivity behavior in the direct presence of males. Collectively, control female guppies
preferred bright males on days 1 and 2 (n = 6, P = 0.044), but did not exhibit a preference for
either bright or dull males on all subsequent days. Female guppies exposed to 700 μg/L of
Roundup strongly preferred bright males on day 1, but, they did not exhibit a strong preference
for either bright or dull males on the following days. Additionally, neither group of females
exhibited strong receptivity to males. Although, this study demonstrates that short-term Roundup
exposure (48 hours) could have affected female choice in guppies, further research is needed to
more thoroughly examine the short- and long-term effects of Roundup exposure on fish
behavior. More holistically understanding the consequences of commonly used herbicides on
wildlife is important so these chemicals can be used more safely and conservatively, or so safer
methods of eliminating weeds can be researched for future use.
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Introduction
Herbicides Effect on Fish
Herbicides are commonly used to control weed growth in both terrestrial and aquatic
ecosystems on a global scale today. Monsanto Company first discovered the properties of
glyphosate as a herbicide in 1970 and put the product, Roundup, into use in 1974 (Williams et
al., 2000). Glyphosate, the active ingredient of Roundup, is a nonselective form of weed control
used in the agricultural and forestry industries, preventing plant growth through the inhibition of
an enzyme responsible for the biosynthesis of several key aromatic amino acids found
exclusively in plants (WHO, 2004). The acting enzyme, enolpyruvylshikimate phosphate
synthase, acts directly to inhibit biosynthesis of chorismate, an intermediate in phenylalanine,
tyrosine, and tryptophan production (Williams et al., 2000). These amino acids are found
primarily in plants; thus, animals are not considered primary targets of the herbicide Roundup.
Glyphosate acts most efficiently on direct application to leaves, and controls growth of long
grasses and broad-leafed weeds specifically. Additionally, Roundup formulations contain POEA
surfactant. POEA facilitates the penetration of active ingredients of Roundup through the plant
cuticle in order to increase the efficiency of the herbicide (Brausch and Smith, 2007; Williams et
al., 2000). Thorough analysis of research conducted on Roundup and the contents of its
formulation have led to the conclusion that Roundup is not a direct health risk for humans, but
that aquatic species including waterfowl and fish may be more susceptible to toxic effects of the
herbicide (Schuette, 1998; WHO, 2004; Williams et al., 2000). Roundup may still negatively
affect the environment due to the extensive use of glyphosate-based herbicides.
Roundup can adversely impact aquatic ecosystems through runoff or direct addition to a
water source. Glyphosate, the active ingredient in Roundup, enters surface or subsurface waters
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through direct use near these areas (WHO, 2004). In Canada, upon direct application of
glyphosate to lakes, ponds, or streams, concentrations are known to be approximately 5153 μg/L.
Additionally, Glyphosate concentrations between 90 μg/L – 1700 μg/L have been found in pond
water in the United States. Fish can encounter glyphosate and the herbicide Roundup by either
living in contaminated water, or by eating other organisms that have come into contact with the
contaminant (Shenoy, 2012). Recent studies have demonstrated some adverse effects of
Roundup and its components on aquatic animals. When exposed to low concentrations of
Roundup between 2.5 mg/L and 20 mg/L for 96 hours, goldfish, Carassius auratus, exhibited
mild oxidative stress in liver and kidney tissues (Lushchak et al., 2009). Moreover, there are
noticeable declines in antibody titres over time in Tilapia nilotica when exposed to 1/1000 of the
field concentration of glyphosate (El-Gendy et al., 1998). Exposure to glyphosate-based
herbicides induced histopathological lesions in the liver and gills of Jenynsia multidentata, a
species of fish (Hued et al., 2011). In this species of fish, glyphosate-based herbicides inhibited
sexual activity which is measured by decreased number of copulations and mating success.
When reproductive success decreases in individuals, more adult fish need to survive to maintain
the number of individuals in a population (Mills & Chichester, 2005). POEA, the non-ionic
surfactant in Roundup, also has a known toxicity effect on fairy shrimp, Thamnocephalus
platyurus (Brausch & Smith, 2007). Three different concentrations of POEA were measured and
found to be extremely toxic to fairy shrimp when tallowamine chain length was reduced during
48-hour trial periods. Based on numerous studies, Roundup and its components are likely toxic
to aquatic animals.
The American Public Health Association and the Organization for Economic Cooperation and Development recommend the use of guppies (Poecilia reticulata) as models for
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toxicology studies (APHA, 1981; Rocha et al., 2015). Glyphosate-based herbicides are mildly
toxic to guppies at a concentration of 3.6 mg/L (Chandrasekera & Weeratunga, 2011; Rocha et
al., 2015). In comparison to aquatic ecosystems however, 3.6 mg/L is a much higher
concentration than what can usually be found in the environment. The highest environmentally
relevant concentration of Roundup is 0.7 mg/L or 700 μg/L while 3.6 mg/L would be equivalent
to 3,600 μg/L (Rocha et al., 2015; Peruzzo, 2008). This concentration does not include the
intensity of direct spills of herbicides to the environment. When introduced to guppies’
environments, glyphosate-based herbicides can adversely affect this species.
Varying concentrations of glyphosate-based herbicides, even at sublethal levels, can
cause morphological and physiological changes in guppies (Chandrasekera & Weeratunga, 2011;
Harayashiki et al., 2013; Rocha et al., 2015). Hyperplasias formed on guppy fingerlings when
exposed to Roundup concentrations ranging from 3.6 mg/L to 18.0 mg/L, with severity of the
condition increasing as the concentration of Roundup increased (Chandrasekera & Weeratunga,
2011). Furthermore, upon exposure to 3.6 mg/L of glyphosate-based herbicides, observers
noticed altered gill protein expression profiles, suppressed energy metabolism, activation of the
stress response, and time-dependent histopathological response in epithelial and muscle cell
types in guppies (Rocha et al., 2015). Roundup is also known to have a direct effect on
reproduction in male Brazilian guppies (Poecilia vivipara). Lower sperm quality is observed in
male fish subjected to Roundup which could directly relate to lower fecundity and decreased
effective population size in fish who experience toxic aquatic environments in nature
(Harayashiki et al., 2013). Previous laboratory experiments have tested the short-term exposure
of glyphosate-based herbicides on guppies and examined their physiological and morphological
status post-exposure. However, longer experimental studies are needed to better test the long-
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term effects of glyphosate-based herbicides. More information is also needed to better determine
how behavior, instead of specific morphological and physiological characteristics, is affected by
these chemicals.
Sexual Selection in Guppies
Pigment coloration and dichromatism play an important role in both natural and sexual
selection in guppies (Kodric-Brown, 1998). The “good genes” hypothesis states that costly traits
in males might reflect male quality. In effect, these traits should reveal social status, good
nutritional status, low parasite load, behavioral vigor, or high viability (Godin & Dugatkin,
1996). Guppies are a model organism for sexual selection and exhibit qualities from the “good
genes” hypothesis. Females tend to preference rare or novel features in males which are
ultimately responsible for the evolution of polymorphic features in male guppies (Farr, 1977).
Female guppies sexually select for males with bright orange coloration in comparison to
duller male fish. Pigment-based color patterns induced by carotenoids and melanin, in addition to
structural color patterns are important to inter- and intrasexual selection in many fish species.
Color patterns are used to signal not only within species, but can be used to communicate
between species (Price, et al., 2008). In guppies, several types of pigments are responsible for
their coloration, including carotenoids (yellow, orange, red), melanins (browns, blacks, and
grays), and structural pigments (iridescent blue and white) (Kodric-Brown & Nicoletto, 2001;
Price, et al., 2008). Due to their carotenoid-based pigmentation and the combined effects of
natural and sexual selection, guppies make a great model organism for different research studies.
Guppies cannot produce carotenoids on their own, and have to rely on other food sources to
exhibit orange coloration (Price et al., 2008). Therefore, more brilliant expression of orange
coloration in males can signal quality and nutritional status to potential mates. In addition, diets
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higher in carotenoids are positively correlated to swimming endurance and indicate good
physical condition in guppies. Carotenoids also play an important role in immune function in
guppies and have been shown to enhance B and T lymphocyte production (Kodric-Brown,
1998). Males who ingest more carotenoids are able to rid themselves of parasites easier at the
sacrifice of orange coloration. Meanwhile, duller males typically do not fight parasites as well
due to lack of nutrition in their diet (Houde & Torio, 1992). Also, male guppies whose diets are
high in carotenoids more strongly reject second-set allografts than those who have low
carotenoid diets (Grether, et al., 2004). Strong rejection of allografts is evidence that carotenoids
boost a component of the male guppies’ adaptive immune system. Yet, a higher-carotenoid diet
did not affect female guppies’ ability to rid themselves of allografts as lower carotenoid diets
support their immune function. Therefore, males with brighter orange coloration who ingest
more carotenoids are healthier and receive more nutritional benefits than males who do not have
diets high in carotenoids and are duller in color. Alternatively, carotenoids are also costly to
acquire and express, as brighter orange coloration increases predation and mortality rates in
males (Godin & McDonough, 2003). Sexual selection and female preference for bright orange
coloration is a tradeoff for increased predation risks.
There are two components to female mate choice in guppies: female preference and
reactiveness (Syriatowicz & Brooks, 2004). Female preference examines the traits females select
for in desirable male mates, while, reactiveness measures how willing female fish positively
respond to males and engage in female choice. Female guppies exhibit reactiveness to males in
several ways. Females will 1) Stop current activity 2) Orient toward the male 3) Obviously swim
toward the male 4) Exhibit a “Glide” response which is a move-toward behavior exhibited by a
distinct gliding motion toward male guppies after their sigmoidal courtship display (Houde,
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1987; Syriatowicz & Brooks, 2004). Possibly, when female guppies’ energy reserves are
depleted, she may choose to save energy to find food instead of mating. Additionally, females
may exhibit decreased responsiveness if conditions are poor, attempting to delay impregnation.
Reluctance of females’ responses to displaying males could potentially raise the standard for
males, and females may only choose to mate with extremely attractive males.
Glyphosate-based herbicides can increase toxicity in aquatic environments due to direct
contamination to the environment or through ingestion of food. Female guppies’ mating
preference functions can be influenced by several factors, and adding glyphosate-based
herbicides into the environment may affect female choice. Research has shown that glyphosatebased herbicides can cause physiological and morphological effects on different species of fish
and guppies alike. However, more research is needed to better evaluate and determine the shortterm and long-term effects herbicides have on animal behavior and sexual selection in aquatic
ecosystems. This study aims to evaluate how the highest environmentally relevant, sublethal
concentration of Roundup, 700 μg/L, affects female choice in guppies. I hypothesized that
female guppies exposed to 700 μg/L of Roundup during a 16-day time period would spend less
time associating with attractive mates. Additionally, I hypothesized that exposure to Roundup
will cause females to respond less to male mating cues when directly placed with other male
guppies. Both behaviors could have a greater impact on guppies’ mating success and
reproduction if guppies are less willing to mate.
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Methods
Pilot Study Experiment
Setup. Preliminary testing began after twelve female guppies were obtained from a stock
population placed outside Steffee B. 108 with permission from biology technician, Liz Caskey.
All fish were selected at random. Then, four females were randomly assigned to one of three,
five-gallon tanks containing varying concentrations of Roundup® Concentrate Plus: 7 μg/L of
Roundup, 70 μg/L of Roundup, or 700 μg/L of Roundup. Each tank was equipped with an
airstone to provide aeration to dechlorinated tap water, but contained no gravel substrate. On
days 3 and 6, the water was changed, and all concentrations of Roundup were refreshed. The
tanks were maintained at room temperature (25ºC – 28ºC) on the same shelf with a photoperiod
of 14 hours of light: 10 hours of dark (USNO, 2016). Fish were fed Tetra® TetraColor Tropical
Flakes once per day.
Fish remained in these conditions for a total of seven days and mortalities were
monitored daily. After seven days of exposure to varying concentrations of Roundup, the eightremaining fish were transferred and reared together in a filtered tank with dechlorinated tap
water and gravel substrate. These fish were then used in all later experimental trials. One
mortality was recorded after trials for the pilot study experiment concluded.
Animal Housing
Approximately 33 female guppies, and 15 male guppies were acquired from a stock
population of guppies located directly outside of Steffee B. 108. The seven female guppies
previously exposed to three different, low concentrations of Roundup during the pilot study
experiment were used in subsequent experiments as well. Male and female guppies were
separated by sex and housed in three different tanks with gravel substrate. All females were
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combined and the 40 female guppies were then randomly assigned to two separate, five-gallon
tanks. In total, 20 females were assigned to a tank with control conditions containing filtered,
dechlorinated water, and 20 females were assigned to a tank containing aerated, dechlorinated
water with the environmentally relevant, sublethal concentration of 700 μg/L of Roundup
(Perruzo et al., 2008). This concentration was chosen due to lack of mortality found during the
pilot study experiment. Male guppies were housed in a 10-gallon tank with filtration and gravel
substrate. Male and female tanks were also placed on different shelves to completely isolate the
sexes of fish from one another.
Water was maintained and circulated between (25ºC – 28ºC). A consistent amount of
lighting was present in each tank with a photoperiod of 10 hours of light and 14 hours of dark for
the beginning of February when experiments took place (USNO, 2017). The water was refreshed
and changed in both the control group tank and treatment group tank on days 6 and 13 of testing.
The 700 μg/L Roundup concentration was refreshed on these days as well. Mortality was
recorded daily, and fish were fed fish food flakes once per day.
Female Guppies Preference Test
To eliminate potential sources of distraction, three sides of a five-gallon tank were
covered with white paper. The tank was split into three sections using two pieces of one-way
glass (Figure 1) to prevent physical contact and sneak copulations from males (Syriatowicz &
Brooks, 2004). Females were able to see into either the left or right side of the tank, but male fish
were not able to see the female in the center section. The female preference zone, measuring 6.35
cm, was established in the center section of the tank on either side of the pieces of one-way glass.
The tank was cleaned before experimental trials began and between each day of testing.
Dechlorinated water was added to the tank at the beginning of each day tests were conducted.
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Figure 1. Skwarko (2015) tank design for live presentation. Female guppies from either
the control group or the treatment group (700 μg/L of Roundup) were placed in the center
of the tank on days 1, 2, 4, 7, 11, and 14 of exposure. A male of brighter color and a male
of duller color were placed on either the left or right. Males were switched to different
sides of the tank between each trial to prevent side bias.
Similar to methods used in Rebecca Skwarko’s (2015) senior project, live video
recordings were used to collect data. Tests were conducted on days 1, 2, 4, 7, 11, and 14 of
exposure to Roundup. On days 1 and 2, three females from the control group and three females
from the treatment group containing 700 μg/L were randomly selected to test female preference.
Then, on days 4, 7, 11, and 14, six females from the control group, and six females from the
treatment group were randomly selected to test female preference. In addition, on each day of
testing a male of dull coloration and a male of bright coloration were selected. To control some
variables, males were selected for specific similarities: body size, gonopodium size, and tail/fin
size. Prior to the start of trials, the scoring method used in Rebecca Skwarko’s (2015) senior
composition project was utilized. Male and female guppies were assigned a number on a scale
from 1 − 5 to indicate brightness. Both sexes were scored between 4 − 5 for the most prominent
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and saturated coloration, 3 for medium saturation, and a 1 − 2 for the least amount of brilliant
coloration.
A random number generator was used to determine whether female guppies from the
control group or treatment group would be tested first. Then, female guppies from either the
control group or treatment group were placed in the center section between the two plates of oneway glass, and were allotted five minutes to acclimate to the environment. After the five-minute
time period for acclimation, male guppies of brighter or duller coloration were added into either
side of the tank. The assignment of bright or dull males to either side of the tank was also
determined using a random number generator. The trial period began following the addition of
both male fish to each side of the tank. Female preference was measured by the amount of time
the female spent in the preference zone and the amount of time she did not spend in the
preference zone. Trial periods were 10 minutes, and were recorded using a cell phone video
camera. During subsequent trials, males were switched from one side of the tank to the other to
prevent female side bias. On days 1 and 2, three females from the control group and three
females from the treatment group were tested, with a total of three trials per fish for a total of 18
trials on day one and 18 trials for day two. On all subsequent days of testing, six females from
the control group and six females from the 700 μg/L Roundup-treatment group were tested for a
total of three trials per fish and a total of 36 trials per day.
After testing fish four of the control group during day 4 of female preference trials, the
bright and dull male used in the experiment were replaced for remaining trials due to their
mortalities. A different pair of males with similar characteristics and saturations as the original
male pair were substituted for the original males used for testing. Subsequently, on days 7, 11,
and 14, three different pairs of males were chosen for testing. Two pairs of bright and dull males
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who were previously exposed to Roundup from a different experimental test were used in this
experiment. Additionally, one set of bright and dull males were taken from the group of
unexposed males originally used in this experiment alone. Scarcity and limited selection of males
from the stock population used during this experiment became a limiting factor. Using exposed
males previously tested in Tara Afjeh’s senior comprehensive project was one of the only
options for testing as they were still active and appeared in good health.
Female Guppies Receptivity Test
Based on methods used by Syriatowicz and Brooks (2004), female receptivity was
measured on day 15 and day 16 of exposure to 700 μg/L of Roundup. A random number
generator was used to determine test order between the control and treatment group. For testing,
two females of similar size and gravid spot coloration were chosen to control for male choice.
Then, four unexposed male guppies were chosen at random from the unexposed males originally
used for this study.
Females were added into one end of a five-gallon tank and were able to acclimate to their
environment for 5 minutes. At the end of this time period, male guppies were added into the
tank. Prior to the initiation of courtship behavior, the attention of the observer was focused from
female to female until courtship behavior was either initiated by the male or female. Upon
initiation of courtship behavior, the female who either initiated courtship behavior or who
courtship behavior was initiated upon, was observed for a five-minute observation period. After
this five-minute time period, the observer’s focus was switched to the other female guppy in the
tank for an additional five-minute observation period. Female and male courtship behavior was
observed during all observation periods.
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Both female and male courtship behaviors were recorded. Female guppies’ responses
were majorly scored using Houde’s (1987) three criteria. Females 1) Stopped current activity 2)
Definitively moved toward the male and followed his movements, and 3) She approached with a
“glide” response and not a jerky swimming motion. Additionally, the number of times no female
response occurred after a male approached the female, and female orientation toward the male
was recorded. The following male guppies’ sexual behavior was also recorded: sigmoid displays,
sneak copulations, and full copulations. In order to minimize stress due to repeated testing, the
same four males were used to test three different pairs of treatment females and three different
pairs of control females for a total of six trials per male pair. There were 12 trials accounting for
each individual female fish from the control group and treatment group. On day 15, one fiveminute observation period per fish was recorded for a total of three trials accounting for the
treatment group and three trials accounting for the control group for a total of six trials. On day
16 of exposure, again five-minute exposure periods were recorded for a total of six trials
accounting for the treatment group and six trials accounting for the control group, for a total of
12 trials.
Upon completion of a trial, females from the control group and treatment group were
moved to rehabilitation tanks to separate them from the fish remaining to be tested. The
rehabilitation tanks, one designated for the treatment group and one designated for the control
group, both contained filtered, dechlorinated water with gravel substrate. Water temperature was
maintained between 25ºC – 28ºC. After all experiments, female and male guppies were returned
to the original stock population from which they were originally, randomly chosen from.
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Statistics
Data for female preference trials were based on the proportion of time females spent in
the bright preference zone in comparison to the total time females spent in both the bright and
dull preference zones. Meanwhile, data for female responsiveness trials were based on counts of
sexual behavior exhibited by male and female guppies. A general analysis utilizing histograms,
standard error, and an effects test using JMP PRO 12 was used to analyze the data. Female
preference data were analyzed with a two-factor analysis in variance in which Roundup
treatment group and day of testing were treated as factors. Female receptivity data were analyzed
with a general linear model in which Roundup treatment was included as a fixed factor and trial
number was included as a random effect.
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Results
Pilot Study Experiment
The pilot study experiment was conducted to determine the most appropriate, yet highest
environmentally relevant, sublethal concentration of Roundup to expose to female guppies
during later experiments. The desired concentration of Roundup was chosen if it caused few or
no mortalities to guppies. Mortalities were observed on days 3 and 5 of a seven-day pilot study.
On day 3, two fish from the 7 μg/L tank were found dead in the tank by the observer, and on day
5, one fish was found dead in the 70 μg/L tank. No mortalities were observed in the tank
containing 700 μg/L of Roundup throughout the seven-day trial period. Thus, the 700 μg/L
concentration was chosen as the most appropriate, highest environmentally relevant, sublethal
concentration of Roundup, and was used in following experiments. After the conclusion of this
experiment, one mortality was recorded, then requiring the procurement of an additional female
guppy from the stock population for preference trials.
Female Preference Test
Bright male preference was tested to determine if female guppies from both the control
group and the group exposed to 700 μg/L of Roundup spent more time with brightly colored
male guppies rather than dull male guppies. Overall, there was no significant effect of either
Roundup treatment (F1,8 = 1.50, P = 0.26) or day of testing (F1,8 = 2.85, P = 0.13) on the
proportion of time female guppies spent in the preference zone (Figure 2). However, there was a
significant treatment x day interaction (F1,8 = 5.71, P = 0.044). On day 1, females from both the
control (n = 3) and treatment groups (n = 3) significantly preferred brighter males to duller males
a greater mean proportion of the time (M = 0.77, SE = ±0.10; Figure 3). However, on day 2,
female guppies from the control groups exhibited a strong preference for bright males, but
Yemc | 17
females treated with Roundup did not (Figure 3). Additionally, females from the control and
treatment groups (n = 12) showed no strong preference for brightly colored males on days 4, 7,
11, and 14, and generally spent less than half the time associating with the brightly colored males
on these days (Figures 2, 3).
Figure 2. Combined bright male proportional preference of female guppies from control
conditions and female guppies exposed to 700 μg/L of Roundup. Each bar represents the mean
proportion time that females spent in the bright male preference zone. Error bars were constructed
using one standard error from the mean.
Mortalities were recorded in all tanks throughout female preference test trials. On day 4,
two males from the unexposed sample population chosen from the stock population died.
Therefore, the unexposed male population sample size was reduced from 15 unexposed, male
guppies to 13 unexposed, male guppies. Additionally, on day 6, one female from the control
group and one female from the 700 μg/L of Roundup treatment were found dead in the tank.
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Thus, sample size was reduced after day six to 19 female guppies in both the control and
treatment groups.
Figure 3. Female proportional preference for bright males by day and treatment group. Each bar
represents the mean proportion of time females from either the control group or the treatment
group spent in the bright male preference zone. Error bars were constructed using one standard
error from the mean.
Female Receptivity Test
There were no significant differences in receptivity behaviors exhibited by female
guppies exposed to sublethal concentrations of Roundup and female guppies who were not
exposed to Roundup (Figure 4). Females in the control group and treatment group both stopped
current activity (Figure 4A), oriented toward the male (Figure 4B), swam toward males (Figure
4C), and initiated “Glide” responses (Figure 4D), approximately the same number of times.
Additionally, no significant differences were observed in male mating behavior initiated toward
Yemc | 19
female fish. Males initiated sigmoid displays (Figure 4E) and sneak copulations (Figure 4F) a
similar number of times in response to females from either the control or Roundup-treatment
group.
Figure 4. Mating receptivity of female guppies. Each bar represent counts of the mean number of
times a female stopped current activity (4A), a female oriented toward a male (4B), a female
swam toward a male (4C), a female initiated a “Glide” response (4D), a male initiated a sigmoid
display (4E), a male initiated a sneak copulation (4F). Error bars were constructed using one
standard of error from the mean.
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Discussion
Globally, widespread and heavy use of herbicides can increase the chance of runoff
contamination or direct application to aquatic or terrestrial environments affecting animals which
are not the chemical’s primary target (Jiraungkoorskul et al., 2001; WHO, 2004). Glyphosatebased herbicides and other acting chemicals in Roundup have toxic effects on fish both
physiologically and morphologically (Brausch & Smith, 2007; Chandrasekera & Weeratunga,
2011; El-Gendy et al., 1998; Harayashiki et al., 2013; Hued et al., 2001; Lushchak et al., 2009;
Rocha et al., 2015). Due to a lack of research on animal behavior in the presence of Roundup,
this experiment aimed to measure the effect of an environmentally relevant, sublethal Roundup
concentration on female mate choice in guppies. I hypothesized that female guppies exposed to
Roundup over a 16-day time period would not spend as much time associating with brighter,
more desirable male mates. Additionally, I hypothesized females in the Roundup treatment
would not strongly respond to male mate cues when directly in the presence of male mates.
Overall, the results for female preference trials indicated no significant effect of Roundup
treatment on mate choice (P = 0.26) and no significant effect of day of testing (P = 0.13; Figure
2). Significant effects of day x treatment interactions exist as female guppies exposed to
sublethal concentrations of Roundup proportionally preferred associating with bright males over
dull males on day 1 of testing (P = 0.044, Figure 3). On day 2, however, female guppies exposed
to sublethal concentrations of Roundup did not proportionally prefer bright males over dull
males, while unexposed, control females proportionally preferred them strongly (Figure 3). For
remaining trial days, female guppies did not prefer bright males to dull males, with the possible
exception of Roundup-treated females on day 14 (Figure 3). Additionally, no significant
differences existed between exposed and unexposed female guppies during receptivity trials
Yemc | 21
conducted on 15-16 days of exposure (Figure 4). These results are not consistent with my
hypotheses. Thus, I failed to reject both my null hypotheses: 1) Female guppies in both the
treatment group and the control groups will spend equal amounts of time with both bright and
dull males, and 2) Female guppies from the Roundup treatment group and control group will be
equally as receptive to male mates. The results obtained through this study leave remaining
questions about the short-term and long-term effects of Roundup on mate choice in female guppy
fish, and indicate a greater need for research on the effect glyphosate-based herbicides have on
animal behavior.
The results of this study demonstrate that female guppies’ mate choice may be altered in
the short-term due to the effect of environmentally relevant, sublethal Roundup. During days 1
and 2 of preference trials, females from both the Roundup treatment group and control group
strongly preferred brighter males more than duller males (Figure 2, Figure 3). Significant
differences exist in treatment x day interactions on days 1 and 2, during which females from the
Roundup treatment group significantly preferred brighter males to duller males (P = 0.044;
Figure 3). On day 2, female guppies from the control group exhibited a strong preference for
brighter males, rather than the Roundup treated females who did not exhibit a strong preference
(Figure 3). The results obtained from days 1 and 2 could be due to several factors.
Roundup-treated female guppies might have strongly preferred bright males on day 1 and
then lost interest due to endocrine disrupting effects of glyphosate. Endocrine disruption refers
to toxicity which prevents cells, tissues, and organs from communicating properly, resulting in
adverse health outcomes (McKinlay et al., 2008). Adverse health outcomes can include any of
the following or more: reduced fertility or fecundity, spontaneous abortion, skewed sex ratios of
offspring, impaired immune function, a variety of cancers, neurobehavioral disorders, etc. Bodies
Yemc | 22
of water in aquatic ecosystems are known as “ultimate sinks” for man-made chemicals, and
typically house endocrine disrupting chemicals (Mills & Chichester, 2005). Because endocrine
disruptors are found in these environments, they are likely affecting the animals living there. For
instance, when Rana pipiens tadpoles are exposed to several different concentrations of Roundup
with acute toxicity, several morphological and physiological changes are observed (Howe et al.,
2004). Tadpoles experienced decreased snout-vent length at metamorphosis, tail damage,
gonadal abnormalities, and greater time to metamorphosis. The level of chemical toxicity can
also influence endocrine disrupting effects (McKinlay et al., 2008). Low dosages may not induce
the mechanisms which would produce toxic lethality, but could disrupt other mechanisms within
the organism. Glyphosate in particular, has been found to disrupt aromatase, in effect inhibiting
the production of estrogens in human placental cells (Richard et al., 2005). This also suggests
that glyphosate can influence mammalian cell lines in addition to plant cell lines. Exposure to
sex hormones has disrupted mating behavior in Zebrafish upon addition of sex hormones to the
aquatic environment (Pradhan & Olsson, 2015). Disruption of the sex hormone estrogen in
female guppies could potentially act to increase females’ desire to mate initially, but then
decrease their desire to mate after initial exposure to glyphosate-based herbicides.
The results of this study can also indicate several variables can affect female mate choice,
expanding on research that male color displays are not the only factor which affect if a female
will show interest in mating. Overall, female guppies from both treatment groups did not
indicate a strong preference for bright males during days 4 through 14 of female preference trials
(Figures 2, 3). Lack of preference for bright males must reflect reasons other than endocrine
disrupting effects of glyphosate. Individuals from the same stock population of guppies used in
this experiment could potentially have their own individual preferences for male coloration.
Yemc | 23
Guppies have considerable inter-individual variability to a degree in their mating preferences
(Godin & Dugatkin, 1995). Variation among the female population is expected, however is not
necessary because selective pressures in female mating are important for selecting exaggerated
male traits. Additionally, it is important to note that male and female guppies used for this study
both came from the same stock population. Females independently prefer males, and have the
cognitive ability to remember male identity and conspecifics (Dugatkin & Alfieri, 1991). Thus,
female guppies may modify their behavior due to past interactions which could indicate why this
study found that females preferred neither bright nor dull males. Female guppies could have
potentially recognized and remembered males they previously came into contact with from the
stock population. If females did not initially prefer the males from the population, they may
continue to be specific about their mating preference and will not prefer a specific male.
Furthermore, female mate preference for male ornaments, such as carotenoid pigment, may also
depend on the age of the female and if she has given birth (Kodric-Brown & Nicoletto, 2001).
Younger female guppies with less sexual experience − approximately 6 months in age −
typically chose mates with a greater area of orange carotenoid pigment when compared with
older, more mature females or females who have given birth to a brood of guppies. In addition,
even though each fish was chosen at random and had an independent chance to be chosen for
testing, there is a possibility the same females underwent testing on multiple days. Undergoing
multiple days of repeated testing could have potentially led to female habituation (Liley &
Wishlow, 1974). In effect, female guppies from the control group and Roundup-treatment group
might have shown no preference for male ornaments due to habituation to the males used for
testing, therefore, exhibiting a preference for neither bright nor dull males.
Yemc | 24
Limitations and weakness to this study include inconsistent sample sizes from day one
and two of female preference tests, and differences in sample size on day 15 and 16 of female
receptivity tests. In hindsight, if sample sizes remained consistent throughout the study, results
obtained would have perhaps remained more consistent throughout the experiment. Additionally,
females from the 700 μg/L of Roundup treatment were placed in dechlorinated, tap water
between 30 minutes to 9 hours while waiting to be tested, thus having enough time to acclimate
to this water. The treatment females should have remained in water containing 700 μg/L of
Roundup while waiting to be tested in order to maximize and maintain long-term exposure to
Roundup as much as possible. Giving the females time to acclimate to dechlorinated tap water,
without the effects of Roundup, could have decreased the effect of Roundup and potentially
influenced preference of the Roundup treated females. Moreover, five-minute test trials during
the receptivity tests could have been increased to maximize time during which mating behaviors
could have been observed. A more holistic and detailed understanding through increasing time
trials of mating behaviors in the presence of Roundup might aid in determining if differences in
receptivity do exist in greater time frames.
Herbicides like Roundup, and its components, may have a harmful impact on the
environment. Both aquatic and terrestrial ecosystems may be affected by exposure to these
contaminants. There is not much information on how these chemicals affect animal behavior or
what kind of endocrine disrupting effects exist due to herbicides. There is vast importance in
understanding how animals behave in response to Roundup. Guppies are a well-researched
model organism for sexual selection and are also recommended for toxicology studies. Future
studies on sexual selection in guppies when exposed to Roundup during short- and long-term
exposure would be useful to help better understand, clarify, and repeat the results found in this
Yemc | 25
study. In addition, it would be useful to study and compare guppies’ behavior when fish are
exposed to glyphosate, POEA, and Roundup during the same time frame. This information could
help indicate which component or components are needed together to create a certain behavioral
effect. Understanding the effects of the chemical components of one of the most commonly used
herbicides today can be most useful to more specifically identify the harm these chemicals have
on the environment in both the short- and long-term. Safer means of herbicide use can be
beneficial for not only aquatic ecosystems, but for humans as well. Proposing safer means of
herbicide use could help decrease the chance the product will be indirectly applied to either
terrestrial or aquatic environments. Taking extra safety measures or inventing a safer, yet
efficient herbicide can also increase safety for humans who directly use the products or who are
indirectly affected by Roundup.
Acknowledgements
First, I would like to thank Dr. Mumme for his continued guidance, support, and
expertise. Most importantly though, I would like to thank him for continuing to push me to try
my best throughout the comping process. I would also like to thank Dr. Webb, my second reader,
for her additional guidance, support, and input on my comp. Additionally, a big thank you to my
friends and family for being there for me while running my experiments. Comping would have
been especially hard if I didn’t have someone to vent to about the occasional death of my fish.
Finally, I would like to thank Vocelli’s because sometimes it’s just a three-slice day.
Yemc | 26
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