Saddleback
Journal of Biology
Budgerigar, Melopsittacus undulatus, on treadmill (see pg. 1)
Published by
Saddleback College Biological Society
Volume 3
Spring 2005
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Advisors, Tony Huntley and Steve Teh
Editor, Grace Tran
TABLE OF CONTENTS
Peer Reviewed Manuscripts
Author(s)
Kyle Stansifer &
Robert Vogel
Stacie Aarsvold
Andrea Acosta &
Jordana Longoria
Mike Beck &
Dmitriy Pastaranak
Deborah Contreras,
Wyatt Sum &
Gabriel Tran
Title
Oxygen consumption during terrestrial locomotion in the
budgerigar, Melopsittacus undulatus
The Effects of Caffeine on Rat (Rattus norvegicus) Memory
Species Competition in Dana Point Tide Pools
Adrena
Gharibjanians
& Melinda Tanabe
Do Caterpillars Think? A Study of the Learning Ability of
Cabbage Loopers and the Effect of Pesticides
16
Jason Gillis &
Terha Trost
Michael Hinson &
Yury Penaloza
Kirby Jacobs
The Effects of Creatine Supplementation on Anolis
carolinensis during Anaerobic Exercise
The Effects of Caffeine on the Metabolic Rate of the Fish
Carassius auratus
Effects of Age on the Cardiovascular Recovery Rate in
Female Aquatic Athletes
Reaction Times are Improved with Caffeine Intake
21
Sara Liechty &
Morvarid Vahdati
Moji Ogunleye
The Effects of pH on the Germination and Growth of Zea
Saccharata
Response of the Anterior and Posterior Osmoregulatory
Mechanisms of the Goldfish, Carassius auratus, at Three
Temperatures
Goldfish (Carassius auratus) Metabolism in Various
Temperatures
John Phillips &
The Effects of Dehydroepiandrosterone and Creatine
Rudy Scalisi
Monohydrate on the Metabolic Rate of Mice,Mus musculus
Ellyne Dudkowsk
The Effects of Varying Temperatures on the Stridulation
Frequency of Crickets, Gryllus assimilis
Raquel Rosales
The Effect of Temperature on the Growth of Peperomia
obtusifolia
Janelle Silva & Juilet The Effects of Lowing and Raising pH on Goldfish
Hamner & Rigoberto Respiration
Aguilar
James Steele &
The Effect of Olfaction on Hummingbirds’ Foraging
Chenae Dahlstrom Behavior
Ashley Waugh
Effect of Bread Components on Net Mold Production Over
Time
Robert Ward
The Effect of NaCl on the Metabolic Rate of Crassius
auratus
Amir Akhavan &
Effects of Ginkgo biloba on Memory in Mus musculus
Julius Chiu
Page
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44
48
52
55
Abstract Issue
Abstracts of papers presented at the Spring Saddleback College Biology Meeting
The meeting organizers do not assume responsibility for any inconsistencies in quality or errors
in abstract information. Abstracts are in numerical order according to the abstract number
assigned to each presentation. Authorless abstracts appear at the end of all the abstracts,
including non-emailed abstracts. Abstracts begin on page 58.
Note: Author name(s) and abstract titles were printed directly from the abstract form without corrections. The
presentation order was determined by the order in which the emailed abstracts were received.
1.
The Effect of High pH on the Photosynthetic Rate of Elodea canadensis.
Carolyn Dreyer, Katie Bennett, Katheryn Saab
2. Growth of Tomato Plants under Different Wavelengths of Light.
Nam Le and Nina Turk
3. 3. Effects of alkaline pH on the metabolic rate of goldfish (Carassius auratus).
Julie G. Rhodes and Angelina Thompson
4. 4. Is a more symmetrical face more likely to secrete better smelling pheromones and
attract more mates?
Jennifer Quinn
5. 5. The Effects of Ultraviolet radiation on the HA2 yeast strain.
Sinikka Kreuzer and Ethel Soriano
6. 6. Respiration of Mice (Mus musculus) at Differing Altitudes.
Matt Ireland and Jacob Smith
7. 7. Effects of Basic pH on the Metabolic Rate of Comet Goldfish (Carassius auratus).
Jerry Lee and Lacey Meyerhardt
8. 8. Attraction of Fruit Flies (Drosphila melanogaster) to Different Substances. Joseph
Ling and Yama Osmazada
9. 9. The Effects of Varying Ambient Temperature on the Resting Metabolic Rate of
Budgerigars (Melopsittacus undulatus).
John Supance, Shallom Han, Matthew McGeough
10. 10. The Effects of pH on Oxygen Production of Elodea canadensis.
Erfan Shenghur and Shane Zarifzadeh
11. 11. Body size does not affect the metabolic rate in goldfish at ambient temperature.
Nastran Aghazadeh, Supriya Chaudhary, Nam Phuong Nguyen
12. 12. The Effect of Reduced Barometric Pressure on Goldfish Opercular Pumping Rate.
Sheng-Chieh Chang and Albert Trinh
13. 13. The Effects of Polarized Light on the Oxygen Production in Elodea canadensis.
Sean Stanton, Misty Guzman
14. 14. The Effects of Electric Current on Cell Growth in Onions (Allium cepa).
Russell E. Roberson
Oxygen consumption during terrestrial locomotion in the budgerigar,
Melopsittacus undulatus
Kyle Stansifer and Robert Vogel
Department of Biochemistry
Saddleback College
28000 Marguerite Pkwy., Mission Viejo, CA 92692-3635.
Budgerigars, Melopsittacus undulatus, spend as much as 65 percent of daily activity
walking and running on branches. The energetics and morphology of terrestrial locomotion in
this species has not been studied. In this study, we propose that oxygen consumption should
increase proportionally with speed of terrestrial locomotion. Rates of oxygen consumption
were measured in seven birds trained to run on a treadmill. Oxygen consumption was
recorded for all birds at running speeds of 0 m/s, 0.2310 m/sec, 0.4128 m/sec, and 0.6142 m/sec.
Rate of oxygen consumption as a function of running speed was linear. Several birds
exhibited variations in gait morphology when running at higher speeds. Changes in gait
morphology suggest possible advantages of certain forms of terrestrial locomotion.
Introduction
The terrestrial locomotion of budgerigars,
Melopsittacus undulatus has been disregarded for
decades. Previous research of similar species have
analyzed energy expenditure during flight. These data
have been recorded as the rate of oxygen consumption
(VO2), which can be derived by VO2 = aMbb, where a is
the body mass coefficient, Mb is the body mass, and b
exponent is the body mass exponent (Suarez, 1998).
Other research has used alternative ways to relate VO2
and flight specifically in M. undulatus. Behavioral
research has shown that normal activity primarily
consists of a series of social interactions including
biting, chasing, and calling other birds in the wild and
captivity (Diamond and Bond, 2003). These activities
are significantly apparent terrestrially, rather than in
flight, constituting approximately 65%-75% of their
day (A. Huntley, pers. comm. 2005). As important as
terrestrial locomotion is for M. undulatus, no research
has been conducted on actual energy demand of their
locomotion.
VO2 during variable rates of terrestrial
locomotion in other vertebrates has been extensively
studied. Increased speeds of locomotion in horses have
shown an exponential increase in oxygen consumption
(Wickler, et al. 2000). Research on the cost of transport
has shown that horses have an optimal speed at which
cost of transport is at a minimum (Wickler, et al. 2002).
Studies have also been conducted on vertebrates of
different classes that run. One study suggested that
emus exhibit a linear increase when comparing energy
used (EmetabWb-1) and speed at which they run (Roberts
et al., 1998). Other studies have suggested a correlation
between gait and speed of vampire bats (Riskin and
Hermanson, 2005). This shows a direct relationship
between a preferred gait and rate of oxygen
consumption. Furthermore, horses make a transition
from trotting to a canter in order to minimize energy
cost (Hoyt and Taylor, 1981). Therefore, M. undulatus
could show variable VO2 at different speeds of
locomotion, as well as a preferred gait.
Methods and Materials
Animals
Seven budgerigars from a population of thirteen
purchased from Bird Crazy (San Diego, CA) were used
in this study. The body weights of all experimental
subjects are shown in Table 1. Birds were housed in
three cages indoors at Saddleback College, Mission
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Saddleback Journal of Biology
Spring 2005
Figure 1. Budgerigar on treadmill.
Viejo, CA. All birds were given Hartz bird seed (Hartz
Corp) and water ad libitum.
Training
Birds were trained for a total of two months prior
to data collection. Hand training was performed, four
days a week, for the first month. This allowed the birds
to become acclimated to human handling and being
transferred to and from the treadmill apparatus (Figure
1).
The treadmill was incased in the top chamber of
the apparatus, measuring 23.0 x 5.6 x 10.0cm, made of
Plexiglas. A Styrofoam triangular solid, 10.7 x 5.6 x
7.3cm, was inserted in the back of the chamber to allow
proper running morphology (tail lifted) and a decreased
volume of air within the chamber. Total volume of air
within the top chamber was calculated by subtracting
the volume of the triangle and belt, resulting in
approximately 1,000 mL of free air space. The lower
chamber, 24.0 x 6.5 x 21.5cm incased an electric motor
powered by a Dual Range DC Power Supply (Electro
Industries, model 3670). Speed of treadmill belt was
regulated by voltage output connected to motor.
Table 1. Body weight of all subjects
Parakeet
Body Mass (g)
Green-Blue Nasal
33.35
Gracie
32.40
Bitey
32.58
Lefty
30.05
Bluey
28.48
Femmeril
29.24
Righty
28.73
Birds were trained on the treadmill four times a
week during the second month. Birds ran at speeds
between 0.231 m/s and 0.614 m/s based upon
capabilities. A warm up and cool down session of
0.231 m/s was completed as well. Total running time
per bird per day averaged to approximately four
minutes.
Airflow from the treadmill chamber was directed
9
Gracie
-1 -1
V
O
2 (m
L
O
2g h )
8
Gracie 5/4/05
7
Bitey
6
Femmeril
5
4
Lefty
3
Righty
2
Bluey
1
green blue
nasal
AVG
0
0
1
2
3
4
5
T ime (min)
Figure 2. Basal VO2 for seven budgies at room
temperature (22 to 23 °C). Subject “Gracie” was run
two times. All volumes are corrected to STPD.
to a FOXBOX Oxygen analyzer (Sable Systems, Las
Vegas, NV). Incoming air was passed over a 25 cc
syringe filled with Ascarite and Drierite, prior to
metering or analysis.
Birds were fasted 12-20 hours before
experimental recording. We assumed an RQ of 0.71
and utilization stored fat. Percentage oxygen in the air
stream leaving the treadmill chamber, chamber
temperature, and barometric pressure were recorded by
FOXBOX every five seconds throughout each run.
Flow rates through the chamber were controlled
at 900 to 100 mL/min. Resting oxygen consumption
was recorded for each bird for three minutes following
a five to eight minute habituation period in the
chamber. This recording was made with the chamber
covered and the bird in the dark. Birds were then run at
speed of 0.231 ms/s, 0.413 m/s and 0.614 m/s for
periods of 1one to two minutes. Birds were rested for
five to eight minutes between treadmill runs. Upon
completion of a treadmill session, birds were weighed
and given food. All running activity was recorded
using a model DCR TRV10 SONY digital video
camera, to allow future analysis of gait patterns.
All data recorded by FOXBOX were transferred
to a Microsoft Excel spreadsheet on a COMPAQ
Presario 1692 computer. VO2 was calculated using the
following equation (4A from Withers, 1977).
VO2 (mLO2g-1 h-1) was then converted from laboratory
atmospheric pressure and temperature into VO2 at STP
by utilizing the ideal gas law.
Results
All VO2 values from the birds were graphed as
a function of the varying speeds (0 m/s (basal), 0.2310
m/s, 0.4128 m/s, and 0.6142 m/s). Data at 0 m/s
indicate that the basal metabolic rate for the M.
undulatus was around 2.5 mLO2g-1 h-1 (Figure 2).
Data above 2.5 mLO2g-1 h-1 are due to bird activity in
the chamber and do not represent VO2 at rest. Data at
0.231m/s show that most birds had a VO2 rate above
5.0 mLO2g-1 h-1 (Figure 3). Data at 0.4128m/s show
that most birds had a VO2 rate above 5.0 mLO2g-1 h-1
(Figure 4). Data at 0.6142m/s show that the birds had
a VO2 rate above 6.0 mLO2g-1 h-1 (Figure 5).
The combined average rate of oxygen
consumption from all experimental birds versus
running speed is shown in Figure 6. The results show
a linear function (R2=0.8279) with an increase in the
rate of oxygen consumption as speed increased.
ANOVA testing indicated that significant differences
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Saddleback Journal of Biology
Spring 2005
existed among the data at all speeds. A post-hoc
test (Bonferroni Correction) demonstrated a
significant statistical difference between all rates of
VO2 consumption rates at all speeds.
8
Gracie
Femmeril
Righty
Bluey
Green Blue Nasal
4
2
12
10
-1
6
h )
VO2 (mL O2 g-1 h-1)
10
Green et al. (2001) found that heart rate and
mass-specific rate oxygen consumption increased
linearly with increasing walking speed. It is therefore
shown that as walking and/or running speeds increase,
birds metabolic pathways, such as heart rate, also
increase. This is due to higher demands of energy
8
0
0.5
1
VO2 (mLO 2 g
-1
0
1.5
Time (min)
Figure 3. VO2 at 0.231m/s for five budgies at room
temperature (22 to 23 °C). All volumes are corrected
to STPD.
Gracie
Gracie 5/4/05
Bitey
bluey
green blue nasal
AVG
6
4
2
0
0
0.5
Figure 5. VO2 at 0.6142m/s for four budgies at room
temperature (22 to 23 °C). Subject “Gracie” was run
two times. All volumes are corrected to STPD.
9
7
6
9
Lefty
y = 4.9951x + 4.4118
R2 = 0.8279
8
5
green blue nasal
AVG
6
-1
3
7
-1
4
VO2 (mL O2 g h )
-1
h )
8
-1
1.5
Time (min)
10
VO2 (mL O2 g
1
2
1
0
5
4
3
2
0
0.5
1
1.5
1
Time (min)
Figure 4. VO2 at 0.413m/s for two budgies at room
temperature (22 to 23 °C). All volumes are corrected
to STPD.
Discussion
The rate of oxygen consumption in M. undulatus
increased linearly as running speed increased. The
amount of oxygen inhaled is directly related to the
bird’s metabolic system. These data suggest that if the
birds were to run at higher speeds, oxygen consumption
would continually increase, as would its metabolic
activity. It is also suggested that there is no preferred
speed where energy utilization is lowest at the highest
possible speed.
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Speed (m/s)
Figure 6. VO2 at four running speeds. Points are
averages, ± s.e.m.
utilization to allow the bird to continuously run.
It is proposed that M. undulatus have a
maximum allotted energy expenditure because the
increase of oxygen consumption is a linear progression.
It is presumed that a typical M. undulatus in the wild
has enough available metabolic energy to successfully
run in short bursts in order to evade predators and climb
on tree branches, as well as other specific behavioral
activities.
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Saddleback Journal of Biology
Spring 2005
Budgerigar terrestrial locomotion varied between
birds. Some birds changed their running morphology
with increasing speeds. At faster speeds it was noticed
that some of the bird’s legs moved closer together,
causing its body to lift higher. Simultaneously the bird
lowered its head and raised its tail. The significance of
this behavior is unknown, however it could be in
response to various stimuli. This might be an example
of differentiations between gait at varying speeds, very
similar to that of a horse (Hoyt and Taylor, 1981).
However, M. undulatus differs from a horse in that the
changing of gait, or stride, does not increase its
locomotive efficiency. In actuality its VO2 increased
linearly with speed during the morphological change in
running. Therefore data suggests that change in
morphology might be linked to past events, possibly an
efficient running ancestor, such as the emu.
Furthermore, there has been enough years to separate
the two to allow M. undulatus to reside in the trees as
well as the land, but the connection has not been lost.
Wickler et al. (2002) analyzed horses with
variable stride length, and concluded that stride length
has no effect on the rate of oxygen consumption. This
could also explain the tendency for change in running
morphology with no change in VO2. M. undulatus that
ran with their body and tail lifted seemed to have much
more consistent strides and higher capabilities of
running longer. Because only some birds have the
natural ability to vary running morphology, it is
suggested that various forms of gait is an advantageous
trait to evade predators.
Acknowledgements
The authors would like to acknowledge the
assistance of Mr. S. Teh and Dr T. Huntley for help
with experimental design, data analysis and equipment.
Two anonymous reviewers greatly improved the quality
of the manuscript.
Literature Cited
ANOVA. (2005). Analysis of variance between
groups. College of Saint Benedict Saint John's
University's Physics Department. Retrieved May 05,
2005 from the World Wide Web:
http://www.physics.csbsju.edu/stats/anova.html
Diamond, J. and Bond, A.B. (2003). A Comparative
Analysis of Social Play in Birds. Behaviour, 140:10911115.
Hoyt, D.F. and Taylor C.R. (1981). Gait and the
energetics of locomotion in horses. Nature, 292:239240.
Green, J.A., Butler, P.J., Woakes, A.J., Boyd I.L., and
Holder R.L. (2001). Heart rate and rate of oxygen
consumption of exercising macaroni penguins. J. expt.
Biol., 204:673-684.
Nudds, R.L. and Bryant, D.M. (2000). The Energetic
Cost of Short Flights in Birds. J. expt. Biol., 203:15611572.
Riskin, D.K. and Hermanson, J.W. (2005).
Independent evolution of running in vampire bats.
Nature, 434:292.
Roberts, T.J., Kram, R., Weyand, P.G., and Taylor, R.
(1998). Energetics of Bipedal Running. J. expt. Biol.,
201:2745-2751.
Suarez, R.K. (1998). Oxygen and the Upper Limits to
Animal Design and Performance. J. expt. Biol,
201:1065-1072.
Wickler, S.J., Hoyt, D.F., Cogger, E.A. and Hirschbein,
M. (2000). Preferred speed and cost of transport: the
effect of incline. J. expt. Biol., 203:2195-2200.
Wickler, S.J., Hoyt, D.F., Cogger, E.A., and McGuire,
R. (2002). The cost of transport in an extended trot.
Equine vet. J., Supple., 34:126-130.
Withers, P.C. (1977). Measurement of VO2, VCO2, and
evaporative water loss with a flow-through mask. J.
appl. Physiol, 42:120-123.
Bishop, C.M. and Butler, P.J. (1995). Physiological
Modeling of Oxygen Consumption in Birds of Flight. J.
expt. Biol., 198:2153-2163.
Bonferroni Correction. (2005). Post test calculator.
GraphPad Software, Inc. Retrieved May 05, 2005 from
the World Wide Web:
http://graphpad.com/quickcalcs/posttest1.cfm
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Saddleback Journal of Biology
Spring 2005
The Effects of Caffeine on Memory in the Rat (Rattus norvegicus)
Stacie Aarsvold
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Memory is one of the most important functions of the brain, however, the loss of
memory can lead to a decrease in the quality of life. Many people try to reverse memory
problems with drugs and other herbal remedies, however, they often do not account for
common, everyday drugs, such as caffeine. This experiment tested hooded rats’ memories
before and after the consumption of caffeine. The hypothesis tested was caffeine would
have a positive effect on memory. Ten rats were tested two separate times in a water maze.
They were first tested before any caffeine had been consumed. They were then tested again
after having received 1 mL of caffeine. The experiment showed no difference in memory
between the control group (without caffeine) and the experimental group (with caffeine).
The average time to do the maze for the non-caffeinated rats was 18.65 seconds and the
average time for the caffeinated rats was 18.04 seconds which produce a p value of 0.86.
These results do not support the hypothesis that caffeine has a positive effect on memory.
Introduction
Memory is one of the most important
functions of the brain. In recent times however, this
has been one of the first things to go as people age.
This loss of memory can lead to many diseases and
disorders, one of the most common being Alzheimer’s,
which is a type of senile dementia. Memories are
stored in different parts of the brain depending on the
type of memory that it is. In this experiment, the
memories will be stored in the cerebellum, which is
where memories involving movement are stored.
(Hockenbury, 1998).
Due to this increasing decline in mental
health, many people have turned to drugs or herbal
remedies to help with these symptoms. What many
people don’t realize is that caffeine is one of the most
commonly used drugs. It is found in many foods and
drinks including coffee, tea, and soda. On almost
every corner there is someone drinking a caffeinated
drink. With the amount of people consuming caffeine
it is important to know if it is one of the factors that is
leading to the decline in mental health.
In 1958, the Food and Drug Administration
(FDA) and the American Medical Association (AMA)
have found caffeine to affect some aspects of
physiology, but none of the effects are serious. They
have found no relationship between caffeine and
osteoporosis, cancer, or cardiovascular disease. The
most common side effect of caffeine is increase heart
rate and increased alertness, but caffeine leaves the
system quickly, so it doesn’t cause any long-term
effects.
Methods and Materials
This study was performed in Laguna Beach,
CA. The study measured memory in hooded rats
(n=10) before and after the consumption of caffeine.
The rats were tested in a water maze in which the rats
are taught where a platform is underwater and how to
swim there. The rats learn where the platform is after
swimming around the outside of the tub only to find no
escape. They then swim around other parts of the tub
until they find the platform. After finding the platform
on their own, the rats will swim there immediately after
being put in the water. They are able to find the
platform because they are able to triangulate while on
the platform so that they can find the platform based on
the surrounding environment. The rats were tested in
three trials before being given any caffeine. The rats
were then fasted for 24 hours and fed a 1 mL of a
teaspoon of caffeine. The caffeine came from a Rock
Star energy drink and was soaked into a Ritz cracker.
The rats were tested at the same time everyday to make
sure that time and alertness did not affect the outcome.
The data were then analyzed using a Microsoft Excel
run unpaired t-test.
Results
The average times for the control and
experimental groups showed no statistical difference.
The average time for the rats to find the platform in the
water maze for all three trials for the non-caffeinated
group was 18.65 seconds. For the caffeinated group
the average time was 18.04 seconds (Fig. 1). The data
was run through an unpaired t-test which showed
5
Saddleback Journal of Biology
Spring 2005
p=0.86 which showed that no statistical difference
between the two groups can be established.
Rat Memory with and Without Caffein
60
50
40
No Caffeine
Caffeine
30
20
10
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
0
1
2
3
4
5
6
7
8
9
10
Rat and Trial Numbe
Figure 1. Results of memory Tests.
Discussion
The results of this experiment showed that
there was no difference between the two groups. The
very slight difference could be due to the increase in
metabolism, which can lead to an increase in activity.
Many other tests have shown similar results, in which
there was no change in memory, but reaction time
shortened when they were given caffeine.
Kelemen and Creeley (2001) did an
experiment in which they studied sustained attention
and free recall after caffeine consumption. They tested
142 people who were either given caffeine or a
placebo, and they found that caffeine did not affect the
accuracy of memory but did slightly improve free
recall. However, no substantial impact of caffeine on
memory was found.
A similar experiment performed by Smith et
al. (1999) studied the effects of caffeine on memory,
attention, mood, and cardiovascular systems. They
compared the effects of eating breakfast and the effects
of a caffeinated vs. a decaffeinated coffee on the above
factors. They found that caffeine had no effect on
memory or mood initially, but did help to reduce levels
of fatigue and improved encoding of new information.
They also found that caffeine increased blood pressure
and pulse.
Walach et al. (2002) tested the effects of
caffeine of physiological systems such as heart rate and
blood pressure. The subjects were also tested using a
cognitive test, but the results did not show any change
in any of the tests done. Finally, Oei and Hartley
(2005) tested the effects of caffeine on attention and
memory. Participants were given either a placebo or a
caffeine beverage and were then given a memoryscanning test. They found that there was no difference
between to two groups for memory, but they did find
that the reaction time for participants who had been
given caffeine was faster. They also found that
caffeine did not have any effect on free recall tests. All
of the above experiments support the results that
caffeine does not affect memory but does affect
reaction time and metabolic systems.
There are many ways this experiment could be
improved. Factors that could have influenced the
results are the amount of caffeine given and the sample
size. The rats may not have received enough caffeine
for their weight. One way this could be remedied by
giving a known amount of caffeine for a given weight.
The type of cracker used, or the specific caffeine drink
used may also have affected the amount of caffeine
given. The other factor that could have influenced the
results is sample size. While ten s a typical sample
size, a larger sample size might have shown more
diversity within their results.
Overall, the results of this experiment
supported the results of other experiments. The small
differences in results could be due to a number of
factors including caffeine intake, sample size, and
differences in physiology.
Literature Cited
Hockenbury; Hockenbury.
1998.
Discovering
Psychology. New York: Worth Publishers.
Kelemen, WL, Creeley, CE. 2001. Caffeine (4mg/kg)
influences sustained attention and delayed free recall
but not memory predictions. Hum Psychopharmacol.
Jun; 16(4):309-319.
Oei, A, Hartley, LR. 2005. The effects of caffeine and
expectancy on attention and memory.
Hum
Psychopharmacol. Mar 1;20(3):193-202.
Smith, AP, clark R, Gallagher J. 1999. Breakfast
cereal and caffeinated coffee: effects on working
memory, attention, mood, and cardiovascular function.
Physiol Behav. Aug 1; 67(1): 9-17.
Walach, H, Schmidt, S, Dirhold, T, Nosch, S. 2002.
The effects of a caffeine placebo and suggestion on
blood pressure, heart rate, well-being and cognitive
performance. Int J Psychophysiol. Mar: 43(3): 24760.
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Saddleback Journal of Biology
Spring 2005
Species Competition in Dana Point Tide Pools
Andrea Acosta & Jordana Longoria
Department of Biological Science
Saddleback College
Mission Viejo, CA 92692
In order to observe possible effects of biological and physical factors on the
horizontal and vertical distribution of rocky shore organism’s two different species were
sampled at the Dana Point tide pools. Ten 100m horizontal transect lines and ten 5m
vertical transect lines were taken on April 1, 2005 from noon until 4:00pm at a tide of 0.1
ft. The species observed were the Owl limpet (Lottia gigantean) and Volcano keyhole
limpet (Fissurella volcano). These species were chosen because of the known availability of
these species on the Southern California coast line, also these species are not fast moving
animals that may skew the accuracy of the data. By observing these two species the
possible effects of environmental factors, physical and disturbance was observed. Small
tides are susceptible to extreme heating, salinity, and lower oxygen levels which therefore
house species with a high stress level. In the rocky shore tide pool community zone-nation
and adaptations were qualified. Within the total area measured the horizontal and vertical
distribution density, frequency and possibility of association amongst the two species were
also analyzed to help determine what specie was more stress adaptive to this particular tide
pool community. Our hypothesis is that the Volcano keyhole limpet will have a high stress
tolerance and be found in small and large tide pools horizontally and vertically on the
shore line.
Introduction
California boast over 1,100 miles of coastline
and within it, some of the world’s richest tidal life.
The cold, nutrient rich water and rocky shoreline
California possesses provide the perfect habitat for
hundreds of species (Moring, 2001). The areas where
the sea meets the land is called the intertidal zone, the
intertidal is an ecologically unique environment.
Species that live in the intertidal must have the ability
to withstand dryness, waves, storms, wind and rain.
The ability to withstand weather conditions and
interaction with each other such as eating, being eaten,
competing for space and reproduction, will determine
who dominates within the rocky intertidal areas. The
species that live in tide pools that are exposed longer
during tide changes are subject to more specific
conditions: for example; high salinity, low oxygen
concentrations, and high temperatures (Szabo, 2002).
Any biological and (or) physical effects on the tide
pool environment will enhance adaptations. In general
a species that has a lowered population density
enhances its resource heterogeneity and the strength of
growth between variables over time; autocorrelation,
can occur without invoking size dependant advantages
(Hofmann, 1999). Understanding variability in a
species performance, independent of size, is
fundamental to evolutionary theory. Variation in
species performance can affect the dynamics of
populations and how they exhibit characteristics in a
model (Pfister and Peacor, 2003).
Limpets are classified within the phylum
Mollusca, class Gastropoda. Different species inhibit
the tide shores differently; some are located on the
sides of the rock while others are directly on top of the
rocks. Different characteristics demonstrated by varied
limpet species allow one limpet species to be more
advantageous over another (Dudgeon et al., 1999).
The Volcano keyhole limpet has a central opening on
top of its shell which allows it to breathe as the water
currents pass through it. This allows for respiration
and retention of sea water inside their shell body. The
Owl limpet has its shell that is sealed tightly helping it
to adapt to high temperatures, this characteristic allows
it not to dry out. The competition in the tide pools deal
with the amount of resources that are available and the
space which the individual limpets have.
Materials and Methods
Two different species of limpets, the Owl
limpet (Lottia gigantean) and the Volcano keyhole
limpet (Fissurella volcano) were studied. Each
individual established a 100 meter horizontal transect
7
Saddleback Journal of Biology
Spring 2005
line, which started from the north end of the coastline
to the south end. Every ten meters of each of the
horizontal transect lines was sampled. It was laid out
on the water’s edge directly at the lowest tidal
zone; the tidal zone was 0.1 ft. At every 10 meter mark
along the tape, the individual recorded the number of
each limpet species. This was done by having a 0.5 m2
quadrant centered on each 10 meter mark, and counting
the total number of Volcano keyhole limpets and Owl
limpets inside the quadrant. For the ten vertical
transect lines at every 0.5m the species were counted
for a total of ten quadrants for each line. For the
horizontal and vertical transect lines the Cole’s
coefficient was calculated by the following formula;
Ca= ad-bc / (a+b) (b+d) (Teh and Huntley, 2004). The
Ca is the possibility that in one 10 quadrant transect the
particular Volcano keyhole limpet is associated with
the Owl limpet. The variable “a” represents the
number in which both species are present. The
variable “b” represents the number that the Volcano
keyhole limpet is only present; “c” is the number that
the Owl limpet is only present and “d” is the number of
times that neither species is present. If Ca is greater
than zero, there is an indication of an association
between the two species. If Ca is less than zero, there is
an indication of avoidance. A value of zero for Ca
indicated independence between the two species.
Results
A total of 100 quadrants were calculated. Ten
quadrants every 10 meters for 100 meters horizontally.
Also, ten quadrants were calculated every 0.5 meters
vertically. Association was calculated by the Cole’s
Coefficient. The result for the vertical transect line
indicated that there was avoidance and association on
the northern side of the coastline most likely due to the
rigid landscape. As the landscape gradually leveled
out, the Owl limpet and Volcanic keyhole displayed an
association. With each transect line after the seventh
line became independent and (or) association between
the two species of limpets was revealed (Figure 1).
The result for the horizontal transect line indicated that
in the area closest to the water the limpets associated
with each other. Once the limpets were in the splash
zone where they were exposed to wave, wind, and sun
the data indicates that there was an oscillation of
association and avoidance between the two limpets.
After the seventh quadrant going horizontally the
limpets worked coherently due to the calmer conditions
(Figure 2). The density for the Volcano keyhole limpet
was 0.386 species/m2. For the Owl limpet the density
was 0.110 species/m2. The frequency for the Volcano
keyhole was 0.58 species/total quadrant.
The
frequency for the Owl limpet was 0.34 species/total
quadrant. The value for density and frequency support
our hypothesis that the Volcano keyhole limpet proves
to have a high stress tolerant capability. Within the
Dana Point Harbor intertidal rocky coast not only did
we observe patterns of association using the Cole’s
Coefficient but the density and frequency of the
Volcano limpet are greater than the density and
frequency of the Owl Limpet.
1.
5
1
Cole's Coefficient
Vertical transect
line
(Ca)
0.
5
0
1
2
3
4
5
6
7
8
0.5
9
1
0
Transect
Number
Figure 1. Display of the ten vertical transect lines with
the Cole’s Coefficient value.
0.
4
0.
3
0.
2
Cole's Coefficient
(CA)
0.
1
Horizontal
Transect
Lin
0
e
1
2
3
4
0.1
5
6
7
8
9
1
0
0.2
0.3
Transect
Number
Figure 2. Display of the ten horizontal transect lines
with the Cole’s Coefficient value.
Discussion
General adaptations can be categorized into
three main groups: adaptations for tides, waves, and
salinity. First, adaptations for tides are important
because a species is exposed to air when the tide is out
and must not dry out. So certain species have a
protective body structure like a shell such as the two
mollusks sampled. Species will also gather in masses
to reduce the body surface area exposed to the air. The
Volcano keyhole limpet was notably observed in
clusters. More specifically an adaptation a limpet will
have is to fit into a small depression; they ground
8
Saddleback Journal of Biology
Spring 2005
themselves into a rock (Francis L., 2004). This
particular adaptation was clearly observed in the Owl
limpet. The next group of adaptation is for waves.
Waves are dangerous for species because a species can
be slammed against adjacent rocks or carried outside of
the intertidal area. Two major adaptations for wave
protection are: species will fasten themselves securely
to rocks and hide by crawling underneath or between
rocks. Both limpets displayed the first adaptation for
wave protection because they were all firmly fastened
to rocks. The Volcano keyhole limpet displayed the
second adaptation because they were clustered on the
sides of the rocks. Lastly, species need adaptations for
salinity in order to survive in an intertidal rocky
habitat. Most species retain sea water inside their
shells or quickly adjust their internal salt balance such
as pool fishes. However, an adaptation for salinity was
not observed with the Volcano limpet or the Owl
limpet. The pattern seen in the vertical transect line of
distribution seen in the results is explained by the
increasing mean standard length with decreasing
distance above mean tidal height. This is because this
distribution is related to intercohort competition for
food and (or) cover (Bustamantle et al., 1995). It is
important to understand species competition because it
will determine what species will dominate over the
other and against the elements such as weather and
human interference. Tides are important because it can
be an indicator of coastal health and the species within
the tides are indicators of that particular geographical
area.
Two Species of Sculpins in Maine Tidepools.
Northeastern Naturalist Steuben. Maine. 8: 207
Pfister C and S. Peacor ( 2003). Variable Performance
of Individuals: the Role of Population Density and
Endogenously Formed Landscape Heterogeneity.
Journal of Animal Ecology. Chicago, IL. 72: 725-735
Szabo A. (2002). Experimental tests of intercohort
competition for food and cover in the tidepool sculpin
(Oligocottus maculosus Girard). Canadian Journal
Zoology. Canada. 80: 137-144
Literature Cited
Bustamantle, R.H., G.M. Branch, S. Eekhout. (1995).
Maintenance of an Exceptional Intertidal Grazer
Biomass in South Africa: Subsidy by Sutidal Kelp.
Ecology. 76: 2314
Dudgeon, Stevenson R., Robert S. Steneck, Ian R.
Davinson, Robert Vadas. (1999). Coexistence of
Similar Species in A Space- limited Intertidal Zone.
Ecological Monographs. 69: 331
Francis L.( 2004).
Microscaling: Why Larger
Anemones Have Longer Cnidae. The Biological
Bulletin Woods Hole. Maine 207: 116-129 (14 pp.)
Hofmann, Gretchen E. (1999). Ecologically Releant
Variation in Induction and Function of Heart Shock
Proteins in Marine Organisms. American Zoologist. 39:
889
Huntley, Tony. (2004) Biology 3B Laboratory: The
Rocky Intertidal Ecosystem. Saddleback College. 1-9,
available at:
http://www.saddleback.edu/faculty/thuntley/bio3b/labs/
Moring J.(2001). Appearance and Possible Homing of
9
Saddleback Journal of Biology
Spring 2005
The Effects of pH on the Germination and Growth of Sweet Corn,
Zea saccharata
Mike Beck & Dmitriy Pastaranak
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Zea saccharata is one of the world’s staple crops, and in recent times has been the
subject of genetic engineering. Many plants grow better under specific conditions, and
sweet corn is no different. In this experiment, the effects of pH were tested on the overall
length of growth of germinating Zea saccharata seeds. One hundred and eighty seeds were
assigned to three sets of moist paper towels, sixty in a pH of six, sixty in a pH of seven, and
sixty in a pH of eight. The number of seeds that germinated was counted over the next
seven days, and on the seventh day growth of the roots and stems was measured. The
results showed growth at pH of eight was significantly better than growth at a pH of six.
One reason for this may be that the internal workings of the seeds of Zea saccharata work
better at a higher pH. More probable, is the presence of mold and fungi that took over the
plants slowly from day one.
Introduction
Zea saccharata is another name for one of
America’s favorite foods, sweet corn. It is a staple
crop seen at every supermarket and food stand across
the nation. A recent variety called BT-11 is one of
recent debate because it is currently genetically
engineered.
Plants often need very specific conditions to
grow maximally. Many factors go into whether a plant
will grow including amount of water, sunlight, food,
temperature, and pH. Neighboring plants can also
influence growth by restricting room for expansion of
roots.
These different conditions also apply to
germination, and account for why different seeds need
to be germinated in different ways (Bugbee and Frantz
2002). Corn seeds are proven to have a high tolerance
to many environmental conditions. The large size of
corn seeds makes it easy to monitor the progress of
germination. The condition of pH is known to function
a variety of plant processes including ATP Synthesis,
the opening of guard cells, and water absorption (Fox
2003). In many books regarding plant growth one will
find recommendations about optimal pH’s (Pleasant
1994). The purpose of the present experiment was to
determine the effects of different pH’s on the overall
growth of germinating Zea saccharata over a time
period of one week.
Materials and Methods
One-hundred eighty seeds of Zea saccharata
were germinated over a period of one week on soaked
paper towels of three different pH solutions. The three
different solutions were at a pH of six, seven, and
eight. The pH’s were determined with pH tester strips,
and the pH was altered with three molar sodium
hydroxide and three molar hydrochloric acid. The
seeds were watered a minimum of three times a day, to
insure moistness. The seeds were placed in an
environment that went through a normal days
temperature changes in San Clemente, California.
After the week of germination, the number of
germinated seeds was counted, and lengths of both
their roots and sprouts were measured. The lengths of
the roots and sprouts of each seed were added together
to give a total growth length. An ANOVA was run on
the total lengths for the seeds to determine if there was
any statistical difference between the growths at the
different pH’s.
Results
The number of germinations occurring
throughout each day stayed consistent with one
another. The seeds in the pH six solution germinated
the most at the beginning, but by the end of the
experiment the other seeds had caught up and
surpassed them (Figure 1). At the end of the
experiment very similar amounts of all pH seeds had
germinated, and had followed a similar progression.
Twenty-eight seeds germinated from the pH of six,
thirty-one germinated from the pH of seven, and thirty
germinated from the pH of eight. Forty-seven percent
germinated from pH of six, fifty-two percent from pH
10
Saddleback Journal of Biology
Spring 2005
of seven, and fifty percent from pH of eight. There
was no statistical difference in the number of
germinations at the different pH’s. Most of the seeds
that germinated grew both roots and sprouts over the
time period, and the lengths were added together to
give an overall growth length. The average growth for
a seed that germinated in the pH six solution was
23.5mm, 44.6 mm in the pH seven solution, and
39.7mm in the pH eight solution. The longest growth
for a single pH six seed is 54 mm where the longest for
pH seven is 141mm and the longest for pH eight is 161
mm (Figure 2). Analysis was done on the total growth
for the seeds and statistical difference was found in the
growths at different pH solutions. Further analysis was
done to prove which ones were statistically different.
It was proven that the growth of pH seven and pH eight
were not statistically different from one another, pH six
and pH eight were not statistically different from one
another, but pH six and pH seven were statistically
different from one another.
Number of Seeds Germinated Over a One Week Span for Zea saccharata
35
30
N u m b e r o f G e rm in ate d S e ed s
25
20
pH 6
pH 7
pH 8
15
10
5
0
1
2
3
4
5
6
7
Day
Figure 1. Number of seeds germinated in one week
Average Growth and Longest Growth at Three Different pH's for Zea saccharata
180
160
Discussion
The data for root growth and stem growth
over a period of one week’s time for the germination
shows that Zea saccharata grows better in a pH of
eight than in a pH of six. There might be several
reasons the seeds at pH six did not grow as well as the
seeds at pH eight. The definition of pH is the
concentration of hydrogen cations and anions (Norden
2003). PH is a key factor in getting biological
processes to work optimally. The right hydrogen ion
concentration is required for many enzymes in cell
membranes to function (Campell and Reese 2002).
Another reason that certain enzymes work better in
different pH’s may be due to the fact that pH is also a
key factor in expressing specific genes in some plants
(Mendrinos et al 2002). A more likely explanation for
the outcome would be the effect of pH on the growth of
mold on the seeds. Since day two, some seeds had
exhibited a pink, fuzzy mold which was later identified
as snow mold (Casler et al 2005). Snow mold is
known for infecting plants and depleting their
carbohydrate storage (Mohammad et al 1997). This
mold infected the seeds at all pH’s at various times
along the way. At the end of the experiment all seeds
that had not germinated had exhibited signs of mold
growth. It is possible that snow molds work best at a
slightly acidic pH, and were thus able to hinder the pH
six seeds more. Various molds have been reported to
work optimally at acidic pH’s (Marquina and Santos
2004), so it seems fitting that the mold was what
caused the growth hindrance. The acidic pH did not
hinder the beginning germination, as there was more
germination at the pH of six than any other through
most of the experiment. Further research is required to
see if this is truly the outcome of germination growth at
different pH’s. The inadvertent addition of a second
variable, the mold, creates many doubts. It should be
determined if the specific snow mold found on the
seeds is most active at acidic pH’s, and a much larger
sample of seeds should be used so the numbers are
more statistically stable. Preventative action against
mold should be taken, if it is at all possible, to insure
pH is the only variable in the future.
140
Length (mm)
120
100
pH 6
pH 7
pH 8
80
60
40
Literature Cited
Bugbee, Bruce and Frantz, Jonathan.
(2002)
Anaerobic conditions improve germination of a
gibberellic acid deficient rice. Crop Science, v42 i2
p651(4)
Campbell, Neil and Reece, Jane. (2002) Biology 6th
Edition. Pearson Education Inc. San Francisco p
20
0
Average Growth
Longest Growth
Figure 2. Average growth and longest growth
Casler, M.D; Gregos, J.S; Millett, S.M; Stier, J.C and
Wang, Z. (2005) Genotypic variation for snow mold
11
Saddleback Journal of Biology
Spring 2005
reaction among creeping bent grass clones. Crop
Science, v45 i1 p399(8)
Pseudomonas syringae pv. Phaseolicola. Journal of
Bacteriology, V174 n11-12 p3499(9)
Fox, Theodore. (2003) Handbook of Plant Growth: pH
as the Master Variable. Crop Science, v43 i4 p1575(2)
Mohammad, F; Souza, E and Windes, J.M. (1997)
Total non-structural carbohydrates in winter wheat
populations segregating for snow mold tolerance. Crop
Science, v37 n1 p108(5)
Marquina, D and Santos, A. (2004) Killer toxin of
Pichia membranifaciens and its possible use as a
biocontrol agent against grey mould disease of
grapevine. Microbiology, v150 i8 p2527(8).
Mindrinos, Michael; Panopoulos, Nickolas and Rahme,
Laurence. (2002) Plant and environmental sensory
signals control the expression of hrp genes in
Norden, Randy. (2003) Do you really know what pH
is? Journal of Environmental Health, v65 i6 p48
Pleasant, Barbara. (1994) Have a super sweet corn
summer! Organic Gardening, v41 n5 p50(6)
Response of the Anterior and Posterior Osmoregulatory Mechanisms of the
Goldfish, Carassius auratus, at Three Temperatures
Deborah Contreras, Wyatt Sum and Gabriel Tran
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Osmoregulatory homeostasis is critical to the survival of freshwater fishes. In the
goldfish, the gills are the primary anterior osmoregulatory organ while the kidney provides
osmoregulatory function in the posterior. We designed a Plexiglas™ chamber divided by a
thick latex sheet into anterior and posterior sections. Goldfish were placed halfway
through the latex material, so that the head (gills) region was isolated from the tail region.
With the fish in place, 450ml of 0.1% salt solution and 450ml of deionized water filled
anterior and posterior chambers respectively. Osmolality of the anterior and posterior
water was then measured at ten minute intervals for 60 minute trial (n=10) at three
temperatures 23°C, 33°, and 13°C. ANOVA and post-hoc testing showed no significant
differences in osmolalities of the two chambers at the different temperatures.
Introduction
Osmoregulation is the control of the body’s
water and solute concentration to surrounding
environment to maintain equilibrium. Osmosis is the
process of solvent passed from a dilute side of a
semipermeable membrane to a more concentrated side,
thus affecting both water and salt excretion and uptake
within an organism. Hydromineral balance of fluids
deals with the relation between internal and external
fluids. Dialysis differs from osmosis through diffusion
of solutes across a semipermeable membrane. In
optimal osmotic conditions, fluid uptake both orally
and through gill site enters the fish and is secondarily
(renal activity is secondary to gill function) removed.
Further characterized by direct elimination of ammonia
to conserve metabolic energy, these species are
commonly referred to as ammonotelic. In kingdom
Animalia, family Cyprinidae, FW (freshwater)
Goldfish (Carassius auratus), like many other
vertebrates have a closed circulatory system consisting
of cells bathed in interstitial fluid, indirectly controlled
by the branchial epithelium. In fish, this transport
epithelium is located at the gills and in the kidneys.
The branchial epithelium is the main transport site that
balances the effects of osmotic and ion gradients
(Evans, 2003). Ions move across the epithelium
actively via ATPases and passively via ion transporters
(Kultz, 2001). Gill structure functions in homeostasis
by passage of sodium (Na+), chloride (Cl-), water
(H2O), and carbon dioxide (CO2) (Evans, 2002).
Varying extremes of temperature and ion
concentrations (more specifically salinity) in different
12
Saddleback Journal of Biology
Spring 2005
Materials & Methods
Thirty goldfish (Carassius auratus) were
purchased at PETCO in Mission Viejo, California.
Goldfish placed in a 10 gallon aquarium and fasted for
2 days prior to data collection. The goldfish were
placed in a bisected holding tank constructed out of
0.64cm thick Plexiglas. For each chamber two
cylindrical pieces of Plexiglas were cut with a height of
7.62cm and a radius of 5.08cm. A hole with radius of
2.54cm was cut out from each cylinder to allow for
pouring of 450ml of 0.1% salt solution and 450ml of
pure deionized water to fill anterior and posterior
chambers respectively.
Both pieces were glued
independently to a separate Plexiglas base using acrylic
cement. Four holes drilled in each corner of each base
allowed for attachment of four 0.64cm all-thread
support rods. Heavy duty 12.7cm x 12.7cm cut sheet
of thick gauge latex with a centered 10cm cut slit was
stretched across one of the Plexiglas cylinder openings
and secured by a thick rubber band bisected the two
chambers and allowed for the goldfish anterior
region(including gills) and posterior region (from
pectoral fins to tail) to be suspended between two
chambers. Open ends of the cylinders were tightly
secured together by washers and nuts on the all-thread
rods (Figure 1).
A control group of ten goldfish were each
placed into the chamber; 450ml of deionized water was
poured into the posterior end while 450ml of a 0.1%
salt solution was poured into the anterior end. Data
collection was carried out at a temperature of 23°C in
both the anterior and posterior portion of the chamber
for a total of 60 minutes. A micropipette was used to
collect a sample of 10µl from both the posterior and
anterior chambers every 10 minutes. Samples were
then placed in the Wescor Vapor Pressure Osmometer
and readings were recorded in units of mmol/kg. The
same process was carried out at higher temperatures
where the chamber was submerged into a 33°C water
bath. Another experimental group was carried out, this
time submerged in a 13°C water bath. Statistical
significance was determined between the four groups
by using an ANOVA and Post-hoc test.
Figure 1. Holding Chamber.
Results
Both control group at 23ºC and experimental
cold group at 13ºC had very similar results with mean
posterior osmolality readings of 19.2 mmol/kg and 18.9
mmol/kg. Experimental group at 33ºC had a slightly
higher mean posterior osmolality reading at 24.5
mmol/kg. Completion of an ANOVA and post-hoc test
concluded that the three values were not statistically
different (Figure 2).
Average Posterior Osmolality
Osmolality (mmol/kg)
aquatic environments directly influence sensitive
physiological responses of aquatic ectotherms (Haney,
2003). Despite eurythermal mechanisms of goldfish,
extreme changes in physiological processes tend to
disrupt internal homeostasis thus increasing
vulnerability to mortality (Metz, 2003).
Fluctuations in ambient temperature trigger
adaptations to maintain homeostasis and may strongly
influence osmoregulation mechanisms, including salt
excretion and osmolality values. Stressors increase
antagonistic responses including permeability of the
fish’s integument to ions and water thus disturbing
equilibrium (Wendelaar Bonga, 1997). If stenohaline
animals are oppressed by substantial levels of either
two factors outside of their livable parameters the
consequences could potentially be fatal (Campbell and
Reece, 2002). The purpose of the present study was to
observe the transport of salinity in varying ambient
aquatic temperatures as osmoregulatory function in
FW. It is hypothesized that at higher ambient
temperature, C. auratus would increase salinity
excretion and osmolality values, and decrease at lower
ambient temperature to maintain homeostasis in an FW
aquatic environment.
30
25
20
15
10
5
0
13ºC
23ºC
33ºC
Temperature in Degrees Celsius
Figure 2. Average Posterior Osmolality: Osmolality
readings for the three temperatures were shown not to
be statistically different.
13
Saddleback Journal of Biology
Spring 2005
Change in Anterior Osmolality Over Time
Osmolality (mmol/kg)
Osmolality (mmol/kg)
Average Anterior Osmolality
47
46
45
44
43
42
41
40
13ºC
23ºC
55
50
40
35
33ºC
Figure 3. Average Anterior Osmolality: Osmolality
readings for the three temperatures were shown not to
be statistically different.
Anterior osmolality for both the control
(23ºC) and the experimental cold group (13ºC)
exemplified similar results with both the osmolalities
of 42.4 mmol/kg. Experimental warm group (33ºC)
showed a slightly higher average anterior osmolality
reading of 45.9 mmol/kg. The ANOVA and Post-hoc
test for the three anterior groups showed that there was
no statistical difference between the three means
(Figure 3). Both control group (23ºC) and experimental
cold group (13ºC) showed a slow and gradual increase
in both the average anterior and posterior osmolality
readings. The experimental warm group (33ºC) showed
an increase in the osmolality that was much higher than
that of both the control group (23ºC) and the
experimental cold group (13ºC) for both the anterior
and posterior osmolality readings (Figures 4 and 5).
Osmolality (mmol/kg)
Change in Posterior of Osmolality Over Time
30
25
Control
Warm
Cold
15
10
5
0
10
20
30
40
50
10
20
30
40
50
60
Time (min)
Temperature in Degrees Celsius
20
Control
Warm
Cold
45
60
Time (min)
Figure 4. Change in Posterior of Osmolality Over
Time: gradual and steady increase of osmolality over
the course of one hour.
Figure 5. Change in Anterior Osmolality Over Time:
gradual and steady increase of osmolality over the
course of one hour.
Discussion
This osmoregulation study exemplifies
[importance of consideration] the effects of handling
stress on physiological functions and reactions. The
data that was obtained from the experiment suggested
that when the goldfish were submerged into a salt
solution with variant temperatures, there was no
increase or decrease in their osmoregulatory output.
When comparing the data that was collected it was
found that the average osmolality of both the anterior
and posterior samples were not found to be statistically
different. However, although not statistically
significant, it should be noted that posterior osmolality
readings of the experimental warm group (33ºC) had an
overall higher value than that of both the control (23ºC)
and the experimental cold (13ºC) groups. It should also
be noted that the overall osmolality of both the anterior
and posterior continued to increase as time progressed
(Figures 4 and 5).
The slow and gradually increasing trend of
osmolality values over time could be due to the very
dilute urine (ammonia) that is constantly being
excreted by the fish as it takes up ions over the gill
epithelium and kidneys to maintain high ion levels in
the blood (Marshall, 2002). This also may explain why
the results suggest there was no statistical significance
between data sets. Over the allotted time, the ammonia
solution that was being released by the fish may not
have been concentrated enough to produce increasing
osmolality readings. A longer test period may result in
statistically significant osmolality values.
Slight increase in posterior osmolality values
of the experimental warm group (33ºC) potentially
could have occurred due to the change in the
homeostasis of the fish. The temperature can have an
impact on the physiological processes and ultimately
disrupted homeostasis (Metz, 2003). This change in
homeostasis can increase osmoregulatory organ
14
Saddleback Journal of Biology
Spring 2005
activity located in the gills, intestines, and kidneys
(Metz, 2003). This may explain why the data set
suggested higher osmolality values for both the anterior
and posterior experimental warm groups. The slight
increase in anterior osmolality values might be due to
the idea that ion exhange is mostly completed at the
branchial epithelium rather then the kidney (Claiborne,
2002).
Abrupt introduction and brief acclimation
period to ambient temperature(s) and other
environmental factors induce shock on freshwater
fishes (Van den Burg, 2004).
Stressors disrupt
common ectothermic responses triggering an increase
in hormonal activity and mortality rate (Wendelaar,
1997). Advantages of and extended acclimation period
in normal environmental conditions allows for better
adaptation thus reducing stress factors in ectothermic
fishes.
Further research is required to determine if the
temperature has an influence on the osmoregulation of
Carassius auratus. Haney and Walsh (2003)
constructed a similar experiment except with the use
Limia melanonotata. They were studied over a period
of weeks, which is a method that should be
implemented in further exploration of osmoregulation.
Kultz Dietmar, Chakravarty Devulapalli, Adilakshmi.
2001. A novel 14-3-3 gene is osmoregulated in gill
epithelium of the euryhaline teleost Fundulus
heteroclitus. The Journal of Experimental Biology,
204; 3975-2985.
Metz R. Juriaan, Van den Burg H. Erwin, Wendelaar
Bonga E. Sjoerd, Flik Gert. 2003. Regulation of
branchial NA+/K+-ATPase in common carp Cyprinus
carpio acclimated to different temperatures. The
Journal of Experimental Biology, 206: 2273-2280.
Marshall W.S. 2002. Na+, Cl-, Ca2+, and Zn2+ Transport
by Fish Gills: Retrospective Review and Prospective
Synthesis. Journal of Experimental Zoology, 293: 264283.
Wendelaar Bonga E. S.1997. The Stress Response in
Fish. Physiological Reviews, 77:591-625.
Van Den Burg H. Erwin, Peeters R. Ronald, Verhoye
Marleen, Meek Johannes, Flik Gert, Van Der Linden
Annemie. 2004. Brain Responses to Ambient
Temperature Fluctuations in Fish: Reduction of Blood
Volume and Initiation of a Whole-Body Stress
Response. Journal of Neurophysiology, 93: 2849-2855.
Acknowledgements
The authors would like to thank Mr. S. Teh, Dr. T
Huntley and Mr. T. Burrows foe their skilled technical
support, osmometer and laboratory use.
Literature Cited
Campbell, Neil. and Reece James. 2002. Biology.
Benjamin and Cummings, California. 936-942
Claiborne B. James, Edwards L. Susan, MorrisonSherlar I. Allison. 2002. Acid-Base Regulation in
Fishes: Cellular and Molecular Mechanisms. Journal of
Experimental Zoology, 293:302-319
Evans H. David. 2002. Cell Signaling and Ion
Transport Across the Fish Gil Epithelium. Journal of
Experimental Zoology, 293:336-347.
Evans H. David, Piermarini M. Peter, Choe P. Keith.
2005. The Multifunctional Fish Gill: Dominant Site of
Gas Exchange, Osmoregulation, Acid-Base Regulation,
and Excretion of Nitrogenous Waste. Physiological
Reviews, 85: 97-177.
Haney C. Dennis, Walsh J. Stephen. 2003. Influence of
Salinity and Temperature on the Physiology of Limia
melanonotata (Cyprinodontiformes: Poeciliidae): A
Search for Abiotic Factors Limiting Insular
Distribution in Hispaniola. Caribbean Journal of
Science, 39, 3: 327-337.
15
Saddleback Journal of Biology
Spring 2005
Do Caterpillars Think? A Study of the Learning Ability of
Cabbage Loopers and the Effect of Pesticides
Adrena Gharibjanians & Melinda Tanabe
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Larval forms of some insects damage plants by eating through and creating holes in
any vegetables they come across, destroying an entire crop. Therefore different pesticides
have been created to attract caterpillars in different ways. An experiment was performed
to study the learning capability of Trichoplusia ni in order to introduce a different
approach for pesticide production. Our hypothesis was that caterpillars can learn,
therefore they are capable of avoiding any kind of obstacle present in the soil, making
traditional pesticides inefficient. One-hundred cabbage loopers were fasted for a period of
24 hours and were individually placed in the center of a maze. Roots and leaves were
placed in the four exits, while insecticide was placed all around the maze. Each individual
caterpillar was timed (in seconds) from the moment it was placed in the box to the moment
it reached the food. Ten trials were performed with each caterpillar under light and dark
conditions. Vision was tested by using half the sample size of worms and placing them in a
green maze. By using cotton soaked in different solutions of caffeine, vinegar-salt, lime and
garlic, olfactory senses were tested. An ANOVA test for multiple comparisons and a
Bonferroni correction (post-hoc test) were performed. The results showed that there is a
significant difference between the first and last trial of the light the dark environment in
both colored mazes. Through research, it was found that worms’ simple nervous system
can’t perform higher functions like human ones, not enabling them to think. One of the
reasons for the results is the presence of chemo and photoreceptors all along their bodies
and their good sensory structures. No significant difference was found between the
experiments in the white maze the green maze, which means that their sight is
underdeveloped. However, results proved that there was a statistical significance between
the scented cotton, with coffee as the preferred scent. The olfactory sense of the cabbage
looper is the sense they rely on most, preventing them from getting into dangerous
situations, and allowing them to find food. The caffeine found in coffee acts as a
neurotoxin, making caterpillars confused and hyper after ingestion, ultimately leading to
their death. We can conclude that caterpillars do not learn and that caffeine could be a
very effective and efficient organic pesticide, as well as a very good tonic for plants.
Introduction
The larval form of Trichoplusia ni, better
known as cabbage looper or cabbage worm, has a light
green color with light stripes on its body. The size of
the cabbage looper ranges from about 1 to 3.2 cm long.
The middle section of its body does not have
appendages, but cabbage loopers do have three pairs of
slender prolegs (fleshy auxiliary legs), which are
located near the head. They also have three other pairs
of larger prolegs located at the rear end of the body,
which allow them to move in a serpentine type of
motion. This type of motion occurs when the cabbage
looper draws the front of its body forward, straightens
its entire body, and then brings in the back portion of
its body, which ends up creating the characteristic loop
(of serpentine motion) in the middle of the caterpillar.
Over a span of time, the cabbage looper enters into the
adult form and becomes a brown moth with white spots
on each wing.
Before metamorphosis can take place within
the caterpillar, it must undergo an arrested
developmental stage called diapause. During this stage,
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Saddleback Journal of Biology
Spring 2005
the larvae are protected by a previously secreted
cocoon lying on the underside of the leaves. The worm
in the larval stage feeds upon the underside of leaves at
night, creating holes in them, and then depositing into
those holes dark little balls of excrement that may stain
the head leaves. Cabbage loopers can consume a wide
variety of plant leaves such as lettuce, cotton, tobacco,
soybeans, tomatoes, and any other plant in the cabbage
family. In 2002, Ausubel and others studied the
different responses of plants against these worms.
Plants can be tremendously defoliated and there growth
can be stunted, affecting the production of head leaves
or becoming unsuitable for consumption (Berenbaum
et al, 2002).
Due to the large range of plants the cabbage
loopers can eat, and the damage they can cause to
plants, they are recognized worldwide as one of the
most harmful and annoying pests. When they are
segregated into specific groups, they can destroy a
whole crop, causing notable physical and economical
damage. The physical damage can be the loss of
quality or yield of the crop, while the economical
damage can be how far its qualitative or quantitative
value will drop. Therefore, the farming community is
implementing different ways in which to destroy these
caterpillars.
There are many different ways to get rid of
cabbage worms, all of which have various pros and
cons. One way is the use of Carbamate and
organophosphate insecticides. These insecticides work
by inhibiting vital enzymes (Cholinerase) that are
fundamental for the communication through nerve
impulses, which affects the respiration of the worm and
leads to its eventual death. One of the disadvantages of
this method is the fact that other species such as birds
and even humans are affected. The overexposure of
this insecticide in humans can cause paralysis, different
heart diseases, respiratory failure, and other damages.
For example, the residue of a specific insecticide,
Cygon (dimethoate), could potentially infect plants and
fruits, making the food that humans eat toxic.
Another method useful in getting rid of
cabbage worms is using natural enemies that are
predators of caterpillars. The problem which this
method is that an ideal environment, full of natural
resources and protection against diseases and
chemicals, needs to be provided for these natural
enemies (organisms) to be able to reproduce and
function as predators, in order to significantly reduce
the population of cabbage worms. Another issue with
this method is that these natural organisms can also be
susceptible to the pesticides used to control the worms.
An example of this situation would be that if the
Chrysoperla carnea (insect that feeds primarily upon
eggs and small caterpillars) were to attempt to catch a
cabbage looper where there is some pesticide present,
being potentially affected by the poison.
The final method that can be used to control
the cabbage loopers are microbial insecticides, which
are very effective, but whose results do not last very
long. Most of the available commercial products are in
this category (microbial insecticides). This method is
almost flawless, for it is harmless to humans and to
non-target, beneficial insects that would normally be
affected by other insecticides. Since they tend to be
host specific, complementary pesticides may need to be
used along with the insecticide already being used. One
example is Bacillus thuringiensis (BT) which activity
spectrum depends on the strain of the bacterium. These
toxins are produced by the plant through sporulation,
depositing them around the surface of the spores. Once
inside the caterpillars’ gut, these crystals dissolve and
release toxic compounds (Aronson, 1986). This
bacterium does not disperse very well and is easily
broken down by ultraviolet light; therefore continuous
applications are needed for it to have a significant
effect. Microbial insecticides are very expensive,
especially since they must be continually replenished.
All of the methods for elimination of the
cabbage loopers require a control on the population of
the pest throughout the season. Through attempting to
control the population, the money that the farmer
would normally spend on buying more seeds to grow
more crops ends up going towards other things, which
in turn makes the crops sold more expensive for the
consumers. Also, through this process, the different
substrates that could potentially kill the caterpillars are
discovered, as are the most effective pesticides. Today,
most greenhouses use microbial insecticides because
they are practically harmless to humans and very
effective.
The purpose of this experiment is to determine
whether or not cabbage loopers have the ability to think
and learn in order to attempt to discover a new, more
effective type of pesticide. It is important to realize that
the immune system of these caterpillars will not be
taken into consideration since no resistance to the
poison can be developed during the time frame of the
experiment. The hypothesis tested was that cabbage
loopers can learn; therefore they are capable of
avoiding any kind of obstacle present in the soil,
making traditional pesticides inefficient.
Materials and Methods
Over the span of time between March 12th to
20th, 2005, one-hundred specimens of Trichoplusia ni,
better known as cabbage loopers, were obtained from
different individuals at the Farmer’s Market in Irvine,
CA, USA. Once received, the worms were fasted for a
period of 24 hours inside a glass container with air
holes on the lid. Two mazes of the same dimensions
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Saddleback Journal of Biology
Spring 2005
(Figure 1) were constructed using white poster board
for one and green poster board for the other. The inner
box of the maze was a 16cm X 16cm square. The four
areas of poison (on each side of the inner box) were
each 13cm X 3cm. The four exits of the inner box,
leading to the outer box were each 4cm X 4 cm. The
outer box of the maze was a 27cm X 27cm square. The
four areas of poison (on each corner of the outer box)
were each 5cm X 3cm. The four exits of the outer box,
leading to the case of food were each 5cm X 5cm. The
case of food itself was 6cm X 6cm.
Figure 1. The maze (with measurements)
The poison used was “Ant killer granules,”
made by Gardener’s choice. The active ingredient was
permethrin, which is very effective in killing ants,
boxeleder bugs, brown dog ticks, centipedes,
cockroaches, sow bugs, spiders, and other types of
insects. The purpose for which we need this poison will
be fulfilled, for the insecticide that was chosen does not
very effectively kill the cabbage looper, but it
definitely is not good for the worm and will eventually
harm it. One of the reasons this insecticide was chosen
is that the worms needed to continue living even after
contact with the poison.
In order to keep the poison granules in their
respective places, double sided tape was used to ensure
that they would not be moved from their spot on the
poster board. The source of nutrients placed in the food
cases was roots and some leaves. In order to have more
accurate results, all the cases had the same amount of
leaves of approximately the same size.
Once inside the maze, a chronometer was used
to time the cabbage loopers to see how long (in
seconds) it would take them to find and get to the case
of food. Ten trials were performed for each worm in
two different light intensities (day and night). An
ANOVA and Post-hoc test was performed with the
data gathered to see if there was a significant difference
between the first and last trial and fifth and tenth trial
of both light intensities. This data was then used find
whether or not our sample of worms could learn,
telling us whether there was or there wasn’t a statistical
difference between the trials.
By then using the green maze, the cabbage
loopers’ sense of sight was tested. The same onehundred cabbage loopers were again fasted and
individually placed in the center of the maze. The
cabbage looper was timed in seconds to determine its
color preference and visual memory in, again, both the
light and dark. Also, to test the caterpillars’ sense of
smell, the white maze was used. In this case, four
different pieces of cotton (placed into the corners
where the food was previously placed) were drenched
in four different liquids (homemade pesticides). The
four different pesticides consisted of coffee (made by
diluting one teaspoon of coffee grains with 500mL of
water), garlic spray (one teaspoon of chopped garlic
diluted with 500mL of water), lime spray (one
teaspoon of lime juice diluted with 500mL of water),
and vinegar-salt (half of a teaspoon of salt and vinegar
diluted with 500mL of water). These solutions were
chosen because they all have very strong and distinct
odors, and are also, at the same time, tonics and good
soil sterilizers for plants. The caterpillars were
individually timed in seconds, under daylight, to see
which odor they preferred. Three trials were performed
with each caterpillar.
The data gathered from the different tests was
analyzed by performing an ANOVA test for multiple
comparisons and a post-hoc test (Bonferroni
correction), with the p-value for statistical significance
at p ≤ 0.05. The outcome of the experiment determined
the odor preference of cabbage loopers, which when
combined with the outcome of the entire experiment,
determined whether or not a new economical and more
productive approach to the production of pesticides
was possible.
Results
The average time that it took the sample of
one-hundred cabbage loopers in the control group (in
the white maze and under high intensity light) was 71.3
seconds. According to the post-hoc test, there was a
significant difference between the first and final trials
of putting the worms in the maze (t = 2.937), but no
significant difference was found between the first and
fifth trials (t = 1.635) or between the fifth and final
trials (t = 1.303).
The average time that it took the sample of
one-hundred cabbage loopers in the group placed in the
white maze under near complete darkness was 95.6
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Saddleback Journal of Biology
Spring 2005
seconds. As in the group with light, there was a
statistical difference between the first and final trials of
putting the worm in the maze (t = 3.261), but no
significant difference was found between the first and
fifth trials (t = 0.785).
The cabbage loopers placed in the green maze
(under different light intensities) were compared with
those in the control group. When compared with the
green colored maze under intense light, a t – value of
0.554 was found, and when compared under no light, a
t –value of 0.222 was found, showing no significant
difference between the different colored mazes. Figure
two and three show a comparison between the average
times it took the cabbage loopers in the maze between
trials one to ten under different conditions.
Figure 2: average times from Trial 1-10 of white
and green mazes under light
Time ( sec)
100
White maze in light
80
60
Green maze in light
40
Linear (White maze in
light)
20
0
1
2
3
4
5
6
7
8
9 10
Trial #
Figure 2.
Time ( sec)
Figure 3: average times from trial 1-10 of white
and green mazes under darkness
140
120
100
80
60
40
20
0
White maze in dark
Green maze in dark
Linear (White maze in
dark)
1
2
3
4
5
6
7
8
9 10
Trial #
Figure 3.
Different smells played an important role in
this experiment. The time it took for the sample of
hundred cabbage loopers to reach the differently
scented food cases and time it took the sample of onehundred caterpillars to reach the food in the control
group were significantly different (t = 3.101). The
mean time that it took the sample of one-hundred
cabbage loopers in the group with scented cotton
placed in the food cases was 51.7 seconds. Of the onehundred cabbage loopers, an average of forty-four went
directly to the coffee container, seven went to the garlic
container, thirty-seven went to the lime container, and
ten went to the vinegar-salt container. During our
experiment, two worms died between trial one and two.
Discussion
The data for how long the cabbage loopers
took to reach the food containers show that there was a
significant difference between the first and last trial (t =
2.937), but not between any of the other trials. After
much research, it was found that worms do not have
brains, but that they do in fact have a simpler nervous
system than humans do. The significant difference in
our data resulted from the fact that worms have a fairly
rapid response to different stimuli, due to their good
sensory structures and simple nervous system. The
central nervous system of cabbage loopers is very
similar to that of worms. It consists of a cerebral
ganglion (the brain with nerves bundled up together
into knot-like things called ganglia) and a ventral nerve
cord that extends throughout the body by rings formed
from segmental nerves. Their function is primarily to
receive stimuli, such as temperature, light, dryness, and
even food, or different predator’s stimuli. They cannot
do higher-function activities that human brains do, like
thinking and learning.
A significant difference was also found
between the times that it took the cabbage loopers to
get to a container of food exposed to high and low
intensities of sunlight (t = 3.845). This can be
explained by the presence of chemoreceptors and
photoreceptors on the tails of cabbage loopers,
combined with their exceptionally large nerve fibers
(located in the ventral nerve cord), which are
responsible for the quick responses of caterpillars. Of
these fibers, one is located in the anterior segment of
the worm and the other is located in the posterior
segment. When one type of fiber called medium giant
fiber (MGF) is excited by the touch sensory stimuli on
the anterior end, impulses are conducted through the
ventral nerve cord to the segmental motor neurons. The
longitudinal muscle segments are then excited, which
results in a contraction in the anterior end of the worm.
The other two lateral giant fibers (LGF) follow a
similar pathway, but respond on the posterior end of
the worm. The worm reacts not only with sensory
touch stimulation but also with shadow stimulus due to
the presence of photoreceptors on its tail. In our
experiment, when the cabbage loopers received too
much light (as a source of heat), their LGF was excited,
which conducted impulses to the ventral nerve cord,
affecting their motor neurons and longitudinal muscles,
causing rapid movement and shortening of the cabbage
loopers’ posterior segments.
In 2004, Arbucci and others performed an
experiment involving the intake of sour substances in
worms. It was found that the most important
chemosensory organ of the worms is the amphid,
which is in charge of many functions, such as olfaction,
taste, thermosensation, and mechanosensation.
Cabbage loopers communicate by means of chemical
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Saddleback Journal of Biology
Spring 2005
compounds more specifically called pheromone
systems, which is the reason why they need good
chemoreceptors (Grant et al, 1987). Some neurons
were identified that are responsible for the transmitting
of these senses to the ganglia. In our experiment, the
cabbage loopers began avoiding dangerous particles,
such as poison, by smelling them as a response to their
stimuli. This is called a behavioral strategy. When the
cabbage loopers were first introduced to the maze, they
started to investigate the different areas. After a couple
of trials they became used to the smell of the poison
and began avoiding it, by the activity of
chemoreceptors on their body, and looking for a path
that would lead them directly to the food.
The data show that there was a significant
difference in the preference of odors of the worms (t =
3.101). Coffee was the preferred above all others and
the smell of lime was the second most preferred, while
vinegar-salt and garlic were so undesirable that the
worms even began avoiding them. This happens
because of the presence of neurotransmitters that are
receptive to different chemicals, located specially on
the ganglia (Caveney, 2002). In 2003, D.E. Dussourd,
reached the same conclusion using different chemicals,
attributing the effect to the presence of neurological
activity in the worms. Although cabbage loopers
preferred the smell of coffee, once they began digesting
it, they started becoming more and more hyper, to the
point where they became confused and eventually died.
Caffeine acts as a neurotoxin, causing twitching
movements within the worms before they die.
Results from data involving the vision of
cabbage worms show that there was no significant
difference between the two different colored mazes (t =
0.554). But, results were similar when the cabbage
loopers were placed in a lit environment in the white
maze when compared to when they were exposed to
both light and dark environments in the green maze.
There was a significant difference of t = 4.177.
Apparently their sense of sight is not as developed as
the rest of their sensory organs. The fact that cabbage
loopers posses chemoreceptors all along their body
makes way for them to survive without vision.
In conclusion, cabbage loopers posses a
distinct central and peripheral nervous system, similar
to ours, which makes them capable of some type of
memory, but their nervous system is not as complex
and advanced as ours is. Their memory, like in the case
of our maze, consisted of the avoidance of repeated
electrical stimuli. However, with their good sensory
organs, they can smell distinct odors, like pesticides,
for example. Therefore, we can conclude that no
commercial pesticide that come in granules or liquid
insecticide will be effectively eliminate cabbage
loopers. One of the most used and effective pesticide
today is Bacillus thuringiensis, or pheromone traps, but
a lot of control is required in order to affect its
productiveness (R. G. Van Driesche, 1995). The fact
that our data proved that cabbage loopers have a good
response to odors, especially to caffeine, helped
determine that it is possible to create a very efficient,
inexpensive, home-made pesticide by using used,
ground coffee. Previous studies have shown that
caffeine affects the growth and overall development of
plants, acting as a good plant tonic (Devasagayam et al,
2001). This is very convenient because between the
years 1998 and 2000, approximately 1.7 million tons
(29 million bags) of coffee were consumed. And
according to data provided by Food and Agriculture
Organization of the United Nations, the amount of
coffee consumed in 2010 is expected to reach 1.9
million tons (32 million bags). Further research will
have to be done in order to determine the optimal
concentration of caffeine that can act as an organic
pesticide and can be beneficial to plants at the same
time.
Acknowledgements
We would like to thank the people and places
that helped us conduct our experiment. First, we are
grateful to the Armstrong Garden Center in Dana Point,
CA and Home Depot in Ladera Ranch, CA for
providing us with helpful information about worms and
different insecticides used in the experiment. Also, we
would like to thank the specific farmers from the
Farmer’s Market in Irvine, CA for providing us with
the cabbage worms. Finally, we appreciate Steve Teh
and the anonymous peer-reviewers, who contributed
much of their knowledge to the writing of our paper.
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The Effects of Creatine Supplementation on Anolis carolinensis during
Anaerobic Exercise
Jason Gillis and Terha Trost
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Creatine supplementation is commonly used as a performance enhancer for many
sprint-employed sports. Creatine is phosphorylated by creatine kinase to form
phosphocreatine which serves as an energy store during anaerobic respiration. Thus,
creatine supplementation would be expected to increase duration of fast twitch muscular
contraction in anaerobic conditions. An experimental group of Anolis carolinensis
specimens, which rely on anaerobic respiration for mobility, were introduced to creatine
monohydrate through diet. Endurance was represented as the duration of sprinting time of
the Anolis carolinensis on a treadmill. Comparison between average run durations of the
two groups were recorded and found statistically significant with a P = 0.008. The
consumption of creatine supplements did indeed increase endurance of the Anolis
carolinensis during anaerobic respiration. Sprint mobility is ceased when ATP is no longer
available to fuel the reaction pathway of muscle contraction. It is postulated that the
creatine monohydrate supplementation acted as a buffer for depleted ATP by
phosphorylating ADP.
21
Saddleback Journal of Biology
Spring 2005
Methods and Materials
Twelve mature Anolis carolinensis (green
anoles) were separated based on diet into two, six
specimen study groups. The control group was fed two
crickets per a day. The experimental group was fed the
same amount of crickets, but they were also
supplemented with “Great Body” pure creatine
monohydrate. The creatine was introduced to the
anoles via dusting the crickets. Dusting was achieved
by spraying the crickets down with water and then
completely covering them in the creatine powder. The
average amount of creatine adhering to the crickets was
found to be 6.4 milligrams. By virtue of each
experimental anole consuming two crickets daily,
creatine intake was 12.8 milligrams per specimen.
A period of four days was spent feeding the
two groups there respective diets. This loading period
allowed the experimental group to ingest creatine into
muscle cells. On the fifth day of the experiment, the
first of six anaerobic endurance trials began. The
anoles were placed in a plexi-glass container on
wheels. The container was placed on a conveyer belt
which rotated and forced the anoles to run at a rate of
30 cm/sec. The anoles ran at that rate until a qualitative
analysis deemed muscular failure (the point when the
anoles could not keep up with the rate of the belt). The
anoles performance was quantitatively recorded by
measuring the time between the first muscular
contractions of the run and muscular failure. The
anoles were tested at various times between 10am and
5pm.
Results
Overall, the supplemented Anolis carolinensis
and the control Anolis carolinenesis showed similar
endurance patterns while sprinting on the treadmill.
Both groups illustrated an improvement in the ability
to maintain mobility on the treadmill under anaerobic
conditions from trial one to trial six (Figure 1).
Effects of Creatine on Sprint Duration of Anolis Carolinensis
60
50
Mean Sprint Duration (sec)
Introduction
The free energy released by the degradation
of adenosine triphosphate (ATP) to adenosine
diphosphate (ADP) is used as energy currency for
muscle contraction. In anaerobic conditions seen in
strenuous activity of fast twitch muscle fibers, ATP is
in limited supply. Once the store of ATP in a muscle
cell is consumed, the muscle will fail to contract until a
new supply of ATP can be brought back via aerobic
pathways such as oxidative phosphorylation (Branch et
al).
Phosphocreatine is a molecule that resides in
the muscle cell and acts as a buffer for ATP energy
stores. Once ATP is used in a muscle contraction the
resulting ADP can be rephosphorylated by
phosphocreatine. The newly synthesized molecule of
ATP can be used in the next muscle contraction,
therefore prolonging activity without the use of
oxygen. Phosphocreatine is scientifically accepted as a
determining factor in sustaining short term strenuous
muscular activity (Cupp et al). In theory, if the supply
of phosphocreatine in the cell was in increased, the
duration of anaerobic fast twitch muscle contraction
would increase as well.
A product that has become a staple in many
fitness regimens is creatine monohydrate. It is
advertised in the fitness market as a pre-cursor for
ATP. Small amounts of creatine are introduced to the
body via production in the liver, or consumption of
meat and fish products (Bemben). This amount can be
vastly multiplied with creatine monohydrate
supplementation products that are manufactured in pill
and crystalline powder form.
Orally ingested creatine monohydrate crystals
disassociate into creatine molecules which then enter
the cell. Once in the cell creatine kinase phosphorylates
the creatine molecule which becomes phosphocreatine
(Branch). Studies have had varying results in the
effectiveness of orally ingested creatine monohydrate
on anaerobic muscular activities. One study found the
performance of forearm muscles in 12 healthy men was
improved via creatine supplementation (Chance) .
While another study found that there was no
performance improvement of 15 college soccer players
tested on a treadmill at high intensity intervals (Biwer
et al).
The Anolis carolinensis rely almost solely on
anaerobic respiration for skeletal movement such as
running. The emphasis on keeping ATP stores
available to sustain skeletal muscular contraction
makes Anolis carolinensis perfect candidates to test the
postulation that oral consumption of creatine
monohydrate
will
supply
extra
store
of
phosphocreatine, hence prolong anaerobic exercise.
40
Experimental Group
Control Group
Linear (Control Group)
Linear (Experimental Group)
30
20
10
0
0
22
Saddleback Journal of Biology
Spring 2005
1
2
3
4
Trial No.
5
6
7
Experimental Vs. Control
60
Average Sprint Duration (sec)
50
40
Experimental
Control
30
20
10
0
Experimental
Control
Group Type
The control group had an initial trial average
of 38.6 seconds and a final trial average of 44.9
seconds, improving by 6.3 seconds. The experimental
group had an initial trial average of 50.0 seconds, and a
final trial average of 56.0 seconds, improving by 6
seconds.
The creatine supplemented group consistently
demonstrated longer sprint durations than the control
group. The combined means of all six trials for the
experimental and controls groups were 49.1 and 38.0
seconds respectively (figure 2). Statistical analysis via
a one tailed unpaired t-test yielded a 0.008 P value.
Discussion
The hypothesis that the creatine supplemented
group would yield on overall longer average sprint
duration than the control group was proved statistically
significant by the one-tailed unpaired t-test (P=0.008).
Utilization of creatine monohydrate by the
experimental group allowed the Anolis carolinensis to
supersede the expected average sprint duration set by
the control group. The experimental group exhibited
the mechanical effects of excess phosphocreatine stores
in active skeletal muscle cells. During sprinting
periods, the readily available ATP decreased as time
increased. At the average time of 38.0 seconds the
mobility of the control group ceased due to depleted
ATP stores. The experimental group was able to stretch
the average sprint duration to 49.1 seconds by
converting ADP in muscle cells back to ATP,
replenishing the fuel source for muscle contraction.
The overwhelming evidence that creatine
supplementation was a factor in enhancing anaerobic
muscular performance of the anoles is congruent with
several studies that used human subjects in similar
circumstances. One such study found that squat
exercise, which utilizes fast twitch muscles, was
improved with male subjects on a creatine
monohydrate regimen of 3.0 grams per day (French et
al). Another study found slight, but significant
improvements in calf muscle performance from
muscular dystrophy patients who ingested 5.0 grams of
creatine monohydrate per a day. Interestingly,
muscular analysis derived from this study found no
increase in phosphocreatine levels in the muscle cells
due to supplementation. It would be advantageous to
conduct a study that measures levels of
phosphocreatine in anoles before and after creatine
monohydrate supplementation and simultaneously
measure anaerobic muscular endurance. This type of
study would confirm or deny the theory that creatine
monohydrate supplementation extends anaerobic
activity through building up phosphocreatine stores..
Both specimen groups showed a rather
significant climb in times between trials 1-6. Two
explanations can be given for these results. First, the
Anolis carolinensis were newly trained specimens that
were not adjusted to the mechanics of the treadmill.
Further trials yielded higher cooperation of the
specimens. Second, both groups may have increased
sprint duration times simultaneously because of an
overall elevation in endurance. It would be expected
that frequent exercising would show an increase in
longevity capabilities. Fluctuation in data caused by a
build in muscle endurance was controlled in a similar
study by using creatine supplementation on 15 college
soccer players who were previously conditioned for
sprinting exercise (Biwer et al).
It has been proposed that creatine
supplementation, along with ergogenic effects, may
promote an increase muscle mass. This proposal was
tested at the Washington University School of
Medicine, where athletes who orally ingested creatine
showed an increase in lean body mass (Racette). A
similar experimental method could be explored using
the Anolis carolinensis species.
A continuation of this study could be
improved by using a reptile species that produce a good
number of hatchlings. The snapping turtle for instance
can lay 40 hatchling eggs per nesting period (Zuppa).
Using the hatchlings for the study would ensure that
the specimens are the same age and have similar
genetic traits, in turn limiting the number of variables.
Another improvement to the methods could include
training the subjects prior to the time trials, eliminating
the learning curve variable. Along with training,
conducting more trials would give more concrete
results.
Literature Cited
Bemben MG, Lamont HS. Creatine Supplementation
and Exercise Performance:
Recent Findings. Sports Med. 2005; 35(2): 107-25.
Biwer CJ, Jensen RL, Schmidt WD, Watts PB. The
Effect of Creatine on Treadmill Running With High
23
Saddleback Journal of Biology
Spring 2005
Intensity Intervals. Strength Condition Res. 2003 Aug;
17 (3): 439-45.
Branch David J, Kreider Richard, Williams Melvin H.
1999. Creatine: The Power
Supplement.
Champaign, Illionois: Human Kenetics. Pg. 34-35
Chance B, Katsumura J, Kimura N, Kurosaway L,
Sako T. Creatine Supplementation Enhances Anaerobic
ATP Synthesis During a Single 10 Second Maximal
Handgrip Exercise. Mol. Cell Biochem. 2003 Feb; 244
(1-2): 105-12.
Cupp Melanie, Romanchak Nancy, Tracy Timothy.
2001. Dietary Supplements. Totowa, New Jersy:
Human Press. Pg 91- 101.
French DN, Kraemer WJ, Hakkinen K, Volek JS. The
Effects of Creatine
Supplementation
on
Muscular Performance and Body Composition
Responses To Short-term Resistance Training
Overreaching. Eur J Appl Physiol. 2004 May; 91(5-6):
628-37.
MatsumuraT, Nozaki S, Saito T, Shinno S, Yokoe M.
A Clinical Trial of Creatine Monohydrate in Muscular
Dystrophy Patients. Rinsho Shinkeigaku. 2004 Oct;
44(10): 661-6.
Racette SB. Creatine Supplementation and Athletic
Performance. Orthop Sports Phys. Ther. 2003 Oct; 33
(10): 615-21.
Ruggeri P. 2000. Creatine: From Basic Science to
Clinical Application. Milan, Italy: Fondazione
Giovanni Lorenzini. Pg 59-70.
Zuppa Steve. Common Snapping Turtles: Chelydra
serpintina.
May
1,
2005.
http://www.kyherpsoc.org/services/snappingturtlecare.
htm.
The Effects of Caffeine on the Metabolic Rate of the Fish Carassius auratus
Michael Hinson and Yury Penaloza
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Caffeine is becoming a more and more popular ingredient in commercial products.
While its has the ability to increase awareness and physical activity, it also may have some
severe side effects. The purpose of this experiment was to determine the effects on a
metablolic system by testing its effects on goldfish Carassius auratu. The metabolic system
of goldfish is similar to humans so testing on goldfish will provide a good idea of the affects
of caffeine on humans as well as detail possible pollution effects in aquatic habitats. By
giving goldfish a controlled amount of caffeine (5mg) each day over a period of 10 days,
caffeine was determined to increase the metabolic rate compared to goldfish without
caffeine exposure. The mean opercular pumping rate for unexposed goldfish was 178.57
pumps/2min while the opercular pumping rate for the goldfish subjected to caffeine was
208.17 pumps/2min. Running a t-test on the observations revealed a statistical difference
between the two groups. This increase in the metabolic rate can be characterized by
increases in heart rate, blood pressure, and respiration rates. From the results of the
experiment, future experiments will be able to more closely identify how caffeine reacts
within the human body both short term and long term, as well as how it can be harnessed
for better health and environmental benefits.
Introduction
Stimulants are a kind of drug that have the
ability to increase alertness and physical activity.
Medicinally they are used to treat ailments such as
attention deficit disorder and hypertension. They
achieve these results by having a profound affect on the
central nervous system and the sympathetic nervous
system. Stimulants can increase heart rate, increase
24
Saddleback Journal of Biology
Spring 2005
respiration rate, increase blood pressure, and decrease
appetite. It is these adverse effects that make certain
stimulants like cocaine, nicotine, and amphetamines
highly controlled substances (Emboden W 1979).
One of the most common stimulants found in
everyday life is caffeine, trimethylxanthine (Figure 1).
Caffeine is part of a class known as alkaloids, since
they contain nitrogen and have properties of an organic
amine base (Pavia DL 2005). Caffeine is the strongest
stimulant in the xanthines family. The xanthines are the
oldest known stimulants and have been found to have
several beneficial uses in the medical field. For
example, theophylline is used to dilate the coronary
artery; as well as serve as a bronchodilator to treat
bronchial asthma (Ritchie, J.M 1990).
Caffeine is found in several common
commercial products. Caffeine can be extracted from
the cocoa tree and tea plant (Encarta 2005). The
quantity of caffeine tends to vary from product to
product. In a serving of brewed coffee there is 30 mg
of caffeine, 70 mg in an expresso and 45.6 mg in a
Coca-Cola. Other popular products with caffeine
include chocolate, tea, and cocoa beverages. A typical
male will consume around 1150 mg of caffeine over 72
hours and a female will consume around 1273 mg (Ray
O.S 1996). Caffeine is also the active ingredient in
several over the counter tablets designed to help people
stay awake and be more alert.
Figure 1. Structure of Caffeine
Caffeine is found in several common
commercial products. Caffeine can be extracted from
the cocoa tree and tea plant (Encarta 2005). The
quantity of caffeine tends to vary from product to
product. In a serving of brewed coffee there is 30 mg
of caffeine, 70 mg in an expresso and 45.6 mg in a
Coca-Cola. Other popular products with caffeine
include chocolate, tea, and cocoa beverages. A typical
male will consume around 1150 mg of caffeine over 72
hours and a female will consume around 1273 mg (Ray
O.S 1996). Caffeine is also the active ingredient in
several over the counter tablets designed to help people
stay awake and be more alert.
With more and more products using caffeine
as an active ingredient, there are more and more
concerns about its health affects. Abnormally high
levels of caffeine may promote cancerous conditions
and mental side effects (Miller 1997). Certain birth
defects seem to be related to high concentrations of
caffeine. There could be an increased risk of heart
ailments. There is also major concern the people are
becoming dependent of the drug. Withdrawal from
caffeine has shown symptoms of fatigue, headaches,
and depression (Ray O.S 1996). The International
Olympic Committee has even placed caffeine on the
list of banned substances.
To get a better understanding of caffeine and
its physiological influences, this experiment was
designed to test the effects of caffeine on the metabolic
rate of the goldfish, Carassius Auratus.
The
observations of opercular contractions, which are an
indirect measure of the metabolic process, will help
determine what kind of effect is present. The caffeine
will be absorbed by the goldfish through the gills. One
way the gills become ventilated are through the active
process of opercular pumping. Opercular pumping
occurs when the goldfish pulls in water through the
mouth and then pushes it over the gills. The gills
function when water flows over the epithelial surface
and gas exchange occurs. Since caffeine is found to
increase the metabolic rate, by increasing the caloric
intake (Sarah Marshall Hons), it should increase their
opercular pumping rate. Contraction of the operculum
is innervated by a dorsal group of cranial nerves termed
brachial nerves (Taylor et all, 1999). These brachial
nerves should also be influenced by caffeine’s affect on
the sympathetic nervous system (Busselen P 1980).
The hypothesis for the experiment is that caffeine will
cause an increase in the metabolic rate of the exposed
goldfish compared to the goldfish without caffeine
absorption. The results will give a better idea of how
caffeine immediately stimulates the metabolic system
and will provide a foundation for future caffeine
experiments. The experiment will also illustrate if there
is an effect of polluting an aquatic environment with
products containing caffeine.
Materials and Methods
Twenty individual eight-ounce containers
were prepared with 16 ounces of deionized water.
Twenty goldfish of similar size and weight were
separated and given an individual container. Then ten
fish were chosen at random and were separated into a
control group. The remaining ten goldfish were then
grouped into the experimental group. The goldfish for
both groups were fed 0.1g of goldfish flakes at 8:00 am
every morning. Feedings were performed in the
morning as to not have an effect on the data. Past
experiments have shown a direct correlation between
25
Saddleback Journal of Biology
Spring 2005
Results
After all the data was obtained, the data from
the observations over the 10 days were inserted into a
spreadsheet in Excel. Then the average opercular
pumping rate for each day was determined and
displayed on Figure 2.
To determine if there was any statistical
differences between the control group and
experimental group a two-tail t-test assuming unequal
variance was performed. The results of the t-test
revealed a value of P(T<=t) 1.56 x10-22. With a t-value
well below 0.05, there is a statistically significant
difference in the values obtained between the control
group and experimental group subjected to caffeine. To
confirm the t-test, an ANOVA single factor for
repeated variables was run to compare the results. The
results of the ANOVA gave a p value of 9.63 x 10-23.
The ANOVA confirmed the results of the t-test and
that there is a significant statistical difference between
the two groups. The mean opercular pumping rate for
the 100 observations of the control group was 178.57
contractions per two minutes and for the 100
observations of the experimental group the mean
opercular pumping rate was 208.17 contractions per
two minutes.
Opercular Pum ping Rates of Goldfish
Each Day
Contractions / 2
min
feeding and increased metabolic rates. Every night at
11:00 PM, the water for every fish was changed to
prevent a buildup of ammonia and control the pH. This
process of water replacement was performed for 3 days
as to allow the goldfish to become accustomed to the
fatigue and stress of constant water changes.
During the acclimation phase of three days,
the opercular pumping rate was measured at 10:00 PM
every night. The opercular pumping rate was
determined by counting the number of times the gills
on the goldfish opened and closed during a 2-minute
interval. These contraction rates will serve as the
foundation to test the hypothesis. After the three-day
resting period, the experimental group was given 5mg
of caffeine at 10:00 PM every night for 10 nights. The
caffeine was retrieved by crushing 200mg Rite Aid
“Stay Awake” tablets into a fine yellow powder and
weighing out 5mg doses. The caffeine powder was
added to the water, without excessive stirring as to
stress the goldfish, and allowed to dissolve. The
goldfish were able to absorb the caffeine through their
gills for a period of 25 minutes. While the experimental
group was absorbing the caffeine, the opercular
pumping rate was determined for the control group.
The contractions were counted over the 2-minute
period and recorded in the control column. Once the
25-minute period for absorption expired, then the
opercular pumping rate of the experimental group was
determined.
After all the opercular pumping rates for all
the fish were determined, the water for each container
was changed. The water was changed to prevent
ammonia buildup, control pH, and make sure that no
caffeine remained in the water overnight as to skew the
results for the next trial. Also by changing the water
after the data was recorded it provided a 24-hour
window for the fish to readjust to their new
environment and have amble time to overcome the
stress of the water change.
230
210
190
170
150
1
2
3
4
5
6
Day
Experimental
7
8
9 10
Control
Figure 2. Average opercular pumping rates for each
day of the goldfish in both groups.
Discussion
From the two-tail t-test, it is evident that the
hypothesis was correct and that caffeine did have a
statistical effect on the opercular pumping rate. The
increase in the opercular pumping rate directly
correlates to an increase in the metabolic rate of
goldfish exposed to caffeine compared to goldfish at
rest or in normal conditions. The caffeine in the water
was absorbed by the goldfish through countercurrent
gas exchange in the gills. The caffeine proceeded by
traveling throughout the body of the goldfish in the
circulatory system. Once in the system, the caffeine
increased the heart rate and blood pressure and in turn
increased the respiratory rate. With a higher respiratory
rate, the amount of water flowing through the gills
increased and thus the incoming supply of caffeine
increased. This cycle quickly allowed for the
noticeable difference between the two groups.
Further analysis of Figure 2 can also be
revealing. It is interesting to note that there was no
pronounced trend of increased metabolism over the
entire ten-day period. If the metabolic rates showed an
increasing trend from day 1 to day 10 then there would
be severe concern whether the fish could sustain
prolonged exposure to caffeine. If there was a
decreasing trend, then there might have been less
reason for concern considering the effects of caffeine
would have diminished over time meaning the fish
adjusted to the exposure of a foreign chemical.
However, without showing a consistent trend on the
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Saddleback Journal of Biology
Spring 2005
metabolic rate, future experiments will need to be
performed to identify long-term effects of caffeine on
metabolism. These long-term effects could be the most
interesting to see. If caffeine losses effectiveness after
prolonged exposure then people will have to increase
the intake of caffeine in order to produce their desired
results. If the results are the other way, then caffeine
could play a huge role in several growing medical
ailments on today’s society.
Knowing that caffeine can affect the
metabolic rate of goldfish, allows for a better
understanding of how caffeine affects the human body.
Once in the circulatory system, caffeine affects the
receptors of the central nervous system by stimulating
the sympathetic nervous system forcing the body to
perform the “flight or fight” response (Johnson JD
1999). This response can raise blood pressure and
potentially cause heart problems. The heart problems;
therefore, can be caused through extensive stimulation
by caffeine. Identifying the severity of caffeine related
problems and the abundance or quantity of caffeine
needed for these negative ailments will need to be
determined. The results of these experiments will help
develop a diet that will have a safe know quantity of
caffeine to avoid over stimulation.
Ray O.S. Caffeine. Drugs, society and human
behavior. 7th ed. St Louis: C. V. Mosby, 1996.
Ritchie, J.M. Central Nervous System Stimulants. II:
The Xanthines. The Pharmacological basis of
therapeutics, 8th ed. New York: Macmillan, 1990.
Taylor EW, Jordan D, Coote JH. 1999. Central Control
of the cardiovascular and respiratory systems and their
interactions in vertebrates. Physiology reviews 79: 815916.
Literature Cited
Busselen P, Van Kerkhove E. The effect of caffeine on
the calcium distribution in goldfish ventricles. PMID:
7406588 1980
“Caffeine,” Microsoft Encarta Online Encyclopedia
2005.
Emboden W. The stimulants. Narcotic Plants, rev ed.
New York: Macmillan, 1979.
Hons, Sarah Marshall
http://www.myfit.ca/archives/viewanarticle.asp?table=s
upplements&ID=18.
Johnson JD, Wong CJ, Yunker WK, Chang JP.
Caffeine-stimulated GTH-II release involves Ca(2+)
stores with novel properties. Department of Biological
Sciences, Biological Sciences Building, University of
Alberta 1999.
Miller, C. S. Toxicant-Induced loss of tolerance-an
emerging theory or disease? Environ Health Perspect,
105(1197): 445-453
Pavia DL, Lampman GM. 2005. Essay: Caffeine.
Introduction to Organic Laboratory techniques 2nd
edition. 78-82
27
Saddleback Journal of Biology
Spring 2005
Effects of Age on the Cardiovascular Recovery Rate in Female Aquatic
Athletes
Kirby Jacobs
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Heart rate is affected by parasympathetic and sympathetic actions. When
sympathetic action is stimulated heart rate goes up. When parasympathetic action is
stimulated heart rate goes down. During exercise, parasympathetic action is suppressed
and sympathetic action is stimulated. This causes heart rate to go up. As mammals age,
the heart muscle stretches out. This increases cardiac output and decreasing heart rate.
The sino-atrial node also responds slower to stimulus. These factors should show an
increase in the time it takes to go from a post-exercise heart rate back to a resting heart
rate, or the cardiovascular recovery rate. However, in this experiment, the older group,
18-29 year old women, had a significantly lower mean recovery rate at 110. 941 seconds.
The younger group, 14-18 year old girls had a mean of 235.542 seconds. The two groups
both were tested using a Polar A1 series heart rate monitor. The results do not support the
research found, therefore, some factors that were not controlled such as difficulty of
workout and outside activities would need to be controlled to more accurately test this
hypothesis.
Introduction
During exercise, there is an increase in heart
rate due to increased sympathetic activity and
decreased parasympathetic activity. The increase in
heart rate allows for the cardiac output to be increased
by up to 7 times from the normal 5 liters/minute to 35
liters/minute in elite athletes (Vander et al. 2001). The
increase in cardiac output as well as the vasodilation in
the muscle tissues allows for the muscles to perform at
a higher level than at rest. This is because there is
greatly increased blood flow so more oxygen is
flowing to the muscles and more waste products are
flowing out of the muscles. After exercise, the
sympathetic nervous system is suppressed and the
parasympathetic nervous system is stimulated. This
causes the heart rate to go back to normal and is called
the recovery rate. The cardiovascular recovery rate has
shown in studies to be an accurate indicator of cardiac
function. For example in stress tests the recovery rate
is what doctors have used to show risk factors for heart
disease (Nishime 2000). My intention in conducting
this experiment was to determine what effect age has
on the cardiovascular recovery rate of high school and
college age, female aquatic athletes. The research
shows that increased age decreases heart function
thereby; increasing heart rate and cardiovascular
recovery rate therefore it can be hypothesized that the
high school age athletes will have a shorter recovery
time.
Materials and methods
On 10 days starting on March 14th 2005 a
Polar A1 model heart rate monitor was used to collect
data from 51 girls at El Toro high school and 25 girls at
Saddleback College. The data was collected by putting
the heart rate monitor on the subject while they were
resting. The monitor was read at this point for the
initial resting heart rate. The subject then swam 50
yards as fast as possible. At the point when they
touched the wall at the end of the 50 yards a stopwatch
was started. The monitor was observed until the heart
rate was back at the original resting rate, at which point
the watch was stopped. This was done for all of the
girls tested. The data was then entered into a database
and an unpaired t-test was run.
Results
From the first day of data collection there was
an apparent difference in the results (Figure 1). The
average recovery rate for the high school girls, 14-18
years old, was 235.542 + 91.7 seconds. This is
statistically different then the 110.941 + 62.2, which
was the average for the college girls, 18-29 years old.
The t-test resulted in a p-value of 4.936 x 10^-8, this is
less than 0.05 and is therefore statistically significant.
28
Saddleback Journal of Biology
Spring 2005
Average recovery time
(seconds)
exercise score as predictors of mortality in patients
referred for exercise ECG. Journal of the American
Medical Association 284: 1392-1398.
250
200
150
100
50
0
High School
College
Figure 1. Average recovery time for high school and
college age, female aquatic athletes.
Discussion
Studies have shown a slow decline in heart
function as age increases (Stein et al. 1999), as well as
a decline in responsiveness to sympathetic stimuli
(Kreider et al. 1984). The decreased heart function can
be related to cardiac dilation. This increases end
diastolic volume and stroke volume as well as a
decrease in heart rate (Rodeheffer 1984). With this
information one would assume that the recovery rate
for the older group would be longer due to the slow
reaction to stimulus. It was shown in this experiment
that age had the opposite effect on the recovery rate.
The college age girls’ times were statistically shorter
than the high school age girls. There are many reasons
ways to account for this. The research that was found
discussed a slow decline in heart function, it is possible
that the ages that were tested were too close together to
see a difference in function. Also, the oldest subject
was 29 years old; this might not have been old enough
to see a decline in heart function. The main difference
is training regimen. Although the two groups both
workout two to three hours per day, five to six days per
week, their practices are much different. The college
group’s workouts were much harder, with more
strenuous time intervals and more yards swam per
workout. Due to the more strenuous workouts, the
college age athletes could have been in better
cardiovascular shape to begin with. This would cause
higher stroke volume and a more efficient heart in
general. In order to more accurately depict the
cardiovascular differences in these 2 groups, they
should be put on the same training schedule.
Rodeheffer R, Gerstenblith G, Becker L, Fleg J,
Weisfeldt M, and Lakatta E. 1984. Exercise cardiac
output is maintained with advancing age in healthy
human subjects: cardiac dilation increased stroke
volume compensate for a diminished heart rate.
Circulation 69: 203-213.
Stein P, Ehsani A, Domitrovich P, Kleiger R, and
Rottman J. 1999. Effect of exercise training on heart
rate variability in healthy older adults. Boletim
Cientifico Diario.
Vander A, Sherman J, and Luciano D. 2001. Human
Physiology: the mechanisms of body function. New
York: McGraw-Hill. 442-446.
Literature cited
Kreider M, Goldberg P, and Roberts J. 1984. Effect of
age on adrenergic neuronal uptake in rat heart. Journal
of Pharmacology and Experimental Therapeutics 231:
367-372.
Nishime E, Cole C, Blackstone E, Pashkow F, and
Lauer M. 2000. Heart rate recovery and treadmill
29
Saddleback Journal of Biology
Spring 2005
Reaction Times are Improved with Caffeine Intake
Sara Liechty and Morvarid Vahdati
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
It has been known that caffeine is a stimulant. The effects caffeine has on reaction
times have been previously studied. Reaction times measure the time it takes to respond to
a stimulus. Caffeine increases alertness and attentiveness by stimulating the sympathetic
nervous system. This can be witnessed by seeing a decrease in reaction time to a stimulus
upon oral consumption of caffeine. In this experiment the response times of individuals
were measured by using a meter stick with pre-measured time intervals, and dropping it.
The response times were measured by how fast the individual grabbed the meter stick once
it was dropped. The results showed a significant difference between the reaction times
before and after caffeine intake, having a p-value of 0.0262. This difference validates the
notion that caffeine improves on the ability to focus and be more alert, thus stimulating the
nervous system to react more rapidly.
Introduction
The nervous system of the human body is a
giant network of nerves that are affected by various
stimulants in the environment. These stimuli elicit a
response through a chain of events. A sensory receptor
collects the information, and this information is then
conveyed to the brain, the central nervous system.
There the information is processed. A network of
nerves in the peripheral nervous system carries this
message to the designated muscle or gland to obtain a
response. The elapsed time it takes for the information
to be perceived and then generated into a response
fluctuates according to each individual. Different
factors can spur and motivate various responses.
Response time can be altered when certain
drugs come into play and affect the nervous system.
Society uses stimulants to achieve a sense of increased
alertness and energy. In a world where people are
constantly on the go and time is a valued commodity,
caffeine is greatly valued. Caffeine is one of a group
of plant alkaloids that are found in sixty different
species of plants. Plant alkaloids are positively
charged and therefore water soluble. This allows for
the quick diffusion across cell membranes, and is the
reason why the effects of caffeine are seen quickly
after consumption. It is found in the many items used
on a regular basis, including cocoa, tea, coffee, cola,
and some energy drinks (Gray, 1998). Caffeine is one
of the best examples of a substance that provides
“mental energy.” It improves cognitive performance,
mood, and enhances alertness and attention (Bridle et.
al, 2004). These affects are only achieved at doses of
caffeine equal to the amount found in typical foods.
Just like any other luxury in life, too much caffeine can
be detrimental (Lieberman, 2001).
The purpose of this experiment was to
demonstrate the effects of caffeine on the nervous
system and how it alters the response time to a given
stimulus.
Materials and Methods
The experiment was conducted over a period
of three weeks, where individuals were tested at
various locations and times. It was required of the
participants to avoid the intake of caffeine two hours
prior to the experiment. The reflexes of individuals (n
=60) were tested before and after the consumption of
caffeine over a period of ninety minutes. A meter stick
with pre-measured time markings from 0 to 400
milliseconds was held at a position comfortable for the
participant to place his or her thumb and index finger at
the bottom of the meter stick while seated. Each
individual was told to look at the bottom of the meter
stick and wait to grab it until he or she saw it drop. Not
seeing the tester release the meter stick eliminated the
error of anticipation. The measurements on the meter
stick determined how fast the individual grabbed the
stick from when it was initially dropped. Each trial
consisted of ten drops. One Vivarin pill, containing
200mg of caffeine, was given to each participant after
the first trial. This experiment contained a total of four
trials, with thirty minute intervals. An ANOVA and
post- hoc test were performed on the data points
obtained to see if there was a significant difference on
reaction times after the intake of caffeine.
30
Saddleback Journal of Biology
Spring 2005
Results
By performing an ANOVA test, a p-value of
0.0262 was obtained. This showed that there was a
significant difference in reaction times before and after
caffeine consumption (Fig. 1).
195.0000
R e a ctio n T im e (m s )
190.0000
185.0000
180.0000
175.0000
170.0000
165.0000
Before Caffeine
30 min.
60 min.
90 min.
Elapsed Time After Caffeine
Figure 1. The average reaction time before and after
caffeine consumption. * Statistically different between
Before Caffeine group and 30 minute group. **
Statistically different between Before Caffeine group
and 60 minute group.
The post- hoc test showed a significant
difference between the thirty minute trial and the
Before Caffeine Consumption (BCC) trial obtaining a
p-value of 0.0081. There was also a significant
difference between the 60 minute trial and the BCC
trial, obtaining a p value of 0.0185. There was no
significant difference between the 90 minute trial and
the BCC trial. A p-value of 0.0668 proves this result.
Discussion
The consumption of caffeine had a significant
difference on response time to a given stimulus. The pvalue for the ANOVA test was less than 0.05, which
supports the hypothesis. The result obtained is that
caffeine had an immense effect on response time, thirty
minutes after ingestion. The reason why it begins
working so fast, is because once caffeine is digested, it
is almost completely absorbed (Hawley, 1998). The
amount of caffeine that was given can also improve
alertness and enhance concentration (Gray, 1998). The
autonomic nervous system controls these involuntary
behaviors and is divided into parasympathetic and
sympathetic divisions. Caffeine causes a rise in
sympathetic and parasympathetic nerve activity. In
this sense the leading factor for the alertness comes
from the excitement of the nervous system and the
attentiveness is caused by the calming of the same
system (Hibino et al, 1997).
Caffeine is a stimulant, and stimulants tend to
mimic adrenaline. Adrenaline is released during
stressful situations, and increases heart rate and blood
flow to elicit a fight-or-flight response. This explains
why an individual who takes caffeine is apt to improve
mental performance directly from cognition and
indirectly from increased awareness (Femstrom, 2001).
Caffeine also has drug related effects that influence the
sympathetic nervous system (Hibino et. al, 1997). The
decrease in the reaction time is attributed to
sympathetic nerve responses. Because each individual
is in the “fight or flight” mode, they are more alert and
ready to receive the receptor. The next trial followed
the same trend as the first, and the reaction time was
still significantly quicker than before caffeine was
administered. After ninety minutes the caffeine began
to wear off. At that point the sympathetic nervous
system’s
activity
was
depressed
and
the
parasympathetic nervous system’s activity was
stimulated. Though the p-value was close to 0.05, it
showed no significant difference from the first trial.
Even though caffeine has desired effects, these effects
are only temporary. Because caffeine wears off
quickly, dependence seems predictable. Taking high
dosages of caffeine (more than 15mg/kg of body
weight) can lead to tachycardia (Hawley, 1998).
Therefore like anything else, caffeine has its pros and
cons.
Errors in this experiment could be due to
individuals anticipating the receptor.
With the
experiment being conducted multiple times per person,
a person could learn the response to grabbing the meter
stick before it was dropped. By anticipating the
receptor, the reaction time most likely would decrease
(Etnyre and Kinugasa, 2002). There was an attempt
made to avoid people’s anticipation to the receptor by
releasing the meter stick right after the person grabbed
it due to expectation. This extended period of time
might have changed the learned response, avoiding this
error.
Further research can be done on this topic by
determining what caffeine amount to give to an
individual based on their age and body mass, so that all
individuals have equivalent levels of caffeine in their
body. This will give a better comparison of how
caffeine affects the reaction time to a stimulus.
Literature Cited
Bridle, Leisa, June Remick, Evelyn Duffy. 2004. Is
Caffeine Excess Part of Your Differential Diagnosis?
Nurse Practitioner. 29: 39-41, 43-44.
Etnyre, Bruce, Takashi Kinugasa.
2002.
Postcontraction Influences on Reaction Time.
Research Quarterly for Exercise and Sport. 73: 271281.
31
Saddleback Journal of Biology
Spring 2005
Femstrom, John D. 2001. Diet, Neurochemicals, and
Mental Energy. Nutrition Reviews. 59: S22-S25.
Gray, Juliet. 1998. Caffeine, Coffee and Health.
Nutrition and Food Science. 98: 314-319.
Hibino, Gaku, Toshio Moritani, Teruo
Fushiki. 1997. Caffeine Enhances
Parasympathetic Nerve Activity
Quantification Using Power Spectral
Journal of Nutrition. 127: 1422-1427.
Kawada, Tohru
Modulation of
in Humans:
Analysis. The
Hawley, John A. 1998. Fat Burning During Exercise;
Can Ergogenics Change the Balance? The Physician
and Sportsmedicine. 26: 56-62.
Lieberman, Harris R. 2001. The Effects of Ginseng,
Ephedrine, and Caffeine on Cognitive Performance,
Mood and Energy. Nutrition Reviews. 59: 91-102.
Goldfish (Carassius auratus) Metabolism in Various Temperatures
Moji Ogunleye
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Goldfish (Carassius auratus) are quite popular in the U.S. They came from China
originally and are mutations of the Gibel Carp. In this experiment, Goldfish were tested in
three different water temperatures: 11 degrees Celsius, 21 degrees Celsius and 31 degrees
Celsius, to determine their metabolic rates. It turned out that the metabolic rates of the
goldfish varied because of their weight. As a result, the goldfish were separated into two
groups: Group A (1- 10 grams) and Group B (11- 20 grams). They were tested for two days
(so that not to kill them from the water extremes of 11 and 31 degrees Celsius in one day)
on the hypothesis that Group A. would have larger opercular pumping rates and faster
metabolic rates overall. The results showed that Group B had larger opercular pumping
rates and faster metabolic rates overall. Group A’s average opercular pumping rates at 11,
21, and 31 degree Celsius were 46.888 o.p.r. per minute, 79.666 o.p.r. per minute and
128.888 o.p.r. per minute. Group B’s average opercular pumping rates at 11, 21, and 31
degree Celsius were 48.166 o.p.r. per minute, 83.833 o.p.r. per minute and 181.333 o.p.r.
per minute. Group A’s average metabolic rates at Q 10 (+ 10 degrees Celsius) and Q 10 (10 degrees Celsius) were 1.647 and 0.835. Group B’s average metabolic rates at Q 10 (+ 10
degrees Celsius) and Q 10 (- 10 degrees Celsius) were 2.086 and 0.5876.
Introduction
Goldfish (Carassius auratus) are classified as
heterothermic ectotherms. This means that their body
temperature is determined by the temperature of their
environment (in this case, the water the goldfish swim
in). Goldfish originally came from China and were
introduced to the U.S. in the late 1800’s (wetpetz.com).
Goldfish are the most common household pet and are
notoriously filthy (fishindex.com). Goldfish produce
ammonia (NH3) as their waste product. Goldfish live
in water between 10 to 20 degrees Celsius and prefer
the pH of the water to be 6.5 to 7.5. Goldfish metabolic
rates vary depending on the temperature of the water
they live in. The opercular pumping rates of the
goldfish are used in determining the metabolic rates by
the Q 10 factor. The Q 10 factor is an equation used to
find the normal body process like metabolism. The two
Q 10 equations used in this experiment were:
(For 31 degrees Celsius)
Q 10 = Rate (21 + 10 degrees) Celsius
Rate (21 degrees) Celsius
(For 11 degrees Celsius)
Q 10 = Rate (21 -10 degrees) Celsius
Rate (21 degrees) Celsius
The opercular pumping rates in goldfish
increase for four reasons: Temperature increase of the
32
Saddleback Journal of Biology
Spring 2005
water, Ammonia waste (NH3), The goldfish’s weight,
and amount of Oxygen consumption. Oxygen
consumption is increased with temperature (Sollid et
al., 2005). Muscles relax when the actin and myosin
separate from each other and no more calcium ions are
released (Moshktov et al., 2005).
Materials and Methods
In order to find the opercular pumping rates of
the goldfish, they were tested in three beakers (200 ml)
that held 100 ml of water in each. Each beaker had a
different water temperature of 11 degrees Celsius, 21
degrees Celsius and 31 degrees Celsius. The Q 10
factor was used in determining what temperature
intervals to use and finding the metabolic rates of the
goldfish in each temperature. The Q 10 factor is an
equation used to rates of Biological processes.
Temperature change is said to effect metabolic rates in
goldfish. Fifteen goldfish were separated into two
groups, determined by their weight in grams (Group A:
1- 10 grams and Group B 11-20 grams). The beakers
and water used in the experiment were preweighed, to
get an accurate weight of the goldfish. Each goldfish
was tested three times, once in every water temperature
of 11 degrees Celsius, 21 degrees Celsius and 31
degrees Celsius. This was done to find the opercular
pumping rates. The goldfish were timed in one minute
intervals for each temperature. Meanwhile, a clicker
was used to find the total times the goldfish’s
operculum flapped during that minute. A thermometer
was used to keep the water temperature in check.
Results
The goldfish in Groups A and B showed
surprising evidence, proving my original hypothesis to
be wrong. The hypothesis was that the goldfish in
Group A would have larger opercular pumping rates
and faster metabolic rates overall. Under statistical
analysis however, it was completely the opposite.
Group A’s results showed that the averages of the
opercular pumping rates were 46.888 per minute at 11
degrees Celsius, 79.666 per minute at 21 degrees
Celsius, and 128.888 per minute at 31 degrees Celsius.
Group A’s individual opercular pumping rates results
can be seen (Table 1). Group B’s results showed that
the averages of the opercular pumping rates were
48.166 per minute at 11 degrees Celsius, 83.833 per
minute at 21 degrees Celsius, and 181.333 per minute
at 31degreesCelsius. Group B’s individual opercular
pumping rates results can be seen (Table 2). The
Standard deviance for Group A was P= +/- 37.522. The
Standard deviance for Group B was P= +/- 69.292. The
results for Q 10 (+ 10 degrees Celsius) and (– 10
degrees Celsius) can be seen in Tables 3 and 4. Grooup
A’s average metabolic rate in Q 10 (+ 10 degrees
Celsius) was 1.647 and Q 10 (- 10 degrees Celsius) was
.5835. Group B’s average metabolic rates in Q 10 (+ 10
degrees Celsius) were 2.086 and Q 10 (- 10 degrees
Celsius) was .5876. Human error could have played a
role in how the results turned out the way they did.
Only Fifteen Goldfish were used in the experiment and
Group A had more goldfish than Group B. group A had
9 goldfish and Group B had 6 goldfish.
Goldfish
1
2
3
4
5
6
7
8
9
Weight
(g)
5.11
3.69
3.01
4.21
1.5
2.6
7.53
3.87
8.35
o.p.r 11 c
o.p.r 21 c
o.p.r 31 c
46
51
46
75
65
24
30
49
36
75
81
78
103
92
63
84
90
51
148
108
115
152
132
117
160
125
103
Table 1. Group A’s Opercular Pumping Rates in each
temperature.
Goldfish
1
2
3
4
5
6
1
Weight
(g)
16.68
10.88
13.1
19.49
15
11
16.68
o.p.r 11 c
o.p.r 21 c
o.p.r 31 c
51
39
56
42
48
53
51
91
66
80
105
89
72
91
163
252
155
123
293
102
163
Table 2. Group B’s Opercular Pumping Rates in each
temperature.
Goldfish
1
2
3
4
5
6
7
8
9
Q10 (+10)
1.973
1.333
1.474
1.455
1.434
1.857
1.904
1.388
2.01
Q10 (-10)
0.613
0.629
0.59
0.728
0.706
0.38
0.357
0.544
0.705
Table 3. Group A’s Q 10 results.
Goldfish
1
2
3
4
5
6
Q10 (+10)
1.791
3.818
1.987
1.171
3.292
1.4616
Q10 (-10)
0.56
0.591
0.7
0.4
0.539
0.736
Table 4. Group B’s Q 10 results.
Discussion
The goldfish in Group B had larger opercular
pumping rates and faster metabolic rates overall.
33
Saddleback Journal of Biology
Spring 2005
Goldfish from both groups had their own way to
regulate breathing in various temperatures of water (11,
21, 31 degrees Celsius). Group A and Group B results
came out the way it did because of their different
weights, oxygen consumption, opercular pumping rate
reaction to temperature increase and the Ammonia
(NH3) waste.
One reason why the goldfish in Group B had
higher opercular pumping rates is because of the
greater demand of oxygen consumption. Group A had
smaller goldfish, which needed less oxygen to
consume. Oxygen consumption is increased with
temperature (Sollid et al., 2005). Both groups had only
100 ml of water out of a 200 ml beaker to obtain
oxygen. Group B’s goldfish consumed the oxygen
faster. That was why the goldfish had to stay close to
the surface to have a continuous supply of oxygen and
created higher opercular pumping rates.
During the experiment, both Groups A and B
produced more ammonia waste at 31 degrees Celsius
water than in 11 or 21 degree Celsius water. The hotter
the water, the more likeliness for the protein in the
goldfish to denature and cause the goldfish to die. The
Ammonia waste (NH3) was produced at this
temperature interval because the muscles of the
goldfish relaxed more in 31 degrees Celsius water than
21 degrees Celsius water. This makes expelling waste
easier. The muscle release was caused from the actin
and myosin unbinding from each other and calcium
ions not being produced anymore (Moshkov et al.,
2005). The Ammonia waste (NH3) in turn, causes the
surrounding water to become more basic (between 8
and 14pH). But, goldfish prefer to live in water of a pH
between 6.5 and 7.5, which is slightly acidic/slightly
basic. When the water becomes basic, this causes the
goldfish to fight to obtain oxygen and increase the
opercular pumping rate.
In this experiment silly, small mistakes were
made that could have influenced the results. First, I
tested the goldfish in two days doing half of the project
one day and the other half another day. Second, I did
not indicate which data belonged to each fish. So, I
weighed them both days, and matched the data with the
fish that were most similar in weight. Third, Group A
had 9 goldfish and Group B had 6 goldfish, since they
were separated by their weight in grams.
My hypothesis of the goldfish in Group A having
larger opercular pumping rates and faster metabolic
rates turned out to be wrong. The opercular pumping
rates of the goldfish increased by temperature,
ammonia waste, the goldfish’s weight and oxygen
consumption. The Q 10 equation was used to find the
metabolic rates of 11 and 31 degrees Celsius of the
goldfish in Groups A and B. Goldfish (Carassius
auratus) metabolism is affected by various
temperatures.
Literature Cited
Jorund Sollid, Roy E. Weber, Goran E.
Nilsson.Temperature alters the respiratory surface area
of crucian carp Carassius carassius and goldfish
Carrassius auratus.The Journal of Experimental
Biology 2005:1109-1116. Retrieved April 28, 2005
from the World Wide Web: <http://jeb.biologists.org>
Moshkov DA, Bezgina EN, Pavlik LL, Mukhtasimova
Nf, Mavlytov TA. Distribution of calcium ions in the
mixed synapses of Mauthner neurons in the goldfish in
normal conditions, in exhaustion, and in conditions of
adaptation to exhaustion.Pub Med 2005;35(2):117-122.
Retrieved April 28, 2005 from the World Wide Web:
<http://www.ncbi.nlm.nih.gov>
<http://species.fishindex.com/species_3193carassius_a
uratus_auratus_goldfish.html>
<http://wetpetz.com/goldfish.htm >
34
Saddleback Journal of Biology
Spring 2005
The Effects of Dehydroepiandrosterone and Creatine Monohydrate on the
Metabolic Rate of Mice, Mus musculus
John Phillips and Rudy Scalisi
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Taking performance enhancing drugs has become a part of many athletes daily
routine. Supplements such as Dehydroepiandrosterone (DHEA) and creatine monohydrate
are used to enhance performance. Mus musculus, also known as the common house mouse,
were fed creatine monohydrate and DHEA to test its effects on metabolic rate. An initial
and final metabolic rate was tested for all three groups, with each group consisting of 6
Mus musculus. It was presumed that creatine and DHEA will increase the metabolic rate
of Mus musculus. Each mouse in the DHEA group was force fed 0.06 grams of DHEA and
each mouse in the creatine group was forced fed 0.125 grams of creatine. This feeding
continued for a total of eleven days. Metabolic rate was measured by using respirometers.
The initial mean metabolic rate was 5.65 mlO2/gm/hr for the control, 5.33 mlO2/gm/hr for
the creatine group, and 5.44 mlO2/gm/hr for the DHEA group. The Mus musculus were
ran on a treadmill for 3 minutes per group for six out of the eleven days. The final mean
metabolic rate was 4.52 mlO2/gm/hr for the control, 6.31 mlO2/gm/hr for the creatine
group, and 6.36 mlO2/gm/hr for the DHEA group. There was a statistical difference
between the initial control groups and the final groups, having a p value of p< 0.0167. The
increase in muscle mass increased the metabolic rate because there is a higher demand for
oxygen. The results show that the metabolic rates for Mus musculus, who were fed creatine
and DHEA, did have an increase in metabolic rate. While the control decreased in
metabolic rate because the overall weight of the mice increased, thus meaning that less
oxygen is needed to maintain homeostasis.
Introduction
Staying in shape is a goal that many people in
today’s world have in common. One method includes
people partaking in resistance training. To enhance
results, many people take supplements. Some of these
supplements are legal, while others are illegal. Within
the world of sports, the issue of performance enhancing
drugs is high.
Different supplements enhance
performance by increasing the production of hormones
and proteins already produced in the body, while others
supply more of what is already being produced.
Supplements such as dehydroepiandrosterone (DHEA)
and creatine monohydrate are supplements taken by
many athletes. DHEA is a supplement that increases
the production of testosterone (Pergola 2000). Creatine
monohydrate is produced naturally in the body and
used to transfer phosphate to ADP, making ATP in
anaerobic respiration (Klotter 2003). With the use of
creatine, muscle mass can be increased (Chilibeck, et
al. 2004). Supplements can affect metabolic rate by
increasing muscle mass.
Metabolic rate is the amount of energy that an
animal consumes over a period of time. This is the
energy required to maintain a stable body temperature
and perform basic bodily functions. Metabolic rates
have many variables including age, sex, activity level,
time of day, and the type of food that is eaten.
Metabolic rate can be measured using respirometers,
which measures oxygen consumption. By measuring
oxygen intake, this gives the amount of oxygen
consumed over time, which is directly related to the
metabolic rate. The higher the metabolic rate, the
higher the amount of oxygen consumed because more
energy in the form of ATP is needed to maintain
homeostasis.
The purpose of this experiment was to test the
effects of DHEA and creatine on metabolic rate of Mus
musculus. It was proposed that consumption of DHEA
and creatine would increase the metabolic rate of Mus
musculus.
35
Saddleback Journal of Biology
Spring 2005
approximately 0.125 grams of creatine per day and the
DHEA group received approximately 0.06 grams of
DHEA per day. Each group was force fed using a
medicine dropper that was marked at ¼ mL (5 drops).
After each group received their solution they were run
on the treadmill for three minutes, along with the
control, for a total of six times.
On April 19, 2005 the feeding was completed
and final metabolic rates were determined (see above).
To find the metabolic rate of each mouse, the following
formula was used:
10cc/time (sec) x 60sec/1 min x 60min/1 hour x
1/weight (grams)
The average metabolic rates were found for each group
and entered into Microsoft Excel. This was done for
the initial and final metabolic rate results. Once all the
data had been entered, an ANOVA and POST-HOC
tests were performed with p<.05 in order to determine
if there was a statistical difference.
Results
The initial metabolic rates for Mus musculus
groups had no statistical difference. The initial mean
metabolic rates were 5.65 mlO2/gm/hr, 5.33
mlO2/gm/hr, and 5.44 mlO2/gm/hr, for the control,
creatine and DHEA groups respectively. The final
mean metabolic rate was 4.52 mlO2/gm/hr for the
control, 6.31 mlO2/gm/hr for the creatine group, and
6.36 mlO2/gm/hr for the DHEA group. The final
metabolic rates were higher than the initial metabolic
rates in the DHEA and creatine groups and lower in the
control group (Fig. 1). A statistical difference was
found between the initial control and final in all three
groups (p < 0.0167).
Effect of Meatabolic Rate on Mus musculus due to
respectable dietary supplements
7
A v e r a g e M e ta b o l i c R a te
(m l s / O 2 / g m / h r )
Materials and Methods
On March 28th 2005, eighteen male Mus
musculus were purchased at the Pet Co. in Mission
Viejo CA. On April 4th 2005 the experimental
procedure began at Saddleback College in the Science
Math building room SM108. Six Mus musculus were
placed in three separate containers. In order to
distinguish between different Mus musculus within
each group, tails were marked with different color
sharpies. Each mouse was weighed in grams by
placing them on a scale in a tared medicine bottle.
Once the weights for each mouse were measured, six
respirometers had to be set up.
The respirometers were set up by adding soda
lime to cottage cheese paper, and then wrapped in wire
mesh to prevent consumption. Once the respirometers
were ready, the first group of mice was placed
individually into respirometers. The temperature was
recorded in degrees Celsius after equilibrium had been
reached by using a thermometer with a plug for its
base. Six manometer stoppers were placed on the top
of the respirometers containing the Mus musculus. The
manometers were prepared by marking manometer
with a clay pencil and then filling each manometer with
a green liquid just below the line.
Using a syringe, 10cc of air was injected into
each respirometer via the dropper bulb on the injection
port. Just prior to the injection of air, the green liquid
in the manometer was set to the mark. After air had
been injected, a stopwatch was used to record the time
for the fluid in the manometer to return to its initial
level. When the liquid reached the initial level the
stopwatch was stopped, the time was recorded and final
temperature was taken. Each mouse was removed
from the respirometer and weighed. This process was
repeated two more times, for a total of three trials per
group.
The mice were run on a treadmill for three
minutes. This was done by cutting out a shoe box with
only the four walls remaining in order to keep them
confined on the treadmill. This was repeated for the
three groups. Each group was placed into their
separate containers, which had water and Kaytee Fortidiet mouse and rat food; this was supplied at ad
libitum. Water was not given to any of the Mus
musculus groups overnight (10pm-10am), so mice
would consume the solutions.
One of the three Mus musculus groups was
then fed creatine monohydrate, one was fed DHEA,
and the third group was the control with no solution
given. The creatine solution was made by mixing 50
mL of water with 25 grams of creatine. The DHEA
solution was made by mixing 50 mL of water with
11.97 grams of DHEA. Each mouse in each group was
given 5 drops of the solution. From April 7, 2005
through April 18, 2005, the creatine group received
6
5
4
Initial
3
Final
2
1
0
Control
Creatine
DHEA
Figure 1. Difference in initial and final metabolic rate
for Mus musculus.
Discussion
The data collected showed that the feeding of
DHEA and creatine had an effect on the metabolic rate
of Mus musculus. Along with the DHEA and creatine
36
Saddleback Journal of Biology
Spring 2005
groups the control group also had a significant
difference in its initial and final metabolic rate.
There are many factors that contribute to these
results. DHEA is a fat reducing hormone that
stimulates resting metabolic rate (Pergola 2000). The
Mus musculus group that was fed DHEA showed both
of these symptoms. Rats that were injected with
DHEA showed to have a greater rate of oxygen
consumption (Berdanier 1989). Since there is an
increase of oxygen consumption due to the intake of
DHEA, metabolic rate increases. DHEA has also been
found to increase mitochondrial respiration in rats
(Mohan and Cleary, 1991). With the increase of
mitochondrial respiration within the muscle cells,
further increase in metabolic rate would occur.
Observations indicated that the heart rate of the mice
increased with the consumption of DHEA. It was
shown in penguins that an increased heart rate
positively effects oxygen consumption (Green et al.
2001), supporting that the mice that were given
supplements had an increase in their metabolic rate.
Creatine is taken to increase muscle mass and
to enhance performance (Klotter 2003). When male
and female humans were given creatine supplements
when training their arms, the results showed an
increase in muscle thickness of the arms (Chilibeck et.
al 2004). Increased lean muscle mass requires more
oxygen so that anaerobic respiration does not occur
(Davos, et al 2003), resulting in lactase build up. Since
creatine has been found to positively affect muscle
mass and oxygen consumption, increase in metabolic
rate will occur.
A possible reason the metabolic rate for the
control decrease is a result of weight gain. A larger
mouse needs less oxygen than a smaller Mus musculus
because the smaller Mus musculus’ tissues need a
higher rate of intake of oxygen in order to maintain a
stable body temperature.
In both the DHEA and creatine groups, the
metabolic rates increased. However, the metabolic rate
for the control decreased. This outcome agrees with
the initial hypothesis that was stated. In order to
improve upon results, different techniques could be
taken when feeding the supplements to the Mus
musculus such as injecting the mice with a syringe.
This would better help monitor the exact amount that
they receive. Further tests could be taken in order to
find the exact correlation between muscle mass,
oxygen consumption, and metabolic rate.
Literature Cited
Berdanier CD, Mcintosh MK.1989. Further studies on
the effects of dehdroepiandrosterone on hepatic
metabolism in BHE rats. Proc Soc Exp Biol
Med.192(3):242-247.
Chilibeck PD, Stride D, Farthing JP, Burke DG. 2004.
Effect of creatine ingestion after exercise on muscle
thickness in males and females. Med Sci Sports Exerc.
36(10):1781-1788.
Davos C, Doehner W, Piepoli M, Coats A, Anker S.
2003.
Lean
Tissue-Adjusted
Peak
Oxygen
Consumption in Chronic Heart Failure.
De Pergola G. 2000. The adipose tissue metabolism:
role of testosterone and dehydroepiandrosterone. Int J
Obes Relat Metab Disord 2:59-63.
Green JA, Butler PJ, Woaks AJ, Boyd IL, Holder RL.
2001. Heart rate and rate of oxygen consumption of
exercising macaroni penguins. Journal of Experimental
Biology 204(4):673.
Klotter J. 2003. Creatine for muscle building.
Townsend letter for Doctors and Patients (1): 26
Mohan PF, Cleary MP. 1991. Short-term effects of
dehydroepiandrosterone treatment in rats on
mitochondrial respiration. Journal of Nutrition
121(2):240-250.
Acknowledgements
We would like to thank Steve Teh and Tony
Huntley for providing us with the materials and a
locality to complete this experiment.
37
Saddleback Journal of Biology
Spring 2005
The Effects of Varying Temperatures on the Stridulation Frequency of
Crickets, Gryllus assimilis
Ellyne Dudkowsk
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Gryllus assimilis (field crickets) are notorious for the chirping sound that becomes
abundant during the evening, since crickets are nocturnal. In China, crickets were kept as
pets in decorative cages because of their singing. The crickets would chirp at night, singing
the owner to sleep peacefully. Crickets are able to produce high kHz frequencies called
stridulates. They stridulate monotonously by rubbing their scrapper along the file between
the base of one front wing and the bottom side of the other front wing. Only male Gryllus
assimilis have the ability to stridulate. They stridulate in order to attract females of
different species, to call an alarm, or while fighting. Stridulate frequencies fluctuate with
temperatures and depending on the reasons of stridulating. The male Gryllus assimilis’
stridulate frequencies are predicted to increase at 25°C, doubling stridulates/min after
adding +10 °C and to decrease at 5°C, by one half stridulates/min after subtracting -10 °C.
Forty-five male Gryllus assimilis’ stridulate frequencies were observed at 5 °C, 15 °C, and
25 °C; forty-five crickets were separated into three temperature variables. The Gryllus
assimilis were held in three separate small Bug Houses 7cm x 12.1cm. The cricket’s
stridute frequencies were individually recorded while placed in a sealed medium plastic
cup for one minute. The cup’s temperature was altered by submerging the cup into cold
and hot water with a thermometer. An ANOVA: Analysis of Variance Between Groups
Test was conducted comparing three unequal variances revealed a insignificant p-vale of p
≥ ± 0.05. The average stridulate frequency for the 5 °C had an average of 90.4 ± 0.0001
stridulates /min, the 15 °C had an average of 140.2 ± 0.0001 stridulates /min, and the 25 °C
had an average of 212.6 ± 0.0001 stridulates/min. The data proves that stridulate rates did
decrease at a rate of 55.08 % stridulates/min per (-) 10 °C and an increased at a rate of
51.64 % stridulates/min per (+) 10 °C change of temperature.
Introduction
Male Gryllus assimilis (Field crickets) produce a
chirping sound called stridulate, at different
frequencies, depending on the size of the cricket and
the temperature. Gryllus assimilis stridulate tediously
by raising their forewing at a 45 degree angle above
their abdomen, rubbing their scrapper along the file.
The file is placed between the base of one front wing
and the bottom side of the other front wing. A vein
with evenly spaced small teeth is positioned under the
file assisting in the production of pulsating sounds
from the vibrating membranes.
The patterns of
pulsations are segmented by the closing of the
forewings which produces the “chirping” sound.
Females lack a file vein and therefore are mute. A
male Gryllus assimilis generates loud stridulates during
territorial fights with other males. Higher frequency,
softer stridulating frequencies lure sexually responsive
females during courtship. Each species have their own
pattern of stridulate songs which attract females of the
same species. The best cricket’s courtship song yields
a females consent to mate. A universal stridulate call
awares other crickets of danger. Cricket’s retrieve
stridulate vibrations in their ears, located right below
what would be their “knees” of its front legs. The ear
openings are within the exoskeleton, connecting to
chambers inside the leg. Changing body positions
allows the cricket to tract the direction that each
stridulate frequency is coming from. In 189, A.E.
Dolbear, a physics professor at Tufts College, created
an equation that solves for temperature by the
frequency of chirps in field crickets. Dolbear’s Law:
T = 50 + (N - 40 / 4).
38
Saddleback Journal of Biology
Spring 2005
“T” refers to the temperature in degrees Fahrenheit and
“N” refers to the amount of chirps per minute.
Dolbear’s Law produces inaccurate estimates of the
temperature outside, because different species of
crickets have different patterns, size, and also the
reason of stridulate changes the frequency.
Materials and Methods
Forty-five male (2.54 cm ± 0.2cm on average)
Gryllus assimilis (field crickets) were purchased at
Petco© and housed in Bug Houses 7cm x 12.1cm.
Fifteen crickets stridulate frequencies were observed at
variable temperature groups; 5 °C, 15 °C, and 25 °C.
One cricket at a time was placed into a medium plastic
drinking cup 19.05 cm height with an 8.89 cm diameter
and sealed with square 12.7 cm Saran wrap timed
during one minute. The plastic cup was submerged
into the temperature variables 20 fluid ounces of water
in a 12.7 cm x 15.24 cm bowl for one minute. The
temperature was monitored by a Celsius thermometer.
The amount of stridulates per minute were counted and
recorded with a stopwatch. Stridulates were counted
based on amount of stridulates produced by a single
cricket per minute. Each cricket was monitored once
within its temperature condition equating to forty-five
trials total.
Results
Temperature greatly affected the crickets stridulate
frequencies. An ANOVA: Analysis of Variance
Between Groups Test was conducted comparing three
unequal variances revealed the 5 °C had an average of
90.4 ± 0.0001 stridulates/min, 15 °C had an average of
140.2 ± 0.0001 stridulates/min, and 25 °C had an
average of 212.6 ± 0.0001 stridulates/min (See Figure
1).
Figure 1: Gryllus assimilis stridulate rates
S trid u la te s
(s trid u la te s /m in )
250
200
150
100
50
0
5C
15 C
25 C
Temperature (C)
Figure 1. Clearly represents the division of increasing
stridulates/minute verses the degree Celsius of
temperature. Between the 15 °C and the 5 °C, a
reduction (-) 10 °C, shows a decreased rate of 55.08%
stridulates/min. Between the 15 °C and the 25°C, an
addition of (+) 10 °C, shows an increased rate of
51.64%. The different affects of temperature did
significantly affect the amount of stridulates per min.
Discussion
The frequency of crickets stridulates have also
been experimented on with Rufocephalus campestris, a
small brachypterous cricket that produces a call with a
carrier frequency of approximately 3.2 kHz from its
burrow. The size of the cricket determines the call
frequency, the Rufocephalus campestris’ length is 9.6
mm. It is suggested that the high purity of the songs
results from close entrainment of the sound-producing
mechanism of the insect’s wings to the sharply
resonant burrow (Bailey, 2001).
Acoustic
communication occurs in vertebrates and arthropods,
and both groups possess specific organs for the
emission and reception of acoustic signals. Sound is
produced when the right file hits the left plectrum
during FW closure and is radiated by the mechanical
resonator made of enlarged areas of both FWs.
Phylogenetic studies have analyzed the acoustic
evolution in crickets and have demonstrated that
acoustic behavior could be particularly liable in some
clades (Desutler-Grandcolas, 2004). The spectrum of
an individual chirps generally contains two frequency
components: the principal component, which was
related to the plectrum-file strike rate, and a second
component, which was related to the free vibration of
the wings (Stephen, 1995). The sublar-tegminal
resonance/ auditory feedback hypothesis attempts to
explain how crickets control their carrier frequency, the
loudness and the spectral purity of their calls. It
suggests that crickets are capable of driving their harps
to vibrate at any frequency and that they use a tunable
Helmholtz-like resonator consisting of the tegmina and
the subalar spaces air amplifies and filters the
frequency (Prestwich, 2000). The frequency of chirps
is associated with compensatory behavior, such as
perching, to permit the propagation of high frequencies
in the natural environment of the species (Robillard,
2004). In conclusion, the frequency of chirps was
affected by the cricket’s ability to change frequencies
through air filtrations during warmer and colder
temperatures. The crickets stridulate frequencies did
double while increasing and did decrease less than one
half, as predicted.
Literature Cited
Bailey W, Benet-Clark H, Fletcher N. 2001. Acoustics
of Small Australian Burrowing Cricket: The Control of
Low-Frequency Pure-Tone Songs. The Journal of
Experimental Biology 204: 2827-2841.
39
Saddleback Journal of Biology
Spring 2005
Desutler-Grandcolas Laure, Robillard Tony. 2004.
Acoustic Evolution in Crickets: Need for Phylogenetic
Study and a Reappraisal of Signal Effectiveness.
Annals of the Brazilian Academy of Sciences 76: 301315.
Robillard Tony, Desutler-Grandcolas Laure. 2004.
Evolution of Acoustic Communication in Crickets:
Phylogeny of Eneopterinae Reveals and Adaptive
Radiation Involving High-Frequency Calling. Annals
of the Brazilian Academy of Sciences 76: 297-300.
Prestwich Kenneth, Lenihan Kristy, Martin Deborah.
2000. The Control of Carrier Frequency in Cricket
Calls: A Refutation of the Subalar-Tegminal
Resonance/Auditory Feedback Model. The Journal of
Experimental Biology 203: 585-596.
Stephen R, Hartley J. 1995. Sound Production in
Crickets. The Journal of Experimental Biology 198:
2139-2151.
The Effect of Temperature on the Growth of Peperomia obtusifolia
Raquel Rosales
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Plant growth is affected by many things such as nutrients, light and temperature.
Temperature seems to be the one we least control. The purpose of this experiment was to
determine whether temperature has a significant effect on plant growth or not. In this
experiment the temperature was increased from around 21˚C (room temperature) to
around 27˚C to determine if higher temperatures have a significant affect on the growth of
Peperomia obtusifolia. The height and diameter of 20 plants, ten placed at room
temperature and ten at a higher temperature, were taken on day one and on day 28. The
average growth in height and diameter was slightly greater in the plants that were placed
under higher temperatures than those at room temperature. This suggests that plant
growth is affected to some extent by temperature.
Introduction
Due to the increasing “greenhouse” gases the
earth’s temperature will increase by several degrees
Celsius within the next one hundred years (Price and
Waser, 1998) and will affect places of higher altitude
more because of the longer snow-free season which
can affect plant growth and reproduction (Price and
Waser 1998). Elevated temperatures can cause a
stimulation of mineralization within the soil helping the
plant grow (Chapin et al. 1995). This could be a
positive outcome especially for crop development. This
is because temperature is a major influence in the
development (Baker and Reddy, 2001). The purpose of
this experiment was to determine whether an increase
in temperature would increase the average growth of P.
obtusifolia as predicted or not. P. obtusifolia was used
because it is a plant that does not require direct sunlight
and can therefore be kept inside where temperature is
more stable and easier to control.
Materials and Methods
In order to calculate the average growth of P.
obtusifolia at room temperature (around 21˚C) and at a
higher temperature (around 27˚C), ten P. obtusifolia
were placed in a cardboard box (56cm x 56 cm) and ten
other plants were put into another cardboard box (56cm
x 56cm) with two heat lamps suspended 26 centimeters
above the plants with a 120v ceramic bulb in each to
raise the temperature. The boxes were placed in a room
where there was no direct sunlight. The height was
measured with a ruler from the top of the pot to the
highest point of the plant. The diameter was taken by
measuring from one end of the plant through the
middle and to the opposite side. The plants were
watered once every four days with 60 milliliters of
water. The average height and diameter was calculated
in order to determine if there was a significant
difference in the average growth at a higher
temperature compared to room temperature.
40
Saddleback Journal of Biology
Spring 2005
Results
Both the heated plants (at around 27-28˚) and the plants
at room temperature (21-22˚C) grew slightly over a
period of 28 days as shown in figures one and two. The
plants that were placed at room temperature had an
average height growth of 1.0 centimeter and an average
growth in diameter of 1.6 centimeters while the heated
plants had an average height growth of 1.15
centimeters and an average growth in diameter of 1.85
centimeters. Although the average growth in height and
diameter is greater in the heated group there is no
significant difference in height (P= 0.346314) and in
diameter (P= 0.311878).
Average Change in Growth Of Peperomia obtusifolia at Room
Temperature
L en g th (cm
25
20
15
Height
10
Diameter
5
0
day 1
at different temperatures one can predict how they
might be affected and how they might react when the
planet itself heats up because of global warming.
Global warming is also more likely to affect plants
growing at higher elevations because it will result in a
longer snow-free season which could potentially affect
plant growth and reproduction (Price and Waser,
1998). Higher temperatures might be more beneficial
during the summer but during the winter it could have
drastic negative consequences on plant survival
(Bertrand and Castonguay, 2003). Although
temperature may help with plant growth by enhancing
nutrient availability, other things such as nutrients and
light have a greater impact and are more critical to
plant growth (Chapin et al. 1995). Overall, many
different plants at different altitudes and places are
affected by temperature differently. Responses to
changes in temperature vary form plant to plant making
it hard to specify a trend in growth depending on
temperature (Chapin et al 1995).
Literature Cited
Baker J.T., Reddy V.R. 2001. Temperature Effects on
Phenological Development and Yield of Muskmelon.
Annals of Botany 87: 605-613.
day 28
Bertrand A, Canstonguay Y. 2003. Plant Adaptations
to Overwintering Stresses and Implications of Climate
Change1. Ottava 81 (12): 1145-1153.
Time (days)
Figure 1.
Chapin F Stuart III et al. 1995. Responses of Artic
Tundra to Experimental and Observed Changes in
Climate. Ecology 76 (3): 694-702.
Average Change in Growth of Peperomia obtusifolia at Higher
Temperatures
L en g th (cm
25
20
15
height
10
Diameter
Price M, Waser N. 1998. Effects of Experimental
Warming on Plant Reproductive Phenology on a
Subalpine Meadow. Econlgy 79 (4): 1261-1272.
5
0
day 1
Liu X, Huang B, Gary B. 2002. Cytokinin Effects on
Creeping Bentgrass Responses to Heat Stress: I. Shoot
and Root Growth. Crop Science 42 (2): 457-466.
day 28
Time (days)
Figure 2.
Discussion
The growth of P. obtusifolia did not seem to
be as affected by temperature as predicted. In a similar
experiment, the artic tundra also shows relatively little
growth response to an increase in temperature (Chapin
et al. 1995). The raise in temperature did not cause any
signs of heat stress such as wilting most likely because
the temperature wasn’t too high for this type of plant,
also because a the air temperature was changed instead
of the soil temperature which can cause more damage
or harm (Liu et al. 2002). By experimenting with plants
41
Saddleback Journal of Biology
Spring 2005
The Effects of Lowing and Raising pH on Goldfish Respiration
Janelle Silva, Juilet Hamner and Rigoberto Aguilar
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
The steady rise of carbon dioxide since the 1960s has caused alarm in the form of
global warming. Carbon dioxide readily dissolves in water forming bicarbonate and
resulting in lowering of the pH. This experiment tested the significance of raising and
lowering the pH (by a factor of 1.0 to 1.5 units) on the respiratory rates of common goldfish
Carassius auratus. Seventy percent of the goldfish tested exhibited higher respiratory rates
in the pH treated water.
Introduction
The common goldfish is one of the tougher of
the fish species. It is actually so hearty, that many
recreational lakes and waterways have put restrictions
on fisherman concerning the use of goldfish as bait
(Lake MV rules/regulations). These restrictions are
trying to protect native fish from deprivation of their
resources due to over population of the goldfish. It is
this heartiness that led this experiment to test pH
differences on the common goldfish.
In a previous experiment, Rainbow T
rout
(Oncorhynchus mykiss) were exposed to water for 30
days from the Rhine River, which has been cleaner in
recent years but is still polluted to a level that caused
significant weight loss beginning after the first seven
days (Nolan DT et al.). Data revealed that the cause
was due to inhibition or exhaustion of the
hypothalamic-pituitary-interrenal (HPI) axis (Nolan
DT et al 2003). Hydro mineral balance of Na+, Cl-, and
K+ were not significantly altered, but activities of Na+,
K+ and ATPase in the intestine and kidneys were. Fish
native to Rio Negro in Brazil were tolerant of waters
with a pH as low as 4.0 to 3.5, and less hardy species
have been observed in Australia in water with 4.5 pH.
In the Brazilian species a pH of 3.25 or lower killed the
fish within six to eight hours. Common goldfish
(Carassius auratus) usually inhabit water with a pH of
7.0-7.8. The resilience of goldfish to water with a
raised, neutral and then lower pH was sought to give
insight into the importance and possible ecological
problems and effects of increasing atmospheric levels
of carbon dioxide on fish.
Materials and Methods
Ten large sized Petsmart goldfish were placed
in a 500mL container with neutral, acidic (pH 5), and
basic (pH 9) solutions one at a time, opercula beats
were counted for one minute. Jungle Labs pH
increaser and decreaser, Aquarium Pharmaceuticals pH
indicator, and Freshwater pH color card were used to
alter and figure the pH of the three solutions. Water
was taken out of the 500mL container and placed in a
testing vile. The pH indicator solution was added to
the vile, and the resulting color of the water was
compared to the test card to determine the pH.
Results
Opercula rates varied from 50 beats per
minute (BPM) in neutral water to 94 beats per minute
in acidic water (figure 1). Means were 70.8 BPM in
neutral, 69.7 BPM in basic, and 83.6 BPM in acidic
(figure 2). A t-test was run between neutral-basic and
neutral-acidic. The difference between neutral-basic
was .4 and the neutral acidic was .87 which is greater
than .05 resulting in a significant difference. The
majority of the fish had higher opercula rates in both
the acidic and basic water. However all the fish
exhibited signs of stress once removed from the bowl
containing the rest of the fish. Stress was determined
by sporadic swimming and jumping out of the water.
Due to the presence of scales and slime floating on top
of the acidic and basic water it can be assumed that the
goldfish lost or experienced damage to these protective
coatings. Three of the goldfish died about two hours
after the tests were done.
Discussion
The observation of higher respiratory rates in acidic
water was the expected outcome of this experiment because
the fish would try to maintain its normal homeostasis. A
possible explanation for this may be due to the activities of
Na+ and Ca2+. At a pH of 3.5 and 6.5 Na+ uptake was
stimulated in Neon-tetras of the Rio Negro, Amazon and
Ca2+ played a role in ion permeability in high pH whereas
alternate Ca2+ independent mechanisms control Na2+ efflux
(Gonzalez, Preest). The tetras used in the Gonzalez, Preest
experiment normally inhabit a slightly acidic river instead of
neutral water like that of the goldfish which may result in
different ion concentration occurring for different pH’s.
42
Saddleback Journal of Biology
Spring 2005
Further testing on the goldfish might reveal similarities in
Na+/Ca2+ activity. In another experiment Blackskirt Tetras
inhabiting acidic waters of the Amazon experienced massive
loss of Na+ at a pH of 5.0 and an 80% inhibition of uptake of
Na+ 9Gonzalez, et al.). Both of the tetra species are native to
and thrive in acidic water yet when the pH is lowered slightly
they experienced ion loss and inability to re-obtain ions.
Tetras inhabiting the acidic water experienced Ca2+ leaching
out from tight junctions that stimulated ion loss, while Na+
effluxed out of the cells when pH was lowered slightly
(Gonzalez, et al.). Our goldfish probably experienced the
same occurrence.
Goldfish usually inhabit water with a pH between
7.0-7.8. The Tilapia living in geothermal springs around
Lake Magadi, Kenya inhabit water with a pH of 9.9.
Experiments done on the Tilapia showed urea production was
inhibited by low pH but does not appear to play a role in
normal acid/base regulation (Wood et al.).
Other
experiments at a lower pH resulted in an intense acidosis
(Wood et al.). Whether or not our fish underwent acidosis in
the lower pH and massive efflux of Na+ like that of the tetras
in a higher pH we don’t know because we only tested for
visual physical effects. . The removal of the slime coat and
deaths of the fish would suggest that the goldfish would not
survive in acidic or basic water if allowed to try and dwell in
it. If pollution of waterways persists these results suggest
goldfish and other fish species will have to evolve
mechanisms like that of the slightly acidic water inhabiting
tetras.
Acidic Waters in the Neon Tetra (Paracheirodon
innesi). 72.
Gonzalez RJ, Dalton VM, Patrick ML.1997. Ion
Regulation in Ion-Poor Acidic Water by the Blackskirt
Tetra (Gymnocorymbus Ternetzi), a Fish Native to the
Amazon River.70 (4):428-35.
California Department of Fish and Game.
http://www.dfg.ca.gov/mrd/fishing.html
Lake Rules and Regulations.
http://www.lakemissionviejo.org/06rules/fishrule.html
Literature Cited
Ellis B, Morris S. 1995. Effects of Extreme pH on the
Physiology of the Australian ‘yabby’ Cherax
Destructor. 198(Pt 2): 395-407.
Wood C, Bergman H, Laurent P, Maina J, Narahara A,
Walsh P.1994. Urea Production, Acid-Base Regulation
and Their Interactions in the Lake Magadi Tilapia, a
Unique Teleost Adapted to a Highly Alkaline
Environment. 189.
McKenzie DJ, Piraccini G, Felskie A, Romano P,
Bronzi P, Bolis CL. 1999. Effects of Plasma Total
Ammonia Content and pH on Urea Excretion in Nile
tilapia. 72.
Wood CM, Part P.2000. Intracellular pH Regulation
and Buffer Capacity in CO2/HCO3-Buffered Media in
Cultured Epithelial Cells from Rainbow Trout Gills.
170.
Nolan DT, Spanings FA, Ruane NM, Hadderingh RH,
Jenner HA, Wendelaar Bonga SE. 2003. Exposure to
Water from the Lower Rhine Induces a Stress
Response in the Rainbow Trout Oncorhynchus Mykiss.
45.
Gonzalez RJ, Preest MR.1999. Ionoregulatory
Specializations for Exceptional Toleranceof Ion-poor,
43
Saddleback Journal of Biology
Spring 2005
The Effect of Olfaction on Hummingbirds’ Foraging Behavior
James A. Steele and Chenae R. Dahlstrom
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
One aspect of the symbiotic relationship that has evolved between angiosperms and
their hummingbird pollinators was tested experimentally. This study examined whether or
not olfactory cues are used by hummingbirds to locate nectar sources. Theoretically,
flowers which attract the highest number of pollinators increase their reproductive success
by increasing the probability of pollen dispersal. The time spent feeding, and the number of
bill probes by Anna’s hummingbirds, were recorded using standard Gardensong® liquid
hummingbird feeders. For five days prior to the experimental testing, the birds were
provided a 20 % sucrose feeder with an ethyl butyrate attractant and a plain water feeder
with no attractant. For these five days hummingbirds were trained solely to associate a
nectar reward with the scented feeder, the feeders were rotated every two hours to assure
position was not a factor. Then, during 30 minute experimentation bouts, the
hummingbirds were provided a feeder supplied with plain tap and no attractant (control
feeder), and a plain water feeder supplied with an ethyl butyrate attractant (test feeder).
Feeding visits where recorded using video and only visits from the downwind side were
recorded. Mean duration times at the test feeder were 6.9±4.7 seconds while the average
duration times at the control feeder were 2.2±1.7 seconds. The test feeder received an
average of 3.6±2.1 bill probes while the control feeder averaged 1.6±0.8 probes. The results
support the hypothesis; the scented feeder had a significantly higher average feeding
duration time (t-test, p = 0.000329) and received a significantly greater number of bill
probes (t-test, p = 0.000949). A 32% increase in the mean duration, and a 45% increase in
the average number of bill probes, suggests hummingbirds favored the test feeder. Results
suggest that olfaction plays a role in helping Anna’s hummingbirds spatially remember a
nectar sources locality. Hummingbirds are also insectivorous, supplementing there diet of
nectar with insect protein. The ethyl butyrate attracted many insects which may also be
important to the hummingbirds.
Introduction
Hummingbirds
have
coevolved
with
angiosperms to form a symbiotic relationship;
flowering plants provide sugars, in the form of nectar,
while hummingbirds act as pollinators to promote
angiosperms’ reproductive success. Hummingbirds,
which consume dilute nectars from many different
flowers, can ingest up to six times their body mass per
day (McWhorter et al., 2003). The black-chinned
hummingbird has an average metabolic rate of 29.1 ±
6.3 kilojoules per day (Chambers, 2004).
Hummingbirds have an extremely high metabolic rate,
a low thermal inertia, and high rate of heat loss (Chai et
al., 1998). Requiring a lot of energy to sustain a high
metabolic rate, hummingbirds choose the most
productive flowers, those which provide a regular
supply of nectar high in sugar concentration (Steele
and Dahlstrom, 2004).
Hummingbirds have learned what time of year
and day different flowers bloom and use both temporal
and spatial memories to locate productive nectar
sources. (Cubie, 2002). They are very efficient feeders
and use sensory cues to locate and pinpoint food
sources. Hummingbirds’ exhibit learned behaviors:
Melendez-Ackerman, Campbell, and Waser (1997)
showed that red flowers containing large amounts of
nectar were preferred over smaller white flowers with
lesser rewards. A hummingbird’s brain makes up 4.2
percent of its body weight, which is nearly twice the
human brain-to-body-weight ratio (Aziz, 2001).
Olfaction plays a major role in bird behavior; even
birds with relatively small olfactory bulbs can sense
odors and use them to find food sources (Malakoff,
44
Saddleback Journal of Biology
Spring 2005
Materials and Methods
This experiment was conducted from 4/12/05
to 4/27/05 in Mission Viejo California, USA. Three
clean Gardensong® hummingbird feeders were filled
to 400 ml; two were filled with tap water while one
was filled with a 20% sucrose solution. 80 grams of
granulated white pure-cane sugar were mixed with 400
ml of tap water to make the 20% sucrose solution. Four
drops of the chemical ethyl butyrate, having a sweet
floral smell, were placed on the attractant feeder
containing the 20% sucrose solution and the test feeder
containing H2O. The control feeder was unscented and
contained only H2O. All 3 feeders were identical, red
with four yellow flowers. Four pea sized cotton balls
were placed between the flowers on all 3 feeders to
absorb drops of ethyl butyrate although no chemical
was placed on the control. Hummingbirds were trained
for the first 5 days to associate the ethyl butyrate scent
with a nectar reward, having only the control and the
sucrose feeder with the chemical attractant hung.
Feeders were hung 30 inches apart on a metal clothes
rack 65 inches from the ground. The positions of the
two feeders were rotated every two hours, between
7:00 and 19:00 PST daily, along with the addition of
the ethyl butyrate attractant to the sucrose feeder. Then
for the next 10 days, during the experimental phase of
the project, the 20% sucrose feeder was switched with
the plain water feeder still bearing the ethyl butyrate
attractant at 9:00 PST. At this time half-hour video
recordings were taken using a Kodak DX6490 digital
camera. Videos were analyzed to see if the scented test
feeder giving no sucrose reward attracted
hummingbirds more than the control. The time spent
within 6 inches of each feeder, along with the number
of bill probes into each flower, were recorded. The
mean times spent at each feeder, and the mean number
of bill probes into each feeder, were analyzed using
unpaired t-tests for unequal variances using Microsoft
Excel. P values ≤ 0.05 were accepted as being
statistically significant. Wind direction was taken into
account and measured using a wind sock and only
hummingbird visits from the downwind side of feeders
were recorded. Two different hummingbird species
were observed, however only visits from Anna’s
hummingbirds, Calypte anna, were recorded, n = 3.
Results
The longest mean duration of time spent by
hummingbirds was 6.9 ± 4.7 seconds at the scented test
feeder, while the control received visits lasting only 2.2
± 1.7 seconds (Figure 1). The feeder treated with ethyl
butyrate had a 32% increase in duration time spent by
hummingbird pollinators when compared to the
control. Statistical analysis using Excel calculated a p
value of 0.000329 and duration times spent at each
feeder were accepted as being statistically significant.
Mean Duration Time (sec)
1999). Ruby-throated hummingbirds have un-fused
olfactory bulbs which were measure 0.81 mm long and
0.57 mm wide, and they have a bulb to hemisphere
ratio of 14% (Goldsmith, 1982). Anna’s hummingbirds
are believed to have similar brain anatomies.
Hummingbirds have keen audiovisual senses, the can
see ultraviolet light along with the three primary colors
(Kevan, 2001). Little research has been done on
hummingbirds’ sense of smell.
The purpose of the present experiment was to
determine if having a floral scent increases a flowering
plant’s potential reproductive success. Each
hummingbird visit or bill probe into a flower
theoretically increases the chances of pollen adhering
to the birds feathers to later be dispersed to other
flowers. Prior to the experimental procedure, a
hummingbird was witnessed feeding from the nectar of
Antirrhinum majus, a scented angiosperm. From this
observation a hypothesis was developed and tested
during a pilot study; do angiosperms attract by scent?
The pilot study showed that a 20% sucrose solution
feeder rubbed with flowers from Antirrhinum majus
received the longest duration times spent by
hummingbird pollinators (Steele and Dahlstrom, 2004).
A significant 52% increase was observed when
compared to the 20% sucrose solution feeder alone.
Adding an organic variable to the synthetic
hummingbird feeder seemed to act as an olfactory
mechanism to attract both hummingbirds and insects
alike. Hummingbirds require protein which they obtain
by eating insects (Yanega, 2002). Scented angiosperms
may be favored by hummingbird pollinators as they
promote the birds overall health and fitness by
attracting insects and providing sensory cues tied to
memory.
8
6.9
7
6
5
test feeder
4
3
2.2
control
2
1
0
Hummingbird Feeders
Figure 1. Average duration time spent by
hummingbirds at feeders; a 32% increase in duration
time was witnessed at the test feeder scented with ethyl
butyrate.
45
Saddleback Journal of Biology
Spring 2005
Along with the duration of time spent at each
feeder, the number of bill probes into each feeder was
recorded. As hypothesized, the test feeder received a
higher mean number of bill probes, averaging 3.6 ± 2.1
probes. The control obtained a mean number of 1.6 ±
0.8 hummingbird probes, a 45% decrease when
compared to the test feeder scented with ethyl butyrate
(Figure 2). Statistical analyses resulted in a significant
p value ≤ .05 (p = 0.000949) and the hypothesis that
hummingbirds use olfaction to aid in locating food
sources was accepted.
4
3.6
Mean Number of Probes
3.5
3
2.5
2
test feeder
1.7
control
1.5
1
0.5
0
Hummingbird Feeders
Figure 2. The mean number of hummingbird probes
into the scented feeder increased 45% when compared
to the control feeder.
Discussion
As the accepted hypothesis suggests; Anna’s
hummingbirds visited the scented plastic water feeder
more than the unscented plastic water feeder. The
results show that the chemical scent clearly induced a
significantly larger number of feeding probes and
increased time spent feeding.
As expected,
hummingbirds have been experimentally determined to
favor plants that provide more nectar (Mitchell et al.,
1998). Olfaction and other sensory cues help
hummingbirds locate productive nectar sources.
Although natural variation in pollinator behavior and
composition may lead to spatial or temporal differences
in hybridization and fitness of plant hybrids (Campbell
et al., 2002), a general trend was observed. An
angiosperms reproductive success could potentially be
benefited by the flowering plants release of a floral
fragrance. Results suggest that angiosperms giving off
a floral odor can attract hummingbirds more
frequently, for longer periods of time, and receive more
probes from hummingbird pollinators, although further
testing with scented and unscented angiosperms must
be done.
Although more hummingbird visits to a
flower increase the flower’s chance of pollen dispersal,
many other isolating mechanisms such as floral
morphology and geographic range affect the flowering
plant’s overall reproductive success. Hummingbirds
are highly territorial (Coniff, 2001). Hummingbird
territoriality may have altered test results as some birds
were witnessed probing at much faster rates when
looking out for competitors and at times where scared
away by competitors. Likewise, hummingbirds were
recorded having many more probes, and staying for
longer durations of time, when no competitors were
present. The hypothesis was accepted using the most
accurate data; the average time spent at each feeder,
and the average number of bill probes into each feeder,
were used. It is speculated that with an increased
spacing of the feeders, even better results may be
obtained.
Hummingbirds have a highly specialized
physiology that is extremely adapted to their
environment, most specifically to the nectar-filled
flowers they feed from. The family Trochilidae has an
average heartbeat of 10 times per second (Doyle,
2004). In order to sustain a heart rate of 1,260 beats per
minute and powerful wing beats up to 200 beats per
second these amazing birds must intake large quantities
of nectar (Aziz, 2001). Having a spatial memory,
hummingbirds can project their cognitive maps to
locate sources of nectar. The hummingbird’s cognitive
memory is important for finding flowers with nectar
throughout the different seasons of the year and of
varying plant species. Knowing where to find
productive nectar sources, hummingbirds have more
time and energy for mating, nest building, and other
important behaviors. Hummingbirds learned to
associate the ethyl butyrate scent with a sweet sucrose
reward by day five and visits to the control almost
ceased although the feeders were continually rotated.
Hummingbirds have played a major role on the
adapting physiology of flowers upon which they feed,
and continue to do so today. Angiosperms release of
odors may be a co-evolutionary adaptation to attract
hummingbird pollinators.
Scented angiosperms may have a selective
edge in attracting hummingbird pollinators. Results
suggest that olfaction may play a major role in helping
hummingbirds locate sources of nectar. Scented feeders
were experimentally shown to attract more
hummingbirds than unscented feeders, although this is
not the only factor in attracting hummingbirds.
Identical plastic feeders were used to account for
flower morphology and color since vision is very
important in the foraging behavior of hummingbirds.
The ethyl butyrate attracted flies, bees, and mites
which could have increased the attractiveness of the
test feeder to hummingbirds. Hummingbirds ingest
arthropods to satisfy their amino acid and nitrogen
requirements (Lopez-Calleja et al., 2003).In a pilot
46
Saddleback Journal of Biology
Spring 2005
study a 20% sucrose solution feeder was rubbed with
sweet smelling flowers from Antirrhinum majus, the
feeder attracted hummingbirds 52% more than a 20%
sucrose solution feeder alone (Steele and Dahlstrom,
2004). From these results it was hypothesized that the
rubbed on organic variable may have provided added
nutrients or have provided an olfactory mechanism for
attracting hummingbirds. This experiment provided
added support for the hypothesis that hummingbirds
use smell to locate food sources, although the organic
variables’ nutritional value was not established.
Angiosperms
with
higher
sucrose
concentrations in their nectar attract a greater number
of hummingbird pollinators, for longer time durations,
than angiosperms with lesser concentrations of sugar in
their nectar (Steele and Dahlstrom, 2004).
Angiosperms use attractants such as color and scent to
further attract pollinators. Angiosperms have
coevolved with hummingbirds; flowering plants utilize
the hummingbird’s senses to attract them and further
their own reproductive success while providing energy
in the form of sugars. Hummingbirds use sensory
mechanisms such as olfaction to locate and remember
the localities of food sources to better sustain there
high metabolism while flying from flower to flower.
The increase in hummingbird visits to the scented
feeder implies that scented angiosperms receive more
visits from hummingbird pollinators, allowing for an
increased probability of a plant’s reproductive success
due to greater chances of pollen adhesion and dispersal.
Many factors besides the olfactory mechanism that was
tested may act to attract hummingbirds. These factors
are embedded in every flower’s unique physiology and
have evolved in angiosperms to promote each species
overall fitness.
To conclude, the experimental procedure will
be duplicated and further studies will be conducted for
the sole purpose of developing a new hummingbird
feeder that promotes the overall fitness of the bird.
Many scientists believe that plastic feeders may harm
hummingbird species by creating synthetic sugar
dependencies while taking away vital nutrients.
Hummingbirds may visit and pollinate lesser flowers
upon having an ample supply of nectar, and may die
when the supply of nectar runs out. This ideal
hummingbird feeder will be one that utilizes all of the
hummingbird’s senses and provides the healthiest
return. Feeders will mimic the ideal sugar
concentration of angiosperms found in the
hummingbird species’ territory, give off the scent of
locally known flowers that hummingbirds are attracted
to, and provide the proper nutrients needed to sustain a
healthy, very active, hummingbird lifestyle. Very little
research has been conducted on Hummingbirds’
olfactory senses. Although this experiment concludes
that hummingbirds do tune into the smell released by
flowering plants, more research must be done to
examine the role olfaction plays in finding food
sources. Hummingbird olfaction may be as important
as sight.
Acknowledgments
The authors thank Steve Teh at Saddleback
College, for laboratory use, references, materials, and
internet links used during the experimental procedure.
Literature Cited
Aziz, Laurel. 2001. First Flower on the Left. Canadian
Geographic 121 i4: 17.
Campbell, Diane R.; Nickolas M. Waser; and Gregory
T. Pederson. 2002. Predicting Patterns of Mating and
Potential Hybridization from Pollinator Behavior. The
American Naturalist 59 i5: 438(13).
Chai, Peng; Andrew C. Chang; and Robert Dudley.
1998. Flight Thermogenesis and Energy Conservation
in Hovering Hummingbirds. Journal of Experimental
Biology 201 n7: 963(6).
Chambers, Lanny. 2004. In-flight Meal. New Scientist
182 i2446: 65(1).
Conniff, Richard. 2001. So Tiny, So Sweeet... So
Mean. Reader's Digest 158 i949: 132(6).
Cubie, Doreen. 2002. Creating a Haven for
Hummingbirds;
(hummingbird-friendly gardens).
National Wildlife: NA.
Dickinson, Mary B.; and Melissa Hunsiker. 2002.
National Geographic; Field Guide to the Birds North
America. Washington D.C.: National Geographic
society. 3p.
Doyle, Brian. 2004. Joyas Volardores. American
Scholar 73 i4: 25(3).
Goldsmith, Kenneth M.; and Timothy Goldsmith.
1982. Sense of Smell in the Black-Chinned
Hummingbird. Condor 84: 237 (2).
Kevan, Peter G.; Lars Chittka; and Adrian G. Dyer.
2001. Limits to the Salience of Ultraviolet: Lessons
from Colour Vision in Bees and Birds. Journal of
Experimental Biology 204 i14: 2571.
Lopez-Calleja, Victoria M.; Maria Jose Fernandez; and
Francisco Bozinovic. 2003. The Integration of Energy
and Nitrogen Balance in the Hummingbird
Sephanoides sephaniodes. Journal of Experimental
Biology 206 i19: 3349(11).
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McWhorter, Todd J.; Donald R. Powers; and Carlos
Martinez del Rio. 2003. Are Hummingbirds
Facultatively Ammonotelic? Nitrogen Excretion and
Requirements as a Function of Body Size.
Physiological and Biochemical Zoology 76 i5: 731(13).
Malakoff, David. 1999. Following the Scent of Avian
Olfaction. Science 286 i5440: 704.
Melendez-Ackerman, Elvia; Diane R. Campbell; and
Nickolas M. Waser. 1997. Hummingbird Behavior and
Mechanisms of Selection on Flower Color in
Ipomopsis. Ecology 78 n8: 2532(10).
Mitchell, Randall J.; Ruth G. Shaw; and Nickolas M.
Waser. 1998. Pollinator Selection, Quantitative
Genetics, and Predicted Evolutionary Responses of
Floral
Traits
in
Pestemon
centranthifolus
(Scrophulariaceae). International Journal of Plant
Sciences 158 n2: 331(7).
Pezzullo, John C. 2004. John C. Pezzullo's Home
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Steele, James A.; and Chenae R. Dahlstrom. 2004. The
Effects of Varying Sugar Concentrations of Nectar on a
Plant’s Reproductive Success Due to Hummingbird
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Yanega, G.; and M. Rubega. 2002. The Hummingbird
Bill as a Utensil for Insectivory: Prey-capture and
Transport in the Ruby-throated Hummingbird.
Integrative and Comparative Biology 42 i6: 1340(1).
Effect of Bread Components on Net Mold Production Over Time
Ashley Waugh
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Kingdom Fungi is a diverse group of organisms that range from basic water molds
to gigantic portabella mushrooms. Common molds such as Penicillium and Rhizopus also
belong to the Kingdom Fungi. This study was performed in order to determine whether
different types of bread contained components that inhibited mold growth. Four types of
bread: Albertson’s brand white bread, Don Francisco Sourdough bread and Sara Lee
100% Whole Grain Wheat Bread both purchased at Albertson’s, as well as George’s
Lavash purchased at the Jordan Market, were moistened with water and left out to be
exposed to any spores in the air (n=10). The individual pieces of bread were sealed and put
in boxes to develop mold. After the 7 days it took for all pieces to develop mold it was
found that the white bread had the greatest area of growth (36.1±12.2 cm2), lavash yielded
the second highest area (31.6±18.1 cm2), sourdough (31.6±21.0 cm2) and the wheat bread
(14.9±16.1 cm2). An ANOVA and Bonferoni correction test was performed to determine
statistical significance among the overall area of mold growth on the 4 types of bread. It
was found that there is no statistical difference among the overall mold growth (p=0.057),
nor was there a statistical difference between any of the two types of bread. It was found
that there was no statistical difference in average Penicillium growth (p=0.108), Rhizopus
(p=0.904), Aspergillis (p=0.943), yellow (p=0.224), or red (p=0.372). The only statistical
difference was found in the growth of the orange mold (p=0.001). This could be due to
water content or retention, the presence of preservatives such as calcium propionate, or the
presence of the lactic acid bacteria present in sourdough bread. Mold contamination
during production and packaging of bread and other baked goods can have detrimental
effects on one’s health as well as unfavorable economic ramifications.
48
Saddleback Journal of Biology
Spring 2005
Introduction
Molds are a member of the Kingdom Fungi
along with the millions of other different species of
fungus known to scientists, and there are potentially
many more waiting to be discovered. Some of these
species can be deadly; others aid our body in simple
functions such as digestion, while others serve as a
form of food (mushrooms). Molds, and usually fungus
all together, is not a favorite house hold item,
especially when food is concerned. Although some
molds, such as certain species of the genus Penicillium
are used to ripen cheeses such as Roquefort or Brie,
other molds, even other species of Penicillium, can be
harmful or even deadly if ingested. According to Suhr
et al, the spoilage of most bakery products (breads) is
mostly cause by molds and yeasts and sometimes
bacteria found floating in the air. He goes on the say
that molds in the dough are killed during the baking
process, this means that all contamination must occur
after the bread is baked, the most common contaminant
being various types of Penicillium species (68). The
spores then settle on their substrate (the bread) and
begin to mature into a haploid (2n) structure known as
hyphae. The hyphae then begin to spread along the
surface and burrow into the substrate for nutrients and
anchorage, this is the “white fuzz” that is commonly
seen on the surface. Dependent on whether the fungi
will undergo sexual or asexual reproduction will
depend on the next structure produced. In the case of
Penicillium, the common bluish-green mold that the
common antibiotic Penicillin is derived from, from the
Phylum Deuteromycota, asexual reproduction is the
only form that has been charted. When the conditions
are ideal and the hyphae have matured a haploid
structure known as a conidiaphore is produced (Perry,
31). The condiaphore then produces conidia, the site of
spore production via mitosis; this is the dark bluishgreen fuzz.
Lavermicocca et al, states that
contamination can be prevented by using a modified
atmosphere or by adding certain fungal inhibitors or
preservatives such as ethanol, propionic acid and acetic
acid and their salts shelf-life can be extended (634).
She goes on to say that there are several components of
sourdough the show signs of antifungal activity. For
example, a lactic acid bacteria, Lactobacillus
plantarum, along with phenyllactic acid, lactic acid and
acetic acid are main components in sourdough’s natural
fungicidal properties (634).
In this study, several different types of bread
were exposed to the naturally occurring spores in an
average household and were let to spoil or mold for
approximately one week in order to determine what
ingredients or properties of certain types of bread
would cause it to mold faster or slower than others.
Methods and Materials
On April 9, 2005 at 9:00 PM in Dana Point,
Orange County, California, ten pieces of Albertson’s
Brand white bread, ten pieces of Don Francisco’s
Sourdough Bread purchased at Albertson’s, ten pieces
of Sara Lee 100% Whole Wheat Bread also purchased
at Albertson’s, and ten pieces of George’s Lavash
purchased at the Jordan Market were all spread out in a
random order on a table. All forty pieces were then
sprayed with water five times on each side with a
house-hold spray bottle in order to ensure the bread
remained moist throughout the experiment. The bread
was then allowed to sit out to be exposed to any spores
or bacteria in the air for twelve hours. After the twelve
hour period, April 10, 2005 at 9:00 AM, each piece of
bread was individually sealed in a Zip-Loc™ sandwich
bag and labeled with the bread type and piece number
(ex: White 1). The sealed bread was then carefully
placed in only one layer inside 5 boxes. The boxes
were used in order to filter out as much light as
possible, as well as block wind that could alter the
temperature more than day to day fluctuation. While in
the box, the pieces of bread were left to sit and grow
for as long as it was necessary to have growth on every
slice of bread. Each piece was thoroughly inspected
every twenty-four hours for beginning signs of growth.
After each piece of bread showed signs of growth,
April 17, 2005, the bread was thoroughly inspected and
each colony was counted and the diameter was taken
with a centimeter ruler, keeping each species separate.
Using Microsoft Excel the area of each colony, the
total area of each species, the total growth for each
piece of bread, the average growth for each type of
bread per species and the average overall growth for
each type of bread was calculated. An ANOVA and
Bonferoni correction test were performed in order to
establish whether or not there was a statistical
difference in the total growth of each kind of bread and
the total Penicillium growth for each type of bread.
Since only the white bread and the lavash had other
species growing besides Penicillium, an unpaired t-test
was used to establish statistical significance among the
other species found.
Results
The first signs of mold growth appeared on
the white bread on Thursday, April 14, 2005.
However, immature (white) mold may have begun
growing earlier and was not visible against the white
bread. It was not until Sunday, April 17, 2005, that
every piece of bread had mold on it. When the bread
was checked on April 15, the wheat bread had finally
begun to show the first signs of visible growth. When
each kind of bread was inspected it was found that the
Albertson’s brand white bread had the most area of
49
Saddleback Journal of Biology
Spring 2005
60
50
A re a (c m ^ 2 )
40
30
20
10
0
-10
White
Lavash
Sourdough
Wheat
Bread Type
Figure 1. Average Overall Area of Mold Growth (cm2).
Graph shows the average total area of mold growth for
each type of bread (n=10), also shows the standard
deviation for each type of bread. The white bread had
the highest overall growth, the lavash had the second,
the sourdough yielded the third most and the wheat had
the least amount.
60
50
A r e a (c m ^ 2 )
growth (36.1±12.1 cm2), George’s Lavash showed the
second highest area of growth (31.6±18.1 cm2), the
Don Francisco Sourdough showed the third highest
average area of growth (31.6±21.0 cm2), and the Sara
Lee Wheat had the smallest area of growth (14.9±16.1
cm2) (Fig. 1). When the specific species were
examined it was found that the all four types of bread
had Penicillium growth; however, only the white bread
and lavash showed signs of other species. Fuzzy black
mold, Rhizopus; a very dark black mold, Aspergillis; a
yellow colored mold, an orange mold and a red mold
were all found on both the white bread and the lavash.
The average area for each species and each type of
bread was calculated. It was found that Albertson’s
white bread produced 1.17±1.52 cm2, and George’s
Lavash had 1.31±3.26 cm2 of growth for the Rhizopus
species. When the area of Aspergillis growth was
measured it was found that Albertson’s white bread
had 0.733±0.489 cm2 and George’s Lavash had
0.706±1.07 cm2. The yellow colored mold produced
an average of 3.47±2.44 cm2 on white bread and lavash
had 2.07±2.50 cm2; the orange mold showed and
average of 2.35±1.79 cm2 on white bread and the
lavash showed an average of 0.03533±0.112 cm2.
Finally, the red colored mold produced an average of
0.01256±0.03972 cm2 on the Albertson’s white bread
and 0.154±0.487 cm2 on the lavash. Figure 2 illustrates
the average growth of each species on each type of
bread.
Penicillium
40
Rhizopus
30
Aspergillis
20
Yellow
Orange
10
Red
0
-10
Albertson’s
White Bread
George’s
Lavash
Don
Francisco
Sourdough
Bread Type
Sara Lee
100% Whole
Grain Wheat
Figure 2. Average Area of Growth per Species on 4
Bread Types. Graph illustrates the average area of
growth ± the standard deviation for all 6 mold species
found. Only Penicillium was found on the sourdough
and wheat bread, and only very small amounts of the
orange and red mold were found which makes them not
visible on the scale of the graph (values included in
Table).
The ANOVA tests performed revealed that
there was no significant difference in the average
overall mold growth in all four types of bread
(p=0.057). The Bonferoni correction test showed that
the was no statistical difference in the total average
growth between any of two types of bread (white v
sourdough, white v lavash, white v wheat, sourdough v
lavash, sourdough v wheat, lavash v wheat). The
second ANOVA test performed showed that there was
no significant difference in the average total
Penicillium growth either (p=0.108), the Bonferoni
correction test performed for this set of data also
revealed no statistical difference in the average
Penicillium growth when comparing two types of bread
at a time. Since the sourdough and wheat bread only
had Penicillium growth, an ANOVA test could not be
used to determine statistical significance for the other
species. The unpaired t-test showed that there was no
statistical significance in the average area of growth of
any of the other species of mold on the white bread and
lavash except the orange mold (p=0.001); Rhizopus
(p=0.904), Apergillis (p=0.943), yellow (p=0.224), red
(p=0.372).
Discussion
Based on the data obtained and the results
from the statistical analysis it was found that the four
different types of bread did not have a significant
difference in the overall mold growth over the given
period of time (7 days, p=0.057). The analysis of the
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Saddleback Journal of Biology
Spring 2005
data also showed that there was only a statistical
difference among the growth of the orange colored
mold (p=0.001). Even though there was no statistical
significance among the area of mold growth on the
different types of bread the white bread did show the
greatest area of mold growth over the lavash,
sourdough and wheat bread (Fig. 1). The white bread
and lavash were the only types of bread that showed
growth of other mold species besides Penicillium
species (Fig. 2). While the white bread had a greater
area of mold growth and was the first to mold, once the
lavash began (one day later) it grew at a much faster
rate then tapered off rather quickly. This could be due
to the lack of preservatives such as calcium propionate
in the lavash, which was present in all the other kinds
of bread. According to a study by Suhr et al, propionic
acid (an its salts) inhibit molds and Bacillus spores, but
not yeasts to the same extent, which is why it has been
a common choice for bread preservatives (68). A
possible reason why the white bread would have
molded first even though it contains the preservatives
is because of its spongy consistency, which would
allow it to absorb and retain a greater amount of water
than the others. Although sourdough is a spongier
bread than the lavash and would retain more water
(resulting in more ideal conditions), the sourdough
contains antifungal components that have been found
in many studies.
For example, in a study by
Lavermicocca et al, it was found that acetic acid
concentration was strictly related to antifungal antivity
and that other bacterial metabolites also have inhibitory
affects. She goes on to report that the antifungal
activity of sourdough lactic acid bacteria varies, but is
found mainly in heterofermentative Lactobacillus,
specifically the Lactobacillus sanfranciscensis CB1
due to the production of a mixture of organic acids
(4084). Since the wheat bread does not contain the
lactic acid bacteria and other organic acids that inhibit
mold growth, rather it only contains the preservative
calcium propionate, the wheat bread would be
predicted to mold before the sourdough; however, the
wheat bread is not able to absorb and retain as much
water because the bread is much coarser and denser
than the sourdough. This lack of water absorption as
well as less processed whole wheat grains could
potentially attribute to the later presence of mold
growth. In order to help support whether water content
had a significant effect on overall mold growth an
additional analysis of water content of the fresh bread
as well as the water content of the bread after it was
moistened an exposed to determine the amount of
water each piece of bread absorbed or retained over the
exposure period would have been helpful.
Determining proper conditions for bread
production and storage in order to protect against
harmful contamination by mold and bacteria is
necessary to protect consumers against potential death
due to the ingestion of the harmful mycotoxins
produced by many types of molds. There are millions
of mold spores circulating in the air, hiding in soil and
the damp environment between walls of a house
waiting for ideal conditions so that it can move to
another area with more nutrients, food for example.
Even though the health effects of mold infection can be
devastating, the contamination of food products can
also be extremely costly economically to bakeries and
fruit producers around the world. We will never be
able to completely eliminate contamination; however,
many steps could be taken to reduce the risk, such as
more sterile environments and better transportation
conditions. Even with appropriate precautions, the
burden will always lie on the consumer to check
bakery, produce and all other products for signs of
spoilage before consumption.
Literature Cited
Lavermicocca, Paola; Francesca Valerio, Antonio
Evidente, Silvia Lazzaroni, Aldo Corsetti and Marco
Gobbeitti. 2000. “Purification and Characterization of
Novel Antifungal Compounds from the Sourdough
Lactobacillus plantarum Strain 21B.” Applied and
Environmental Micobiology. Vol. 66: 4084
Lavermicocca, Paola; Francesca Valerio and Angelo
Visconti. 2003, “Antifungal Activity of Phenyllactic
Acid again Molds Isolated from Bakery Products.”
Applied and Environmental Microbiology.
Vol.
69:634.
Meroth, Christiane B.; Walter P. Hammes and
Christian Hertel. 2003. “Identification of Population
Dynamics of Yeasts in Sourdough Fermentation
Processes by PCR-Denaturing Gradient Gel
Electrophoresis.”
Applied and Environmental
Microbiology. Vol. 69:
Perry, James W. and David Morton. 1996. Photo
Atlas for Biology. Wadsworth Publishing Company.
Pg 31.
Petersson, Stina; and Johan Schnürer.
1995.
“Biocontrol of Mold Growth in High-Moisture Wheat
Stored under Airtight Conditions by Pichia anomala,
Pichia guilliermondii, and Saccharomyces cerevisiae.”
Applied and Environmental Microbiology. Vol 61:
Suhr, K.I. and P.V. Nielsen. 2004. “Effect of Weak
Acid Preservatives on Growth of Bakery Product
Spoilage Fungi at Different Water Activities and pH
Values.” International Journal of Food Microbiology.
Vol. 95: 67-68.
51
Saddleback Journal of Biology
Spring 2005
The Effect of NaCl on the Metabolic Rate of Crassius auratus
Robert Ward
Department of Economics
Saddleback College
Mission Viejo, CA 92692
Oxygen is the key to life for every living organism, for without one shall parish.
Crassius auratus is no exception and as a substance such as NaCl disrupts oxygen intake the
organism will die slowly. Forty Crassius auratus were evenly divided into four groups of
ten and placed one by one into specific NaCl concentrations to demonstrate the necessity of
oxygen and disruption of NaCl on a fresh water organism. A sample size of n=10 Crassius
auratus for each NaCl concentration ranging from 0%, 1%, 3.5%, and 15% was used. For
each Crassius auratus the metabolic rate was determined by each gill opening and closing.
Each rate was averaged for the specific concentration and an average opercular rate per
minute was derived and used to represent the metabolic rate of Crassius auratus in each
NaCl concentration. After concluding this experiment it was recorded that the mean
opercular beat per minute was 70.8 in the 0%, 53.87 in the 1%, and 22.48 in the 3.5%. An
ANOVA test and a Bonferroni test confirmed the statistical difference between each
metabolic rate, confirming that in fact the NaCl was disrupting oxygen intake. Introducing
NaCl to a freshwater environment will disrupt the oxygen flow to Crassius auratus
disabling cellular respiration, halting ATP production, and slowly killing the organism.
Introduction
The environment of any species defines the life
style and health of any organism (Brauner et al., 2004).
In the case of a Crassius auratus (goldfish) an
appropriate environment, allowing for an optimal
metabolic rate, consists of room temperature (22.0°C),
and unadulterated H20 (Lee et al., 2003).
An
appropriate environment is desired for the purpose of
respiration. Respiration is the ultimate necessity of
Crassius auratus, for without, no other functions are
capable.
Respiration occurs only to conduct cellular
respiration. Cellular respiration is the determinant of
life and death for a living creature. Cellular respiration
allows the organism to trigger ATP production through
the use of proteins and hydrogen. When NaCl is
introduced to Crassius auratus’s environment it has a
direct effect on the cellular respiration. NaCl is used to
dehydrate and the same principle is affective in respect
to Crassius auratus. NaCl is inevitably forced into the
fish through each intake of the gill. Once the NaCl
enters Crassius auratus’s system the proteins are
dehydrated losing their function to act as a transport for
hydrogen, hence all ATP production has ceased. With
out ATP production the fish will die. Such a process
was conducted using rainbow trout ending with similar
results (Hawkings et al., 2004).
It is expected that for Crassius auratus, an
increase in the salt concentration from 0% to higher
concentrations such as 1% and 3.5% will effectively
decrease the metabolic rate, decreasing the opercular
rate. Such a result will derive from the decrease in
ATP production of cellular respiration as seen the
Hawkings et al., (2004) experiment of 2004.
Materials and Methods
Six one-gallon round fish bowls were
obtained from Petco in Rancho Santa Margarita, CA,
along with a small fish net and forty Crassius auratus
(goldfish) seven cm. in length. Crassius auratus
were immediately brought into my kitchen in Mission
Viejo, CA and transferred into one of the one-gallon
fish bowls, which was filled completely with tap H2
O at 22.0°C. The remaining empty bowls were
clearly labeled one through five to prevent future
confusion. Two liters of tap H2 O at 22.0°C were
measured and poured into a clean two-liter coke
bottle creating a perfect mixing device suitable for
dissolving NaCl. Twenty grams of NaCl, table salt,
were carefully measured and placed in the two liter
coke bottle using a funnel conveniently purchased at
Lowes Hardware in Rancho Santa Margarita, CA.
The mixture, 1% NaCl, was shaken until no salt
particles were visible. The one-gallon fish bowl
labeled 1 was filled with the latter solution. Using
52
Saddleback Journal of Biology
Spring 2005
the small fishnet a single Crassius auratus was
extracted randomly from the forty and placed
carefully into bowl number one. Using a timer, five
minutes were allowed for proper acclimation to the
new environment. Immediately after acclimation the
timer was set for five minutes and a tally of gill
openings began in order to determine Crassius
auratus’s metabolic rate. When the five minutes
concluded Crassius auratus was carefully retrieved
with the small fishnet and placed into bowl 5, filled
completely with tap H2O and used as a storage bowl
to separate the used fish. The solution containing
twenty grams of NaCl was stirred once again with the
small fishnet to prevent settling and accumulation of
NaCl at the bottom of the bowl. The processes was
repeated nine more times including the acclimation of
Crassius auratus, the tally of gill openings, and the
stirring of the solution. The completed process will
accumulate ten tally readings.
A similar process was conducted for bowl
2. Two liters of tap H2O at 22.0°C were measured
and poured into the two-liter coke bottle. Seventy
grams of NaCl were measured and funneled into the
two-liter coke bottle. The two-liter bottle was
shaken until the salt particles were not visible. The
solution, 3.5% NaCl, was poured into the Bowl
labeled 2. Using the small fish net a random
Crassius auratus was extracted and placed carefully
into the solution of bowl 2. An acclimation period of
five minutes was conducted and the five minute
metabolic tally began. At the conclusion of five
minutes the small fishnet was used to retrieve
Crassius auratus and it was placed into bowl number
5. The solution containing seventy grams of NaCl
was stirred once again with the small fishnet to
prevent settling and accumulation of NaCl at the
bottom of the bowl. The processes was repeated
nine more times including the acclimation of
Crassius auratus, the tally of gill openings, and the
stirring of the solution. The completed process will
accumulate ten tally readings.
This familiar process was repeated for bowl
3. Two liters of tap H2O at 22.0°C were measured
and poured into the two-liter coke bottle. Three
hundred grams of NaCl were measured and funneled
into the two-liter coke bottle. The two-liter bottle
was shaken until the salt particles were not visible.
The solution, 15% NaCl, was poured into the Bowl
labeled 3. Using the small fish net a random Crassius
auratus was extracted and placed carefully into the
solution of bowl 3. An acclimation period of five
minutes was conducted and the five minute metabolic
tally began. At the conclusion of five minutes the
small fishnet was used to retrieve Crassius auratus
and it was placed into bowl number 5.
Bowl 4 was used as the control group. Two
liters of tap H2O at 22.0°C were measured and
poured into the two-liter bottle. The H2O was
poured straight into bowl 4. Crassius auratus was
randomly extracted from bowl 6 and transported to
bowl 4 with the small fishnet. Five minutes of
acclimation were allowed and the five minute
metabolic tally followed immediately.
At the
conclusion of five minutes Crassius auratus was
retrieved by the small fishnet and placed into bowl
five. The solution was stirred with the fishnet and
the experiment conducted nine more times for a tally
of ten readings.
All individual metabolic tallies for all five
minute intervals were divided by five to find the
metabolic rate per minute for each Crassius auratus.
Using the metabolic rate per minute data, an
ANOVA (MS Excel) test was run on to find
statistical
difference
the
different
NaCl
concentrations; p≤ 0.05 was considered statistically
different. After the ANOVA, a Bonferroni correction
was run to conduct a multiple comparison between
the different NaCl concentrations to see where the
differences
of
the
ANOVA
occurred
(http://graphpad.com/quickcalcs/posttest1.cfm), p≤
0.0167 was considered statistically different.
Results
After analyzing data recorded from all
groups of NaCl concentration, a large variance
becomes obvious in the metabolic rate of Crassius
auratus. An average for the 1% NaCl group was
53.9 opercular beats per minute, 22.5 opercular beats
per minute for the 3.5% NaCl concentration, and
70.8 opercular beats per minute for the control
concentration of 0% (figure 1). Note that bowl 3’s
concentration of 15% NaCl was fatally toxic to
Crassius auratus and results were not included.
Utilizing the average opercular beats per minute
from the 0% concentration; a comparison to the
NaCl solutions presents an immediate 24% decrease
in opercular beats per minute in Crassius auratus
from the 1% NaCl concentration, and a 64%
decrease in opercular beats per minute in Crassius
auratus from the 3.5% NaCl concentration.
Discussion
While following trends in the average
opercular beats per minute for Crassius auratus in
the various NaCl concentrations a clear pattern
exposes itself. As the NaCl concentration increases
as the opercular rate decreases. It becomes evident
that the NaCl is decreasing oxygen supply to the
fish therefore decreasing its metabolic rate. As the
metabolic rate decreases the production of ATP
decreases essentially killing Crassius auratus with
53
Saddleback Journal of Biology
Spring 2005
Crassius auratus and eventually kill the fish.
Crassius auratus will also have a very hard time
regulating a normal pH balance considering the
regulatory organ for maintaining pH balance is the
gills (Hawkings et al., 2004).
Another possible explanation for the
decreasing metabolic rate while the NaCl
concentrate increases is a chain reaction that
corresponds with the latter explanation.
As
proteins lose their function due to dehydration,
ATP production is greatly decreasing. As ATP
production
decreases
organs
functioning
decreases, hence gill movement decreases
significantly halting any oxygen consumption.
Metabolic Rate (op. beats/min)
Average Opercular Beats Per Minute in
Varying NaCl Concentrations
80
70
60
50
40
30
20
10
0
0%
1%
3.50%
NaCl Concentration
Figure 1. Crassius auratus averaged 70.8
opercular beats per minute in the 0% NaCl
concentration, 53.87 in the 1% NaCl solution,
and 22.48 in the 3.5% NaCl solution. All
comparisons were statistically different from each
other (p≤ 0.0167).
every gill intake (Alexander 1999). The steep
decline of 16.93 opercular beats per minute when
comparing the average Crassius auratus in the 0%
NaCl solution to the average Crassius auratus in
the 1% Nacl solution is dramatic considering 99%
of the solution remained unchanged. Even more
dramatic is the 64% decrease of opercular beats per
minute (decrease of 48.32 ob/m) when comparing
Crassius auratus in the 3.5% NaCl solution to
Crassius auratus in the 0% NaCl solution.
The NaCl must be causing a disruption in
oxygen intake considering the gill is the primary
site for aquatic respiration (Evans et al., 2005).
Such a rapid decrease in opercular rate per minute
suggests that NaCl can be fatally toxic for Crassius
auratus. NaCl introduces a possible two-fold effect
on Crassius auratus. In other words the NaCl is
harming Crassius auratus in two separate ways.
NaCl is greatly affecting Crassius
auratus’s pH level. The environment in the 1%
and 3.5% solutions contains high levels of NaCl
compared to the normal environment for Crassius
auratus. During aquatic respiration an intake of
high levels of NaCl will dehydrate proteins of
Literature Cited
Alexander, R. M. 1999. Energy for Animal Life.
New York. Oxford University Press. p32-43.
Brauner, C.J., V. Matey., J.M. Wilson., N.J.
Bernier., and A.L. Val. 2004. Transition in an
organ function during the evolution of air-breathing;
insights from Arapaima gigas, an obligate air
breathing teleost from the Amazon. Journal of
Experimental Biology 207: 1433-1438.
Evans, D.H, P.M. Piermarini, and K.P. Choe. 2005.
The Multi Fish Gill: Dominant Site of Gas
Exchange, Osmoregulation, Acid-Base Regulation,
and Excretion of the Nitrogenous Waste. Physiology
Rev. 85: 97-177.
Hawkings, G.S, F. Galvez, and G.G. Goss. 2004.
Seawater acclimation cause independent alterations
in Na/K and H-ATPase activity in isolated
mitochondria-rich cell subtypes of the rainbow trout
gill. Journal of Experimental Biology 207: 905-912.
Lee, C.G, A.P. Farrel, A. Lotto, M.J. MacNutt, S.G.
Hinch, and M.C. Healey. 2003. The Effect of
Temperature on Swimming Performance and
Oxygen Consumption on Adult Sockeye
(Oncorhynchus nerka) and coho (O. kisuthc) salmon
stocks. Journal of Experimental Biology 206: 32393251.
54
Saddleback Journal of Biology
Spring 2005
Effects of Ginkgo biloba on Memory in Mus musculus
Amir Akhavan & Julius Chiu
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
With a significant rise in many memory related problems caused by variety of
factors such as aging, an inherent need arises to mitigate this problem. Ginkgo
extract is one of the most widely used supplements for memory improvement.
Millions of people take ginkgo hoping it would boost their memory. However,
results of many studies done on the effectiveness of ginkgo on memory in the past
few decades have been conflicting. The purpose of this experiment was to determine
the effects of ginkgo on memory in Mus musculus (feeder mice). 10 healthy adult M.
musculus of relatively same size were randomly chosen for this study. A maze was
constructed and 10 M. musculus were introduced to the maze once a day for a
period of 14 days without ginkgo. Then the maze was reoriented and M. musculus
were introduced to the new maze once a day for another 14 days with ginkgo added
to their diet. The result indicate that the average solving time of the maze for the
experimental group decreased 169s over the period of 14 days where as the average
solving time for the control group decreased 129s over the same period. Therefore
there was a slightly higher decrease in average solving time for the group with
ginkgo as part of their diet. However a t-test was run and a p value of equal to 0.48
was obtained witch suggests no significant differences between these two groups.
Therefore it is concluded that G. biloba had no effects on memory of M. musculus
over a period of 14 days.
Introduction
Currently in today’s society memory is one
of the key factors of determining a normal life. It
plays a major role in the way people interact with
each others, how they perform simple tasks, and how
they adapt to their environment. With the increasing
number of individuals who suffer from Alzheimers,
dementia and other degenerative, memory-related
disorders, an inherent need arises among modern
society to mitigate this problem. Also, with the large
workload students are put under in certain highdemand majors, a need for improving memory is
once again apparent.
It is believed by neuroscientists that
memory is located in specific sets or circuits of
neurons, and that brain chemicals may be the ink
with which memories are written. It is known that
actually specific parts of the brain are responsible for
memory function. One section is the hippocampus,
located in the temporal ventral sides of the brain,
which is responsible for explicit memory and spacial
memory (Beldarrain et al, 2002). A sufficient blood
supply to the brain is essential for the brain to
function properly. When aging, the amount of blood
supply to the brain decreases, which leads to
chemical changes and degeneration of brain cells by
free radical. Comparing observation of a normal
brain with that person suffering from Alzheimer
reveals that hippocampus is significantly smaller in
the brain with Alzheimer compare to a normal brain.
This is due to degeneration of cells in hippocampus
by free radicals (Grandstorm, 2000). According to
Grandstorm, an increase of blood flow to the brain
will reduce degeneration of the hippocampus, and
memory may even be improved among healthy
adults.
New studies of dietary supplements are
published every day and science is discovering new
uses for different plants and herbs. One of these
plants which has drawn many researchers’ attentions
is the Chinese plant G. biloba. Traditional uses for
ginkgo extract included the alleviation of cough,
asthma, bronchitis, and allergic inflammation (Singh,
2004). Additionally, in the past decade more than
300 studies have been done to show how this plant
provides many benefits including enhancing
acquisition, retention and recall of memory.
55
Saddleback Journal of Biology
Spring 2005
Millions of people take the herbal
supplement of ginkgo, hoping it will boost their
memory. However, scientific evidence of its
effectiveness on memory has been conflicting. While
some research shows an effectiveness of ginkgo on
memory enhancement, other studies suggest no
difference at all. According to Solomon et al. 2002,
based on a six-week study of 203 participants,
ginkgo did not facilitate memory retention or
improve learning processes. Many doctors, herbalists
and nutritionists believe that the doses of ginkgo
extract given to the subjects, and also the short
period of the experiment, might have affected the
outcome of this study.
Since there is still much debate on the
effectiveness of this supplement on memory, the
purpose of this experiment was to determine any
affects of ginkgo on memory in M. musculus. This
species was particularly chosen for this experiment
because they are small and have similar
fundamental biological principles and physiology to
humans.
Materials and Methods
A maze with the dimensions of 24x18x4
inches was constructed using Styrofoam and metal
nails. Two inches of space between all the walls of
the maze was allowed for the M. musculus to move
forward and backward. The top portion of the maze
was securely covered with a large piece of
plexiglass to make sure outside environmental
factors such as smell and heat do not interfere with
the experiment. Five grams of cottage cheese was
placed at the end point of the maze to signify
completion.
A total of ten adult M. musculus (6 males,
4 females) were chosen randomly, and each
specimen was kept in a separate cage to ensure the
same amount of food and water availability. Every
evening at 8:00pm, each specimen was weighed
with an electrical scale and the data was recorded.
The specimen would then be placed at the starting
point of the maze. The total time to solve the maze
and reach the cheese at the endpoint was recorded in
seconds using a digital stop watch. After each run,
all parts of the maze were treated with “C-dox”, a
scientifically advanced odor eliminator, to eliminate
any odor left behind from the previous mouse. Also,
each specimen was given two grams of “Forti-Diet”
and five mL of tap water at 8:45pm every evening.
The food was taken away next morning at 8:00am
and the M. musculus fasted for a period of almost 13
hours until 8:45pm.
All ten specimens were run inside the maze
once a day at 8:00pm for a total period of 14 days.
After the data was collected from the first trial, the
maze was reoriented so the M. musculus would solve
the maze backward, essentially creating a new maze.
Then for another 14 days, all ten specimens were run
inside the reoriented maze once a day. The amount
of food and water given and the time in which they
were given were kept constant, with the only
difference being that one drop of ginkgo was added to
the 5 mL water supply each day. After the second 14day trial, the data was collected again to run a pair of
t-tests and a p-value of 0.48 was obtained.
Results
The daily average solving time of the maze
for 14 days for both control group (without ginkgo)
and experimental group (with ginkgo) is constructed
on the graph in figure 1. The daily average solving
times decreases over a 14 days period for both groups.
Figure 1 indicates that from day 4 to 5, daily
average solving time of the maze for the group not
subjected to ginkgo decreased significantly from 202s
to 127s and then reached 230s on the 6th day. This
huge fluctuation is assumed to be associated with error
or some environmental factors. A similar, though not
at dramatic, pattern was observed for the group
subjected to ginkgo from days 8 to 10. The total
difference in daily average solving time from the first
day to day 14th was equal to -120s for the group not
subjected to ginkgo and -169s for the group subjected
to ginkgo. The p-value calculated is equal to 0.48
which suggests no significant difference between the
daily average maze completion time of the control and
experimental group.
Daily Average completion Time for Fourteen Days Trials
300
T im e (s e c ond )
250
200
Without Ginkgo
150
With Ginkgo
100
50
0
Da
y1
Da
y2
Da
y3
Da
y4
Da
D
D
D
D
D
D a D a Da
Da
y 5 ay 6 ay 7 ay 8 ay 9 ay
1 0 y 1 1 y 12 y 1 3 y 1 4
Days
Figure 1. Daily average completion time for group
subjected to ginkgo and the group not subjected to
ginkgo for 14 days
56
Saddleback Journal of Biology
Spring 2005
Discussion
The daily maze completion time decrease
for both groups over 14 days period. However, the
group subjected to ginkgo showed a more gradual,
yet slightly faster, decrease than the group not
subjected to ginkgo. This might be due to increase
of blood flow to the brain in M. musculus. Ginkgo
extract dilates arteries such as carotid artery, which
supplies blood to the head. This results in increase
of blood flow to all parts of body including head. As
a result, the brain will receive more oxygen and
glucose and will function more efficiently (Peppiatt
et al 2004).
However, the fact that p=0.48 and is
less than 0.05 alpha level indicates no significant
difference between daily average completion time in
each group. Therefore, it can be concluded that
although ginkgo extract might have slightly
improved blood flow in M. musculus, it fails to
enhance memory in significant degree over a period
of 14 days.
Few side factors might have affected
the outcomes of this study. One reason could be
the maturity (age) of the M. musculus . When the
M. musculus were first purchased, they were
extremely skittish. After a few weeks however,
they became more settled. Also when tested the
older the M. musculus
became the more
methodical the M. musculus became, instead of
wandering around every possible route within
the maze. A theory for this nervous behavior can
be from the fact that M. musculus are a prey
species, and are constantly looking out for
predators (Rop, 2001). Possibly since threat from
other animals is eliminated the M. musculus
became more comfortable, which also links back
to being acclimated to the training schedule.
To improve this experiment, an
adequate amount of time is needed to train the
M. musculus , for more consistent results. Also
another factor to be taken into consideration
should be the amount of time the M. musculus
are tested with the ginkgo. As seen in other
studies mentioned earlier, the short time could
have an affect on the outcome. In addition, a
larger sample size with an equal amount of male
and female would be ideal to test not only if the
ginkgo has an effect, but sex has one as well.
Acknowledgments
We thank Saddleback College for
providing us with important equipment such as
electrical time watch and balance; and also we
would like to thank Professor S. Teh for his help
and support.
References
Beldarrian GM, Gafman J, Ruiz De Velasco J,
Pascual Leone, A. 2002. Prefrontal Lesions
Impair the Implicit and Explicit Learning of
Sequences In Visuomotor Tasks. Experimental
Brain Search 142: 529-538.
Grandsom JK. 2000. Ginkgo Biloba: A Treasure
From The Past. Life Extension 6(5): 32-40
Higbee, KL. 2004. What Aspects of Their
Memories Do College Students Most Want to
Improve?. College Student Journal 38(4):5
Peppiatt C, Attwell D. 2004. Neuroscience: On
Blood Floe In The Brain. Nature 431:137
Rop C. 2001. Mouse Behavior; Conjectures
About Adaptations For Survival. The American
Biology Teacher 63(5): 346-352
Singh B, Song H, Liu X, Hardy M, et al. 2004.
Dangshen (Codonopsis Pilosula) and Bai Guo
(Ginkgo Biloba) Enhance Learning and Memory.
Alternative Therapies in Health and Medicine
10(4):52-27
Sommer BR, Schatzberg AF. 2002. Ginkgo
Biloba and Related Compounds in Alzheimer’s
disease. Psychiatric Annals 32(1):13-23
Solomon PR, Adams F, Silver A, Zimmer J,
DeVeaux R. 2002. Ginkgo for memory
enhancement: a randomized controlled trial.
JAMA 288:835-840
57
Saddleback Journal of Biology
Spring 2005
1.
The Effect of High pH on the Photosynthetic Rate of Elodea canadensis. Carolyn Dreyer,
Katie Bennett, Katheryn Saab. Department of Biological Sciences, Saddleback College, Mission
Viejo, 92692, USA.
Oxygen is a byproduct of photosynthesis. Therefore, by measuring the oxygen production rate
the photosynthetic rate can be indirectly measured. In earlier photosynthesis experiments, the
pH level of the water surrounding the Elodea plants was lowered. Lowering pH levels creates an
acidic environment, which significantly affected the photosynthetic rate. In this experiment, the
pH of the water surrounding the Elodea was raised to 9 and 10. First, the water was adjusted to
1.0 N sodium bicarbonate to provide carbon dioxide for the photosynthesis process; then sodium
hydroxide was added until the desired pH level was reached. Eight 17-cm long Elodea segments
were placed in 8 large test tubes. Each test tube was then filled with the pH 9 water and capped
with a manometer. All experimental tubes were paired with a control test tube containing only
the pH treated water capped with a manometer. The eight Elodea segments were placed under
incandescent light at about 5000 Lux for 30 minutes. At two minute intervals, the movement of
the water in the manometers was measured. This process was repeated for a second set of Elodea
plants at pH 10. There was a significant difference in oxygen production between plants at the
two pH levels. The average production of oxygen at pH 9 was 0.0588 milliliters, whereas the
average production of oxygen at pH 10 was 0.0135 milliliters.
2.
Growth of Tomato Plants under Different Wavelengths of Light. Nam Le and Nina Turk,
Department of Biological Sciences, Saddleback College, Mission Viejo, 92692, USA.
Thirty tomato plants were tested to see growth under different colors of light. Colored
cellophane was used to produce blue, red and white light. Plants were divided evenly among the
three categories. The mean initial height of the plants in the blue light was 16.59cm and had a
growth to 17.87 cm. Plants in the white light had a mean initial height of 17.3 cm and had a final
height of 18.7 cm. Lastly the plants under red light had a mean initial height of 16.22 cm and a
final mean height of 23.85 cm. Though data suggest that plants absorb the red light the most and
therefore can undergo photosynthesis more efficiently than the plants under the other colors, it is
inconclusive because white light should have the same effect since it has all wavelengths of light.
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3.
Effects of alkaline pH on the metabolic rate of goldfish (Carassius auratus). Julie G. Rhodes
and Angelina Thompson. Department of Biological Sciences, Saddleback College, Mission
Viejo, CA 92692 USA.
Goldfish are heterothermic ectotherms. Ectotherms are animals that match their body
temperature to their living environment. The purpose of this experiment was to measure the
effect of an alkaline pH environment on the metabolic rate of goldfish. Earlier experiments have
established the effects of lower pH on the opercular pumping rate in goldfish. At low pH
respiration increases; supporting the hypothesis that the fish’s respiratory control center sees this
as in increase in dissolved carbon dioxide. Based upon these data, we hypothesized that the
metabolic rate will decrease as the pH level increases above neutral pH. The opercular pumping
rates of six fish were observed and recorded after acclimating for 10 minutes in neutral water and
increased pH water. To obtain the desired pH level of the test environment, 1mL of 4% sodium
hydroxide was added to each experimental beaker. Contrary to our hypothesis, the opercular
pumping rates of goldfish in increased pH water were significantly higher (113.6 per min) than
those in neutral water (80.3 per min, p <0.5, one tailed t-test). We suggest that shifts in pH
(either higher or lower) may result in an increase in activity, which results in increased
respiration.
4.
Is a more symmetrical face more likely to secrete better smelling pheromones and attract
more mates? Jennifer Quinn Department of Biological Sciences, Saddleback College, Mission
Viejo, CA 92692 USA.
Pheromones and symmetry are relatively new areas of study in both the biological and
psychological fields. In this study, five females ages 18-25 were asked to wear the same shirt for
three days. The only exterior products allowed were ones given to them. Then fifty males ages
18-25 were asked to rate each shirt’s smell from best to worst or 1 to 5. These results do show a
significant difference in attractiveness in relation to symmetry. The most symmetrical girl
received a higher frequency for best smell. Her frequency rate was p=0.36 and she only had 1
mm difference in her facial lines of symmetry. The rest of the girls fall in order of symmetry as
well. There was also an interesting result for the 5th spot or worst smelling shirt. The order that
the shirts were returned from first to last is shown also in the data for this rating. Based on these
results it would appear that the pheromones do send out signals to mates that the body is more
symmetrical, and therefore attracts more mates.
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5.
The Effects of Ultraviolet radiation on the HA2 yeast strain. Sinikka Kreuzer and Ethel
Soriano. Department of Biological Sciences, Saddleback College, Mission Viejo, CA 92692
USA.
HA2 strain yeast colonies were grown on YED agar Petri dishes and counted. Twelve cultures
were created all together. These cultures were then exposed to ultraviolet radiation for different
periods of time; two were not exposed as a control. For every time period two cultures were used
the results for the two cultures were then averaged for more accurate results. After being exposed
to the ultraviolet radiation the cultures were incubated so that the colonies surviving could grow
and show the effect the ultraviolet radiation had. The ultraviolet radiation had two major effects
on the yeast colonies; number of colonies surviving and occurrence of mutant white colonies
among the red colonies. With longer ultraviolet radiation exposure the percent of surviving
colonies decreased; 100% of the control survived with no ultraviolet radiation, 94% survived at
10 seconds of exposure, 72% survived at 20 seconds of exposure, 52% survived at 30 seconds of
exposure, 21.5% survived at 45 seconds of exposure, and 6% survived at 1 minute of exposure.
The ultraviolet radiation also caused the occurrence of mutation in the yeast colonies, the longer
the colonies were exposed to the ultraviolet radiation the more frequent the occurrence of white
mutants was in the surviving colonies; there was about 1% mutation at 10 seconds, 6% mutation
at 20 seconds, 12% mutation at 30 seconds, 26% mutation at 45 seconds, and 50% mutation after
one minute.
6.
Respiration of Mice (Mus musculus) at Differing Altitudes. Matt Ireland and Jacob Smith.
Department of Biological Sciences, Saddleback College, Mission Viejo, CA 92692 USA.
Mus musculus, or the common house mouse, exists virtually worldwide due to their introduction
by humans to nearly every habitat. Mice are homoeothermic endotherms, meaning that their
body temperature does not readily change relative to the ambient temperature. In this
experiment Mus musculus was exposed to different altitudes. We hypothesize that metabolic
rates should increase at high altitude as the mice work harder to get oxygen. The weight specific
basal metabolic rate was measured at sea level and at 1,945 meters. Manometric respirometers
were used to measure the oxygen consumption of 2 male and 2 female mice over a period of
time. The time taken to respire 10 cc of air was recorded. In every case, the oxygen
consumption was less at the higher altitude. The average oxygen consumption at sea level was
7.61 mL O2/g/hr. The average oxygen consumption at 1,945 meters was 5.82 mL O2/g/hr. All
measurements were converted to STPD. Oxygen consumption was significantly less at higher
altitudes (one tailed t-test, p<0.05). The pressure decreased from 759.8 mm Hg at sea level to
604.6 mm Hg at 6,380 ft. This decrease in pressure caused the partial pressure of oxygen to
decrease from 158.8 mm Hg to 126.4 mm Hg. A decrease in the partial pressure of oxygen
causes a decline in percentage of hemoglobin saturation (oxygen’s affinity for hemoglobin
decreases). There was no increase in respiration to compensate for the decrease in oxygen.
Paradoxically, there is less oxygen being consumed per unit time at the higher altitude.
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7.
Effects of Basic pH on the Metabolic Rate of Comet Goldfish (Carassius auratus). Jerry Lee
and Lacey Meyerhardt, Department of Biological Sciences, Saddleback College, Mission Viejo,
CA 92692 USA.
Goldfish are heterothermic ectotherms which means that their metabolic rates are directly
affected by ambient temperature. The metabolic rate is controlled by the opercular structures
which contract and force water over the gills allowing the diffusion of oxygen and carbon
dioxide. By measuring the opercular pumping rate, the respiratory metabolism of goldfish can be
estimated. However, factors such as pH may also affect the metabolic rate of goldfish. In this
study, the opercular pumping rate of Carassius auratus (Oranda goldfish) was measured in water
with a pH of 10. Goldfish were first placed in water with a pH 7 and the rate of opercular
pumping was measured for three intervals of 120 seconds to serve as a control. Subjects were
then placed in water with a pH 10 and the rate of opercular pumping was measured again in three
intervals of 120 seconds. The mean operculum pumping rate at pH 7 was 151.5 ± 27, whereas
the mean opercular pumping in pH 10 was 123.5 ± 53. A paired t-test indicated that a significant
decrease occurs in opercular pumping rates between pH 7 vs. pH 10. From these data we infer
that the metabolic rate of Carassius auratus (Oranda goldfish) decreases as pH increases.
8.
Attraction of Fruit Flies (Drosphila melanogaster) to Different Substances. Joseph Ling and
Yama Osmazada. Department of Biological Sciences, Saddleback College, Mission Viejo, CA
92692 USA.
The purpose of this experiment was to identify the source that attracts fruit flies
(Drosophila melanogaster). In an effort to further understand which component draws these
flies, a fish tank containing five different vials filled with five different items was used. The flies
were placed into the fish tank and the tank was sealed off to prevent the flies from escaping. The
fruit flies were allowed to roam freely in the container for a number of hours. After a certain
amount of time, each fly was counted and the results were assessed. This procedure was
conducted a total of three times. The percentage of animals drawn to each substance was 82.1%
yeast, 5.7% phenylthiourea, 0.8% water, 10.6% pineapple, and 0.8% control. In each trial, the
vial that attracted most of these flies, approximately eighty-two percent, was the one which
contained the yeast. We have concluded from these results that yeast is the main element that
draws in the fruit flies. Although there were flies that flew into the other vials, there were not a
sufficient number of them to draw any conclusions that those items can attract the fruit flies.
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9.
The Effects of Varying Ambient Temperature on the Resting Metabolic Rate of
Budgerigars (Melopsittacus undulatus). John Supance, Shallom Han, Matthew McGeough.
Department of Biological Sciences, Saddleback College, Mission Viejo, CA 92692 USA.
This experiment measured the change in metabolic rate (oxygen consumed per unit time) of
budgerigars, Melopsittacus undulatus at different ambient temperature. The metabolic rate of
eight subjects was measured at ambient temperatures between 0oC and 50oC. It was
hypothesized that metabolic rate in these animals, being homoeothermic endotherms, would be
unaffected by its ambient temperature within the zone of thermoneutrality. At temperature above
or below that zone, we hypothesized that metabolism would increase. As ambient temperature
approached 0oC, the metabolism of the bird increased. Contrary to our hypothesis, however, as
ambient temperature increased, the metabolism of the bird did not increase. These results
indicated that the environment in which budgies are acclimated is susceptible to ambient
temperatures of greater amount than the environment studied. Therefore, the upper critical
temperature for these animals would be much greater than anticipated for their survival in warm
environments.
10.
The Effects of pH on Oxygen Production of Elodea canadensis. Erfan Shenghur and Shane
Zarifzadeh Department of Biological Sciences, Saddleback College, Mission Viejo, CA 92692
USA.
Elodea canadensis has a great impact on the aquatic environment of many lakes by providing
habitat for many aquatic invertebrates as well as cover for young fish and amphibians. Elodea is
also of economic importance as an attractive and easy to keep aquarium plant which can provide
oxygen to its surrounding habitat. This study examined the oxygen production of Elodea under
three different pH conditions: pH 7.0, pH 6.0, and pH 8.0. Oxygen production was measured
using a manometer and a syringe fitted onto a rubber test tube stopper. The rubber stopper,
along with the syringe and manometer, was then fitted onto a large test tube containing Elodea in
either an acidic, neutral, or basic solution. The length of Elodea, intensity and amount of light,
pH of solution, amount of sodium bicarbonate, and temperature were all controlled. Eight trials
were run for thirty minutes each for each of the three pH solutions. The mean oxygen produced
in thirty minutes for pH 7.0 was 0.635 mL, pH 6.0 was 0.502 mL, and pH 8.0 was 0.332 mL.
The results showed that there was a significant decrease in oxygen production between the three
different pH solutions (ANOVA, p = 4.71 x 10-8). The most oxygen was produced by Elodea in
neutral pH and there was more oxygen produced by Elodea placed into an acidic solution than
Elodea placed into a basic solution. A possible explanation for the results could be that
enzymatic activity in Elodea might have an effect on oxygen production because certain
enzymes may only function within certain pH range and most enzymes in Elodea are better
suited to a slightly acidic to neutral environment rather than a basic environment.
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11.
Body size does not affect the metabolic rate in goldfish at ambient temperature. Nastran
Aghazadeh, Supriya Chaudhary, Nam Phuong Nguyen. Department of Biological Sciences,
Saddleback College, Mission Viejo, CA 92692 USA.
Metabolic rates of poikilothermic animals such as fish are dependent on a number of
environmental factors such as temperature, oxygen, light, and season. In addition, differences in
metabolic rates are influenced by the size of the individuals, their activity, and sex, even under
similar environmental conditions. We hypothesize that size of fish should affect the metabolic
rate, measured as the opercular pumping rate. In the present experiment the opercular pumping
rate of goldfish of various sizes and constant ambient temperature was measured. Fish size was
determined by weight and length. The results did not support our hypothesis; neither mass nor
length of the goldfish affect significantly the opercular pumping rate.
12.
The Effect of Reduced Barometric Pressure on Goldfish Opercular Pumping Rate. Shengchieh Chang and Albert Trinh. Department of Biological Sciences, Saddleback College, Mission
Viejo, CA 92692 USA.
One key characteristic of life is the ability to respond to environmental change in order to
maintain homeostasis. While there are many different types of environmental variables, the
effect of barometric pressure on the metabolism was the primary concern of this study. Pressure
changes in aquatic environments are significant due to their effect on the diffusion of gases such
as oxygen. The survival of fish in environments that vary from 8000m deep ocean trenches to
mountain lakes over 2000m high was the starting point for our hypothesis. In this study, fish
respiration at high altitudes was simulated. The rate of opercular movement over a period of 2
minutes at 3 pressures created inside a vacuum desiccator was measured. At 0.75 atm, roughly a
2km increase in elevation from sea level, the fish exhibited a small but significant increase
(4.31%) in rate of opercular movement. However when pressure was decreased to 0.5 atm,
approximately a 5km elevation above sea level, the opercular movement rate dramatically
decreased by an average of 30.8%. One-way analysis of variance (ANOVA) with post-hoc
testing showed that minor pressure reduction has no significant effect on the opercular activity of
goldfish while the 50% pressure drop caused significant reduction in this rate on the big and
medium fish. For the smallest fish, the pressure drop from 0.75atm to 0.5atm caused a significant
decrease in opercular pumping rate; however, no significant difference was seen between the
measurements at 1atm and 0.5atm.
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13.
The Effects of Polarized Light on the Oxygen Production in Elodea canadensis. Sean
Stanton, Misty Guzman. Department of Biological Sciences, Saddleback College, Mission Viejo,
CA 92692 USA.
Photosynthesis requires light, and the quality and quantity of light influences photosynthetic
activity and cellular respiration. By measuring the rate of oxygen production of Elodea we can
estimate the photosynthetic rate. Oxygen production was measured in Elodea exposed to
coherent (polarized) and incoherent light. Two trials were conducted using control and
experimental tubes containing Elodea. Each tube was filled with a 0.1 N sodium bicarbonate
solution. Tubes were then exposed to polarized or non-polarized incandescent light. A 50%
decrease in oxygen production was recorded in tubes exposed to polarized light. Lux
measurements indicated the total light flux reaching the plants was reduced 50% by the
polarizing filter. This could have accounted for the reduction in oxygen production.
14.
The Effects of Electric Current on Cell Growth in Onions (Allium cepa). Russell E.
Roberson. Department of Biological Sciences, Saddleback College, Mission Viejo, CA 92692
USA.
Electrostatics is the primary governing force involved in the various movements within a
growing cell during the mitotic cycle. In this experiment, the significance of these forces was
tested through the application of an electric current on a group of pearl onions (Allium cepa) over
four days. Four sets of two onions each were placed in electrophoresis apparatuses containing
positive and negative electrodes, with one onion placed on either side. The onions were partly
submerged within the apparatus under 150mL of tap water, for the dual purpose of supplying
oxygen to the plants, as well as carrying the electric current. The experiment was run twice, with
the voltage raised by fifty percent for the second run (10V to 15V, respectively). A control set of
four onions were grown during the same periods of time within a similar apparatuses, without the
electric current. The results showed a 2.6% increase in growth during the first run (10V), but a
1.3% decrease in growth during the second run (15V). There was no significant statistical
difference in the growth between the positive or negative sides.
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