Download Department of Life and Earth Sciences (Alphabetical Order)

Department of Life and Earth Sciences
2006-2007 ABSTRACTS OF SENIOR RESEARCH PROJECTS
(Alphabetical Order)
AMANDA K. APPLEGARTH
The Role of Epitope Specific CD8+ T Cells in T1 Black Hole Formation and Motor
Dysfunction in an Animal Model of Multiple Sclerosis
The presence of MRI T1 black holes, a type of brain lesion, in MS patients correlates
with disability through an unknown mechanism. A murine model of T1 black hole
formation using the Theiler’s murine encephalitis virus (TMEV) was developed. In the
TMEV model previous studies have determined that CD8 T cells are mediating T1 black
hole formation. Injection of the VP2121-130 peptide prior to infection with TMEV causes a
depletion of VP2121-130 specific CD8 T cells and preservation of motor function. The
purpose of this pilot study was to determine if these Db: VP2121-130 specific CD8 T cells
contributed to T1 black hole formation in our IFN-γR-/- mouse model system. It was
hypothesized that antigen specific CD8 T cells would contribute to T1 black hole
formation in the TMEV infected IFN-γR-/- mice and these T1 black holes would correlate
with disability. By treating mice with VP2121-130 peptide prior to and during TMEV
infection expansion of Db: VP2121-130 epitope specific CD8+ T cells was inhibited. The
total volume of T1 black holes per mouse was measured using T1-weighted MRI brain
scans and Analyze 7.0 at days 14 and 49. Rotorod was used to assess motor
dysfunction twice a week. At days 14 and 49 no correlation could be seen between T1
black hole lesion volume and motor dysfunction. Injector error and age effect of the
mice were considered to skew the results.
STEVE BEELEY
Effects of Respiratory Muscle Training on Forced Expiratory Volume and Peak
Flow in Competitive Male Athletes
In competitive exercise it is crucial that the volume of air passing through the alveoli per
unit time (alveolar ventilation) is maximized in order to optimize oxygen uptake and
therefore athletic performance. Expand-A-Lung™ (Miami, FL) is a device that purports
to increase ventilatory capacity by training respiratory muscles against airflow
resistance during a specified daily regimen, but there is little research to support this
claim. The hypothesis of this study was that such training of ventilatory muscles over
four weeks in male track athletes would produce an increase in ventilatory capacity as
measured by peak expiratory flow rates (PEFR) and forced expiratory volume in one
second (FEV1). Fifteen male track athletes were recruited as the experimental group
with fifteen age–matched male track athletes as the control group. Baseline ventilatory
data were collected: FEV1 and Vital Capacity (VC) using respiratory transducers (Biopac
Student Lab Inc., Goleta, CA), and PEFR, a measurement of ventilatory flow (L/min),
using a professional peak flow meter (Wright™, Louisville, CO). A specific training
regimen for using Expand-A-Lung™ was developed. All subjects performed their daily
athletic practice; the experimental group additionally used Expand-A-Lung™ daily. Data
collection was repeated at two and four weeks. Analysis by repeated measures ANOVA
compared the means of the experimental group’s ventilatory function with those of the
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control group at baseline, two and four weeks. No significant increase in FEV1/VC or in
PEFR was observed from the use of Expand-A-Lung™ over two or four weeks. This
could be because the respiratory muscles of athletes (although not specifically trained)
have little margin for increased power, or alternatively because the training regimen
used was insufficiently rigorous in the resistance used and/or the training time. Studies
using increased resistance and training time would determine which of these applies.
ADAM BLALOCK
The Development and Analysis of an Assay for DNA Adduction and PAH
Metabolites
Polycyclic Aromatic Hydrocarbons are potentially harmful organic substances that are
cytotoxic, mutagenic, and genotoxic. A PAH is a substance that contains two or more
conjugated rings made of carbon and hydrogen atoms that contain no heteroatom or
side chains. Exposure to humans can come from eating food, drinking liquids, breathing
air, or even skin contact to substances that contain PAHs. In our experiment, we
examined the reaction between 1,2-naphthoquinone and calf thymus DNA (ctDNA). It is
our belief that when these two compounds react, they will form such adductions. An
assay was also developed to detect DNA adducts formed by the known PAH
metabolite, 1,2-naphthoquinone. This assay is based on tetramethylammonium
hydroxide (TMAH) thermochemolysis, which hydrolyzes and methylates the adducted
metabolites so that they can be detected through gas chromatography-mass
spectrometry (GC/MS) and High Performance Liquid Chromatography (HPLC). The
TMAH thermochemolysis assay shows promise as a means for detecting DNA adducts
formed by PAH quinone metabolites. It does allow for the identification and
quantification of reaction peaks formed by ctDNA and 1,2-naphthoquinone through the
use of GC/MS.
CHERISH BRAM
Growth Analysis of Haloalkaliphilic Bacteria
Mono Lake is a hypersaline alkaline lake with an average pH of greater than 10.0,
located in the Sierra Nevadas of California. It is home to extremophiles bacteria called
haloalkaliphiles. Samples were collected from thirteen locations including samples from
dry limestone tufa, salt grasses and iron precipitates near the shore line. All samples
were cultured on SW10 minimal media in a range of alkaline pH broths, and then
analyzed for optimal pH through growth curves over a four day period. Analysis of four
different pHs (7.2, 8.0, 9.5 and 10.5) revealed that the optimal pH for most bacteria
cultured from Mono lake was between 8.0 and 9.5. Growth at higher pH was limited for
most bacteria, however many bacteria did sustain slow growth at pH 10.5. Although not
optimal, the ability to sustain growth at this alkaline pH gives alkaliphiles a unique niche.
Gram stain show a range of bacteria, with most classified as gram positive bacilli. One
spirochete was identified, that shows a phenotype similar to bacteria previously
identified at Mono Lake Spirochaeta americana.
DENISE BUNGER
Identification and Comparison of Microbial Gut Populations of Wild Freshwater
Turtle Digestive Tracts
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Herbivorous vertebrates require microbial gut symbionts to ferment plant cell walls.
Although these symbionts are fairly well studied in mammals, few studies have been
done in reptiles. The purpose of this research is to describe microbial gut populations in
wild, freshwater turtle species, and to identify a possible relationship between microbial
populations and diet. Turtles were trapped using floating basking traps and hoop traps
at Otterbein Pond in Westerville, OH. A total of thirty turtles were sampled: ten painted
turtles, Chrysemys picta, twelve map turtles, Graptemys geographica, seven red-ear
sliders, Trachemys scripta elegans, and one yellow-bellied slider, Trachemys scripta
scripta. A sterile plastic pipet tip was inserted into the cloaca of each turtle to obtain a
bacterial sample from the large intestine. Bacteria were then inoculated on Tryptic Soy
Agar (TSA) plates and grown in candle jars at room temperature, ensuring an anaerobic
environment. Colonies grown on TSA plates were then isolated and identified as grampositive or negative using Phenylethyl Alcohol (PEA) and Eosine and Methylene Blue
(EMB) plates respectively. Colonies were then isolated from these plates and inoculated
in enterotubes, designed to perform multiple biochemical assays at once. These test
results were then used to help determine the identification of bacteria. Initial analyses
indicate that approximately six unknown species were found, two of which were species
of the Megashaera genera. Further biochemical assays, as well as DNA sequencing,
is needed to be performed in order to identify the remaining bacteria species.
Additionally, according to these results, no specific relationship can be established
between turtle diet or species and bacterial species found in the gut at this time.
DOMINIC M. DEPOMPEI
An Analysis of Chlorophyll Concentration in Relationship to Water Depth, And
Turbidity, In Thalassia testudinum in Belize Reef Environments
Turtle grass (Thalassia testudinum) adapts to decreasing light (increasing depth) by increasing
area specific chlorophyll content. This study examined two aspects of turtle grass chlorophyll
adaptations. The first was whether Thalassia was light saturated in shallow water and the second
was how chlorophyll content varied with environment, in particular between windward and
leeward sites. At each of three patch reefs, central Belize Barrier Reef system, ambient
illumination was measured at various depths. Fifteen turtle grass leaves at each depth were then
harvested and their chlorophyll concentration was recorded and no saturation point was found.
Windward sites were significantly brighter at 3 and 5 meter depths and had significantly lower
chlorophyll concentrations than leeward sites. These results explain why turtle grass
productivity could be higher and deeper in windward settings.
JIMMY FEE
Culturing and Isolating of Microbes from Acid Mine Drainage in Perry and
Hocking County Ohio
Acid Mine Drainage is the environmental problem of acidic contamination in water
systems caused by abandoned coal mines. The Monday Creek in Perry and Hocking
County Ohio has a problem with acid mine drainage causing the water to have an
extremely low pH varying from 6.1 to 2.2. Samples were collected from two sites on
Monday Creek; Little Monday Creek and Rock Run. The samples were inoculated in a
9-K basal salt solution and SW10 solution liquid media to culture acidophilic microbes.
Microbes grew in the SW10 solution liquid media at a pH 2.6 and pH 4.0 and were then
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cultured on LB agar plates at pH 4.0. Pure culture isolation streaks were performed and
three different types of microbes were isolated. No bacteria were successfully isolated
from the acidic environments, which was unexpected. However three types of fungi
were successfully cultured. Two of the isolations were from the Zygomycota fungi
phylum based on sporangium and one of the isolated cultures was identified as a
pseudohyphae budding yeast. The results show that there are microbes living in the
extreme acidic environments within the Monday Creek acid mine drainage sites. Studies
conducted by Cal-Berkley University and The Ohio State University have shown these
types of microbes, as well as others, growing in them. Further work using different
media agars, metabolic biochemistry and RNA sequencing techniques could further
identify species of microbes located in this water region. A number of different fungi and
bacteria have been used in wetland construction in order to filter out heavy metals and
return the pH to normal ranges. Identification of these microbes can help in the
bioremediation of acid mine drainage areas.
MARSHALL H. GOODMAN
A Mussel Index of Biotic Integrity (M-IBI)
Freshwater mussels (Unionidae) are often the largest and longest lived members of the
benthos of streams. They are essentially immobile, filter water continuously for food
and oxygen, and have a complex life cycle, including a parasitic larval stage.
Freshwater mussels integrate water quality, habitat quality, and symbiotic function
throughout their lifetimes and often leave behind a record of their presences in the form
of a dead shell. All of these suggest mussels could be valuable indicators of the health
of a stream or its ecological integrity. This study examined the relationship between
mussel community structure and water resource integrity of the Little Miami River
system from 1990 to the present. The analysis resulted in a Mussel Index of Biotic
Integrity (M-IBI). An Index of Biotic Integrity uses the biological community to help
determine the health of a system. The index is composed of ten metrics that represent
rarity (three metrics), reproductive potential (three metrics), and community structure
(four metrics). The validity of M-IBI was determined by using mussel data from
reference sites from various Ohio streams. Reference sites (for excellent, good, fair
and poor) were selected based on their mussel communities alone. The M-IBI scores
calculated from these sites were correlated with predicted scores for the M-IBI. This
correlation was found to be significant by means of regression analysis. Once the MIBI was field-truthed in this manner, it was used to calculate M-IBI scores for the Little
Miami River mussel community data collected in 2006. The resulting M-IBI scores were
compared with individual species richness and species diversity data as well as water
chemistry and fish health data for the Little Miami River. The previous mussel (1990
and 2006) data and fish data demonstrate areas of degradation in the Little Miami River
system. The M-IBI scores capture this degradation for the Little Miami River system.
B. ANN HOEDT
Macroboring along Depth Gradients at Enriquillo Valley, Dominican Republic and
Wee Wee Caye Patch Reef, Central Belize
Macroboring, a main component of bioerosion within coral reef systems, was
investigated at two reef systems: a living reef around Wee Wee Caye, Central Belize
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and a fossilized, turbid reef in Enriquillo Valley, Dominican Republic. Macroboring
patterns at each site were evaluated in four or five facies (lagoon, branching, mixed,
massive, and platy) to discover how macroboring intensity and type varies with depth in
the Caribbean. Coral samples were collected from surface rubble in Belize and in situ
pieces along horizontal transects in the Dominican Republic. Pieces were evaluated
for taphonomic grade (surface condition) and then examined in cross section to
determine macroboring intensity, and main borers. Average macroboring intensity
varied between 13-23% in different facies in the Dominican Republic, while the Belize
average was much higher (31-38%) with little difference between facies. Highest levels
in the Dominican samples occurred in both very shallow and very deep facies. Types of
borers also differed markedly between sites. The Dominican Republic site was
dominated by boring lithophagid bivalves followed by sponge borings with notable worm
and barnacle traces. Belize macroboring was mainly a result of sponges, especially
Entobia mammillata, with minimal amounts of bivalve and worm borings. These results
mirror patterns previously found in the Pacific, but so far unstudied in the Atlantic.
Boring type and intensity is more heavily correlated with turbidity (nutrients and
sedimentation) than with the local effects of depth (ex. wave energy and diminishing
light). By knowing boring patterns within each facies, the carbonate budget can be
found and used to determine whether the reef is accreting or diminishing.
LORRAINE M. HOLLABAUGH
Adaptation of Common Teasel, Dipsacus fullonum to Arsenic-Contaminated Soils
In this experiment, growth rate and percent germination were investigated in an effort to
examine the effects of arsenic on an extant population of common teasel, Dipsacus
fullonum, and to determine variation in tolerance levels between populations collect
from contaminated and non-contaminated sites. Seed and soil samples were collected
from two sites located in Westerville, OH during the late summer early fall 2006. The
first site (i.e. Tussic Avenue) is a property previously used for munitions burning and is a
known source of contamination including arsenic. The second site (i.e. Cleveland
Avenue) had not been exposed to high levels of arsenic. Seeds were grown in plates
containing an arsenic amended media and were monitored over a period of nine days in
order to determine changes in germination, radical length, shoot length, cotyledon
length and overall appearance. Arsenic effected germination rate and growth in
samples collected from both the contaminated and non-contaminated sites. Seeds from
the Tussic Avenue site showed more tolerance to arsenic than seeds from the
Cleveland Avenue sites at concentrations of 50 and 100pm. Results indicate that
populations of teasel, D. fullonum previously exposed to arsenic were more tolerant
than populations that were not.
AMANDA HOUSTON
Arsenic Hyperaccumulation in Gametophtyes of the Fern Pteris vittata as
Measured by Inductively Coupled Plasma Mass Spectrometry
Gametophytes of Pteris vittata, the Chinese break fern, have been shown to
hyperaccumulate arsenic (As) to a similar degree as their sporophyte counterparts. To
date, there have been no quantitative studies describing the effects of arsenic on growth
or arsenic content in the first 3 weeks following germination. The objective of this study
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is to quantify biomass (μg) and arsenic accumulation (ppm) in P. vittata gametophytes
from day 0 to 21 after germination. We grew P. vittata gametophytes in arsenicamended medium (500 ppm) and no arsenic for 21 days under controlled conditions.
Gametophytes were harvested on days 3, 5, 7, 9, 12, 15, 18 and 21 and dried (30
relative humidity). The weight and arsenic content as measured with inductively
coupled plasma mass spectrometry (ICP-MS) was determined. The presence of
arsenic did not affect the growth of P. vittata gametophytes. Spores produced typical
rhizoids and prothallia in the presence of arsenic. In addition, the rates of biomass
increase were statistically the same for the arsenic and non-arsenic treatments.
Between days 7 and 9 (roughly the time for initiation of prothallia development), the
gametophytes began hyperaccumulating arsenic above the background medium level of
500 ppm. Arsenic content asymptotically approached 4600-5000 ppm on days 15 to 21.
Our study shows that despite their small size and morphological simplicity, P. vittata
gametophytes are able to not only tolerate and grow at normal levels in arsenic
amended conditions, but they can also hyperaccumulate to a similar degree as their
sporophyte counterparts. We believe the ability to hyperaccumulate arsenic provides
the gametophyte with an ecological advantage in arsenic rich environments.
MEGAN E. KONERTH
The Effect of Vitamin E Supplementation on Antioxidant Status, Blood Variables,
and Exercise Tolerance in Otterbein Equestrian Horses
Exercise-induced muscle damage is a common problem among horses (1, 4, 7, 17, 19).
Research regarding the effects of Vitamin E supplementation on muscle longevity has
primarily revolved around racehorses and endurance horses. The objective of this study
was to evaluate the effects of Vitamin E supplementation on exercise-induced muscle
damage in school horses. Eleven horses of mixed breeds were selected from the
Otterbein College Equine Science Facility. Horses were divided into two groups:
supplemented and non-supplemented. The study was conducted as a crossover design
in which during the first phase one group was supplemented and the other not. Then
these treatments were reversed in the second phase. Each phase consisted of a two
week basal period in which horses were fed diets low in Vitamin E, less than 50 IU/day;
and a three week treatment period in which supplemented horses received an additional
5000 IU/1000 lbs Vitamin E supplement. In between each rotation, the horses received
a five to six week washout period in which they returned back to their normal dietary,
exercise, and turnout regimen. During the course of both phases, horses were
exercised three to five hours per week and restricted from grass turnout. Blood
samples were taken one day before and one day following treatment periods to assess
Vitamin E, CK, and AST serum concentrations. Blocking the variability among horses,
Vitamin E concentrations between supplemented and non-supplemented horses, as
well as pre vs. post supplemented horses, were significantly different (P≤ 0.05).
Though supplementation significantly increased Vitamin E concentrations of the blood,
effects of Vitamin E supplementation regarding CK and AST enzymes were insignificant
(0.05≤ P). Results of this study suggest that Vitamin E supplementation had no affect
on exercise-induced muscle damage in Otterbein school horses.
R. ALEXANDER MACK
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Microbial Diversity in Metal Contaminated Soils of Kilgore Farm, Delaware Co.,
OH, Using Microbiology and Metagenomic Sequencing Techniques
The Kilgore Farm, located in southeastern Delaware Co., OH, was home to ammunition
manufacturing facilities during World War II. The destruction and burial of wastes took
place on the property in a marked ‘burn pit’. The microbial diversity of the burn pit was
examined by using metagenomic sequencing and BLAST searching databases.
Organisms from the burn pit and control sites were examined in order to determine
whether the species differences could be attributed to different levels of metal
contaminants present in the soils. A total of ten soil samples were taken from three sites
within the burn pit and two sites out of the pit within the same soil type. From each site,
two samples were taken from depths of 6-12in and 18-24in. ICP-MS analysis was used
to determine the metal composition of the soil samples. Elevated concentrations of Cr,
Mn, and Ca were found in almost all samples taken. Sample Control One at 1ft showed
elevated concentrations of five different metals. Metagenomes of bacterial DNA were
isolated from the samples using a genomic DNA isolation kit from Epicenter®. Bacterial
‘universal’ 16s rRNA gene primers were used to amplify extracted DNA samples.
Denaturing Gradient Gel Electrophoresis (DGGE) was then used to separate the DNA
of individual organisms from the metagenomic samples. The 16s rDNA subunits of the
separated organisms were sequenced and analyzed by using the National Center for
Biotechnology Information’s BLAST program. A profile of bacterial diversity including
unculturable Acidobacteria, Proteobacteria and Eubacteria, as well as numerous other
uncultured unclassified soil bacteria was obtained.
KURT J. MARKS
A Survey of Turtles in Metro Parks and Residential Ponds in Central Ohio
Recent studies have shown that introduced non-native turtle species, such as red-eared
sliders (Trachemys scripta elegans) can disrupt native turtle ecology by competing for
food and basking areas. Other studies have found that these introduced turtles can
create viable populations. The goal of this study was to survey residential and Metro
Park pond ecosystems in Central Ohio for non-native turtle species. Nine ponds were
included in the study; five were residential ponds located in housing subdivisions and
four were located in Metro Parks. At each of the nine study sites, turtles were trapped
using basking and baited hoops traps set in areas where turtles may feed or bask.
Traps were set in the early morning or the previous night and were checked once a day
for five days at each site. Turtles were identified to species, measured and marked to
prevent recounting. A total of twenty-one turtles were trapped in six of the nine ponds
(three residential ponds and three Metro Park ponds). Four native and two non-native
species were identified. Significantly more non-native species, (T. s. elegans) and
(Trachemys scripta scripta) were caught in residential ponds than Metro Park ponds
(x2=7.84, df.=1, 0.01>p>0.005). These are common pet store species and were likely
released by local pet owners. Future studies should determine if these non-native
turtles comprise a viable population and if they are negatively affecting pond
ecosystems.
AVINASH C. MINHAS
Coral Reef Microboring Trends: Enriquillo Valley, Dominican Republic
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One important type of coral reef bioerosion is microboring by photosynthetic green
algae, fungi, and cyanobacteria. The ultimate goal of this study is to characterize
microboring patterns in different reef facies and to relate these to exposure time of coral
skeleton prior to burial. Four facies were defined within the Enriquillo Valley Holocene
(5000-8000 year old) reef in the Dominican Republic. Three 12 m transects were made
in sections within branching, mixed, massive, and platy coral zones from five localities.
Every coral along each transect was characterized by orientation, taphograde, and
encrustations. Transect results suggest that 76.3% of the mixed coral facies, 88.3% of
the massive facies, and 79.6% of the plate coral facies were in life position with little
surface alteration, suggesting high live coral cover in the Holocene reef. Corals of three
taphogrades from each transect were collected for microboring analysis.
Representative samples were vacuum impregnated with epoxy resin and ichnotaxa will
be identified and the relationship between exposure time and microboring bioerosion
will be quantified. Upon initial examination of amount of microboring, it is clear that
samples which were of a poor taphograde show almost 100% microboring far beyond
the outer margin. Samples of a good taphograde showed significantly less microboring
in those areas. Our observations of high live coral cover contrast dramatically with
relatively poor conditions in modern degraded Caribbean reefs, suggesting that the reef
was in good health prior to its burial.
MEGAN MYERS
The Effects of Plant and Animal Diets on Microbial Gut Symbionts in Juvenile
Pond Slider Turtles, Trachemys scripta
The gastrointestinal tracts of vertebrate herbivores are populated with bacteria which
ferment cellulose, converting it to short-chain fatty acids that the herbivore absorbs for
energy. In herbivorous turtles, such as the yellow-bellied slider, Trachemys scripta,
these microbes play a critical role in the digestion of plant diets. High concentrations of
short- chain fatty acids have also been found in the large intestines of turtles consuming
shrimp diets. Little is known about microbial function on such diets, which do not
contain cellulose. One possibility is that the microbial symbionts are digesting chitin, a
carbohydrate found in invertebrate exoskeletons. In this study, the effect of plant and
animal diets on gastrointestinal microbial composition was assessed in T. scripta.
Additionally, we determined if certain bacteria present in turtles fed both diets were
chitinolytic. Juvenile turtles were inoculated with gastrointestinal bacteria by feeding
feces from wild caught turtles. A feeding trial was conducted with eight turtles fed the
plant, Vallisneria americana, and eight turtles fed crickets, Acheta domesticus, for one
month. Fecal samples were collected from each turtle, and bacterial populations were
isolated. Studies were conducted to identify bacteria and determine substrates for
fermentation. This research is especially relevant to juvenile turtle nutrition because
many turtle species undergo an ontogenetic diet shift from carnivory to herbivory. The
results of this study will provide a better understanding of how the diet shift influences
gastrointestinal microbial populations and helps demonstrate the importance of these
populations to different turtle life stages.
MOLLY A. MYERS
The Effects of Antibiotics on the Gastrointestinal Microflora in Slider Turtles,
Trachemys scripta
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Vertebrate herbivores do not have endogenous enzymes capable of breaking down the
β-1,4- linkages of cellulose, the major component of plant cell walls. Therefore, they
must rely upon microbial gut-symbionts that ferment cellulose and produce short-chain
fatty acids that the herbivore can use as an energy source. The main objective of this
study was to determine which antibiotics would successfully reduce the microbial
symbionts of juvenile freshwater turtles, Trachemys scripta. Destruction of these
microbial gut symbionts would enable the exploration of their importance and potential
sources of inoculation. Three possible antibiotic treatments (Streptomycin with
Bacitracin, Chloramphenicol with Bacitracin, and Enrofloxacin) were considered for this
study. An antibiotic screening was conducted on wild turtle fecal samples to determine
which antibiotics were potentially the most effective. Based upon screening results, we
hypothesized that Enrofloxacin would most likely result in gut sterilization. Twenty four
turtles, inoculated with feces from wild turtles, were assigned to one of four treatments
(n=6 per treatment) in which Enrofloxacin was (1) administered orally in a gelatin based
food, (2) introduced directly into tank water, but not in food (3) administered both in food
and directly in tank water, and (4) not administered in either food or water. Antibiotic
effectiveness was determined by culturing anaerobic bacteria from fecal samples that
were collected before and after the Enrofloxacin treatment. All Enrofloxacin treatments
successfully reduced gastrointestinal microflora. Administration of Enrofloxacin in the
food and water caused the greatest reduction. These results lay the methodological
foundation for future studies of chelonian microbial symbionts.
KARI M. PARKS
Enhancement of Genetic Engineering Laboratory Student Comprehension: A
Comparison of Hands-on and Computer Based DNA Modeling
Students’ comprehension of laboratory experiments are composed of two factors, first
how well they understand the experiment they are about to execute, and second on how
well they can interpret the results they obtain during the experiment. In the Otterbein
College Life Science course, Introductory Biology I: Molecules, Genes, Cells… students
participate in an experiment that challenges them to construct and analyze a
recombinant DNA molecule which contains genes encoding resistance to both ampicillin
and kanamycin antibiotics. However, for the past few years the results of their
experiments have been less than optimal. In order to enhance students’ command of
this experiment, comprehension and technical aspects were varied and analyzed.
These included measuring the students pre-laboratory understanding via a
questionnaire distributed among three groups of students. Each group was evaluated
with identical questionnaires, however one group received no additional instruction, one
group used a computer animation of the recombinant DNA lab, and one group was
given “hands-on” models of the plasmids to reenact digestion and ligation. The
techniques of the experiment were adjusted to compensate for their predictable
inaccuracies so that better instruction could be given on particularly important yet
intricate portions of the experiment. The pre-laboratory instruction which included the
“hands-on” models proved to be a superior learning mechanism over the computer
animation. Multiple common technical errors were identified as possible sources of the
sub-optimal results obtained by students. With the identification of these common
errors additional emphasis can be placed on those areas during the experiment. We
conclude that the “hands-on” learning mechanism should be added as a permanent pre-
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laboratory exercise. Also, with the implementation of the improved techniques the
laboratory experiment should provide the students with the more optimal results for
interpretation. These results, when combined with the pre-laboratory exercise should
significantly enhance students’ perception of this essential laboratory experience.
RACHELLE R. RAMSEY
An Association Study of Single Nucleotide Polymorphisms of the Dopamine
Receptor Gene DRD4 and Sensation-seeking
How much of our personality is due to genetic components within us, “nature,” and how
much is due to circumstances that surround us, “nurture?” The fact that people differ
from one another in behavior is obvious and the variation in personality traits makes a
difference in who we are, how we live our lives, and the people we become. This project
attempted to determine if different levels of sensation-seeking in humans were
associated with differences in the single nucleotide polymorphisms (SNPs) of the
dopamine receptor (DRD4) gene. The HapMap which characterizes sequence variants,
their frequencies, and correlations between groups of SNPs and was used to determine
the location of three focus SNPs. Cheek swab DNA samples and Zuckerman's (1979)
Sensation Seeking Scale V (SSSV) were collected from a sample population of 73
college students. Polymerase chain reaction was used to amplify the dopamine
receptor gene and each DNA sample was genotyped by using the single base
extension technique. Although no statistically significant correlation was found within
this population at any of the SNP locations, variability of the nucleotide at location
rs3758653 suggests that further research should be completed in this area.
Danijela Regulic
Reasons for Blood Culture Contamination Rates among Different Floors or on
Different Types of Drawing Techniques Used in Collecting, Processing and
Analyzing Blood Culture Samples
NICKI SMITH
Analysis of Parrotfish Abundance, Grazing Rates and Bioerosion Rates Relative
to Location and Time of Day on a Caribbean Patch Reef
Stoplight parrotfish, Sparisoma viride, are an important factor in the erosion of coral reef
and coral reef degradation. This study investigates the grazing rates of parrotfish as a
factor of time of day, on the patch reef surrounding Wee Wee Caye, Central coast,
Belize as well as the rate of bioerosion. Grazing rates at three sites around the island
were investigated as well as parrotfish abundance at those sites. Increased grazing
rates per fish occurred during the afternoon hours in addition to an insignificant
difference in the location of the grazing around the island. Rover Diver analysis
indicated a larger concentration of parrotfish near the eastern site; whereas Transect
analysis indicated that more parrotfish could be found at the north site. Feeding rates of
stoplight parrotfish on Wee Wee Caye in 2006 were higher than that of combined
herbivore feeding rates reportedly in 2003. Erosion rates on Wee Wee Caye were
highest at the eastern site and are comparable to rates reported along other reef
locations.
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SARAH SZYMANSKI
The Identification and the Physiological Growth Determinations for Owen’s Dry
Lake Bed and Areas of Death Valley for Haloalkaliphilic Bacteria
Extremophiles are archea and eubacteria which reside in environments at the extremes
of normal environmental conditions, including pH, salt levels, temperature and pressure.
This study looks at a specific division called haloalkaliphiles which have a high affinity
for saline and alkaline environments. Samples were taken from Owen’s Lake and from
the areas of Death Valley and grown in SW10 media at pH ranges of 7.2, 8.0, 9.5, 10.5.
The bacteria grown were analyzed for optimal growth curves as well as gram stained for
phenotypic characteristics. Optimal pH was sample dependant. Owen’s Lake samples
grew best in the 10.5 range. The samples from Bad Water also grew best at 10.5 and
8.0. This correlates with the known alkalinity of Owen’s Lake and Bad Water basin in
Death Valley. Interestingly, many extremophiles are evolutionarily successful because
they tolerate pH levels that other bacteria cannot, yet when surveyed for optimal growth
most extremophiles within standard pH ranges. However the bacteria from Owen’s Lake
and Bad Water Basin not only have adapted to a pH of 10.5, but thrive under these high
alkaline conditions. The Death Valley soil samples grew optimally at 8.0, while the soil
conditions require tolerance to heat and dessication, pH was not a factor. Bacterial
morphology and classification by Gram stain found that the most common phenotype
was gram negative rod and cocci, in contrast to many known haloalkaliphiles which
have been predominantly categorized positive coccus and spirochetes. Further
classification by biochemical analysis and 16srRNA sequencing should be pursued to
provide identification at the level of Genus species, which would allow comparisons to
known haloalkaliphiles.
ALEXIS M. TAYLOR
Utilization of Resources by Two Species of Plethodontid Salamanders
The objective of this study was to understand how two sympatric species of
salamanders, Plethodon cinereus (redback salamander) and Plethodon glutinosus
(slimy salamander) utilize resources in a woodlot located within a mountain bike path
park at Alum Creek State Park. The question of interest was; were these two species of
salamanders using resource partitioning to overcome interspecific competition. It was
hypothesized that the two species were partitioning their habitats in order to allow for
coexistence. To test this hypothesis, the woodlot was divided into six transects, five of
which included manipulated habitat material (rock, board, leaf, tunnel, and bare), which
was selected to mimic their natural environment. The sixth transect was used as a
control and contained no manipulated habitat. The study was conducted from the
beginning of July to the end of September; collections of salamanders were made
weekly. Upon collection species identification, body length, snout-vent length, tail
length, habitat type, and sex were recorded. Although the study did not provide data
that verified resource partitioning of habitat, it did indicate three environmental
preferences by the two species; habitat preference, distance down slope, and monthly
activity. Plethodon cinereus and P. glutinosus demonstrated a substrate preference for
leaf litter. This result may have occurred because of the five substrates, leaf litter was
the most non artificial. The two species were also found to be most abundant six meters
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down the slope. It is probable that the desired moisture level for the plethodonts was
found at that distance down slope. In addition, the redback and slimy were most
abundant during August. This may have many explanations including the need for an
acclimation period to the manipulated habitat material, increased number of
salamanders due to reproduction during the time of this study, or more salamanders
coming to the surface during the warmer days and nights. Although the study could not
identify the source of partitioning it did provide more insight into the reclusive lives of P.
cinereus and P. glutinosus.
ADAM R. WALTERS
Effect of Arsenic on the Morphological Development of Gametophytes of Pteris
vittata
Pteris vittata is a hyperaccumulator of arsenic (As), and can be used in the
phytoremediation of arsenic-contaminated soil. Most studies to date have looked at the
sporophyte generation of P.vittata in order to understand its hyperaccumulating
potential. This study investigated the morphological development of gametophyte of P.
vittata when grown in arsenic-amended and non-arsenic mediums. Specifically, we
quantified the percentage formation of rhizoids, prothallia, and anticlinal growth, as well
as rhizoid length and number of cells in the prothallus. P. vittata spores were collected
from greenhouse-grown plants and surface sterilized. Spores were suspended in 4.5 ml
of growth media with or without the addition of potassium arsenate (500 ppm). Spores
were allowed to develop into gametophytes from days 1 to 15 in a controlled
environment (14◦ C, 24 hour light). Gametophytes were harvested on days 3, 5, 7, 9,
and 12 and fixed in formalin. Fixed tissue was examined under brightfield microscopy
(100 to 200× magnification). Data were analyzed as randomized complete block
designs with two treatments and four replications. For percentage formation of rhizoids,
prothallia, and anticlinal growth, non-arsenic treatments developed earlier than arsenic
treatments; however, both treatments reached similar maximum values for percentage
developent. In most cases, values for non-arsenic treatments on intermediate days
(e.g., days 7 and 9) were significantly higher while values on early day (e.g., 3 and 5) or
later days (e.g., 12 and 15) were statistically no different than the arsenic treatment.
There are no differences among treatments for rhizoid length and only on day 12 did
treatment differ for number of cells in the prothallia. Our results indicate that arsenic
does alter morphological development of the arsenic tolerance species P. vittata.
However, the degree of inhibition is slight and does not completely prevent
gametophyte formation.
BRANDT L. WEININGER
Esthetic Evaluation of Materials Used to Fill Access Holes of Screw-Retained
Dental Implant Crowns
The access hole of screw-retained dental implant crowns presents an esthetic obstacle
for the restorative dentist. No studies have compared the different materials used for
this purpose. Objective: To investigate the esthetic values and acceptability of both
commonly used and innovative access hole filling materials from both the layperson and
dentist perspectives. Methods: One cement-retained crown and five screw-retained
crowns were prepared. Access hole filling materials included: dentin composite (DC),
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Fermit® (F), enamel composite (EC), dentin composite with opaquer (DO), and Fermit®
with opaquer (FO). A convenience sample of laypersons (n=50) and dentists (n=25)
were recruited as subjects for this study. All subjects evaluated the six restorations on a
visual analog scale (VAS) and determined acceptability of each. Results: Significant
differences were found between the mean VAS esthetic values and acceptability data in
both the layperson and dentist subject groups for the majority of all casts. Dentists
yielded equal or higher mean acceptability ratings than laypersons for all casts, at an
average of 18% more acceptable. Dentists also yielded higher mean VAS Esthetic
values on all casts, with an average of 7.5 points higher than laypersons. Fermit®
showed marked improvement, both VAS and acceptability data, among both laypersons
and dentists when used with the opaquer. Conclusion: Both laypersons and dentists
are able to detect significant esthetic differences in the materials used to fill the access
holes of screw-retained dental implants. On average, the dentists surveyed viewed
implant restorations as having better esthetic value and more acceptable than the
layperson subjects.
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