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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 1 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 2 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 3 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 4 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 5 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 6 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 7 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 8 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- 9 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. 10 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 11 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), 12 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. 13