Biology 460 (Individualized Research) is the capstone experience for many students in the Biology major. Students spend an entire semester executing a well-planned research project under the direction of a faculty member. The culmination of this project is a scientific paper and a public presentation of the work to an audience of faculty and peers. These are the abstracts of the work of our students.

Click on tab: Spring 2007, Fall 2006, Spring 2006

SPRING 2007

The ability of tufted capuchins (Cebus apella) to choose between food-filled nuts and non-food nuts by weight, sound, and odor

Samantha Bruno
Advisor: Dr. John Winkelmann

Optimal Foraging Theory infers that animals evaluate food items and choose those that maximize net energy gain. Tufted capuchins (Cebus apella) are foraging animals and have been found to adhere to OFT. This research was conducted at the East Coast Exotic Animal Rescue in Fairfield, Pennsylvania. I tested the ability of two brother monkeys to discriminate between pairs of walnut shells (Juglans regia) that had been filled with food (raisins) or non-food items. Combinations of weight, sound and odor were used to create four phases. Bg significantly chose the food-filled nut over the non-food nut in trials where pairs of nuts differed in 1) weight, sound and odor (p < .001), 2) weight alone (p < .001), and 3) sound alone (p < .001). In a fourth phase Bg chose the non-food nut based on odor alone (p < .05). Bg followed the optimal foraging theory based on all variables except odor. In this case the subject may have been conditioned to choose soil, which was used in both food and non-food nuts in phase 4 tests. The older brother (Bg) dominated the trials suggesting that a competition hierarchy affected foraging.

Song structure in the polymorphic white-throated sparrow (Zonotrichia albicollis)

Kathryn Greenfield
Advisor: Dr. Vincent Formica

Sexual selection involves traits, like bird song, which are crucial to defending territories and gaining mating success. Song structure is one variable that has an impact on mating success of males. White-throated sparrow (Zonotrichia albicollis) males have different reproductive strategies that tightly correlate with a genetic polymorphism. Promiscuous white males are expected to be experiencing sexual selection and therefore may have different structural song components. Microhabitat variation in song was also investigated. To determine if song differed between morphs and habitat, a principle component analysis was performed on several song structural components including maximum frequency of the first three notes, frequency difference (Note 2 - Note 1), and frequency ratio (Note 2 ? Note 1). Only one component of song structure was found to be significantly different between white and tan males. No significance microhabitat variation was detected. This suggests that learning behavior may be an important factor in generating song variation and that observed variation may not be correlated to the chromosomal inversion in white-throated sparrows. Also, habitat diversity and fidelity may not be strong enough to cause significant microhabitat variation in song of the white-throated sparrow.

Functional analysis of a Serine/Threonine/Proline-rich motif in snoA^rif1 that mediates association with the nimO^Dbf4 initiator of DNA synthesis

Kristen Leatherbee
Advisor: Dr. Steven James

In the filamentous fungus Aspergillus nidulans, DNA replication is initiated by nimO^Dbf4 and cdc7, the regulatory and catalytic subunits of the universally conserved Dbf4 dependent kinase (DDK). DDK triggers DNA synthesis by phosphorylating the replicative DNA helicase, allowing the DNA to unwind at origins of replication. In addition, the Dbf4 subunit of the DDK contains an N-terminal BRDF motif (BRCT and Dbf4 similarity domain) that is crucial for checkpoint responses. This motif is necessary to inhibit Dbf4 activity, and thereby restrain DNA synthesis, in the presence of DNA damage. In A. nidulans, snoA ^rif1 (suppressor-of-nimO) is a novel inhibitor of nimO^Dbf4, as revealed by observation that loss of snoA rescues nimO18 temperature sensitive lethality and also alleviates the sensitivity of nimO18 to DNA damage agents. We have shown that that snoA may restrain nimO by direct protein-protein association. For example, yeast two-hybrid analysis revealed a physical interaction between the nimO BRDF motif and a short (~100 amino acid) serine/threonine- and proline-rich (S/T/P-rich) region in the snoA C-terminus. To further investigate this S/T/P-rich snoA motif, three in-frame deletions were constructed and used to replace the endogenous snoA locus. Phenotypes of these in-frame deletion mutants were analyzed to determine if specific removal of the S/T/P-rich motif would inactivate snoA and thus prevent inhibition of nimO. Preliminary analysis suggests that two of the three in-frame deletions may compromise snoA function to inhibit nimO. Further studies are necessary to determine whether the snoA^rif1 mutants alter or eliminate physical association with nimO. (Supported by NSF-RUI# 01-14446, and by the Biology Department and Provost Office at Gettysburg College).

The Development of the Optic Nerve in Cyclopic Zebrafish

Christine Knutzen
Advisor: Dr. Ralph Sorensen

Early exposure to ethanol has been shown to cause a variety of adverse defects in embryos. Among these, the central nervous system is greatly impacted by the teratogenic effects of ethanol; one such severe defect is the occurrence of cyclopia. Zebrafish are an excellent organism to study the adverse effects of ethanol on development. In this study zebrafish were exposed to 2.4% ethanol from the dome/30% epiboly stage to 24 hours postfertilization (hpf). Under these conditions 68% of the zebrafish developed with varying degrees of cyclopia. Decreased percentages of cyclopic animals were found when shorter incubation times were used: 3 hours yielded 0% and 6 hours 16% cyclopic zebrafish. In the development of vision, the retinal ganglion axons grow out and connect to the opposite optic tectum region of the brain forming the optic nerve. With a cyclopic animal there is only one retina from which optic nerve enervates. How does the optic nerve connect to the optic tectum in a cyclopic situation? The optic nerve in control and cyclopic zebrafish was viewed at 48 hpf using a Zn-5 antibody labeled with a peroxidase conjugated secondary antibody. Results from cyclopic animals are forthcoming.

Fall 2006

Effects of Copper Sulfate, Cadmium Chloride, and the Pesticide Methoxyclore on Lysosomal Destabilization in the Unionid Clam, Elliptio complanata

Nikolett Molnar
Advisor: Dr. Peter Fong

Lysosomes are regarded as valuable indicators of pollutant-induced injury. These organelles are involved in functions such as nutrition, tissue repair, cellular defense, and turnover of membranes, organelles, and proteins. Lysosomes are also responsible for the sequestration and metabolism of toxins. Lysosomal destabilization of digestive gland cells was used as a biomarker for the toxic effects of metals cadmium chloride and copper sulfate, and the pesticide methoxyclor in the unionid clam, Elliptio complanata. Clams were exposed for 7 and 14 days to cadmium chloride and copper sulfate concentrations of 2.5 ?g/L, 5.0 ?g/L, and 10.0 ?g/L and methoxyclor concentrations of 1.0 ?g/L, 10.0 ?g/L, and 100.0 ?g/L in 0.1% ethanol. Toxin-treated clams were compared to lab controls and freshly-collected clams from the field. Neutral red assay was used to evaluate lysosomal destabilization as stable or destabilized according to neutral red retention. Clams exposed to copper sulfate showed a significant increase in the percent of destabilized lysosomes compared with lab control and freshly-collected clams at both 7 and 14 days exposures for all concentrations. Cadmium chloride-exposed clams did not show a significant difference at 7 days, but at 14 days exposure, cadmium chloride had significantly increased the percent of destabilized lysosomes at all concentrations compared to the freshly-collected clams. Compared to laboratory controls, cadmium chloride increased destabilization at 5.0 ?g/L and 10.0 ?g/L. Methoxyclor showed no significant effects at 7 days. But at 14 days exposure, the pesticide increased percent lysosome destabilization at 10.0?g/L compared to lab controls, and increased destabilization at both of 10.0?g/L and 100.0?g/L compared to the freshly-collected clams.

The Ecological Relationships of Epauletted Fruit Bats and Sycomore Fig Trees in Kruger National Park, South Africa

Christine Brocia and Anya Valdes-Dapena
Advisor: Dr. John Winkelmann

Epauletted fruit bats, Epomophorus wahlbergi and Epomophorus crypturus, were studied to determine their potential roles as dispersers of Ficus sycomorus seeds in Kruger National Park, South Africa. Bat identifications were based on photographs of palatal ridge structure. At Skukuza, a study of feeding behavior was conducted with three newly captured E. wahlbergi. Captives were presented with single ripe figs. The mean number of seeds per a fig was found to be 979 ? S.D. 62. In a single night one bat processed 24.4 figs (132 g) containing approximately 23,888 seeds. Seeds are spit out in pellets of fiber (spats) during feedings, and also are eliminated in feces. Mean time between "spats" was 243 ? 153 s and mean time between defecations was 1207 ? 611 s. Thus, bats process large numbers of seeds nightly, but the seeds in spats are eliminated more quickly than those in feces. At Shingwedzi bats were captured by netting, and radiotransmitters were attached to eleven E. crypturus. Freed bats were radiotracked over a period of ten nights, and GPS/GIS technology was used to create maps of foraging patterns as the bats fed on sycomore figs. Radiotelemetry data also were used to determine the timing of repeated movements between the fruiting fig tree (where a fig was plucked) and the feeding-roost tree (where the fig was processed). This cycle-time was 314 ? 116 s. During these cycles when bat movement are ≤ 100 m from the fruiting tree, seeds in both feces and spats would fall within that radius. However, the mean long axis of nightly foraging ranges along gallery forest was 490 ? 722 m. This indicates that bats change feeding venues during the night, creating longer seed dispersal distances in appropriate fig habitat. During these flights seeds in feces, since they are retained longer, would be dispersed farther. Thus, cage-study data as well as radiotelemetry data support the hypothesis that epauletted fruit bats play a significant role in the dispersal of Ficus sycomorus seeds.

The Aspergillus nidulans snoA^rif1 inhibitor of cell division acts by associating with the BRCT domain of nimO^Dbf4

James M. Barra
Advisor: Dr. Steven James

In the filamentous fungus Aspergillus nidulans, nimO^Dbf4 and cdc7 encode regulatory and catalytic subunits of the conserved Dbf4-dependent kinase (DDK). DDK initiates DNA synthesis by phosphorylating the replicative DNA helicase to trigger DNA unwinding at origins of replication. In addition, Dbf4 plays an important role in the DNA damage response. This role is mediated by an N-terminal BRCT motif (BRCA1 C-Terminus), as revealed by mutations in Saccharomyces cerevisiae Dbf4 that confer enhanced sensitivity to DNA damage agents and failure to restrain DNA synthesis during genotoxic stress. In S. cerevisiae, only the Rad53/Chk2 checkpoint kinase has been shown to associate with the Dbf4 BRCT motif. We identified a novel inhibitor of nimO^Dbf4 called snoA (suppressor-of-nimO), loss of which rescues nimO defects, and over expression of which confers lethal cell cycle arrest. snoA encodes the Aspergillus ortholog of rif1 (Rap1-interacting factor). In budding and fission yeasts, rif1 functions in telomere homeostasis by inhibiting telomere elongation. However, in humans, rif1 has evolved to play an as-yet unspecified role in the S phase DNA damage checkpoint. Here we report a novel interaction between the nimO^Dbf4 BRCT motif and snoA^rif1. Using yeast two-hybrid analysis, we demonstrate that a short (~90 aa) serine- and proline-rich region in the A. nidulans snoA^rif1 C-terminus can associate with the nimO^Dbf4 BRCT motif. This novel discovery suggests that rif1 may exert S phase checkpoint control by directly inhibiting the Dbf4-dependent kinase. (Supported by NSF-RUI #01-14446 to SJ)

Analysis of Drying Methods For Scanning Electron Microscopy of Selected Platyhelminthes

James Gates
Advisor: Dr. Sherman Hendrix

Various methods are available to dry fixed biological specimens for scanning electron microscopy (SEM). Air-drying is unsatisfactory and results in specimen distortion due to the high surface tension of water during the evaporation process. Thus, other modes of dehydration are necessary. Critical-point drying (CPD) using liquid CO₂ is the most common mechanism, and results in relatively distortion-free specimens. However, chemical evaporation using hexamethyldisilizane (HMDS) has been suggested to be a good alternative to CPD for soft-bodies organisms. It is the purpose of this study to discern whether there are any differences in the fine ultrastructure of specimens dried using HMDS compared to those dried using CPD. Selected platyhelminth specimens were dehydrated in ethanol, divided, and dried using either CPD or HMDS. They were viewed using SEM and their ultrastructure evaluated. The resulting micrographs were analyzed for preservation of fine structural detail. HMDS preserved delicate structures as well as or better than CPD with no loss in the quality of the image. Thus, I found HMDS to be a comparable and more rapid method of drying samples for SEM than CPD.

SPRING 2006

The Effects of in Vitro Exposure to 2-Methoxyestradiol on Maturation of Mouse Oocytes in the Presence or Absence of Estradiol and Diethylstilbestrol

Christopher Cox
Advisor: Dr. Ralph Sorensen

Past research has linked an increased instance of aneuploidy with advanced maternal age. The catecholestrogen 2-methoxyestradiol (2-ME) is the only known endogenous aneugen and may be related to increased aneugenic events. The effects of 2-methoxyestradiol on the meiotic division of albino CD-1 mouse oocytes during in vitro maturation were studied. Exposure to 2-ME dose dependently caused a decrease in the ability of oocytes to complete the maturation process with increased arrest at the metaphase I spindle checkpoint (p<.001). Aberrant chromosomal alignment at both the metaphase I and II plates was observed through fluorescence microscopy (p <.001). In order to better explore the drug's effects in vivo, oocytes were subsequently exposed to 2-ME in culture medium containing a 7.5 ?M estradiol background and again with a 7.5 ?M background of the synthetic estrogen diethylstilbestrol. No significant alteration in the ability of 2-ME to disrupt tubulin polymerization was observed in the presence of these compounds.

The Effects of C75 on Diabetic Conditions in Diet-Induced Obese Mice

Anthony Cavallo and Brin Freund
Advisor: Dr. Kay Etheridge

Type II Diabetes mellitus is becoming epidemic, especially in countries where high-fat diets and obesity are on the rise. Recent research involved with the restoration of pre-diabetic conditions via change in diet has been promising. However, no previous research has been conducted using a Carnitine Palmitoyl Transferase-I (CPT-I)/Fatty Acid Synthase (FAS) inhibitor to treat diabetes. C75 (4-methylene-2-octyl-5-oxo-tetrahydro-furan-3-carboxylic acid) is a CPT-I stimulator/FAS inhibitor that could potentially be used as a therapeutic agent in treating diabetes by breaking the link between a high-fat diet and synthesis of body fat. In this study three groups of 15-week old male mice of the strain C57BL/6J were placed on either a normal control diet (NCD group, n=11) or a high-fat diet (HFD group, n=12 and C75 group, n=11). It is well-established that a high-fat diet will induce obesity and diabetes in this strain of diet-induced obese mice, and after four weeks on the high-fat diet, both the HFD and C75 groups showed elevated blood glucose and ketone levels, as well as decreased insulin sensitivity, indicating the onset of diabetes. The C75 group was then treated with C75 injections for a 2-week period, while the HFD and NCD groups received placebo injections. It was hypothesized that C75 would cause a decrease in blood glucose and ketone levels and an increase in respiratory exchange ratio and insulin sensitivity, thereby indicating a reversal of the diabetic condition. After a two-week injection period, the C75 mice experienced a significant decrease in fasting blood glucose levels (244.5 mg/dl to 209 mg/dl, p = 0.014) and a marginally significant decrease in blood ketone concentration (11.3 mg/dl to 6.0 mg/dl, p=0.06), but no significant change in respiratory exchange ratio. From this experiment, it can be concluded that C75 can ameliorate some symptoms of type II diabetes.

Identification of Gene that Codes for Sulochrin Oxidase in Penicillium frequentans

Matthew Wendler
Biology Advisor: Dr. Kazuo Hiraizumi
Chemistry Advisor: Dr. Koren Holland-Deckman

Sulochrin oxidase (SO), an enzyme found in Penicillium frequentans, catalyzes the regio- and stereospecific phenol oxidative coupling reaction to form (+)-bisdechlorogeodin from sulochrin. Because certain fungal metabolites contain anti-angiogenic, anti-microbial, or anti-fungal properties the study of this enzyme is biochemically important. The enzyme sulochrin oxidase (SO) shows high substrate specificity for benzophenone compounds similar to the well studied copper-containing enzyme, dihydrogeodin oxidase (DHGO) found in Aspergillus terreus. As previous studies have shown, SO is also a copper-containing enzyme as shown by the inhibitory effects of copper-chelating agents on activity and its full restoration only with the addition of Cu^2+?. The gene coding for dihydrogeodin oxidase has already been identified and sequenced; therefore, using similar procedures the gene coding for SO could be isolated and sequenced. It was first attempted to amplify and isolate the SO gene sequence from chromosomal DNA using designed primer sequences from DHGO in a polymerase chain reaction. Because this was not completely successful, a separate isolation and amplification experiment was conducted in which the reverse transcription of poly A⁺ RNA from P. frequentans liquid-culture mycelia at the mid-stationary growth phase was used to create a double stranded (ds) cDNA library. The ds cDNA samples were then ligated into plasmid vectors (pDNR-LIB) and electroporated into electrocompetent E. coli. Progress toward generating the cDNA library will be discussed. Future studies include determining the size of the library and screening the library for possible clones of the SO gene.

The Effects of Acute Pulmonary Resistance Exercise on Anaerobic Treadmill Running and Respiratory Parameters

Jamie Heeneke
Advisor: Dr. John Winkelmann, Biology
Dr. Daniel Drury, Health and Exercise Science

For many years, scientists have believed that the pulmonary musculature of the human body was not able to improve with intense training. Recent studies have demonstrated that acute pre-exercise respiratory training can temporarily improve athletic performance. The objective of this study was to determine the effects of acute pulmonary resistance training using the POWERbreathe respiratory trainer on anaerobic power and pulmonary function. We studied 10 female competitive college athletes (age 20.1 + .379 using three separate trials which consisted of: a familiarization trial, a placebo trial and POWERbreathe trial. Pulmonary function was assessed by testing Forced Vital Capacity (FVC), Forced Expiratory Volume (FEV₁) and Maximum Ventilatory Volume/minute (MVV). Anaerobic endurance was assessed using the Anaerobic Treadmill Test (TM-An) which required the women to sprint at a 20% grade at 6.0 mph until volitional failure. The mean MVV and FVC in the active POWERbreathe trial both showed significant (p < 0.05) increases from the control. Additionally a significant increase was observed between the mean MVV of the placebo trial (101.5 + 6.46) and the control MVV value (85.3 + 5.01). The changes observed in these tests were not, however, significantly different between the POWERbreathe and placebo trials. No significant differences were observed in the treadmill time or power statistics. Given the limitations of our research design, these data indicate that the effect of the POWERbreathe on respiratory parameters and anaerobic performance does not exceed that of the placebo trial. Furthermore, our findings suggest that test familiarization may have played a factor among the trials. Although we did incorporate a familiarization trial to account for the potential test re-test variability, future investigators may want to add additional familiarization trials so that any improvements identified can be attributed to the intervention and not the learning curve.

The Effect of Hydration-Dehydration Periods on Two Subspecies with
Contrasting Mating Systems in Clarkia xantiana

Catrina North
Advisor: Dr. V?ronique Delesalle

Seeds from a pair of outcrossing-selfing sister taxa from the genus Clarkia, Clarkia xantiana ssp.xantiana and ssp. parviflora, were treated with varying cycles of hydration-dehydration. Seeds from 10 maternal families per population represented 3 selfing and 3 outcrossing populations experienced six different hydration-dehydration treatments. Percentage of seeds germinated and average days to germination were measured for each maternal family. Seed mass was shown to significantly differ between the subspecies, but seed mass had minimal effect on the variation caused by treatment within each subspecies. The germination response to the various treatments in C. x. parviflora showed a significant effect in average days to germination and number of seeds germinated. No significant germination response effects of treatment were seen in C. x. xantiana. This response to treatment supports the hypothesis that C. x. parviflora is employing a drought avoidance germination strategy, whereas C. x. xantiana is employing a drought tolerance germination strategy.

Telomere Length Control in Aspergillus nidulans

Megan E. Campbell
Advisor: Dr. Steven James

This project explores the possible links between control mechanisms at the telomere and those involved in bulk chromosomal DNA synthesis. Telomeres are structures at the ends of linear chromosomes that serve to cap and protect them. Telomeres are composed of tandemly repeated 6-bp sequences (e.g., TTAGGG in mammals and in the filamentous fungus Aspergillus nidulans). In mammals, the telomere repeat regions are long, ~15kb at birth, and vary in length from chromosome to chromosome and cell to cell. Similarly, the telomeric repeats in budding and fission yeasts range in length from 200-400 bp. Surprisingly, the telomeres in Aspergillus are unlike those of animals, protists, and the budding and fission yeasts in that they are both very short and have discrete lengths. These observations imply that telomere length control mechanisms, which are well known in other systems, must exert very stringent control to maintain the exceptionally short, fixed-length telomeres in Aspergillus. For this reason, we are examining telomere length control (TLC) in Aspergillus by using a Southern blot assay, and have thus far shown that mutations in several key regulators of chromosomal replication do not alter telomere length. Currently, we are deleting or overexpressing orthologs of several known TLC genes, loss of which in budding or fission yeast lengthens telomeres substantially. For example, we have deleted or overexpressed Aspergillus Rap1, Rif1 and Taz1 (equivalent to human TRF1/TRF2), each of which plays a central role in TLC in budding and/or fission yeasts. These deleted and overexpressed strains, along with several others, are being tested for dysregulation of TLC.

Home Range and Foraging Behavior of Wahlberg's and Peter's Epauletted Fruit Bats Along the Mphongolo River in Northern Kruger National Park, South Africa

Brian Musetti
Advisor: Dr. John Winkelmann

This project was part of a larger study that includes the role of epauletted Fruit Bats (Epomohporus crypturus and E. wahlbergi) in the dispersal of the seeds of Sycomore Figs (Ficus sycomorus) in Kruger National Park. Our studies of the foraging and roosting behaviors of these bats were designed to predict the pattern of seed distribution by bats feeding on sycomore figs. Here I report on a radiotelemetry study of epauletted bats along the Mphongolo River drainage in northern KNP. Nine E. crypturus and one E. wahlbergi were netted at a single fruiting fig tree at Babalala. They were equipped with radio transmitters, released, and subsequently followed by radiotelemetry. Spatial data were analyzed using Biotas 2.0 Alpha software from Loas. Based on 663 data points mean Home Range (Minimum Area Probability - 0.95) of the nine E. crypturus was 264 ha, and the mean Core-Use Area (MAP - 0.50) was 23.5 ha. The Home Range and Core-Use Area of the single E. wahlbergi were 1187 ha and 238 ha. Bat position data were superimposed on river and road layers from the KNP GIS database. Activity hotspots were centered on fruiting fig trees which are the primary food resource for these bats during the winter dry season. Four hundred and twenty-seven Sycomore Fig trees were located and added to the KNP GIS database.

Construction and analysis of an epitope-tagged allele of the snoA^rif1 gene in Aspergillus nidulans

Kalin V. Vasilev
Advisor: Dr. Steven James

In the filamentous fungus Aspergillus nidulans, nimO^Dbf4 and cdc7 encode regulatory and catalytic subunits of the conserved Dbf4-dependent kinase (DDK). DDK initiates DNA synthesis by phosphorylating components of the replicative DNA helicase to trigger origin unwinding. A temperature sensitive nimO mutation, nimO18, can be suppressed by mutations in two genes, snoA and snoB. Semi-dominant snoB mutations lie within the cdc7 gene, and suppress by stabilizing the association of cdc7p with nimO18p. Recessive snoA suppressors not only rescue nimO18 ts-lethality, but they also rescue strains hypomorphic for expression of nimO⁺. However, snoA suppressors cannot bypass the complete loss of nimO. These observations reveal that snoA suppression is not allele-specific, and that snoA appears to act by inhibiting the stability or activity of nimOp. snoA was localized within a 340,000 bp interval of the Aspergillus genome and was identified by deletion of candidate ORFs, one of which phenocopied the nimO18 suppressor phenotype of snoA mutants. In this study, two-way fusion PCR was used to insert three copies of the hemagglutinin (HA) epitope at the snoA C-terminus. One-step gene replacement generated a strain carrying snoA::HA3 as the sole copy of the snoA gene. Strains containing snoA-HA3 were evaluated by Southern blotting and PCR. By choosing the correct restriction enzymes it was possible to demonstrate correct integration of the HA3 tag in the chromosomal DNA of the transformed strains. Western blotting will be applied to characterize this tagged snoA protein.