Student Research Projects - 2003-2004
Behavioral changes found in tadpoles as a result of different group size and predator induced environments
Advisor: Geoff Smith
Tadpoles have developed several types of defense mechanism that allow them to avoid predation. In this study I have focused on any changes that may take place in tadpole behavior under the exposure of different types of predators and different group sizes. Through my experiments I have found that predators do have an effect on the behavior of tadpoles. The type of predator also has an effect on the behavior of the tadpoles. As the numbers of tadpoles increase, tadpole activity tends to increase as well. Lone tadpoles tend to avoid the vegetated area in the presence of dytiscid larvae because this is where dytiscid larvae are mostly found.
Isolation and Characterization of hyperosmotically regulated genes in the pulmonary pathogen, Burkholderia cepacia
Advisor: Chris Weingart
Hyperosmotically regulated genes, encoding potential virulence factors have been identified in several bacterial species that colonize the lungs of immunocompromised patients. Specifically, sodium chloride regulated genes have been identified in several pulmonary pathogens including Staphylococcus aureus, Pseudomonas aeruginosa and Burkholderia pseudomallei. Another opportunistic pulmonary pathogen, Burkholderia cepacia inhabits the lungs of cystic fibrosis (CF) patients. However, very little is known about salt regulation in B.cepacia. In this report we use transposon mutagenesis to identify the genes in B.cepacia K56-2 strain that are induced by a hyperosmotic sodium chloride environment. Over 30 transconjugants expressing higher amounts of lacZ gene at hyperosmotic sodium chloride concentration were identified. The β-galactosidase assay was performed on the transconjugants so as to quantify the visually observed variable expression of β-galactosidase in the different salt environments. 4 of these 33 transconjugants showed a 2-5 fold higher β-galactosidase activity in the hyperosmotic sodium chloride environment. Further research needs to be done in order to sequence the gene and also to identify the protein encoded by the gene and its potential function.
Abundance of the northern Redback Salamander (Plethodon cinereus) across a Deciduous/Confiderous Edge
Advisor: Geoff Smith
As global amphibian abundances experience a marked decline, it is important to understand what drives these declines both on the global and local scales. Changes in microhabitat including the effects of habitat fragmentation or loss can drastically alter the ecology and population structure of these organisms. In this experiment, the distribution of the Plethodontid red-back salamander Plethodon cinereus was monitored across a second-growth deciduous/mature coniferous plantation edge at the Denison University Biological Reserve in Granville, Ohio. Of the three seasons observed, fall 2002, spring 2003, and fall 2003, only fall 2003 produced a significant interaction between mean salamander abundance and habitat location across the edge. This may have been the result of newly added coverboards to the transects or more favorable climatic conditions for that particular season.
Testing allozymes for the identification of larval sunfish (Centrarchidae) from Ebaugh and Middleton ponds
Advisor: Jessica Rettig
Although identification guides exist for larval freshwater fish, the variable morphology of larvae leads to uncertainty in identification. Cellulose acetate electrophoresis, an alternate identification method, was used to identify unknown larval fish from two ponds on Denison University’s campus; the ultimate goal being to see the composition of larvae. I experimented with five allozymes for identifying species of sunfish (Centrarchidae): Glucose-6-phosphate isomerase (GPI), alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PDH), lactate dehydrogenase (LDH), and xanthine dehydrogenase (XDH). The adult fish trials showed that GPI and LDH were the best allozymes to distinguish between sunfish. Of the unknown larvae that resolved, all were found to be bluegill. I found that the best procedure used a paper clip for grinding and the GPI stain. Average larval density (#/m3) was compared on each collection date and Middleton pond was found to have more bluegill larvae earlier in the year compared to Ebaugh pond.
Indirect Effects of Endophyte Infection on the Distribution and Abundance of Spiders in Tall Fescue (Festuca arundinacea)
Advisor: Juliana Mulroy
The importance of fungal endophytes in pair-wise interactions between grasses, herbivores and other plants is well established, but ecologists are just beginning to understand the importance of endophytic fungi at the community level. In this research, I review literature regarding the importance of endophytic fungi in pair-wise interactions as well as recent literature on the community level impacts of grass-endophyte symbioses. Following the literature review, I present my own field research investigating the indirect effects of endophytes and structural complexity on the distribution and abundance of spiders in old fields. I experimentally manipulated endophytes and thatch in fields of tall fescue. I found that fungal endophytes strongly reduce the species richness of spiders as well as the abundance of some spider families. I also explore one potential mechanism underlying these results, endophyte mediated plant interactions, in a greenhouse experiment using tall fescue and Conyza canadensis.
Effect of Exogenous Melatonin on Behavioral Sensitization to Cocaine
Advisor: Susan Kennedyv
An ISSR-PCR Assessment of Genetic Variation within the Grapeferns Sceptridium lunarioides and S. jenmanii
Advisor: Warren Hauk
Sceptridium lunarioides and S. jenmanii are species of North American grapeferns found across portions of the southeastern United States. Neither species has been surveyed for within-species genetic variation. For this project, we sampled 13 populations of S. lunarioides across five states and three populations of S. jenmanii across three states. Three ISSR-PCR (inter-simple sequence repeat polymerase chain reaction) primers generated 29 polymorphic loci. Hickory analyses generated an FST value of 0.53 and an FIS of 0.60. Neighbor Joining, Maximum Parsimony, and UPGMA cluster analyses were consistent with FST estimates and showed evidence of greater genetic structuring within S. lunarioides than has been recorded in Ophioglossaceae; distinct clusters corresponded to LA + MS, AL, FL, and GA. S. jenmanii demonstrated similar clustering patterns, but analyses are not yet complete. Based on FIS estimates, S. lunarioides may have a mixed mating system, unlike that of other species of Ophioglossaceae.
Identification of protein interactions of the intracellular domain of Neurotactin by biopanning of a phage display cDNA library
Advisor: Eric Liebl
Neurotactin (Nrt) is a transmembrane cell adhesion molecule that is involved in axon guidance during Drosophila melanogater development. The extracellular domain of Nrt interacts with the secreted protein Amalgam to promote cell:cell adhesion. The interactions of the intracellular domain of Nrt (Nrt-IC), however, are uncharacterized. We attempted to identify the protein:protein interactions of Nrt-IC. To accomplish this goal, we constructed a T7 phage display cDNA library using D. melanogaster embryo mRNA primed with HindIII random primers. Nrt-IC interacting phage were selected for by biopanning over purified Nrt-IC. The screen identified Nrt-IC interactions with phage that encode the neuronally expressed Bangles and beads (Bnb) and the kinase domain of CG1344. However, specific affinity of the phage that encode these cDNAs could not be demonstrated. Future experiments to confirm these interactions include finding conditions where specific affinity for Nrt-IC can be identified and testing for genetic interactions between bnb, CG1344, and nrt.
How does odor sampling change as crayfish grow: High speed video studies of Orconectes immunis.
Advisor: Kristina Mead
Crayfish can interpret their chemical environment by moving their antennules in rapid "flicks" which allow them to sample chemicals in the surrounding fluid. Specimens (N=40) of the local crayfish species Orconectes immunis were collected from Hebron Hatchery, a low-flow lake habitat. Crayfish were collected over a large size range 28-78mm (rostrum-telson length). Crayfish of different sizes were stimulated to flick using cat food pellet extract, and were filmed flicking in a special filming chamber using a high-speed digital video camera. The videos were captured using Dazzle MovieStarTM 5 program, and digitized using ImageJ 1.29x software. The variables that were analyzed included: antennule velocity, flick angle, and antennule structural parameters. These measurements were used to calculate the Re (Reynolds number) describing the flick, the leakiness of flicks, and flow rate through bundles of hairs during flicks. Larger crayfish had larger flick velocities, Re values, leakiness, and flow rate values.
Response of Plethodon cinereus to Chemical Cues from Increasing Numbers of Conspecific Salamanders
Advisor: Geoff Smith
The red-backed salamander, Plethodon cinereus, is a territorial species that generates and responds to chemical cues. Such cues play an important role in establishing territories and, along with many ecological factors, affect interactions among conspecifics. The current study looks at the response of adult male and female P. cinereus to cues from different numbers of conspecifics. Results show a varied response between male and female salamanders. Females tend to avoid substrates containing cues from five conspecifics as opposed to one or zero, while males make no distinction. Additionally, male salamanders move significantly more between substrates than do females. The sex of the donor salamander leaving behind the cues was not significant in this study. These results may indicate a difference in the objectives of males and females regarding their response to cues. Males may use cues to seek a mate while females may seek a safe nesting site.
A Phylogenetic Analysis of Moonwort Species (Botrychium s.s.) Using Three Plastid DNA Sequence Data Sets
Advisor: Warren Hauk
Botrychium s.s., commonly called moonworts, although widespread through most of the world, are small plants and are often difficult to identify just by physical features. Above ground, moonworts are composed of a single leaf, divided into the reproductive (fertile) and non-reproductive (sterile) portions. Because of the small size and relatively simple form of the leaves, there are few morphological characters present that are useful for distinguishing species and constructing classifications. Molecular and genetic data have been useful for distinguishing the 27 North American moonwort species. Previous plastid DNA sequence analyses of 21 moonwort species showed that eight polyploid species have a similar or identical rbcL gene as their chloroplast parent. For this project, we sequenced the chloroplast rpL16 region for 24 moonwort species. For three species not previously sequenced, B. pallidum, B. boreale, and B. gallicomontanum, rbcL and trnL-F sequences were obtained. Analysis of the rpL16 dataset supported species relationships established in the rbcL and trnL-F phylogeny. Three well-supported clades were identified: Lanceolatum (nine species), Lunaria (two species), and Simplex-Campestre (13 species). Simplex-Campestre and Lunaria clades were sister, and these two were sister to the Lanceolatum clade. Autapomorphic substitutions in the rpL16 sequences for species of the Lanceolatum and Lunaria clades were consistent with current species delimitations. Within Simplex-Campestre clade, combined analyses indicated that the Pallidum and Simplex subclades are sister, while members of the Minganense subclade did not group with any known diploid species and their diploid progenitor is either unknown or extinct.
Axon Path-finding in the Central Nervous System of Drosophila melanogaster: Determining Enhancers of the trio Mutant Phenotype from a Random Mutational Screen
Advisor: Eric Liebl
The development of the central nervous system (CNS) through a process known as axon pathfinding involves the correlation of several biochemical signaling pathways. Elucidation of pathway components can be performed through the use of second-site mutagenesis, creating random mutations to genes in a background already containing a known mutation and looking for enhanced or suppressed phenotypes indicative of an interaction. The role of Trio, a guanine nucleotide exchange factor which interacts with GTPases during axonogenesis in Drosophila, is still being uncovered. In this study, a preliminary genetic screen for enhancement was performed on trio mutant background Drosophila which had undergone second-site mutagenesis through exposure to gamma-ray radiation. Three of the stocks were found to be new alleles of previously characterized trio enhancers. Initial phenotypic observation does indicate at least two potentially novel enhancers of the trio mutant phenotype which are worthy of further investigation.
Within Species Variation of Sceptridium biternatum Assessed by ISSR-PCR
Advisor: Warren Hauk
The grapefern Sceptridum biternatum is common in the forests, swamps, fields, and knolls of the southeastern United States. Although well known throughout its geographic range, populations from central Florida exhibit morphological traits not seen in more northern and western states. To examine genetic variation within S. biternatum, ISSR-PCR was utilized. F-statistics documented a low amount of genetic structuring among populations and a high degree of inbreeding. For some populations, Neighbor Joining, Maximum Parsimony, and UPGMA analyses revealed moderate to low correlation between genotype and geographic origin. The Florida populations were not genetically different from more northern populations, suggesting the unique morphology is not a reflection of underlying genetic difference. High spore dispersal may explain the lack of clustering among individuals from the same population. Given the putative inbreeding nature of S. biternatum, these results indicate that dispersal may trump breeding system in determining the overall genetic structure of S. biternatum.
Variation in olfactory chemosensor morphology related to body size and sex in the crayfish Orconectes immunis.
Advisor: Kristina Mead
Crayfish use water-borne cues to interpret their chemical environment. Cues from "distant" sources are sampled using olfaction chemosensors called aesthetascs on the antennules. The arrangement and shape of these chemosensors affect how much odor-containing fluid is able to penetrate close to the antennules during odor sampling. Crayfish of different sizes and sexes have different olfactory needs. This study hypothesizes that olfactory chemosensor structure may change as a function of animal size and gender. The antennules of male and female Orconectes immunis were harvested and analyzed using Scanning Electron Microscopy. Scion Image software was used to measure several structural parameters including the number of aesthetascs bearing segments, aesthetasc length, diameter, insertion angle, and the distance between adjacent aesthetasc bundles. Results showed several parameters varied as a function of size and revealed sexual dimorphisms within and across size classes. There was also parameter variations related to the position along the antennule.
Effect of Forest Floor Characteristics on Salamander Abundance
Advisor: Geoff Smith
Terrestrial salamanders are known to be highly selective in microhabitat characteristics because of their need for an environment with moisture suitable enough to carry out cutatneaous respiration. Using sites of different forest type,I investigated the substrate preferences of the red-backed salamander, beneath a covered microhabitat. Salamanders were positively correlated with bare ground space and negatively correlated with leaf litter. Little research has been done in this particular area and further study is necessary to draw more conclusions.