Abstracts from the Colleges of Agriculture and Marine Studies
Undergraduate Summer Research Symposium August 9, 2006

Ordered alphabetically by student's last name

Cabrera Christopher Cox Demarest Fernandes Madison Madorma Mills Smarsh Wolters

DNA Sequencing Using Nuclear Primers to Determine Genetic Variability among Species of American Oysters.

Leonardo Cabrera
and Patrick M. Gaffney
of Marine Studies, University of Delaware 

Phylogenetic analysis by sequencing DNA helps researchers to catalogue organisms and identify genetic variation among species. American oysters of the genus Crassostrea are    difficult to differentiate because members of the same specie tend to have different morphology depending on the environment that they live in.  By using polymerase chain reaction (PCR), we are able to amplify the DNA of five species of the genus Crassostrea: corteziensis, gasar, rhizophorae, iridescens and virginica. Twenty primer sets were screened for each of the five species. Primers that showed strong amplification for all five species were determined to be the best candidate primers for sequence analysis. Sequence data will be used to assess the variability among species of these five American oysters. This research was funded by  NSF EPSCoR.

Investigation of Nitrate Reductase Activity in Harmful Algae Using Flow Cytometry

Katelyn A. Christopher
and Kathryn J. Coyne
College of Marine Studies, University of Delaware, Lewes, DE 19958 

Two prominent species of harmful algae, Heterosigma akashiwo and Chattonella subsalsa, co-occur in the Delaware Inland Bays.  These toxic species cause devastating harmful algal blooms (HAB’s) that result in significant losses to the aquaculture industry. Heterosigma and Chattonella, along with other HAB species, require nitrogen to bloom.  Heterosigma akashiwo, however, prefers the more metabolically expensive nitrate (NO3-) as opposed to ammonium (NH4+), which is preferred by Chattonella subsalsa. Our research consisted of three experiments investigating the utilization of nitrate by these species.  We monitored the enzyme nitrate reductase (NR) using a flow cytometer with a fluorescent substrate for NR activity. In our first experiment, we measured NR activity in each species when cultured in media with different concentrations of NO3-. The second experiment monitored inhibition of NR for each species after adding NH4+, a NR inhibitor.  Finally, we investigated the diel activity and expression of NR in H. akashiwo over a 24-hour period. These experiments demonstrated species-specific responses to nitrogen source and modes of inhibition of NR activity by ammonium: in H. akashiwo competitive inhibition of NO3- reductase allows this species to utilize NO3- in the presence of NH4+, while in C. subsalsa, inhibition of NR by NH4+ appears to be non-competitive. The diel experiment demonstrated that NR activity in H. akashiwo follows a diel pattern when cultured under 12 hour:12 hour light:dark conditions, but that the pattern of activity is shifted when Heterosigma is cultured in continuous light. This research was funded by Delaware EPSCoR and NOAA-MERHAB.

Physiological and Molecular Characterization of Tellurite Resistant Marine Microbes
Talisha S. Cox
1, 2, 4 and Thomas E. Hanson2, 3
1Lincoln University, 2Delaware Biotechnology Institute, 3Graduate College of Marine Studies,
4Howard Hughes Medical Institute Undergraduate Research Scholars

The overall goal of this project is to understand the role that marine microbes play in the transfer of metalloids from the marine environment to the atmosphere.  Samples from a salt marsh at the University of Delaware’s marine campus were incubated under  aerobic, microaerobic and anaerobic conditions in the presence or absence of potassium tellurite to enrich for tellurite resistant microbes and the population of culturable microbes was followed over time.  New strains were isolated and characterized. Previously isolated aerobic strains were also molecularly characterized by the isolation and sequencing of a section of the 16S rRNA gene.
Under all oxygen conditions, the number of tellurite resistant microbes was higher in tellurite enrichments and increased over time.  A larger number of strains was recovered on rich marine LB agar compared to minimal CP marine medium. Anaerobic isolates were determined to be facultative anaerobes in the presence and absence of tellurite marine LB and marine CP media. The majority of all isolates recovered were Gram positive.  The previously isolated aerobic marine microbes were grouped; revealing that these microbes were marine Bacillus and Bacillus halodurans.  Funding by Howard Hughes Medical Institute and a grant from the National Science Foundation (OCE-0425199 to Prof. T. Church and Prof. T. Hanson).

Land Use Effects on Water Quality in Delaware

Brian Demarest
1, Susan E. White-Hansen2, David J. Hansen3, and Bruce Allison4
Wesley College,
  2Geographic Information Specialist, Research and Education Center, Georgetown, Delaware,
3Delaware Assistant Professor, Department of Plant and Soil Sciences, 4Environmental Studies, Wesley College, Dover, Delaware

Water is a critical resource in Delaware for agriculture, rural water supplies, and the tourism industry.  However, most of the state’s surface waters are not meeting federal or state water quality criteria.  As the state’s population increases there are increasing pressures on the water resource from numerous sources, including housing developments and wastewater treatment facilities.  In July, 2005 a project was initiated that involves collecting water samples upstream and downstream of ten locations; four suburban sites and six wastewater treatment sites.  Water samples have been collected bi-monthly since August, 2005.  Field measurements include stream flow rate, temperature, dissolved oxygen, pH, total dissolved solids, and conductivity.  Laboratory measurements include the major macro and micro nutrients. Results from this project will help to evaluate the impact of housing developments and wastewater treatment facilities on surface water quality in Delaware.  To date more than two hundred and fifty usable data points have been collected.  Initial results indicate tremendous variability in water quality among both the suburban sites and the wastewater sites.  This variability suggests that factors upstream, such as pollutant sources or natural inputs, are an important consideration.  In general, wastewater sites have shown significantly larger changes in upstream versus downstream impacts than suburban sites for the following properties: pH, conductivity, boron, sulfur, and iron.  This work will continue in 2006/2007.  Funding and support provided the Mid-Atlantic Water Quality Program at the University of Delaware and the Experimental Program to Stimulate Competitive Research (EPSCoR) through the University of Delaware and Wesley College.

Offshore Wind Development Project

Jesse Fernandes, Willett Kempton, and Jeremy Firestone
College of Marine Studies

With the threat of global warming and instability of the foreign oil market, Americans are beginning to turn to clean, domestic energy, including wind power. The replacement of fossil fuel burning plants with clean, renewable energy would have innumerable positive effects on the environment, including preventing the release of CO2, SO2, NOx and mercury and reducing the amount of land needed for coal mining and processing. Though wind farms continue to be built on land in the United States, northern European countries have begun to move offshore and a growing body of research suggests there exists a large wind resource offshore. The US offshore wind resource, to date, remains untapped, though wind farms have been proposed in New York, Massachusetts and Texas. As Delaware has a large wind resource offshore (Kempton, et al. 2006), the aim of this project is to determine how big a wind farm would be practical off the Delaware coast and how much fossil fuel generated power would be needed in any event.  Boundary conditions were determined by considering population growth, transmission capabilities, load demands, and non-wind generation.  Through a timetable and map, which considers exclusion zones off Delaware’s coastline, potential viewshed impacts, and the effect previous experience may have on the number of turbines a contractor would be able to install, a progression of offshore wind power development was outlined.  This research indicates that there is more than enough area to build a series of large-scale wind farms off the Delaware coast.  Indeed, by generating enough power to fulfill the needs of the state, offshore wind could relegate the fossil fuel burning power plants to the status of “as needed” or needed only when the wind farm was not generating enough power.

Assessment of Anoxic Conditions in a Waterway of Delaware’s Inland Bay

Andrew Madison, Shufen Ma, Edouard Metzger, and George W. Luther III
                                                                                             Department of Marine Sciences                                                                                                

Seasonal anoxia was studied in waterways at the northern head of Delaware’s inland bay, Rehoboth Bay.  Bald Eagle Creek has tidal exchange with Torquay Canal through a shallow channel.  The mean water depth of Torquay Canal is 2 m, but dredging created depressions 5.5 m deep.  In past years, millions of menhaden have been killed.  While low O2 concentration was assumed to be the problem, recent studies have indicated that production of toxic H2S is likely the cause.  As seasonal anoxic conditions develop because of excess eutrophication and the depressions become stratified, O2 is depleted in deep water.  Because O2 is entirely consumed, sulfate is used as the terminal electron acceptor for organic matter decomposition in sediments with production of H2S, which then diffuses into the water column.  When H2S is mixed into the surface waters by physical forcing (storm events), fish kills can occur.  The purpose of this study was to determine O2 and H2S levels to understand seasonal anoxia and to predict its effects on marine life.  Also, the effect of circulators was studied to determine if this technology could prevent H2S buildup.  In Torquay Canal during June, O2 was not detectable below 4.5 m, whereas in July, O2 was not detectable below 3 m.  H2S levels increased significantly in deep waters rising from 20 µM to 150 µM. In Bald Eagle Creek, H2S was even higher (400 µM).  The circulators have no significant effect in preventing anoxic conditions.  Filling the depressions may be the only way to prevent H2S accumulation.  Funded by Delaware EPSCoR.

Cloning and Transformation of Putative Thiosulfate Oxidation Genes of Chlorobium tepidum into Escherichia coli

Michele M. Madorma 1,2,4 and Thomas E. Hanson 3,4
1Delaware National Science Foundation EPSCoR Grant Scholar, 2College of Agriculture and Natural Resources, 3College of Marine and Earth Studies and the 4Delaware Biotechnology Institute, University of Delaware, Newark, DE.

Chlorobium tepidum is a moderately thermophilic green sulfur bacterium that utilizes reduced sulfur compounds (sulfide, elemental sulfur and thiosulfate) as electron donors to support phototrophic growth.  Based on the complete genome of C. tepidum, a 13 gene cluster, which encodes for sulfur oxidation (Sox) proteins, is predicted to be involved in the oxidation of thiosulfate to sulfate. These putative homologs are highly similar to those found in the α-proteobacterium Paracoccus pantotrophus GB17,where they have been shown both biochemically and geneticallyl to be involved in thiosulfate oxidation. However, the C. tepidum Sox genes have only been identified by similarity.  Furthermore, C. tepidum lacks genes encoding SoxCD, which are responsible for harvesting six electrons from thiosulfate in P. pantotrophus [2]. Therefore, the C. tepidum Sox genes must be further studied to determine how they contribute to sulfur oxidation in this organism.  Prior attempts to clone one half of the C. tepidum Sox cluster, SoxII, in a plasmid in Escherichia coli were unsuccessful.  The purpose of my project was to clone the SoxII fragment as two overlapping gene clusters, SoxII-A and SoxII-B.  These fragments were PCR amplified and cloned into the pCR-XL-TOPO vector and were transformed into E. coli TOP 10 cells by electroporation. Positive constructs carrying either of two gene clusters were identified by PCR, restriction digestion and sequencing.  The resultant constructs will be used to help define functional roles for putative thiosulfate oxidation genes via insertional gene inactivation in the C. tepidum genome. Supported by EPSCoR.

Bacterial Production in the Chesapeake and Delaware Bays

Timothy I. Mills
1, Danielle M. Winget2, and K. Eric Wommack2
1Delaware Technical & Community College, Stanton, DE,
2University of Delaware. Newark, DE

Secondary production occurs from the transformation of dissolved organic matter (DOM) and inorganic nutrients into bacterial biomass. The overall effect of bacterial production (BP) is the transport of DOM from one trophic level to another, recycling material that would otherwise be lost to the system.  Given the ubiquitous nature of bacteria in the sea, the process of bacterial growth is a significant component of the global carbon cycle.  To investigate the magnitude of estuarine BP, we collected samples from several depths at twelve stations in the Chesapeake and Delaware Bays during a research cruise in July 2006. Bacterial production in water samples was determined using the [3H]-leucine micro-centrifuge method. BP averaged 4.6x104 cells ml-1 hr-1 with a std. dev. of +/- 6.0x103 in the Chesapeake Bay (CB), which was significantly higher than average BP in the Delaware Bay (DB), 1.1x104 cells ml-1 hr-1 with a std. dev. of +/- 3.3x103.  The CB showed clear trends in BP whereas the DB showed no clear relationships between BP, sample location, salinity, and temperature. For example in the CB, BP increased as temperature and distance south increased and as depth and salinity decreased. However, BP in the DB only showed a significant correlation of increasing BP with increasing depth, contrary to the pattern in the CB. Reasons for differences in BP in these two estuarine environments may be the higher turbidity and increased storm-water input of the DB, and differences between the circulation regimes of the two bays. Funding for this research was provided by Idea Network of Biomedical Research Excellence of Delaware supported by the National Institute of Health, and the Delaware Biotechnology Institute in affiliation with University of Delaware, and Delaware Technical and Community College.

Validation of a 14K Chicken Integrated Systems Microarray and Its Utility for Other Avian Species

Danielle Smarsh and Larry A. Cogburn
Department of Animal and Food Sciences

Microarray analysis currently plays a pivotal role in the emerging field of functional genomics.  The chicken has recently attained model organism status with the help of gene sequencing of different tissues, the development of microarrays, and the completion of the genome sequence. There are three purposes to this project, the first is to measure the biological variability across four individuals from four different tissues (liver, hypothalamus, breast muscle, and adipose).  From these samples, the number of unique and shared genes among the tissues will be analyzed.  The second purpose is to test the utility of the chicken microarray for gene expression profiling of several different bird species, including turkey, duck, Japanese quail, kestrel, and house sparrow.  Gene expression profiles will be determined from two tissues (liver and hypothalamus) of four individuals.  The final purpose of this experiment is to provide Gene Ontology (GO) annotation for all genes represented by the Del-Mar 14K Integrated Systems Microarray.   RNA has been extracted from the tissues of these birds for hybridization.  We will use a pooled RNA reference design.  The function of the most abundantly expressed genes from each tissue will be determined from Gene Ontology analysis, which will assign genes to three categories-cellular compartment, molecular function, and biological process.  Ultimately, it will be determined which genes are common across tissues, and which are unique to specific tissues. Supported in part by USDA grant.

The Viral Lipase (v-LIP) Gene of Herpesvirus of Turkeys (HVT)

is Nonessential for Replication and Vaccinal Protection
Megan E. Wolters, Erika R. Feierstein, Brewster F. Kingham, Carl J. Schmidt
Department of Animal and Food Science

We have generated a series of 140 transposon insertion mutations in an infectious BAC clone of Herpesvirus of Turkeys (HVT).  The sites of transposon insertion were determined by DNA sequencing.  Three independent mutants containing insertions within the HVT viral-lipase (v-LIP) gene all grew in chicken embryonic fibroblasts with virus particle yields similar to that of the parental HVT-BAC.  One v-LIP insertion mutation was tested for ability to to protect against chellenge with the Marek’s disease virus strain RB1B.  The v-LIP mutant conferred protection against MDV challenge with an efficacy similar to that of the parental HVT-BAC. Supported by USDA grant.

Links: Summer 2006 Undergraduate Research Symposium, Symposium Abstracts from other Colleges and Departments,

Undergraduate Research Summer Enrichment ProgramUnversity of Delaware Undergraduate Research Program, Howard Hughes Undergraduate Program.
Created 1 August 2006. Last up dated 16 August 2006 by Hal White
Copyright 2006, University of Delaware