Performance Report for Cooperative Agreement No: na06oar4810163 for the Period from September 1, 2006 to August 31, 2012 University of Maryland Eastern Shore
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- 3. Project Title: Investigation into persistence and distribution of sediment reservoirs of Hematodinium sp., a protistan parasite of blue crab ( Callinectes
- Thematic Area Addressed
- Planned Start Date
- Application of the ITS2 assay to Indian River Inlet sediment. Summer sediment collections: MD Coastal Bays
- COMB students visit SSU
- How will results be incorporated into NOAA Fisheries operations
- How will results be incorporated into LMRCSC research and curriculum
- Thematic Area Addressed: 1) Essential Fish Habitat Area B and 2) Aquaculture Area A Lead Scientist(s)
- LMRCSC Collaborator(s
Fig. 3: Leukotriene B4 Production by Stimulated Leukocytes at 4 Weeks Post-Injection 
Fig. 3: Cell supernatants from striped bass sampled at 4 weeks have been analyzed via enzyme immunoassay kits for LtB4 production. Values were not significantly different for fish fed either a high (H) or low (L) PUFA diet or injected with M. marinum (+) vs. PBS-injected fish (-). Values are reported as pg of LtB4/ ml cell supernatant. 3. Project Title: Investigation into persistence and distribution of sediment reservoirs of Hematodinium sp., a protistan parasite of blue crab (Callinectes sapidus) Project Description: In 2007 we developed a PCR assay that is suitable for use in environmental samples such as water and sediment. In 2008, the ITS2-based PCR assay was used to uncover evidence of Hematodinium DNA in samples from Indian River Inlet, DE, in two different sampling trips during the summer. One sampling was coincident with a large Hematodinium-associated crab die-off. The crab die-off, and the environmental evidence of the parasite DNA found using the PCR assay was reason to propose a more detailed examination of the Hematodinium signal in the IR Inlet. In addition, it raised questions about whether Hematodinium detected in sediment can cause infections in blue crab. Thus, the two objectives for the 2009 study were to: 1. Define the extent of a ‘hotspot’ in the Indian River Inlet along two perpendicular transects [spatial dynamics], and 2. Compare the persistence of Hematodinium sp. in sediment from known ‘hotspots’ to undisturbed sediment maintained in two different aquaculture regimes [temporal dynamics]. Thematic Area Addressed: Essential Fish Habitat Lead Scientist(s): Eric Schott (COMB) NOAA Collaborator(s): Gretchen Messick, Cooperative Oxford Lab LMRCSC Collaborator(s): Joseph Pitula, UMES; Dennis McIntosh, DSU LMRCSC Research Student(s): Ammar Hanif (Baltimore City Community College, accepted provisionally into the MEES graduate program at UMD), hourly-paid undergraduates in DSU Aquatic Sciences. Joanna R. Donaldson (UMES). Whitney Dyson, UMES (with JS Pitula) Planned Start Date: Nov. 1, 2008 Planned End Date: Oct. 31, 2009 Results of project: Highly accurate standard curve using ‘pseudo-Hematodinium” spiked into sediment. A requirement for meaningful PCR-based quantification is a standard curve based on a dilution of the target organism that approximates the environmental samples that will be later processed. Because the putative life stage of Hematodinium sp. that may inhabit sediment or water is not available, we created a surrogate using the cloned ITS2 target gene in the bacterium E. coli. Q-PCR assays using DNA from “pseudo-Hematodinium” in sediment generated standard curves that parallel standard curves using purified DNA. The real-world standard curve shows that the ITS2 assay is sensitive to as few as 30 copies of the target gene per PCR reaction. Application of the ITS2 assay to Indian River Inlet sediment. Summer sediment collections: MD Coastal Bays: As in 2008, Hematodinium sp. signal was found, but instead of being in most of the sediment samples examined, was found only in two sites. The 2009 sampling design was more thorough than 2008, in that triplicate samples were taken, 3 meters apart, at each location (12 locations in all). Leveraged funding: Planned for submission to the LMRCSC TAB process this year, Dr. Schott and Dr. Ogburn (SSU) are drafting a proposal to study the prevalence of blue crab diseases in megalopae and larval stages of blue crab in the GA coastal zone (anticipated budget of $58,000). Dr. Ogburn’s expertise in crab larval biology and Dr. Schott’s experience with molecular detection of disease agents are combining in a proposal that will fund Ammar Hanif’s training (PhD-track, MEES) for 3 months in 2010. A proposal to study crab disease has been submitted to the NOAA Saltonstall-Kennedy Program, “Monitoring a fatal reo-like virus in the blue crab Callinectes sapidus: molecular tools for the soft shell industry and fishery management” (total for 2 years: $180,000). This submission carries 25% support for Ammar Hanif’s stipend for 2 years. NOAA-NCCOS fisheries biologist Gretchen Messick is collaborator on this proposal. COMB students visit SSU: Ammar Hanif, Joanna Donaldson (UMES) and Eric Schott accepted an invitation from LMRCSC Postdoctoral researcher Matthew Ogburn to visit SSU Biology Department in March, while the COMB contingent was in Savannah for the 101st meeting of the National Shellfisheries Association. The group toured the Dept. of Natural Sciences & Mathematics and attended a master’s thesis seminar. Follow-up collaborations (unfunded) include a shipment of Callinectes similis from SSU to COMB, which were used in a study on cross-infection of the blue crab reo-like virus. This has led to the drafting of a TAB proposal to include SSU and COMB, to examine the prevalence of crab diseases in blue crab larvae. How will results be incorporated into NOAA Fisheries operations? The ITS2 assay accurately detects Hematodinium DNA in sediment, and is useful for screening potential reservoir hosts and for testing blue crabs. This assay is being used in an ongoing collaboration between COMB (Schott) and the Cooperative Oxford Lab (NCCOS, Messick) to assess Hematodinium in samples from COL’s ecological assessments of Chesapeake Bay (the Integrated Assessment). Though the Oxford Lab operations are primarily NCCOS-directed, there are NMFS scientists at COL as well, and this disease detection technology is available to NMFS (e.g., NOAA Chesapeake Bay Office). How will results be incorporated into LMRCSC research and curriculum? The validation of the PCR technique for quantitative detection of Hematodinium in sediment DNA was conducted chiefly by graduate student candidate Ammar Hanif. During the summer projects (June-August), Ammar shared his training and knowledge with summer interns participating in the LMRCSC/COMB program. This training has broadened students’ perspective and links field-based and lab-based investigations into an interdisciplinary approach to understanding disease prevalence. Results of this and previous TABs were incorporated into Dr. Schott’s course MEES608F Diseases in the Chesapeake Bay (offered fall 2008 and fall 2009). 4. Project Title: Programmed Cell Death in Karlodinium micrum (K. venificum), its induction by Environmental Factors and Potential in Bloom Management Project Description: With the discovery of the first toxic dinoflagellate confirmed in the Chesapeake (Deeds et al, 2000) as a result of major fish kills at a hybrid striped bass aquaculture facility (Terlizzi, et al., 2000) there has been increased awareness of the need for management methods for harmful algae blooms in aquaculture operations. We know very little about the factors regulating dinoflagellate bloom mortality. Available data suggest environmental factors e.g. light, CO2 and other nutrients may induce programmed cell death (PCD). In preliminary studies we have evidence that PCD in K. venificum can be induced through dark treatment. If reduced light induces PCD it may be possible to manage blooms in early stages through the use of commercial, environmentally benign dyes. Thematic Area Addressed: 1) Essential Fish Habitat Area B and 2) Aquaculture Area A Lead Scientist(s): D. Terlizzi, D. McIntosh, G. Wikfors, F.Chrest NOAA Collaborator(s): Gary Wikfors Supervisory Research Fisheries Biologist, NOAA, National Marine Fisheries Service, Northeast Fisheries Center, Milford Laboratory, 212 Rogers Avenue, Milford, CT 06460 LMRCSC Collaborator(s): Dennis McIntosh, Assistant Research Professor & Extension Specialist - Aquaculture, DSU LMRCSC Research Student(s): Gregory Oliver, MS Candidate, Delaware State University. Dover, Delaware Planned Start Date: 6/1/09 Planned End Date: 5/1/11 Results of project: Oliver, G., McIntosh, D., Terlizzi, D., & Chrest, F. 2009. Programmed Cell Death in Karlodinium veneficum, its induction by Environmental Factors and Potential in Bloom Management. Abstract. NOAA Education and Science Forum. 11/12-11/14/09. Howard University, Washington, D.C. Oliver,G., Terlizzi, D., Chrest, F. & McIntosh, D. 2009. Current Methods of Controlling Harmful Algal Blooms and Possible Future Solutions. Abstract. World Aquaculture Society Meeting. 4/1-4/5/10. San Diego, CA. How will results be incorporated into NOAA Fisheries operations? Better understanding of PCD in phytoplankton may provide insights into natural bloom termination in the environment and possible application to bloom management. How will results be incorporated into LMRCSC research and curriculum? Gregory Oliver a recent Biology graduate from UMES was selected for the Research Assistantship provided by DSU in support of this Project. Gregory is preparing for a career in commercial fish production and is interested in the training this project provides on fundamental concerns in aquaculture like harmful algal bloom management and the application of flow cytometry in aquaculture. Training: Gregory Oliver visited D. Terlizzi’s laboratory at the UMBI-COMB on July 28, 2009 for a day-long training session on the culture of phytoplankton including media preparation, culture transfer and maintenance. A workshop on the Principles of Flow Cytometry was organized by D.Terlizzi and F. Chrest and presented to faculty, staff and students at DSU on August 26, 2009. F. Chrest presented an overview of flow cytometry which was followed by hands on demonstration of flow cytometry with phytoplankton species used in the research project and the analysis of a preliminary experiment confirming dark induction of PCD in Karlodinium veneficum. This training provided an opportunity to confirm earlier observations on PCD induction in Dunaliella tertiolecta and Karlodinium veneficum. 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