ABSTRACT Title of Document: DEVELOPMENT, VALIDATION AND APPLICATION OF A QUANTITATIVE POLYMERASE CHAIN REACTION ASSAY TO ASSESS ENVIRONMENTAL SAMPLES FOR HEMATODINIUM PEREZI PREVALENCE Ammar W. Hanif, MS, 2012 Directed By: Assistant Professor Eric J. Schott, Institute of Marine and Environmental Technology, University of Maryland Center of Environmental Science Associate Professor Rosemary Jagus, Co- advisor, Institute of Marine and Environmental Technology, University of Maryland Center of Environmental Science Dinoflagellate parasites in the Hematodinium genus have emerged as important pathogens of economically important crustaceans worldwide, causing significant economic losses to fisheries and aquaculture. An understanding of the routes of infection in blue crab (Callinectes sapidus) populations would be facilitated by an improved knowledge of environmental reservoirs. A previously used PCR assay, based on small subunit rRNA sequences, lacked the specificity needed for Hematodinium perezi detection of environmental samples. Therefore a quantitative PCR assay based on the internal transcribed spacer 2 (ITS2) region of H. perezi rRNA genes was developed, validated, and applied to examine temporal and spatial incidences of environmental reservoirs in Delmarva coastal bays. H. perezi was detected in sediment and water in several Delmarva coastal bays, as well as the host, C. sapidus. Results suggest the existence of localized sediment reservoirs in areas where hydrological and geophysical features allow for the formation of cell deposits. DEVELOPMENT, VALIDATION, AND APPLICATION OF A QUANTITATIVE POLYMERASE CHAIN REACTION ASSAY TO ASSESS HEMATODINIUM PEREZI PREVALENCE IN ENVIRONMENTAL SAMPLES By Ammar W. Hanif Thesis submitted to the Faculty of the Graduate School of the University of Maryland College Park, in partial fulfillment of the requirements for the degree of Master of Science 2012 Advisory committee: Professor Eric J. Schott, Chair Professor Rosemary Jagus Professor Joe Pitula © Copyright by Ammar Hanif 2012 Acknowledgements I owe much gratitude to my primary and co-advisor, Dr. Eric Schott and Dr. Rosemary Jagus. They have worked to make sure that I understood the basic aspects of my research and master my technical skills. I am grateful for the support, financial and educational, the NOAA- Education Partnership Program Living Marine Resources Cooperative Science Center (LMRCSC) has provided me. The experiences that I have had as part of the program have been very valuable. I would like to thank Dr. Sook Chung for teaching me basic blue crab biology and physiology and Dr. Feng Chen, Dr. Russell Hill and Dr. Allen Place who have taught me dinoflagellate ecology, principles of molecular techniques and microbiology. While not directly involved in my research, their expertise has helped to enhance my understanding of my research. I would like to thank all the graduate students, lab techs and post docs for helping with experiments, giving feedback on presentations and explaining techniques. I am also grateful for the assistance that LMRCSC summer interns provided with experiments. Lastly I want to thank collaborators from Delaware State; Dr. Dennis McIntosh, Dr. Dewayne Fox, Marissa Brady, Tylor, Johnny Moore, UMES; Dr. Joseph Pitula, Whitney Dyson, and Cooperative Oxford Lab, NOAA-NCCOS; Gretchen Messick. ii Table of Contents Chapter 1……………………………………………………….page 1 Chapter 2……………………………………………………….page 13 Chapter 3……………………………………………………….page 49 References……………………………………………………..page 58 iii List of Tables Table 1.1 BLAST analysis against amplicon generated by Nagle et al primers……………………………………………………….……...page 9 Table 2.1 Oligonucleotides used in this study …………………..page 20 Table 2.2 Levels of H. perezi in Indian River Inlet sediment and water……………………………………………………………..…..page 31 Table 2.3 Levels of H perezi in blue crab during an outbreak in Indian River Inlet…………....…..…………………………………………….…..page 32 Table 2.4 Levels of H. perezi in biotic samples from Indian River Inlet collection sites…………..………………………………………….…………..page 33 Table 2.5a List of Chincoteague Bay sediment samples positive for H. perezi rDNA…………….……………………………………………...……page 36 Table 2.5b List of Chincoteague Bay water samples positive for H. perezi rDNA ………………………………………………………………..………page 37 List of Figures Figure 1.1 Schematic diagram of the in vitro life cycle of Hematodinium sp. from C. sapidus ……………………………………………………..…….…page 4 Figure 2.1 Schematic representation of H. perezi rDNA locus ………………………………………………………………….…….page 27 Figure 2.2 Comparison of qPCR standard curves using pGEMT/HspITS2 DNA in various forms………………………………………………………………….page 29 Figure 2.3 Map of Indian River Inlet sampling sites…………………………………………………………....…….page 30 Figure 2.4 Map of Chincoteague sampling sites …………………………………………………………………...……page 33 Figure 2.5 Temporal variation of the incidence of H. perezi in Chincoteague Bay sediment and water……………………………………................page 39 iv Chapter One: General Introduction Blue crab fishery: The blue crab, Callinectus sapidus, is an ecologically and economically significant crustacean with a habitat range from Nova Scotia to Uruguay, South America. It is a key benthic-pelagic link in the marine environment where it forages for food in the benthos and is a staple in the diet of many pelagic fish. In the United States the blue crab supports a fishery with an estimated worth of $160 million annually. There is a large commercial fishery from Maryland to the Gulf of Mexico. In the Chesapeake Bay the annual value of harvest is over $50 million [1]. From Massachusetts to Delaware it is an important recreational fishery. There was a time when the blue crab was so abundant the harvests were described as “inexhaustible” [2], however prior to 2009 the blue crab population in the Chesapeake Bay had seen a decade long decline [3]. According to the 2010 Chesapeake Bay Blue Crab Advisory Report the historically low population sizes have been associated with high levels of exploitation [4]. Other states such as North Carolina, South Carolina and Georgia have also experienced declines in their blue crab populations. In the Chesapeake Bay, the persistently low blue crab broodstock stimulated the formation of a research consortium to study hatchery-based broodstock enhancement and led to the implementation of unprecedented harvest restrictions [5]. One approach of this research consortium was to assess various areas for their suitability as habitat for entry of hatchery reared blue crabs that would grow into future broodstock. Potential habitat needed to be assessed for disease prevalence to ensure greater success of crab 1 survival. Since 2009, in the Chesapeake Bay harvest restrictions and improved stock management practices have helped to alleviate the pressure on the blue crab population, however it is typical to have year-to-year fluctuations in abundance that cannot be explained by fishing pressure alone [6]. In the context of efforts to sustain a vigorous fishery, it is critical to monitor disease-causing agents. It is now agreed that estimates for natural mortality of blue crab are over- simplified and even though many blue crab diseases have been described, their influence on mortality and shaping of the population are poorly understood [7– 10]. In mid-Atlantic coastal bays of North America, the blue crab population suffers episodic mortalities associated with infections by the parasitic dinoflagellate, Hematodinium perezi [11–13]. It is found in both hemolymph and tissues of the blue crab where the parasites can multiply rapidly, eventually causing organ failure resulting in crab death. These outbreaks generally recur seasonally and are geographically localized in “hotspots,” which are characterized by constrained, high salinity, warm waters. Outbreaks most likely have a varied effect on the blue crab population and most probably go unnoted. For example, Lee and Frischer [14] reported such high mortality rates in a Georgia outbreak that the fishery was affected widely for several years, whereas an outbreak we observed in a Delaware coastal bay(see Chapter 2) was highly localized and transient. The fact that these outbreaks are seasonal and not ongoing shows that there exists some reservoir of the parasite. The presence of Hematodinium sp. is of significant concern since hotspots of this pathogen have 2 been shown to exist in several blue crab fisheries throughout the mid-Atlantic [12, 13, 15]. In particular, release of hatchery crabs should avoid hotspots where this parasite could affect survival. A survey of the parasite in wild blue crabs, blue crab prey, water and sediment samples could help in the understanding of the infection cycle and the impact on survival rates. An understanding of the life cycle, infection dynamics and environmental prevalence and incidence should yield insight into why these outbreaks and recurring infections occur. Life cycle of Hematodinium sp.: The life cycle of Hematodinium sp. within the blue crab is still poorly understood, despite several morphologically distinct stages being reported from the hemolymph and tissues [10, 12, 16–18] and from short-term cultures (7–14 days) of the parasite [19, 20]. These findings suggest that Hematodinium sp. has a very complex life cycle. Naturally infected blue crabs often die over a period of 14 to 35 days depending on the intensity of the infection [12]. Experimentally infected blue crabs start dying 14 to 17 days post- inoculation (p.i.), with cumulative mortalities of 86 % by 40 days p.i. [10]