CILIATE XENOMAS IN CRASSOSTREA VIRGINICA FROM GREAT BAY, NEW HAMPSHIRE By EMILY SCARPA McGURK A thesis submitted to the Graduate School – Camden Rutgers, The State University of New Jersey in partial fulfillment of the requirements for the degree of Master of Science Graduate Program in Biology and approved by ______________________________ Dr. William Saidel ______________________________ Dr. David Bushek ______________________________ Dr. Susan Ford Camden, New Jersey; May 2013 ABSTRACT OF THE THESIS Ciliate xenomas in Crassostrea virginica from Great Bay, New Hampshire By EMILY SCARPA McGURK Thesis Director: William Saidel During routine histological examination of oysters (Crassostrea virginica) from Great Bay, New Hampshire, a high prevalence and intensity of ciliate xenomas has been noted since 1997. Xenomas are hypertrophic lesions on the gills of bivalve molluscs caused by ciliates. Although not known to cause mortality in oysters, xenomas have not previously been reported at this high level of abundance. The objectives of this study were to characterize the xenomas, classify the ciliates, and gather baseline epizootiological data with correlations to environmental and biological parameters. Upon gross examination, xenomas appeared as white nodules located in the gill tissue, up to 3 mm in diameter, occasionally fusing into large masses along the gill filaments. Light microscopy of histological sections revealed xenomas located in the gill water tubes that often occupied the entire cross sectional area. Higher magnification revealed dual nuclei, eight kineties, and conjugation. Transmission electron microscopy ii revealed dual nuclei that vary in density, a maximum of twenty cilia in each kinety radiating from the oral apparatus to the posterior, and a 9+2 axoneme structure within the cilia. Sequencing of the 18S rRNA gene produced a unique sequence not present in GenBank. These oyster gill ciliates are generally listed as Sphenophrya dosiniae (Order Rhynchodida) and although there are no representatives of Rhynchodida in GenBank’s database, all similar matches were within the class Phyllopharyngea. Since 1997, xenoma prevalence has fluctuated with peaks in 2000, 2004, and 2011. Infected oysters generally contained <30 xenomas, but 2.1% contained >100, sharply contrasting the rare prevalence and low intensity reported elsewhere. Prevalence increased with oyster size, leveling off near 50% in oysters >60mm. Infection intensity peaked in 70-90mm oysters. Individually, oyster condition was not associated with xenoma intensity, but sites with oysters in higher condition generally had a greater prevalence and intensity of xenoma infections. Seasonal data indicated an infection cycle increasing from summer to fall, peaking at 55-65% in November and dropping to <10% by spring. The oyster population at Great Bay, NH warrants further examination to understand the mechanisms and conditions controlling xenoma formation, as well as the possible effects of a changing climate. iii ACKNOWLEDGEMENTS I would like to express my deep appreciation to Dr. David Bushek, for his guidance and assistance in my research and writing. I would not have begun this journey without his encouragement. I would also like to express my gratitude to Dr. Susan Ford and Dr. William Saidel, for their mentorship, thought provoking questions, and of course, for their patience! I would also like to recognize Dr. Raymond Grizzle, of the Jackson Estuarine Laboratory, who donated his time and efforts to collect samples for me throughout the year, and Bruce Smith, of the New Hampshire Fish and Game department, who made sure that their annual samples arrived with plenty of extra oysters for me to examine. Both were very accommodating in the accrual of any background information and data that could be of use. I am indebted to my colleagues at the Haskin Shellfish Research Lab, those still around as well as those who have come and gone, who were instrumental in keeping me focused, providing either a second opinion or empathy as needed. Finally, I dedicate this thesis to my family for their endless support and encouragement, my best friend for distracting me when I most needed a break, and my husband for his patience and understanding throughout. iv TABLE OF CONTENTS Abstract ….…………………………………………………..…… ii Acknowledgements ..............….....…………………………………………… iv Introduction: Oyster Parasitology and Xenomas ...............……..….……..…… 1 Background ...........……………………………………………..……. 3 Methods .............…………………………………………….…… 8 Results & Discussion .................….…………………………………..……….. 13 Conclusion ............………………………………………….………. 20 Figures ...........……….……………………………………..…… 24 Tables ...........……….……………………………………..…… 42 References ...........…………………………………………………... 50 1 INTRODUCTION: OYSTER PARASITOLOGY AND XENOMAS Parasites have historically presented problems for shellfisheries and aquaculture. For example, Haplosporidium nelsoni (causative agent of MSX) and Perkinsus marinus (causative agent of dermo disease) have devastated oyster populations on the east coast of the United States (Ford and Tripp, 1996) while pathogens such as Marteilia spp., Bonamia spp. and Perkinsus olseni have caused massive mortality of shellfish worldwide (Bower et al., 1999; Sparks, 2005). When first detected in 1957, MSX had caused 90- 95% mortality in eastern oysters, Crassostrea virginica, which had been planted on leased grounds in lower Delaware Bay. Subsequently, H. nelsoni has been found from Nova Scotia, Canada to Biscayne Bay, Florida (Burreson and Ford, 2004). Dermo disease has caused 50% mortality in C. virginica populations (Carnegie, 2009). The European flat oyster, Ostrea edulis, in Brittany, France experienced 50-90% mortality by Marteilia refringens infection in the 1960s, and then again by Bonamia ostreae in the early 1980s (Sparks, 2005). The 1970’s mortality of the Sydney rock oyster, C. commercialis, in Queensland, Australia was caused by M. sydneyi (Sparks, 2005). Recent publications have attributed marine diseases over the past few decades to both compromised host immunity caused by physiological stress of climate change or pollution, as well as direct disease transmission facilitated by human activity (Harvell et al., 1999; Lafferty et al., 2004; Mydlarz et al., 2006). It is possible that increased vigilance has led to increased reporting of disease events. During routine histological 2 examination of oysters (Crassostrea virginica) from Great Bay, New Hampshire, by the Haskin Shellfish Research Laboratory, a high prevalence and intensity of ciliate xenomas, as described below, has been noted since 1997. The xenoma infections were at a greater prevalence and intensity than observed in other regions, possibly a signal of an emerging disease problem. Despite the abundance of information about several parasites of eastern oysters (C. virginica), relatively little is known about xenomas. Xenomas are not known to cause mortality, but they previously have not been reported at prevalence above 2.5%, and given the devastating effects that parasites can exert on shellfish populations, proactive investigation of changes in parasite prevalence is imperative. Oysters in Great Bay, NH have suffered substantial reductions in both distribution and abundance due to pollution, overharvest, and siltation (Bolster, 2002). During the mid-1990s, an outbreak of MSX and Dermo disease caused further decline of the population. In response to these recent mortalities, and as part of restoration efforts, a program of annual population survey and disease monitoring by the New Hampshire Fish and Game Department was initiated in 1995. As part of this monitoring, oyster samples collected from one to six sites were sent each fall to Rutgers University’s Haskin Shellfish Research Laboratory (HSRL) for histopathology analyses. In the early 2000s, it became apparent that an unusually high prevalence of xenomas was present in the Great Bay oyster population. In 2005, the current study began to more thoroughly document the phenomenon of xenomas in Great Bay oysters. The purpose of this study was to characterize both the ciliates and the xenomas that they form and to gather baseline epizootiological data that would reveal correlations with various environmental and biological parameters to begin to elucidate the cause and 3 effect of the unusual abundance of xenomas in oysters from Great Bay, New Hampshire. The first objective was to characterize the xenomas and the pathological effect on the host through gross examination of the gills as well as light microscopy (LM) and transmission electron microscopy (TEM). The second objective was to classify the species of ciliate involved via LM and TEM, as well as by sequencing a portion of the DNA amplified from the xenomas. The third objective was to gather baseline epizootiological data by identifying temporal and spatial patterns of infection in Great Bay, NH. Patterns were analyzed and correlated with several parameters, including oyster size, condition index, and prevalence of other parasites and disease. BACKGROUND Xenomas are intracellular hypertrophic lesions that can be found on the gills of fish and bivalve molluscs. They occur when a parasite enters a host cell and proliferates, causing the host cell to grow abnormally large. In fish, xenomas are commonly a symptom of microsporidial gill disease and contain microsporidial spores (Morrison and Sprague, 1981). Xenomas in bivalve molluscs, however, are caused by ciliates and have been observed in