Methods in health assessment of freshwater mussels, Amblema plicata and Quadrula spp. Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By K. Hope Valentine, D.V.M. Graduate Program in Veterinary Preventive Medicine The Ohio State University 2011 Thesis Committee: Paivi Rajala-Schultz, Advisor Barbara Wolfe Mary Jo Burkhard Copyright by K. Hope Valentine 2011 ii Abstract Health monitoring that is non-invasive, repeatable, and sensitive is a critical need for North America’s most endangered animal, the freshwater mussel. Ohio is home to a large proportion of these threatened animals and in response has established a captive flow through facility for research and propagation. Currently there is gap in knowledge of mussel physiology and health requirements in the wild and in captivity, representing an urgent need for health assessment methods. Hemolymph, the circulatory fluid of bivalves, and the cellular portion called hemocytes, are just beginning to be investigated in freshwater mussels. Recent studies have shown that hemolymph can be safely and repeatedly drawn. In addition, a schematic for freshwater mussels’ hemocytes was developed using L-cysteine as a novel anticoagulant. Following trends in hemolymph biochemistry and cellular differentials may be a way to monitor the health of freshwater mussel populations in the wild and in captivity; however, current handling protocols for transport of hemolymph are unknown. This thesis aimed to develop hemolymph handling protocols to preserve cellular integrity and function over time and during transport; to develop baseline biochemical and hematologic reference ranges in a population of wild mussels; and to describe population trends in biochemical and hematological parameters over one year following translocation of animals into captivity. ii Due to the marked physiological differences between invertebrate and vertebrate species, handling protocols for hemolymph and cell enumeration cannot be inferred from mammalian based research. Given that the majority of laboratories are 1-4 hours away from the wild locations or captive facility, handling protocols during transport to minimize in-vitro effects were developed. The second chapter of this thesis investigated processing temperatures for hemolymph, the ideal pH for reconstitution of the anticoagulant L-cysteine, and the effects of prolonged exposure to L-cysteine in-vitro. Towards an optimal method for hemocyte enumeration, cytochalasin B, genistein, and a lower dose of formalin than previously published were evaluated. Total hemocyte count, percentage of viable cells, and presence or absence of cellular debris as an indicator of cell lysis and degranulation were compared for each experiment. Chapter 2 found that exposure of hemocytes to temperatures below 10 degrees Celsius increased cellular aggregation in-vitro and decreased cellular viability. Only formalin treatments were found to increase total cell counts. Treatment of hemocytes with formalin, L-cysteine, cytochalasin B, and genistein resulted in a marked production of cellular debris at one hour post exposure. Untreated hemocytes at ambient temperature can maintain a high level of viability (80-93%) for up to 24 hours. Therefore, hemocytes should be transported without fixative or anticoagulant at ambient temperature. It is recommended that hemocytes only be exposed to L-cysteine (25mg/ml, pH 8.0) just prior to slide preparations for morphological analysis. Hemolymph chemistry may be a useful non-lethal indicator of bivalve physiological processes once reference ranges are established to differentiate normal iii from abnormal fluctuations in health. Chapter 3 aimed to establish reference ranges for the following measurable components in hemolymph: sodium, chloride, magnesium, phosphorus, potassium, calcium, glucose, and isoenzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in a population of mussels in the wild and follow changes in the biochemistry of hemolymph from the same population translocated into captivity over one year. Hemolymph from forty animals of three species, Amblema plicata, Quadrula quadrula, and Quadrula pustulosa was collected in July of 2008 from the Muskingum River in Devola, Ohio to develop baseline reference ranges. Thirty of those forty animals were translocated into captivity, with nine captive controls. Animals were sampled biweekly for the first month and then quarterly over one year. Significant differences in sodium, potassium, chloride, magnesium, ALT, AST, and ALP were found between genera, A. plicata and Quadrula spp. at baseline (p<0.05). Both genera showed declines in sodium and chloride in the first month in captivity and had marked increases in all electrolyte values in November, five months after transport to captivity. Calcium and glucose values remained steady in the population until the last collection point in June when values in both parameters declined. Phosphorus levels increased in both genera with significantly higher levels seen in A. plicata in February (p<0.05). Losses occurred during the study in greater proportion after winter sampling time points, potentially indicating that sampling during winter quiescence is not recommended. This study indicates that genus differences are present in biochemical values in freshwater mussel hemolymph in the wild and in captivity over time. iv In mammalian medicine, a complete blood cell count is a crucial piece of the minimal health database for diagnosis of potential disease or routine health checkups. Recent development of a standardized method for hemocyte characterization in freshwater mussels has paved the way for application of this technique to monitor freshwater mussels in the wild and in captivity. However, before interpretations of normal and abnormal fluctuations in cell type can take place, reference ranges need to be developed. This study aimed to provide preliminary baseline reference ranges from two common freshwater mussel genera, Amblema and Quadrula in the wild during the peak summer field season, then translocated animals into captivity for monitoring of hemocytology trends in a captivity over one year. Cell differentials were found to be genus-specific at baseline and for the first month in captivity (p< 0.05). Total hemocyte counts between genera differed significantly at 2wks and in November (p <0.10). Four cell types were identified in each genus studied at each time point. Eosinophilic granulocytes predominated in both genera. The proportion of eosinophilic granulocytes in A. plicata ranged from 53 to73%, large agranulocytes from 19 to 41%, basophilic granulocytes from 1 to 6% and small agranulocytes from 1to 3%, compared to Quadrula spp. that had 44-61% eosinophilic granulocytes, 8-27% basophilic granulocytes, 28-40% large agranulocytes, and less than 1 % small agranulocytes. This study provides a foundation for baseline reference ranges for A. plicata and Quadrula spp. and a preliminary understanding of shifts in blood cells in a population of mussels in captivity over one year. v Dedication This thesis is dedicated to my mentor Dr. Barbara Wolfe who has inspired, encouraged, and pushed me to be and do better whether near or far, and to my parents, Karla Hehl and Val Valentine, without whom none of this would have been possible. vi Acknowledgements I would like to thank Dr. Paivi Rajala-Schultz, for her pragmatic advice and guidance at every step of development of this degree from the inception of the idea to pursue it to its completion and Dr. Mary Jo Burkhard for her expertise in clinical pathology and willingness to study bivalves even though she is deathly allergic to shellfish. To Dr. Amanda Nahlik, my dear friend that really pushed me through the final stages of my writing, providing invaluable support and feedback and reading the umpteenth copy of each chapter. To Casey Pollack for letting me use her primo cubicle space and Dr. Mary Kantula, Steve Cline, Teresa McGee, Dixon Flanders, Phil Kaufmann and Glenn Griffith for their inspiration in the last few weeks of writing. I would also like to thank Sarah Josephine Fannin for making everything more worthwhile and her continuous support to keep going and stay focused. Finally, I would like to thank Trisha Gibson, Brooke Kelly, Kody Kuehnl, and Tom Watters from the Columbus Zoo and Aquarium Freshwater mussel Conservation Research Center for their mussel expertise and dedication to the conservation of these imperiled amazing little creatures. Many thanks to the Morris Animal foundation, the Ohio Division of Wildlife, the Columbus Foundation and Chemical Abstracts for support of this research. vii Vita December 1 1979 ...........................................Born- Raleigh, North Carolina 2002................................................................B.S. Animal Science and Wildlife Conservation, University of Massachusetts- Amherst. Amherst 2007 ...............................................................D.VM., North Carolina State University, College of Veterinary Medicine, Raleigh 2007-2009 .....................................................Conservation Medicine Intern, the Wilds, Cumberland, OH 2008-2010 ...................................................... M.S. Veterinary Preventive Medicine, The Ohio State University, Columbus Publications Valentine, K.H., C.A. Harms , M.B. Cadenas, A.J Birkenheuer, H.S. Marr, R.G Maggi, and E.B. Breitschwerdt.