Myosin heavy chain (MHC) isoform expression in the prehensile tails of didelphid marsupials: functional differences between arboreal and terrestrial opossums by Joseph E. Rupert Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Biology Program YOUNGSTOWN STATE UNIVERSITY May, 2013 Myosin heavy chain (MHC) isoform expression in the prehensile tails of didelphid marsupials: functional differences between arboreal and terrestrial opossums Joseph E. Rupert I hereby release this thesis to the public. I understand that this thesis will be made available from the OhioLINK ETD Center and the Maag Library Circulation Desk for public access. I also authorize the University or other individuals to make copies of this thesis as needed for scholarly research. Signature: Joseph E. Rupert, Student Date Approvals: Dr. Michael T. Butcher, Thesis Advisor Date Dr. Mark D. Womble, Committee Member Date Dr. Gary R. Walker, Committee Member Date Dr. Brian De Poy, Interim Dean, School of Graduate Studies Date ii © Joseph E. Rupert 2013 iii ABSTRACT Prehensile tails are defined as having the ability to grasp objects and may commonly be used as a fifth appendage during arboreal locomotion. Despite the independent evolution of tail prehensility in numerous mammalian genera, data relating muscle structure, physiology, and function of prehensile tails are largely incomplete. Didelphid marsupials make an excellent model to relate myosin heavy chain (MHC) isoform fiber type with structure/function of caudal muscles, because all opossums have a prehensile tail, but tail function varies widely between arboreal and terrestrial forms. Expanding on our previous study in the Virginia opossum, this investigation tests the hypothesis that arboreal and terrestrial opossums differentially express fast versus slow MHC isoforms, respectively. MHC expression and percent fiber type distribution were determined in the flexor caudae longus (FCL) muscle of Caluromys derbianus (arboreal) and Monodelphis domestica (terrestrial), using a combination of gel electrophoresis and immuno-histochemistry analyses. C. derbianus expresses three MHC isoforms (1, 2A, 2X) in the FCL that are distributed as 8.2% MHC-1, 2.5% 1/2A, and 89.3% 2A/X hybrid fibers. M. domestica expresses MHC-1, 2A, 2X, and 2B, distributed as 17.2% MHC-1, 0.7% 1/2A, 7.7% 2A, 73.9% 2A/X, and 0.3% 2X/B hybrid fibers. The distribution of similar fiber types differed significantly between species (P<0.001). Although not statistically significant, C. derbianus was observed to have larger cross-sectional area (CSA) for each corresponding fiber type along with a greater amount of extra-cellular matrix. An overall faster fiber type composition (and larger fibers) in the FCL of an arboreal specialist supports our hypothesis, and correlates with higher muscle force required for tail-hanging and arboreal maneuvering on terminal substrates. Conversely, a broader distribution of highly oxidative fibers is well suited to the tail nest building behaviors of terrestrial opossums. iv ACKNOWLEDGEMENTS I sincerely thank my advisor, Dr. Michael Butcher, for all his guidance and mentoring throughout my Thesis research project and Masters Degree. I thank my graduate committee members, Drs. Mark Womble and Gary Walker for critical reviews of my Thesis and their helpful comments. I am grateful to Dr. John VandeBerg (Texas Biomedical Research Institute) for coordinating muscle harvesting in Monodelphis, Dr. Bernal Rodríguez and Eugenia Cordero Schmidt at the Tirimbina Biological Reserve for coordinating trapping (with MINAET) and muscle harvesting in Caluromys, and Suzanne Peurach for assistance with the mammal collections at the National Museum of Natural History (NMNH-Smithsonian Institute). A very special thanks to Andres Moreira for assistance with field collection and muscle biopsy surgeries in Costa Rica. Also, a special thanks to Manuel Rojas (Tirimbina) for help with animal collection, care and handling of Caluromys. Thanks to Laura Kosiorek (YSU) for assistance with data analysis, and Jake Rose and Marc Gorvet for being terrific lab mates. S. Fatteh (Northside Hospital, Youngstown, Ohio) provided access to the cryostat and Dr. Walker (YSU) assisted with protein analyses. The monoclonal antibodies developed by F. Stockdale (S58, F18), S. Schiaffino (SC71, BF-35, BF-F3) and C. Lucas (2F7, 6H1) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Support by University Research Council funding (URC #02-12) and NSF (IOS-1147044). The Department of Biological Sciences at YSU and the entire staff at the Tirimbina Biological Reserve are also gratefully acknowledged. v DEDICATION I dedicate my Thesis to my family, especially my father Brian and my mother Colette for their unfaltering support, both emotionally and financially, throughout my academic career and during the completion of this Thesis. vi TABLE OF CONTENTS Approval Page ii Copyright Page iii Abstract iv Acknowledgments v Dedication vi Table of Contents vii List of Tables viii List of Figures ix INTRODUCTION 1 MATERIALS and METHODS 3 Animals 3 Muscle biopsy and Harvesting procedures 3 MHC protein analyses: SDS-PAGE 4 Histochemistry and Immuno-histochemistry 5 Data analysis and Statistics 6 RESULTS 7 Electrophoretic identification of MHC 7 Histological determination of MHC fiber types 7 Fiber type distributions and Fiber size 8 DISCUSSION 10 MHC isoform distribution in opossum tails 10 Fiber CSA and Tail function 12 Prehensile tail structure 14 Conclusions and Future directions 14 REFERENCES 16 APPENDIX 27 Literature review 27 vii LIST OF TABLES 1. Morphometric data for experimental animals. 47 2. Monoclonal antibodies (mAbs) used for IHC analyses and their MHC isoform reaction specificity against the tail muscle FCL from C. derbianus and M. domestica. 48 3. Regional mean distributions (%) of MHC isoform fiber types in the tails of C. derbianus and M. domestica. 49 4. Mean percentage distributions (%) of MHC isoform fiber types in the caudal muscle FCL of C. derbianus, M. domestica, and D. virginiana. 50 5. Regional mean fiber CSA (µm2) of MHC isoform fiber types in the tails of C. derbianus and M. domestica. 51 6. Mean fiber CSA (µm2) of MHC isoform fiber types in the caudal muscle FCL of C. derbianus and M. domestica. 52 viii LIST OF FIGURES 1. Diagram of the tail skeletal anatomy of C. derbianus and M. domestica. 54 2. Silver-stained gels identifying MHC isoforms expressed in the FCL of C. derbianus and M. domestica 56 3. Representative fiber type reactivity for mATPase and IHC throughout the FCL of C. derbianus. 58 4. Representative fiber type reactivity for mATPase and IHC throughout the FCL of M. domestica. 60 5. Mean fiber CSA and minimum diameter of MHC isoform fiber types in the FCL of C. derbianus and M. domestica. 62 ix INTRODUCTION Environmental limitations largely influence the morphology of inhabiting species, resulting in selection for specializations that provide species with advantages for resource acquisition. Habitats rich in natural resources and niche space, such as the neotropical rainforests of Central and South America, provide numerous microhabitats for large scale speciation of mammals (Hortal et al., 2008; Qian, 2010). Didelphid marsupials (opossums) are perhaps the best example of species-specific diversity in mammals native to these neotropical regions. Modern opossums descend from a common South American arboreal ancestor (Szalay, 1994; Cozzuol et al., 2006) and are members of the family Didelphidae, representing 19 genera and more than 100 species (Voss and Jansa, 2009), ranging from the southern-most regions of Argentina to southern Canada (Hershkovitz, 1997). Despite marked diversity in their body size and locomotor behaviors (Cunha and Vieira, 2002), all species of opossums share a crouched limb posture, an opposable hallux, and most notably, a prehensile tail (Nowak, 1999). Function of the prehensile tail is specialized for the requirements of either arboreal or scansorial/terrestrial locomotion. Specifically, the degree of tail prehensility changes with the level of vertical strata occupied within the rainforest (Cunha and Vieira, 2002), ranging from fully-prehensile in arboreal species to semi-prehensile in terrestrial species. A fully-prehensile tail is capable of suspending the body from a substrate (Emmons and Gentry, 1983), whereas a semi-prehensile tail is primarily used for gross manipulation of objects, and lacks the ability to suspend the body (Emmons and Feer, 1990; Bezanson, 1999). Arboreal opossums use the tail for maneuvers such as bridging and jumping (Youlatos, 2008) to traverse gaps between terminal branches in the canopy, and also for added stability during postural behaviors (e.g., foot-hanging and tail-hanging) when feeding (Julien-Laferriere, 1999; Schmitt and Lemelin, 2002). Additionally, they rely on their fully-prehensile tail in the occurrence of falls where it can quickly grasp onto a branch and be used as a substrate to pull the body upward (Lemelin et al., 2003). Arboreal specialists such as Caluromys exclusively dwell, forage, and locomote in the forest canopy (Vieira, 1997; Delciellos and Vieira, 2007, 2009), reaching the forest floor only 1% of their life time budget, typically resulting from falls (Hunsaker, 1977; Charles- Dominique, 1983). In contrast, terrestrial opossums spend most of their life on the forest 1