UNIVERSITY OF CINCINNATI Date:____March 3 2006____ I, _______________________Angela M. Horner_________________, hereby submit this work as part of the requirements for the degree of: Master’s of Science in: Biology It is entitled: The effects of viscosity on the axial motor pattern and kinematics of the African lungfish (Protopterus annectens) during lateral undulatory swimming This work and its defense approved by: Chair: _____________Dr._Bruce C. Jayne _____________Dr._Rebecca German ______________Dr.Elke Buschbeck _______________________________ _______________________________ The effects of viscosity on the axial motor pattern and kinematics of the African lungfish (Protopterus annectens) during lateral undulatory swimming A thesis submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of Master of Science In the Department of Biological Sciences of the College of Arts and Sciences 2006 by Angela Margaret Horner B.S., Centre College, 2000 Committee Chair: Bruce C. Jayne, PhD Abstract Individual organisms often must move in different environments, which potentially pose different mechanical challenges. Although separate studies of terrestrial and aquatic locomotion are abundant, research addressing locomotion in transitional environments, such as mud, is scant. Both external and internal mechanical factors are widely recognized as important for understanding mechanisms of propagating waves during undulatory swimming, but few studies of swimming have manipulated extrinsic mechanical factors. The African lungfish (Dipnoi: Protopterus annectens) naturally occurs in ephemeral freshwater, and burrows in mud to escape seasonal drought. Thus, variable viscosity is ecologically relevant for this species, and its capability for air-breathing facilitates testing the biomechanical effects of substances like mud. To determine the effects on locomotion of changing viscosity, such as encountered in a variably muddy environment, I used a non-toxic PHPA polymer in solutions with viscosities of 1, 10, 100, and 1,000 centi-Stokes (cSt). During lateral undulatory swimming, the angle of maximal lateral flexion increased significantly with viscosity, particularly in the most anterior sites tested. The electromyogram (EMG) amplitude and longitudinal extent of muscle activity increased significantly with increased viscosity, although all observed axial motor patterns were posteriorly propagated and alternated between left and right sides. The distance traveled per tail beat cycle from lowest to highest viscosity decreased significantly. The phase shift between EMG onset relative to bending increased with viscosity, so that axial muscles were increasingly active during lengthening phase rather than shortening. Motor pattern and kinematics of lungfish in water did not differ from data on other swimming vertebrate taxa, but muscle activity and kinematics even in the most viscous solution differed extensively from observations of other taxa on land. Keywords: locomotion, lungfish, EMG, kinematics, swimming, viscosity, Protopterus Acknowledgements I would like to thank my advisor, Bruce Jayne, and my committee members for their valuable assistance and insight into this project. Much of the research involved in this project was assisted by Henry Astley, Steve Lochetto, and Lisa Day. Past Jayne lab members provided much-needed moral support and were valuable in shaping the beginning of my graduate experience in biology. I would also like to thank Peter Braun, who is always positive and supportive and never has any doubts in my ability, and my family for financial and moral support. TABLE OF CONTENTS Table of Contents …………………………………………………………………………. i List of Tables ……………………………………………………………………….…….. ii List of Figures …………………………………………………………………………….. iii Introduction …………………………………………………………………………… 1 Methods ……………………………………………………………………………….. 4 Experimental subjects and protocol…………………………………………………….. 4 Kinematics………………………………..……………………………………………….. 5 Electromyography………………………………………………………………………… 6 Muscle strain………….…………………………………………………………………… 6 Statistical analyses………………………………………………………………………….7 Results …………………………………………………………………………………. 8 Morphology……………………………………………………………………………….. 8 Kinematics………………………………………………………………………………… 8 Motor pattern……………………………………………………………………………… 9 Muscle strain…………………………..…………………………………………………. 9 Discussion …………………………………………………………………………………… 10 Morphology and swimming mode…………………………………………………….. 11 Longitudinal variation during steady swimming……………………………………… 12 Environmental effects on locomotion ………………………………………………… 14 Literature Cited……………………………………………………………………………… 19 i LIST OF TABLES Table 1. Summary of electromyography implant sites…………………………………….. 25 Table 2. Summary of F-values from 3 and 2-way ANOVAs performed on kinematic variables………………………………………………………………………………………….. 26 Table 3. Summary of F-values from 3 and 2-way ANOVAs performed on EMG variables for four individuals (sites 5-7) and one individual (sites 2-8)…………………... ……………… 27 Table 4. Summary of strain values estimated for superficial red muscle during steady swimming among four viscosities…………………………………………….………………… 28 ii LIST OF FIGURES Fig. 1. The reconstructed outline and midline of a lungfish from a ventral mirror (effectively dorsal) view. The numbers indicated standard anatomical locations for red muscle EMG sites (see Table 1), with bilateral sites indicated by asterisks, and white muscle sites parenthesized. The midline was partitioned into lengths of vertebrae determined from radiographs of the fish. The large vertical bar indicates the location of the pelvic girdle…………………………..........29 Fig. 2. Grand means of speed (A), cycle duration (B), and distance traveled per cycle (C) for each viscosity. The most similar cycles in each individual were analyzed, but individuals almost always swam faster in the higher viscosities……………………………………………………….30 Fig. 3. Series of lungfish midlines derived from digitized locomotor sequences for one cycle (as shown in Fig. 1) from the same individual in viscosities 1 (A), 10 (B), 100 (C), and 1000 (D) cSt. This individual was traveling at the same average speed (0.22 ± 0.02 L s-1) in all sequences. Note that amplitude increases anteriorly with increasing viscosity………………………………..31 Fig. 4. Mean values ± S.E.M. of maximal lateral vertebral flexion for four individuals swimming in four viscosities for all 8 longitudinal positions along the body. Flexion is always greatest in posterior sites, and anterior flexion increases with viscosity……………………………………….32 Fig. 5. Filtered EMGs from superficial red muscle from four left-side longitudinal sites from a single lungfish. Recruitment of anterior sites increased with increasing viscosity, and overall amplitude generally increased in each site………………………………………………..……….33 iii Fig. 6. Filtered EMGs from superficial red muscle from three longitudinal sites with both left side (black trace) and right side (blue trace) implants……………………………………………………34 Fig. 7. Filtered EMGs from superficial red muscle (black trace) and deeper white muscle (green trace) from a single longitudinal site in a single lungfish in all viscosities. White muscle recruitment increased with viscosity, as indicated by the increasing amplitude in the white muscle signal…………………………………………………………………………………………….35 Fig. 8. Mean values ± S.E.M. of EMG intensity for sites 2-7 among all individuals in all viscosities. ……………………………………………………………………………………………….36 Fig. 9. Lateral vertebral flexion (β) and red muscle activity (horizontal black bars) from sites 1 (top) through 8 (bottom) versus time for two cycles of an individual lungfish swimming in viscosities 1, 10, 100, and 1000 cSt, respectively. Max convexity to the left is represented by positive β values. The filled circles represent raw data of β, and the lines represent a two-point running average (sampling proportion 0.08) of the raw data. ……………………….…………..37 Fig. 10. Comparative summary of bending and red muscle activity for mean values of four lungfish in viscosities 1 (A), 10 (B), 100 (C), and 1000 (D) cSt. ……………..………………….38 Fig. 11. Mean timing of EMG onset and offset relative to lateral vertebral flexion (β). Values are based on a maximum of four individuals at a given site. The timing of both EMG onset and offset is relative to the time of maximum convexity. ………………………………………………..39 iv Introduction Transitions between aquatic and terrestrial environments are common among vertebrates, both on an evolutionary time scale and within an organism’s lifespan. Aquatic animals living in ephemeral pools or streams, for example, must contend with seasonal drought. Some vertebrates, such as eels, avoid drought by migrating over land to another body of water, while others may burrow into the mud to estivate. Drastic changes in the physical environment seem likely to affect the biomechanics of movement, but as yet these transitions are poorly understood. Laboratory studies have compared locomotion on land and water (Ashley-Ross and Bechtel, 2004; Gillis, 1998a; Gillis and Blob, 2001; Jayne, 1988), but how organisms move through a transitional substrate, such as mud, is not well understood. This ubiquitous substance represents the interface of land and water in many environments and was probably the first terrestrial substrate the earliest tetrapod encountered (Clack, 2002). Limbs are often ineffective in mud, and many vertebrates rely on axial musculature to propel them through it. Lateral bending of the axial structure