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
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 is pervasive in the locomotion of ectothermic vertebrates (Gray, 1968), which frequently have correspondingly large amounts of biomass sequestered in axial musculature. For example, the axial muscles of many species of fishes comprise 40-60% of the total biomass (Bone et al., 1978). The axial musculature of all vertebrates demonstrates a metameric pattern of organization
(Carrier, 1990; O'Reilly et al., 2000) despite considerable diversity in the details of anatomical features among and within taxa. Thus, a central issue for understanding axial structure and function in any vertebrate is how motor activity is coordinated among muscular segments as an animal bends.
Axial motor patterns of ectothermic vertebrates during steady aquatic lateral undulatory locomotion share many features. Specifically, for the undulatory swimming of
1 lamprey, sharks, actinopterygian fishes, salamanders, and snakes, the axial motor pattern is unilateral, alternates between the left and right sides, and is propagated posteriorly with a speed faster than that of the wave of bending (Coughlin, 2002; Frolich and Biewener, 1992; Jayne, 1988). Consequently, the same muscle activity pattern seems likely during steady lateral undulatory swimming in lungfish.
The universality of axial motor patterns during steady aquatic lateral undulation suggests evolutionary conservatism, which may be related to grossly similar function.
For example, Blight (1977) posited that the ancestral vertebrate axial body plan is a
‘hybrid oscillator,’ with the anterior-most region providing stiffness and the posterior-most region bending and stiffening in response to extrinsic resistive forces in an aqueous environment. During the water-to-land evolutionary transition in vertebrates, ancestrally aquatic organisms had to cope with increased gravitational forces, change in substrate, and increased ventilatory requirements (Clack, 2002). Despite these changes, vertebrates as disparate as fish and snakes still display the same basic muscle activity patterns during lateral undulatory swimming, which might suggest axial neuromuscular control is an evolutionarily conserved trait.
Lungfish (Dipnoi: Sarcopterygii) move in a variety of environmental conditions, and those in the African genus Protopterus frequently encounter harsh seasonal droughts that gradually dry the shallow rivers and lakes the fish occupy. As the water level decreases, lungfish construct burrows in the increasingly muddy substrate
(Fishman et al., 1987). Protopterus exhibits a morphology secondarily derived for burrowing; the bodies are elongate and the paired fins are diminutive to the point of having no functional role in pelagic locomotion. These behavioral and morphological attributes make lungfish a well-suited taxon for studying how environmental variation affects locomotor function of axial musculature. To date, very little research on any type of locomotion in this group has been performed (Meyers et al., 1998).
2 The muscular basis of both aquatic and terrestrial locomotion in eels, salamanders and snakes is well documented (Frolich and Biewener, 1992; Gillis, 1998a;
Jayne, 1988). Key differences among these taxa occur on land, rather than in water. For example, salamanders exhibit a standing wave of axial muscle activity on land and a traveling wave in water (Frolich and Biewener, 1992). Snakes maintain a posteriorly propagated wave of muscle activity on land for which the speed of propagation matches that of the mechanical wave of bending, described as a phase-locked relationship
(Jayne, 1988). Although eels have demonstrated a posteriorly propagated wave of muscle activity that travels faster than the wave of bending both on land and in water, the timing between the wave of bending and muscle activity on land approaches that of terrestrially undulating snakes (Gillis, 2000). Accordingly, I expected that a very viscous solution might elicit a motor pattern in lungfish more similar to eels and snakes on land when compared to a lower viscosity. Although Gray (1953) and Johnson (1998) have both experimentally manipulated viscosity, the degree to which they did so was marginal compared to the viscosity an organism might encounter in a muddy substrate. My objective was to simulate a range of viscosities that would serve as representative gradations of muddiness.
In the following study, I examined both the kinematics and muscle activity used by the lungfish Protopterus annectens during lateral undulatory swimming in four viscosities. Specifically, I was interested in how lungfish might modulate their motor pattern and kinematics when extrinsic forces were increased. My objectives for this study were (1) to describe lateral undulatory swimming in water in the lungfish and compare data to other vertebrates, and (2) to compare kinematic and electromyographic data among several ranges of viscosity in lungfish to land versus water comparative data gathered from eels, salamanders, and snakes. I predicted that increasing viscosity would
3 cause a corresponding increase in bending, and that at some level or threshold of viscosity motor pattern would shift to resemble a phase-locked relationship.
Materials and Methods
Experimental subjects and protocol
I obtained specimens of the African lungfish, Protopterus annectens, from a commercial dealer located in Baton Rouge, Louisiana. The lungfish were fed a diet of earthworms and goldfish and housed separately in glass aquaria. Water temperatures were maintained at approximately 24 °C, and the experiments were performed at the same temperature (± 1 °C). I performed experiments with a total of eight lungfish and only analyzed data from the four individuals with the best behavior and with the greatest number of electrodes remaining in place after experimentation. The lengths of the four individuals analyzed ranged from 51 cm to 59 cm. The numbers of precaudal vertebrae ranged from 33-35, and numbers of caudal vertebrae ranged from 35-37.
To facilitate implanting electrodes, the lungfish were anesthetized with MS222 (in a concentration of 1.5 g-1L).The fish recovered for at least 12 hours before experimentation. The lungfish were then placed into a 284 L glass tank filled to a depth of 17cm with one of the viscosity mixtures. I used 1, 10, 100, and 1000 centi-Stokes
(cSt) mixtures in a randomized order for each experiment. After five to ten tailbeats of swimming were recorded, the lungfish was moved to a small tank of distilled water while the mixture in the experimental tank was changed to a different level of viscosity. The total transition time to the next mixture lasted no more than 20-30 minutes. For my analyses, I attempted to use only trials of similar speeds and straight trajectories. At the conclusion of each experiment, the lungfish were killed with an overdose of MS222 and fixed in formalin. All individuals were radiographed and dissected to confirm electrode position within the myomere and relative to the vertebral location.
4 I manipulated the viscosity of the medium by using Poly-Bore (Baroid Industries), a PHPA polymer commonly used in offshore oil drilling. The mixtures were clear enough for both lateral and ventral views of the fish. I obtained viscosities of 1 (water), 10, 100, and 1000 cSt by increasing the concentration of Poly-Bore in a water solution and allowing ample time (1-2 months) to obtain a homogeneous mixture. Viscosity was measured prior to the beginning of experimentation, periodically during experimentation
(in between experiments on individuals), and again after all experiments were completed. I used several different sizes of Otswald-style viscometers (Fisher Scientific) to measure viscosities.
Kinematics
The lungfish were videotaped simultaneously from a lateral and dorsal view using a two-camera NAC HSV 500 high speed video system operating at 250 s-1 images.
Video images were digitized at equal time intervals, with at least 20 images per tail-beat.
I digitized 50-60 total points along the outline of the fish using the program Didge Image
Digitizing Software (Cullum, 1999). For each digitized image, additional software (Garr
Updegraff) was used to obtain the angles of bending along the midline. As described in
Jayne and Lauder (1995), the midline was reconstructed then partitioned into lengths representing the head and individual vertebrae obtained from radiographs of each individual fish (Fig. 1). Midline plots were created from x, y coordinates generated from the digitizing software.
Cycle duration was determined by calculating the difference in time between two homologous kinematic events at a particular longitudinal site. The distance traveled per cycle and speeds were converted from centimeters to relative units of total fish length
(L). I determined the angle of lateral bending between adjacent axial segments (β) for each of the eight longitudinal locations corresponding to the electrode implant sites. I
5 also calculated lag times between kinematic events at successive longitudinal locations and converted these values to phase lags by dividing by cycle duration.
Electromyography
After anesthesia, lungfish were implanted percutaneously with a total of thirteen stainless steel bipolar electrodes (California Finewire). Electrodes were bound together with a cyanoacrylate glue, and 0.5 mm of insulation was scraped from the tips of the wire to construct hooks, as described by Jayne (1988). Sutures were sewn into dorsal tissue and the medial fin to affix the electrode wires at each longitudinal location, and all electrodes were glued into a single main cable.
On the left side of the lungfish eight EMG electrodes were implanted in superficial red muscle (Fig. 1, Table 1), two electrodes into deeper, white musculature at sites 2 and 7. In addition, three electrodes were implanted on the right side of the fish in red muscle at locations 2, 4, and 7 (Fig. 1). Data from all thirteen channels were recorded simultaneously. Only electromyograms (EMGs) from electrodes that remained in place throughout the entire experiment and in preservation were analyzed for this study. I confirmed electrode position within a myomere and relative to the vertebral column by post-mortem dissections and radiographs (see Table 1).
EMGs were amplified 5 000 times using Grass model P511 K preamplifiers and then filtered using high- and low- bandpass filter settings of 10 kHz and 30 Hz, respectively, and a 60 Hz notch filter. The analog EMGs were recorded with a TEAC XR-
5000 FM data recorder using a tape speed of 9.5 cm s-1. A square-wave was transmitted simultaneously to both the NAC video system and the TEAC tape recorder, synchronizing the video and EMG data. I converted the analog signal to digital using a
Powerlab 16 channel converter (AD Instruments) at an effective sampling rate of 8.8 kHz.
6 I used Chart 5 software (AD Instruments) to measure EMG burst duration, rectified integrated area, and the times of EMG onset and offset. Relative burst duration
(or EMG duty factor) was calculated by dividing EMG burst duration by the cycle duration. EMG intensity was calculated by dividing the rectified integrated area of a single burst by the burst duration.
The timing of muscle activity relative to lateral bending data was described by the variables On-β and Off-β. The time of maximal convexity (βmax, on the same side of the fish as the electrode) was subtracted from the times of EMG onsets and offsets for each respective longitudinal location. These values were then converted to phase shifts by dividing by the cycle duration. Thus, negative values of On-β indicate that EMG onset preceded maximum convexity at a particular location.
Muscle Strain
Estimates of maximal lateral bending (βmax) were used to estimate per cent resting muscle length, assuming that the superficial red fibers located at the widest part of the fish keep pace with the change in curvature of the fish during swimming (Jayne and Lauder, 1995b; Katz and Shadwick, 1998; Rome, 1990; Rome and Sosnicki, 1991;
Rome et al., 1993). Muscle length change was calculated as the percentage of the ratio of the lateral to midline radii of curvature:
100{[CL/2sin(βmax/2)] – W/2} [CL/2sin(βmax/2)] where CL equals the average length of an adjacent pair of centra, and W is the maximum body width at particular longitudinal site. Table 4 summarizes these values as estimated across individuals for each viscosity.
Statistical analyses
Three to four cycles from each viscosity were analyzed for each individual. For maximal lateral bending (βmax) and EMG intensity, where lack of activity was scored as
7 zero, all eight longitudinal sites were statistically analyzed. The lack of white muscle activity in all but the highest viscosities precluded analyzing these data in a balanced experimental design with all viscosities during which red muscles were active.
Furthermore, the anterior red muscle sites were often inactive in lower viscosities.
Longitudinal site 8 was eliminated from the analysis of variance (ANOVA) due to a dislodged electrode in one individual (Table 1), although there was clear activity prior to the dislodging and site 8 was always active among the other three individuals.
Consequently, an ANOVA with all 4 viscosities was only possible for red EMGs for all fish at sites 5 through 7 for four viscosities. To appropriately account for variation within and among individuals, variables were analyzed in a three-way, mixed-model ANOVA with viscosity (N = 4) and longitudinal site (N = 3) as fixed, crossed factors, and individual (N = 4) as a random factor. The error term for the main, fixed effects was the
2-way interaction term of fixed and random factor. Where possible, such as in the calculation of EMG intensity and βmax (where lack of activity is scored as zero), all sites were used in calculations. I also analyzed an individual fish with two-way ANOVAs to clarify the effects of increased longitudinal site replication (N = 7) in the three highest viscosities.
Results
Morphology
Lungfish are elongate, effectively limbless vertebrates. The trunk is stout and uniformly cylindrical whereas the tail is laterally flattened and thin. Lungfish lack ossified vertebrae, instead having a cartilaginous notochord support (Arratia et al., 2001). The trunk contains 34-36 myotomes with relatively little longitudinal variation in morphology, whereas the tail contains 30-34 myotomes markedly thinner and longer than the trunk axial muscles.
8
Kinematics
I selected cycles of swimming with speed as similar as possible. Speeds tended to increase with increasing viscosity (Fig. 2A), but were not significantly different (Table
2). Cycle duration decreased significantly with increasing viscosity (Fig. 2B). The distance traveled per cycle clearly decreased with increasing viscosity (Fig. 2C) and this value approached significance (Table 2).
With increasing viscosity, the distance from nose tip to tail tip usually decreased, and anterior lateral displacement increased (Fig. 3). Maximum lateral flexion increased significantly both posteriorly and with increasing viscosity (Fig. 4, Table 2).
Motor pattern
The overall pattern of red muscle activity consisted of posteriorly propagated, unilateral EMGs (Fig. 5) that alternated between right and left sides (Fig. 6) at a given longitudinal location. All individuals increased anterior muscle recruitment with increasing viscosity (Fig. 5), and with the exception of one individual, all eight sites were active in the highest viscosity. As viscosity increased, activity at white muscle sites increased correspondingly (Fig. 7).
Burst duration decreased with increasing posterior longitudinal location within an individual among sites 2-8, but no significant effects were evident even when bursts were pooled across individuals for sites 5-7. Duration of absolute EMG bursts decreased significantly with increasing viscosity (Table 3) in sites 5-7 among all individuals and sites 2-8 in one individual. Relative burst duration, or EMG duty factor, did not vary significantly with site or viscosity among individuals in sites 5-7, but decreased significantly within a single subject with greater site replication. Intensity of EMGs increased significantly with viscosity and by site (Fig. 8).
Muscle strain
9 Estimated muscle strain increased significantly with increasing viscosity, and differed among sites (Tables 2, 4). The increase in strain was most pronounced in sites
5 and 6, which represent the sites closest to the pelvic girdle. Although site was a significant factor overall, caudal sites increased only slightly with viscosity in comparison to anterior sites. Muscle strains at sites 1-5 increased two-fold from water to the highest viscosity.
Timing of muscle activity and kinematics
The timing of EMGs relative to bending changed longitudinally, indicating that the
EMGs propagated posteriorly faster than the mechanical wave of bending (Figures 9,
10). If muscle activity corresponded to shortening of muscle tissue, then EMG onset would be synchronous with maximal convexity (Fig. 10 A), and On-β phase shifts would equal zero (Fig. 11). However, as viscosity increased, the EMGs onsets at several longitudinal locations began to precede the timing of maximal convexity, presumably indicating muscle activity during lengthening (Fig. 10). Thus, phase shift of EMG onset relative to bending increased significantly (Table 3) both by viscosity and site (Fig. 11) in sites 5-7 among all four individuals, as well as sites 2-8 in one individual. Though not statistically significant (Table 3), On-β phase shift tended to increase posteriorly with increasing viscosity (Fig. 10). The variation in the longitudinal extent of muscle activity can also be observed in Figure 9.
Discussion
Locomotion can be affected by ultimate causes, such as phylogeny,or proximate causes, such as speed of travel and environment. The complex interaction of all of these factors must be considered any study of locomotion, but parsing out differences can be difficult. In the present study I examined primarily proximate variation in locomotion.
Specifically, I sought 1) to place the locomotion of lungfish into the greater context of
10 vertebrate swimming, and 2) to assess the effects of an environmental perturbation
(viscosity) on locomotion of the lungfish.
Morphology and swimming mode
Elongate animals with greater numbers of vertebrae and shallow, uniform body depths are often expected to be able to bend more per segment length than animals with fewer vertebrae. Hence, animals with fewer vertebrae have fewer waves along the length of the body during swimming and are considered “stiffer” (Blight, 1977). The
African lungfish Protopterus annectens and other Lepidosirenid lungfishes are relatively elongate, and have between 70 to 80 vertebrae. The axial musculature is relatively large and uniformly cylindrical in the trunk. The caudal myomeres are thin, and the tail is thus thinner and laterally flattened relative to the trunk. Like many other elongate vertebrates
(such as lamprey, eels, and snakes), lungfish swim using a mode of lateral undulatory propulsion classified as anguilliform swimming. Axial undulatory swimming mode has frequently been correlated with the number of vertebral segments in an organism
(Coughlin, 2002). Anguilliform swimming has been classically defined as lateral waves of bending traveling from head to tail (Breder, 1926) and is prevalent among organisms with relatively large numbers of vertebrae, such as the American eel (Anguilla rostrata,
103-111 vertebrae). More recent definitions of anguilliform swimming define it further by describing waves of bending and power transfer occurring along the length of the whole body, or very close to it (Lindsey, 1978; Wardle et al., 1995a; Webb, 1975). However, still more recent studies and this study have demonstrated that so-called anguilliform swimmers traveling at slower speeds—or in this case, low viscosity—are propulsed almost entirely by caudal undulation (Gillis, 1997; Liao, 2002). Fishes with fewer vertebrae and deeper bodies tend to be carangiform, or sub-carangiform swimmers
(Wardle et al., 1995b). The many fishes that employ sub-carangiform swimming tend to have a wide range in vertebral number, such as the largemouth bass (Micropterus
11 salmoides) with 34-35 vertebrae (Jayne and Lauder, 1995b) and the trout
(Oncorhynchus mykiss) with 61-65 (Webb and Johnsrude, 1988). Scombrids like the skipjack tuna (Katsuwonus pelamis) have between 40-41 vertebrae, and demonstrate the least flexible swimming mode, thunniform swimming (Syme and Shadwick, 2002).
Longitudinal variation during steady swimming
The lungfish in this study exhibited significant longitudinal variation in morphology, muscle strain (as estimated by bending), EMG burst duration (with increasing viscosity), EMG intensity, and phase shift (EMG onset – max bend).
Longitudinal differences in muscle activity and bending were more pronounced with increased viscosity. During steady swimming, waves of mechanical bending were always greatest in the tail, which conforms to the pattern observed in nearly other fish taxa (Altringham and Ellerby, 1999). Lungfish swimming steadily in the lower viscosities did not exhibit undulatory waves along the whole length of their body, despite this qualification of anguilliform swimming. As viscosity increased, the lungfish increased bending anteriorly, and thus became more “anguilliform”.
Differences in kinematics and EMG patterns among lateral undulatory swimmers have been attributed to differences in morphology, both among species and within a single organism (Gillis, 1997). Variation between trunk and caudal morphology is a significant source of longitudinal variation in any form of swimming, particularly with regard to tail shape. A broad, flat tail with stiff tendinous structures may very effectively transfer forces (e.g. carangiform swimming), whereas a longer more flexible tail would predictably bend more, and be less resistant to hydrodynamic forces. However, some more flexible fishes may construct functionally stiffer tails by altering the intrinsic mechanical properties of the musculature within the tail.
Longitudinal variations in muscle strain are similar across many fish taxa, with caudal strain values always higher than trunk (see Wardle et al., 1995b for review). For
12 example, muscle strain at approximate body length proportions (L) of 0.35 and 0.65 in lamprey, eels, scup, saithe, carp and trout average ± 3% and ± 6% mean fiber length, respectively (Grillner and Kashin, 1976; Rome et al., 1993; van Leeuwen et al., 1990;
Wardle and Videler, 1993; Williams et al., 1989). Interestingly, lungfish swimming in water demonstrated greater strain than what has been observed these other fishes.
Despite similar motor pattern, lungfish averaged strains approximately twice as large
(Table 4) during steady swimming in water at comparable longitudinal locations (sites 3 and 6, this study). Although both eels and lungfish are somewhat similar morphologically, it should be noted that lungfish possess a much stouter trunk and a flatter tail than eels and thus generate more strain by virtue of their width as well as degree of bending. In the highest viscosity, the lungfish averaged ± 23% mean fiber length near the pelvic girdle, demonstrating the extreme effort required to move through the medium.
Perhaps the most physiologically important longitudinal difference is the timing of
EMG progression compared to the lateral curvature along the body (phase shift).
Variation has been described among species with regard to phase shift as well, but there is some amount of disagreement among workers as to how well morphology may predict phase relationships (Gillis, 1998b; Wardle et al., 1995a). Gillis (1998b) found eels varied phase shifts longitudinally, despite predictions to the contrary in Wardle et al (1995b).
Elongate, anguilliform swimmers would not vary phase relationship longitudinally according to this model because force transfer is occurring at a constant rate along the entire length of the body. In actuality, both eels and lungfish are activating caudal myotomes earlier in the cycle than anterior myotomes. Indeed, this pattern of increasing phase shift posteriorly is emerging as a fairly universal trait among lateral undulatory swimming vertebrates (Gillis, 1998b).
13 Coughlin (2002) also observed that in most fishes phase shifts increased posteriorly, causing caudal muscles to be active earlier (relative to bending) than trunk muscles. An established theory to explain this disparity posits that high muscle strains
(per cent change in muscle length relative to rest) and short EMG duty factors (per cent activity in a given cycle) in the tail will be most effective for swimming (Coughlin, 2002;
Long, 1998). To achieve higher strains a muscle at a particular site should be fully active to generate peak force at the beginning of shortening (max convexity on Figure 10). In order for this to be achieved EMG onsets need to occur substantially sooner during a cycle, perhaps as much as 25% as seen in carp caudal musculature (van Leeuwen et al., 1990), or even greater than 30%, as observed in the lungfish at the highest viscosity.
Shorter bursts and earlier activation of muscle fibers during a cycle allow quicker returns to resting length, and consequently require less force to shorten.
Environmental effects on locomotion
Water represents a uniform environment with nearly homogenous density and viscosity, consequently the extrinsic forces acting upon an organism change very little during steady swimming. A key unaddressed question in the field of vertebrate biomechanics has been how animals move through environments at the interface of land and water. Many organisms employ different kinematic locomotor modes in different environments, such as laterally undulating in water and walking on land. However, organisms lacking appendages necessarily laterally undulate both in water and on land.
Studies have demonstrated that even in these instances when axial movements are grossly similar, motor pattern differs significantly between terrestrial and aquatic locomotion. This suggests that environment is a substantial factor in determining locomotor strategy (Frolich and Biewener, 1992; Gillis and Blob, 2001). Viscosity was manipulated in this study to ascertain how a primarily aquatic organism might alter its
14 locomotion to move through an increasingly thicker, “muddier” medium with subsequently increasing extrinsic forces.
Gray (1953) manipulated viscosity in two levels by using Cellofas to increase the viscosity to 7.5 and 64 times greater than water to study the effects on snake locomotion. Gray determined that in swimming snakes when viscosity was increased, the amplitude of the wavelength decreased posteriorly, but dramatically increased anteriorly (Gray, 1953). I found a general trend of increasing amplitude (Fig. 3) along the entire length of the fish, rather than anterior-only, and over a much greater range of viscosity. EMG burst duration decreased and amplitude increased posteriorly with viscosity, but not during steady swimming in water. Eels and other elongate fishes also have not demonstrated significant longitudinal differences with these variables during steady swimming (Gillis, 1998b; Grillner and Kashin, 1976; Wardle et al., 1995a), but fishes such as bluegill and bass demonstrate significant decreases in EMG duty factor posteriorly (Jayne and Lauder, 1993; Jayne and Lauder, 1995a). Bending increased along the entire length of the body, but most notably in anterior segments. During steady swimming lungfish, like eels, bend very little in the trunk. Phase lag between EMG onset and bending was predicted to resemble motor pattern of snakes (phase-locked) or eels
(decreased phase shift) during terrestrial undulation, but in fact lungfish phase shift increased dramatically with increasing viscosity.
Phase lag between EMG onset and bending in eels and snakes on land decreased dramatically compared to lateral undulation in water, and in snakes became fully phase-locked—namely, muscle activity approximated the time of bending, and the relationship remained constant along the length of the animal (Jayne, 1988). Even though lateral undulation is the mode of locomotion employed on land and in water, motor pattern is drastically altered to achieve the same kinematic pattern of movement.
Moving on land represents a different array of external forces acting on organisms, and
15 a much less uniform environment. Viscosity on land is negligible except along the ventral aspect of travel, and efficient transfer of power occurs without increasing the timing of motor activity during lengthening I observed in lungfish. In contrast, the phase shifts observed in lungfish in the higher viscosities drastically increased along the entire length of the body. In some individual cycles analyzed from the highest viscosity, EMG onset occurred in the posterior sites a full half-cycle before maximal convexity (and thus muscle shortening). Increasing speed in the swimming of largemouth bass increased the observed phase shift by almost 10% of a cycle relative to slow, steady swimming (Jayne and Lauder, 1995a). This suggests that the increased hydrodynamic forces due to the increased viscosity are similar but magnified relative to the increase in hydrodynamic force due to increased speed.
In this study increasing viscosity altered the hydrodynamic properties of the medium to increase the external forces acting on the fish. Hydrodynamic forces can also increase with increased swimming speed, therefore fast-swimming fishes often have stream-lined bodies and stiff caudal regions to accommodate the increased drag associated with displacing water at a high velocity (Syme and Shadwick, 2002).
Increasing viscosity represents a similar level of drag with essentially the same quandary; flexible bodies cannot generate enough resistance to push against the medium (Sfakiotakis et al., 1999). For an elongate organism such as the lungfish, the only recourse may be to functionally stiffen the tail by activating muscle fibers earlier in a cycle, as well as to increase tailbeat frequency.
Williams et al (1989) suggested that an increase in the phase lag relationship between the wave of motor pattern and mechanical bending promotes forward movement in a fluid by increasing the stiffness of the tail. In this manner, a fish can “pre- stiffen” the tail before the wave of flexion reaches it, and thus the tail can function as a more effective paddle. When fish increase swimming speeds, so also increasing tail-beat
16 frequency, increased stiffness of the tail is needed to increase the hydrodynamic rate of working (Long and Nipper, 1996). High-performance thunniform swimmers possess very stiff, tendinous tails that bend very little (Syme and Shadwick, 2002). Other fishes, such as the largemouth bass (Micropterus salmoides), functionally stiffen the caudal region during swimming bouts of increasing speed by increasing muscle activity in the tail during lengthening (Jayne and Lauder, 1995a). In bass swimming at higher speeds, muscle strains in the tail approached ± 20% mean fiber length during ‘burst-and-glide’, which approximates the strain observed in the lungfish pelvic girdle at the highest viscosity (Jayne and Lauder, 1993). Anguilliform swimmers, such as eels and lungfish, have a much more uniformly cylindrical morphology and propulsive waves tend to be occur in larger amplitudes over a larger extent of the body (Long, 1998). Thus, ‘negative work’ would play a larger role in anguilliform swimming, as the caudal region is very flexible and lacks the degree of mechanical stiffness observed in other taxa.
During this experiment there appeared to be a threshold effect of viscosity related to speed. While relatively slow speeds were common enough in low viscosities, most of the highest speeds were observed in the highest viscosities. Despite repeated trials within and among subjects in the highest viscosities, the subjects simply did not or could not swim slowly at the highest viscosity. In fact, in many of the unanalyzed cycles, the fish gained no forward displacement despite vigorous and rapid tail-beat cycles. In these instances, the subject increased tail beat frequency and accelerated very rapidly across the tank, often striking one of the walls of the tank. These observations, combined with qualitative data collected from white muscle fiber sites, indicate that white muscle recruitment plays an increasing role in viscous locomotion. Increasingly viscous environments consequently require a minimum speed and power threshold to move through, similar to the minimum flight speed required by aerial organisms to overcome drag (Lighthill, 1974).
17 To summarize, increasing viscosity does not cause motor patterns to more closely resemble motor pattern observed in terrestrial lateral undulation, but rather dramatically amplifies timing differences commonly seen in swimming fishes. The increased phase lag between EMG and bending along the length of the fish with increased viscosity (Figs. 10, 11) suggests that muscle fibers are increasingly active during lengthening, which is associated with ‘negative work’ (Johnson et al., 1994).
Evidently, moving through a viscous medium can not be considered an intermediate between aquatic and terrestrial locomotion, nor does it seem likely that neuromuscular control is changing dramatically in viscosity. The best correlate for the observed motor pattern can be found in fishes swimming at greater speeds. Although the magnitude of phase lag is considerably greater in the lungfish, the pattern remains parallel. For motor pattern to shift to a standing wave, as observed in terrestrial vertebrate locomotion, environmental cues may be playing a key role not accomplished merely by increasing viscosity. This study further expounds the degree of plasticity in motor control and response in vertebrates, but also invites more experimentation evaluating the degree of environmental influence on an organism’s locomotion.
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23
Table 1. Locations of unilateral red muscle implants for
electromyographic recordings
Implant location Fish 1 Fish 2 Fish 3 Fish 4 Mean SD
1 0.18 0.21 0.21 -- 0.20 0.02 (5) (7) (7) (7) (0.9)
2 0.25 0.29 0.32 0.29 0.29 0.03 (10) (13) (14) (13) (13) (1.5)
3 0.35 0.38 0.39 0.38 0.37 0.02 (17) (19) (19) (19) (19) (0.9)
4 0.47 0.45 0.45 0.44 0.45 0.02 (27) (25) (24) (24) (25) (1.2)
5* 0.59 0.53 0.52 0.55 0.55 0.03 (36) (31) (30) (33) (33) (2.3)
6* 0.68 0.63 0.60 0.65 0.64 0.04 (44) (39) (36) (41) (40) (2.9)
7* 0.78 0.72 0.67 0.75 0.73 0.04 (52) (47) (45) (50) (49) (2.7)
8 0.86 0.80 0.77 -- 0.82 0.04 (59) (54) (55) (56) (1.9) Locations are described both as proportions of total length, and intervertebral joint number (in parentheses)
Sites 6-8 represent caudal locations.
-- Empty cells represent dislodged or otherwise defective electrodes
* Indicates sites with discernible EMG activity in all viscosities
24
Table 2. Summary of F-values from 3 and 2-way ANOVAs performed
on four individuals for each variable
Effect Variable Viscosity Site Viscosity X Site (3,9) (7,21) (21,63)
βmax 10.4 * 14.2 * 3.9 *
Muscle strain † 13.5 * 6.7 * 3.7 *
Cycle duration 5.8 * -- --
Speed 0.9 -- --
Distance per 2.75 -- -- Cycle
Degrees of freedom are indicated parenthetically (numerator, denominator) below each effect
† See Table 4 for mean values of muscle strain
* and ** indicate P values of < 0.05 and < 0.0005, respectively
25
Table 3. Summary of F-values from 3 and 2-way ANOVAs performed separately on
each EMG variable for sites 5-7 in four subjects across four viscosities, and sites 2-8 in
one subject across three viscosities; EMG intensity includes all viable sites (sites with
no activity were scored as “0”), all viscosities, and all individuals
Variable Viscosity Site Viscosity X Site
sites 5-7 sites 2-8 sites 5-7 sites 2-8 sites 5-7 sites 2-8 (3, 9) (2, 56) (2, 6) (6, 56) (6, 18) (12, 56)
EMG burst duration (ms) 4.4* 22.4** 2.3 2.0 0.8 2.2*
Duty factor (% cycle) 0.5 5.8* 1.7 8.3** 1.3 1.6
On-β shift 4.4* 0.3 7.3* 52.5** 2.3 0.7
Off-β shift 4.9* 4.0* 3.4 12.6** 3.4* 0.5
EMG intensity 13.5* 2.9* 0.9 (3, 9) (5, 15) (15, 45)
Degrees of freedom are indicated parenthetically (numerator, denominator).
P values of < 0.05 are indicated by one asterisk (*) and two asterisks (**) denote
values < 0.0005.
26
Table 4. Strain of superficial red muscle fibers during steady swimming for four
viscosities estimated from mean morphological values and βmax
Muscle Strain (%) Body Centrum Site width (L) length (L) Viscosity Viscosity Viscosity Viscosity 1 10 100 1000
1 0.11 0.012 4.3 4.1 11.6 8.7 (0.005) (0.0006)
2 0.10 0.012 5.2 5.6 12.1 11.8 (0.003) (0.0010)
3 0.11 0.013 5.8 6.1 14.6 13.2 (0.004) (0.0010)
4 0.10 0.012 8.0 7.8 15.4 16.5 (0.003) (0.0010)
5 0.09 0.012 10.5 13.7 19.9 22.4 (0.008) (0.0017)
6 0.07 0.011 15.2 17.6 21.8 23.2 (0.010) (0.0017)
7 0.04 0.010 16.6 16.0 17.0 19.5 (0.008) (0.0012)
8 0.02 0.008 15.3 15.4 15.2 16.1 (0.003) (0.0015)
Site indicates the longitudinal positions as described in Table 1.
Body width and centrum length are mean values (N = 4).
All values in parentheses are standard deviations.
27 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.
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. Cycle duration and the distance traveled per cycle decreased with increasing viscosity, but only cycle duration was significantly effected (see Table 2).
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.
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.
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.
28 Fig. 6. Filtered EMGs from superficial red muscle from three longitudinal sites with both left side (black trace) and right side (blue trace) implants. EMGs were clearly unilateral and alternating.
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.
Fig. 8. Mean values ± S.E.M. of EMG intensity for sites 2-7 among all individuals in all viscosities. Intensity increased significantly with viscosity, and increased more posteriorly.
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.
All EMG sites are on the left side of the fish. Note the lack of bending and EMG activity in anterior sites in viscosities 1 and 10.
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. Time of all graphs is standardized to zero when maximum convexity occurred at site 5, or 0.55 L. The horizontal bars indicate mean values of muscle onset – offset at a particular longitudinal location. Lateral flexion is relative to the same side (left) of the fish from where EMGs
29 were recorded. Muscle activity between maximum convexity and maximum concavity presumably corresponds to activity during contractile tissue shortening.
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.
30
Fig. 1
5 6 (2)* 3 4* 1 (7)* 8
31
Fig. 2
0.32
0.24
) -1 s L ( 0.16 d e e p S 0.08
0.00
1.6
1.2
0.8
Cycle duration (s) 0.4
0.0
0.3
0.2 )/cycle L
0.1 Distance (
0.0 1 10 100 1000
Viscosity
32
Fig. 3
A
B
C
D
10 cm
33
Fig. 4
8 Viscosity 1 Viscosity 10 Viscosity 100
) x 6
a
m Viscosity 1000
β
(
e
d
u t 4
i
l
p
m
A 2
0 12345678 Longitudinal Site
34
35
36
37
Fig. 8
0.6 Viscosity 1 Viscosity 10 ) V Viscosity 100 ( y t 0.4 Viscosity 1000 i s
n e t n I
G 0.2 M E
0.0
234567 Longitudinal Site
38
Fig. 9
Viscosity 1 Viscosity 10Viscosity 100 Viscosity 1000 4 0 -4 4 0 -4 4 0 -4 4
) 0
e
l g -4
n
a
( 8
n
o i 4
x
e
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l
a r -4
b
e
t
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e -8
v r 8
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t
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I 4 0 -4 -8 8 4 0 -4 -8 8 4 0 -4 -8
0 0.5 1.0 1.5 2 0 0.4 0.8 1.2 0 0.4 0.8 1.2 0 0.4 0.8 1.2 Time (s)
39
Fig. 10 1.0
0.8 ity ex nv o ity 0.6 C av nc Co 0.4
0.2
0.0 1.0
0.8
0.6
0.4
)
L 0.2 ( h
t g n e 0.0 L l 1.0 a t To 0.8
0.6
0.4
0.2
0.0 1.0
0.8
0.6
0.4
0.2
0.0 -0.4 0.0 0.4 0.8 1.2 time (s)
40
Fig. 11
Viscosity 1 Viscosity 10 Viscosity 100 0.1 Viscosity 1000
)
s
e
l 0.0 c
y
c
(
t -0.1
f
i
h
s
β -0.2
-
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-
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-0.1 12345678
Longitudinal Site
41
42