© 2002 by The Society for Integrative and Comparative Biology
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A Molecular Mechanism for Variations in Muscle Function in Rainbow Trout1
1 Widener University, Science Division, One University Place, Chester, Pennsylvania 19013
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Salmonids undergo a developmental transition from parr to smolt that involves a number of physiological and morphological changes. In recent years, my laboratory has studied shifts in red muscle function at this parr-smolt transformation (PST) in rainbow trout, Oncorhynchus mykiss. Parr red muscle has faster contraction kinetics than smolts, including faster rates of activation and relaxation and a faster maximum shortening velocity. At PST, a transition in swimming behavior is also observed, with lower tailbeat frequencies and longer EMG duty cycles in the older smolts. Lastly, there is molecular correlate to changes in kinetics and behavior. During PST, there is a developmental reduction in the number of myosin heavy chain (MHC) isoforms in the red muscle of rainbow trout. Since MHC composition of muscle can determine contractile properties, these molecular results suggest a mechanism for the transition in red muscle kinetics and steady swimming. The red muscle of parr is more likely to contain the fast-twitch or white isoform of MHC, resulting in faster contractile properties of that muscle and higher tailbeat frequencies during steady swimming. Lastly, experimental work supports the conclusion that the shift in kinetics causes the observed shift in swimming behavior.
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Functional morphology, as the study of form and function relationships, typically involves anatomical analysis, biomechanics, electromyography and other physiological techniques (Ashley-Ross and Gillis, 2002
). One area of focus in functional morphology is the function of the musculoskeletal system for feeding and locomotion. In recent years, the use of molecular techniques has become more common for functional morphologists interested in muscle for several reasons. First, muscle is a molecular motor that permits rigorous quantitative analysis of function, such as force production, rates of activation and relaxation and shortening velocity. Second, the molecular structure of muscle has been well described, and there is an ever-growing database on the functional roles of the specific proteins that comprise muscle and on the effects of variations in isoform expression of myofibrillar proteins in a given muscle (see reviews by Moss et al., [1995] and Schiaffino and Reggiani, [1996]).
Since there are spatial and temporal variations in the patterns of expression of different muscle proteins, molecular biological techniques allow the rigorous testing of specific mechanistic hypotheses concerning the implications of changes in the protein composition of muscle (changes in form) on behavioral performance of animals (function). Conversely, when variations in function are identified, the use of molecular techniques allows the testing of hypotheses of the molecular basis for observed variations in function. The integration of molecular biology with traditional functional morphology approaches has been successfully carried out by many researchers, for both invertebrate and vertebrate systems (e.g., Johnson and Bennett, 1995; Saenger, 1997; Fitzhugh and Marden, 1997; James et al., 1998; Brault et al., 1999), as well as for models of human muscle function (e.g., Widrick et al., 1996; Fitts et al., 2000).
This paper reviews recent work in my laboratory on the use of molecular biological techniques to examine developmental changes in red muscle function in steadily swimming rainbow trout, Oncorhynchus mykiss. Steady swimming is powered exclusively by the red or aerobic muscle in most fishes (van Leeuwen et al., 1990; Altringham et al., 1993; Rome et al., 1993; Wardle et al., 1995; Coughlin and Rome, 1999; Coughlin, 2000). Since the red muscle exists as discrete, lateral strips on each side of the body, it provides a convenient system for studying the effects of changes in muscle contractile properties and protein composition on behavioral performance. Red muscle's placement permits reliably in vivo use of electromyography and sonomicrometry during swimming experiments. Further, relatively pure aerobic fiber muscle bundles can be readily dissected from many fishes, useful for both muscle mechanics and molecular biological study. After describing natural life history changes in trout, two types of traditional functional morphology approaches will be examined: analysis of swimming kinematics and the study of muscle mechanics. Then, molecular analysis of trout muscle will be discussed as a means for understanding developmental changes in function.
Salmonids undergo a developmental transition from small juvenile parr to larger juvenile smolts. In anadromous or sea going salmonids, a host of physiological changes associated with life in the sea occur at the parr-smolt transformation (PST or smoltification), including increased ion excretion at the gills, as evidenced by higher Na+ and K+ ATPase activity, reduction in H2O excretion and generally increased metabolism (Hoar, 1988). There are additional changes in the visual system, body morphology and behavior. The ratio of Vitamin A1 to A2 in visual pigments increases with the transition from parr to smolt (Alexander et al., 1994), and there is a loss of ultraviolet visual sensitivity (Hawryshyn et al., 1989; Coughlin and Hawryshyn, 1994). Morphological changes include a loss of parr marks as the body becomes silver (due to purine deposition in dermal layers) and a longer and more slender body (Folmar and Dickhoff, 1980; Hoar, 1988; Damsgård, 1991; Beeman et al., 1994). Besides a typical downstream migration, behavioral changes occurring at PST include decreased aggression and increased schooling (Groot and Margolis, 1991; Koike and Tsukamoto, 1994).
Several studies have also examined the effect of PST on swimming ability or stamina in salmonids. Smolts reportedly show reduced swimming ability with less stamina than parr (Folmar and Dickhoff, 1980; Smith, 1982; Hoar, 1988). In at least one study, however, the swimming stamina of smolts was found to be greater than parr (Muir et al., 1994). In another study, PST in Atlantic salmon (Salmo salar) did not affect maximum sustained swimming speed (Peake and McKinley, 1998).
In non-anadromous salmonids such as rainbow trout, PST can be termed "pseudosmoltification," because the smolts are not physiologically adapted for marine life (Dickhoff and Sullivan, 1987). However, rainbow trout pseudosmolts are morphologically transformed: they are silvery in appearance and their bodies are more slender. In this paper, the term smolt is used for older rainbow trout juveniles that display a number the traits associated with PST, although they are freshwater fish. The parr used in the work described here were 
6–8 months post-hatching and 10–15 cm in total length with a body mass of <30 g, while the smolts were 18–20 mo and 20–25 cm in total length with a body mass of >100 g.
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Functional morphology and steady swimming in rainbow trout
Swimming studies
Steady swimming behavior was first studied via video analysis of basic kinematics in steadily swimming rainbow trout. Video recordings were made of the overhead view of fish swimming in a re-circulating flume at 10°C across of range of steady swimming speeds. Tailbeat frequency could be readily determined, as well as the stride length of each tailbeat. The latter is defined as the distance traveled per tailbeat expressed in units of body length. Tailbeat frequency increases linearly with swimming speed in both parr and smolts (Fig. 1). Parr swim with higher tailbeat frequencies than smolts across the range of steady swimming speeds (Coughlin et al., 2001a
). Further, smolts swim with greater tailbeat frequencies than much larger adult rainbow trout (40 cm fish, Hammond et al., 1998). Adults appear to have a somewhat different relationship of tailbeat frequency to swimming speed compared to smolts: adults display a steeper slope in that relationship and the maximum steady swimming tailbeat frequencies reported for adults are near the minimum values reported for smolts (Fig. 1). The slowing of tailbeat frequency with growth has been reported before by Webb et al. (1984). Stride length also varies with growth, with significantly greater stride lengths observed in smolts. The stride of smolts is similar to that observed in the larger, adult trout (Fig. 1).
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A second round of swimming experiments involved recordings of electromyograms (EMGs) from swimming fish for determination of duty cycle of EMG bursts during each tailbeat. To record the electrical activity of muscle during the tailbeat cycle, twisted wire electrodes were implanted in fish at two locations, anterior = 0.35 BL and posterior = 0.75 BL. Since there are longitudinal variations in muscle activity in most fish species (Altringham and Ellerby, 1999
), data from the anterior and posterior regions of the fish must be considered separately. Fish were again swum across a range of length-specific steady swimming speeds. White muscle activity was also monitored and all data presented are from steady swimming speeds below the recruitment speed for white muscle in each fish. EMG signals were recorded using Grass EMG amplifiers (P511, filtering bandwidth of 10 to 1,000 Hz with a 60 Hz notch filter) and a PC. The duty cycle of EMG bursts was defined as the duration of EMG activity expressed as a proportion of the period of one tailbeat cycle.
Duty cycle increases dramatically during development (Figs. 2 and 3). At equivalent length-specific swimming speeds, the duration of EMG bursts in smolts is clearly longer than parr (Fig. 2). Further, the difference in tailbeat frequency described above based on video analysis can also be observed in these raw EMG traces: there are more EMG bursts per second in parr than smolt at each position. Quantitative analysis of duty cycle shows a significant increase in duty cycle at each position between parr and smolts (Fig. 3). Parr show relatively low duty cycles, with values of 0.15–0.25 for the anterior and 0.1–0.2 in the posterior (Coughlin et al., 2001b). For smolts, longer duty cycles were observed, with values of 0.25–0.40 for the anterior and 0.2–0.25 for the posterior (Coughlin, 2000). Adult duty cycles are considerably longer, close to 0.6 in the anterior and 0.5 in the posterior (Hammond et al., 1998). Developmental shifts in duty cycle of swimming muscle have not been described previously.
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Contraction kinetics
Following swimming experiments, contractile properties of the red muscle of rainbow trout were examined using a muscle mechanics apparatus (Coughlin, 2000
; Coughlin et al., 2001a). Live muscle bundles were extracted from parr and smolt from the two longitudinal positions defined above. These bundles were taken from the red muscle layer, as determined grossly. The bundles were composed almost entirely of aerobic muscle fibers, as indicated by staining with SDH (Coughlin et al., 2001a). As with EMG activity, longitudinal variations in the kinetics of the swimming muscle are common in fishes (Altringham and Ellerby, 1999), therefore contractile properties must be examined separately for the anterior and posterior myotomal muscle. Bundles were maintained in physiological saline (Altringham and Johnston, 1990) at 10°C. For isometric contractions, activation conditions (duration of stimulus pulse train, the duration and amplitude of the pulses, and muscle bundle length) were optimized to maximize force production. At the optimal length, peak tetanic force was measured and the force traces were analyzed for activation time (time from 10–90% of peak force) and relaxation time (time from 90–10% of peak force). Twitch contractions were recorded at the same bundle length using optimized stimulus conditions for the single stimulus pulse. Peak twitch force and twitch time (time from stimulation to 10% of peak force) were determined. The ratio of peak twitch force to peak tetanic force could then be calculated. For some muscle bundles, maximum shortening velocity (Vmax) and peak steady-state power production were determined using the force-clamp technique (Rome and Sosnicki, 1990; Coughlin et al., 2001a). Following mechanics experiments, muscle bundles were processed histologically to determined the cross-section of live muscle fibers (Coughlin, 2000). Isometric force production values (N) could then be converted to isometric stress (kN m–2).
There was no difference between the isometric stress of red muscle from parr and smolts, although the posterior muscle of each stage produced significantly greater force than the anterior (Coughlin et al., 2001a). The anterior muscle bundles of each group generated a mean isometric tension (±SE) of 134.2 ± 11.7 kN m–2, while posterior muscle bundles generated 166.1 ± 14.1 kN m–2. Similar isometric stress values were observed in adults (30 cm fish, Hammond et al., 1998). The ratio of peak twitch to peak tetanic force did not differ between parr and smolts or between positions, with a mean value of 0.575 ± 0.021 for all muscle bundles. In adults, this ratio is much lower, averaging 0.21 for all positions. Parr show faster red muscle contractile properties than smolts (Coughlin et al., 2001a), having faster rates of activation, relaxation and twitch time for both the anterior and posterior positions (Fig. 4). Adults have even slower activation and twitch times than smolts (Hammond et al., 1998).
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There is an additional developmental shift in Vmax of trout red muscle. While there is no difference in Vmax between anterior and posterior muscle, there is a significant shift in Vmax between parr and smolts: parr red muscle has a faster shortening velocity than that of smolts (Coughlin et al., 2001a
). Parr red muscle has a mean (±SE) Vmax of 2.42 ± 0.12 muscle lengths s–1, while the smolt Vmax is 2.05 ± 0.15 ML s–1. Other variables associated with shortening velocity did not differ between the two developmental stages. Parr produce greater peak steady-state power, but the difference between parr and smolts is not significant. The mean value for both groups is 44.0 ± 4.7 W kg–1. The optimal shortening velocity (Vopt) at which power output was maximized also did not differ significantly between parr and smolts. Vopt is expressed a proportion of Vmax, and the mean value of Vopt/Vmax was 0.339 ± 0.010. Swimming and muscle mechanics experiments in rainbow trout show a developmental shift in red muscle function at the parr-smolt transformation. Smolts show slower rates of muscle activation and relaxation and a slower Vmax than parr. Kinetics continue to slow with growth into full adult size. Associated with this shift in kinetics is a change in swimming behavior: parr swim with the fastest tailbeat frequency and the frequency drops throughout growth to the adult size. Smaller fish also swim with higher length-specific swimming speeds but lower stride lengths, than older, larger fish.
Molecular analysis of rainbow trout red muscle
The above results suggest a specific molecular basis for developmental variations in behavioral performance and in contractile properties of red muscle. Shifts in activation, relaxation and Vmax at PST indicate a developmental change in the myosin heavy chain (MHC) composition of the rainbow trout red muscle. MHC contains the binding sites for both actin and ATP, and so it is the ultimate determinant of cross-bridge kinetics (Moss et al., 1995). Since Vmax is the shortening velocity of unloaded muscle, it represents the maximum speed of cross-bridge cycling. Changes in MHC affect the kinetics of crossbridge formation and crossbridge cycling directly, so a change in MHC at the PST could result in changes in muscle function. In Atlantic salmon, PST does involve a change in MHC expression. Two forms of MHC are found in parr red muscle, while only one form is found in red muscle of smolts (Martinez et al., 1993). MHC is the predominant muscle protein and variations in MHC expression are associated commonly with changes in other muscle proteins, such as the regulatory myosin light chains (Schiaffino and Reggiani, 1996).
Two techniques were used to examine the molecular basis of changes in contractile properties. The first was protein analysis using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE, Laemmli, 1970). Purified myofibrillar proteins were separated using this standard protein electrophoresis approach to allow examination of the number and relative abundance of different isoforms of MHC. Protein samples were prepared following the protocols of Talmadge and Roy (1993) and Lutz et al. (1998) and were run on a BioRad Mini Protean II system with pre-cast 5% polyacrylamide gels. Samples of red muscle were isolated from anterior and posterior muscle from parr and smolts. Since no longitudinal variations in protein composition have been observed, data from the anterior and posterior of fish from one developmental stage are combined. MHC bands on the gels could be readily identified due to their large size (245 kD). As previously reported for Atlantic salmon, there is a developmental reduction in MHC isoforms in rainbow trout (Figs. 5, 6). However, the patterns for rainbow trout are less discrete than the description given for Atlantic salmon. Parr typically show two to three bands of MHC on SDS-PAGE gels (Coughlin et al., 2001a). There is typically a dark upper band, indicating a high molecular weight form, and either one or two lower bands. If two lower bands are present, they are very light, while if a single lower band is present it is usually very dense (Fig. 5). Alternatively, smolts typically show one to two bands of MHC (Coughlin et al., 2001a). The upper band seen in parr is commonly present in smolts but is very light, while a single lower band is usually very darkly stained (Fig. 5). Densitometry supports these general observations. There is significant shift in the MHC composition of trout red muscle between parr and smolts (Fig. 6).
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The second molecular technique used was the cloning and sequencing of MHC cDNAs to examine MHC expression patterns. After isolating total RNA from red, white and ventricular muscle, mRNA was reverse transcribed into first strand DNA by priming the RT reaction with oligo-dT. Consensus primers for identifying myosin (Lutz et al., 1998
) were used next in a PCR reaction to amplify MHC cDNAs from each muscle type. PCR products were selected for cloning and sequencing. The 3' tail of a total of three distinct MHC cDNAs (700 bp) were sequenced and were identified as red, white and ventricular isoforms based on the tissue from which they were originally cloned (Weaver et al., 2001). The white form, which was identical to a published sequence (Gauvry and Fauconneau, 1996), was cloned from red muscle samples as well.
To assess the expression patterns of parr and smolt red muscle for the different forms of trout MHC, isoform-specific primer pairs were designed for PCR. There primers would amplify respectively only the red, white or ventricular isoforms (Weaver et al., 2001). PCR using each primer pair would then test presence/absence for the mRNAs of each trout MHC by each producing a different size product. If a product was amplified by PCR using a specific primer set, then the muscle sample was expressing that MHC isoform. The products for each primer pair differed in size to make analysis unambiguous. The red primer pair produced a 280 bp product if the trout red MHC was present, while the white primer pair produced a 224 bp product and the ventricle primer pair produced a 354 bp product.
For five smolts and six parr, red muscle samples were isolated from the anterior and posterior regions of the myotome. These samples were typically 100 mg and contained muscle fibers only from the grossly apparent red muscle layer. Carefully selected samples show a smolt red muscle sample that demonstrates expression of the trout red and white MHC forms but not the ventricular form. The parr example shown expresses all three isoforms of MHC (Fig. 7). This represents the results in a general sense—a reduction in the number of MHC isoforms expressed in rainbow trout red muscle at PST (Weaver et al., 2001). A more rigorous analysis of expression patterns reveals a less discrete developmental shift. Some smolt muscle samples express all three MHC isoforms, and some parr samples express only one or two (Table 1). However, one clear difference is that only 8% (1 out of 12) parr samples expressed only the red isoform, while 30% (3 of 10) smolt samples expressed only the red isoforms. A shift in white expression is partly responsible for the apparent developmental reduction in MHC isoforms: 92% of parr red muscle samples expressing the white isoform versus only 60% of smolt samples expressing the white form (Weaver et al., 2001). Quantitative PCR techniques would allow a determination of the expression levels of each isoform, permitting a more sensitive analysis of changes in MHC expression at PST. However, to date efforts to employ this approach have not been successful.
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Red muscle structure, contractile properties and steady swimming
The developmental shift in MHC expression correlates well with observed variations in contractile properties and swimming behavior. Parr red muscle more frequently expresses the fast (white) isoform of MHC. Parr red muscle bundles also have faster contraction kinetics, with faster activation and relaxation times and a faster maximum shortening velocity. Swimming differs between parr and smolts, too, with higher tailbeat frequencies observed in the younger fish. Lastly, red muscle is used differently in parr versus smolts, as indicated by a dramatic shift in EMG activity from parr to smolt to adult rainbow trout. These results suggest that the shift in MHC expression is a potential mechanism for the shift in kinetics and swimming behavior. This leads to additional questions concerning the nature of the shift in expression.
For now, no data exist on fiber-specific expression of MHC, as the samples analyzed here included a cross-section of many muscle fibers (100s). Are MHC isoforms discretely expressed in individual fibers in trout red muscle? Such inter-fiber variations in expression have been suggested in the past for rainbow trout. Some fibers in the red muscle layer contain a fast type of MHC found in pink and white muscle fibers in rainbow trout (Rowlerson et al., 1985; Mascarello et al., 1986). The change in MHC isoforms observed in the present study could be explained by a developmental shift in fiber type expression involving a reduction of the contribution of fast-twitch or white muscle fibers to the red muscle layer. This has a precedent in salmonids, as Higgins (1990) showed that the pink muscle fibers, which have a morphological intermediate position between the red and white muscle layers, decrease in number with age in Atlantic salmon. An alternative process by which the change in expression might occur is a shift in the intra-fiber contribution of the different myosin isoforms. Individual muscle fibers may contain multiple isoforms of MHC, and the relative contribution of the fast form may decrease at PST. The co-expression of multiple MHC isoforms in a single "fiber type" has been observed in a variety of vertebrates, such as mammals (DeNardi et al., 1993), birds (Page et al., 1992) and amphibians (Lutz et al., 1998). To distinguish between the inter-fiber and intra-fiber explanations for the shift in MHC expression at PST, single fiber analysis is needed, including a comprehensive histological analysis of MHC expression using both myosin ATPase staining along with isoform-specific PCR with single fibers. This would allow for a rigorous determination of the process of by which the change in MHC expression occurs.
As presented above, the relationship of a shift in MHC is correlated with a shift in the swimming behavior of rainbow trout. The data do not show conclusively that the shift in swimming performance is due to anything more than growth—larger fish may simply move their tail more slowly due to inertial effects of larger body mass. More recently, an experimental approach has shown that a change in the kinetic properties of red muscle does lead to a change in swimming behavior, without the confounding variable of fish size. Through the use of the thyroid hormone thyroxine (T4), PST can be induced while fish size is controlled. Using this method, two populations of rainbow trout could be generated, natural parr and induced-smolts, that were the same length and body mass. The hormone-treated fish show the same slowing of muscle contractile properties, slowing of tailbeat frequency, and a lengthening in EMG duty cycle as natural smolts (Coughlin et al., 2001b). All of the changes in induced-smolts mimic those of the natural smolts in the absence of growth, suggesting that the shift in MHC expression observed during natural PST and the associated change in contractile properties of the muscle do contribute to the change in swimming performance in the whole animal.
Demonstrating a developmental shift in the contractile properties and molecular make-up of aerobic swimming muscle and in the kinematics of steady swimming begs the question: how does this shift relate to the ecology of the animal? The parr-smolt transformation typically involves a change in habitat and behavior. In anadromous forms, smolts migrate to the ocean, sometimes a distance of greater than 1,000 km. In non-anadromous forms, smolts can still be migratory, moving towards a lake or towards a stream or river larger than the natal stream. Greater aerobic muscle function presumably is needed for the long periods of swimming associated with migration, and this could be achieved in part through a shift in MHC expression. A decrease in the contribution of the white muscle form of MHC to the aerobic or red muscle layer (regardless of whether the changes are intra- or inter-fiber) would enhance the aerobic capability of the red muscle, potentially improving the migratory swimming performance of the animal. The problem with this speculation is that salmonids typically show a decrease in swimming stamina at PST (Folmar and Dickhoff, 1980; and others). Perhaps the decline in stamina would be even worse without the shift in MHC expression, or perhaps the shift in MHC expression and stamina are not related. The topic merits additional attention.
Other studies that have demonstrated a shift in the contractile properties of muscle with growth in fishes have focused on white muscle (e.g., cod, Anderson and Johnston, 1992; sculpin, James et al., 1998). James et al. (1998) reported a slowing of white muscle contraction kinetics and shortening velocity during growth in sculpin. However, they were unable to find the molecular basis for the developmental change in muscle function. Anderson and Johnston (1992) reported a slowing of white muscle shortening velocity with growth in cod. The anaerobic swimming musculature of both of these species would be interesting targets for the study of the molecular basis for shifts in contractile properties. MHC would be an obvious target of study for both since they each display slowing of shortening velocity, although James et al. (1998) did report no developmental change in peptide maps of MHC (that technique might not be sensitive to the more subtle shifts in MHC expression reported here for trout red muscle). In broader sense, developmental variations in fish muscle might be related to shifts in expression in many other muscle proteins as well, including troponin (I, C and T), myosin light chain, parvalbumin and/or Ca2+ ATPase.
Functional morphology is an inherently integrative endeavor. The use of molecular techniques represents a one more extension of such work. Molecular biology allows us to further our understanding of biomechanics and animal behavior by looking at the contribution of the molecular structure (e.g., analysis of muscle proteins) to the physiological properties of a tissue (e.g., contraction kinetics) and, ultimately, to behavioral performance. The availability of commercial molecular biology "kits" (RNA isolation, RT-PCR, cloning, SDS-PAGE) makes molecular techniques accessible to researchers whose training and expertise is in other areas. In the present study, this integrated approach was used demonstrate a possible molecular basis for developmental changes in swimming behavior in rainbow trout.
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ACKNOWLEDGMENTS |
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I thank Dr. Francis Weaver for collaborating on the molecular analysis of trout muscle and Dr. Gordon Lutz for technical advice and for the design of isoform-specific primers. Thank you to Katherine Saporetti, Jennifer Forry, Jeffrey Burdick, Karen Stauffer and other Widener University undergraduate biology students for their work in my laboratory. Financial support was supplied by an NSF-RUI Grant (Research at Undergraduate Institutions, IBN-9604140) and by grants from Widener University.
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1 From the Symposium Molecules, Muscles, and Macroevolution: Integrative Functional Morphology presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 3–7 January 2001, at Chicago, Illinois.
2 E-mail: coughlin{at}pop1.science.widener.edu
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