Comparison between in vivo force recordings during escape responses and in vitro contractile capacities in the sea scallop Placopecten magellanicus.
Article Type: Report
Subject: Escape behavior (Research)
Scallops (Behavior)
Scallops (Physiological aspects)
Authors: Perez, Hernan Mauricio
Janssoone, Xavier
Cote, Claude
Guderley, Helga
Pub Date: 08/01/2009
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2009 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: August, 2009 Source Volume: 28 Source Issue: 3
Topic: Event Code: 310 Science & research Canadian Subject Form: Escape behaviour
Product: Product Code: 0913070 Scallops NAICS Code: 114112 Shellfish Fishing SIC Code: 0913 Shellfish
Geographic: Geographic Scope: Canada Geographic Code: 1CANA Canada
Accession Number: 206172577
Full Text: ABSTRACT During their escape response, scallops swim, using jet propulsion created by rhythmically opening and closing their valves. Valve closure is powered by the large adductor muscle that acts against the hinge ligament. We compared in vivo force production during escape responses and in vitro isometric contractions measured on fiber bundles from the sea scallop Placopecten magellanicus. The in vivo recordings quantify force development during tonic and phasic contractions and can assess clapping frequency. In vivo responses were measured at les Iles-de-la-Madeleine (QC, Canada) after which the animals were flown to Quebec City where we measured in vitro contractile properties of isolated adductor muscle at 5[degrees]C and 10[degrees]C. Interindividual differences in force production were positively correlated between in vivo and in vitro measurements. However, peak twitch force and peak tetanic force (N [cm.sup.-2]) of the isolated fiber bundles were lower than the maximum phasic force measured in vivo. This difference is likely to reflect damage to fiber bundles during isolation and the mechanical arrangement of the adductor muscle that leads the force measured at the edge of the valve during the acceleration of the upper valve to be greater than that measured near the muscle in vivo. Our comparison of in vitro and in vivo force production by the scallop adductor muscle underscores the advantages of our simple, minimally invasive, in vivo method for assessing the capacities of scallop muscle in natural situations.

KEY WORDS: scallops, Placopecten magellanicus, adductor muscle, escape response, in vitro contractile properties, in vivo force production

INTRODUCTION

The locomotor performance of animals is set by the physiological characteristics of their muscles as well as by the biomechanical system within which the muscles function. Whereas a considerable literature describes the contractile properties of muscles in vitro (Moore & Trueman 1971, Putnam & Bennett 1982, Malamud & Josephson 1991, Peplowski & Marsh 1997, Josephson et al. 2000, Coughlin & Carroll 2006), much less is known concerning muscle performance in vivo (Altringham & Johnston 1990, Girgenrath & Marsh 1997) primarily because of the complex functional interactions between individual muscles, connective tissue, and the skeleton during performance. Thus much remains to be done to clarify how muscle properties determined in vitro are related to muscle performance in vivo.

The simple mechanical arrangement of the scallop adductor muscle makes in vivo study of its activity more feasible than in other organistas. This large muscle powers swimming and jumping. Its rapid contractions close the valves, producing jets of water that exit through two vents near the hinge linking the valves. The internal hinge ligament, which acts like a compression spring, opens the valves during relaxation of the adductor muscle (Alexander 1966). This jet propulsion allows the scallop to advance, ventral edge first, with alternate abductions and adductions. The rapid contractions are produced by the striated part of the muscle that is much larger and morphologically distinct from the adjacent smooth muscle (Lowy 1954). The twitch kinetics, heat production and twitch to tetanus ratio of the phasic adductor muscle of the sea scallop, Placopecten magellanicus, are quite similar to those of frog skeletal muscle (Rall 1981) and resemble those of the phasic adductor muscle of Argopecten irradians (Olson & Marsh 1993). The in vivo mechanical performance of the adductor muscle during natural swimming by Argopecten irradians and Chlamys hastata has been studied by measuring length changes of the muscle and pressure production (Marsh et al. 1992), to estimate the pressure power of fluid in the mantle cavity, the hinge power, and the acceleration power. Because the mechanical performance in vivo was similar to that obtained for isolated phasic adductor muscle working under cyclical length changes simulating in vivo conditions (Marsh & Olson 1994), muscle properties seem to set in vivo performance.

Swimming of scallops during escape responses has been studied by direct observations (Brokordt et al. 2000a, 2000b, 2006) and by more technologically intensive (and expensive) methods including high speed filming (Cheng & Demont 1996, Cheng et al. 1996, Ansell et al. 1998) and by implanted pressure transducers and sonomicrometry crystals (Marsh et al. 1992). Considerable insight into the biomechanical constraints and into how muscle performance sets pressure production is provided by these techniques. Nonetheless, technologically intensive techniques and visual observations have their limitations for the study of scallop performance in vivo, the former are not appropriate for large-scale screening and the latter do not indicate the strength or intensity of contractions.

Measurements of muscle strength using a force gauge provide a simple means of monitoring scallop escape response performance in vivo (Fleury et al. 2005, Guderley et al. 2008). In contrast to visual observations of escape responses, this technique quantifies contractile force and frequency and shows the activity of the phasic and tonic adductor muscles. During these measurements, the valves of the scallops are maintained open at a distance similar to that used during routine respiration, one valve is fixed to the holding tank and the sensor attached to the force gauge is placed under the upper valve. The scallop is then stimulated with a predator and the contractions of the adductor muscle produce changes in force when the upper valve contacts the sensor. Phasic contractions are apparent as rapid peaks, whereas tonic contractions cause gradual changes in force that can be maintained for prolonged periods. In the scallop P. magellanicus, this technique is rapid, reproducible, and sensitive to the physiological status of the animal (Guderley et al. 2008, Perez et al. 2008). The number of phasic contractions during an exhaustive escape response is similar to that obtained during unimpeded escape responses. The frequency ofclaps is in the same range as that recorded for free-swimming Argopecten irradians (Fleury et al. 2005, Olson & Marsh 1993). To assess how the force recorded during such in vivo recordings reflects the contractile capacities of the isolated adductor muscle, we compared the in vivo and in vitro force production of individual sea scallops, Placopecten magellanicus. To this end, we characterized force production during escape responses for the same scallops from which we isolated portions of the phasic adductor muscle to measure the contractile properties in vitro. In vivo measurements were carried out at habitat temperatures (8[degrees]C to 12[degrees]C) whereas laboratory characterization was done at 5[degrees]C and 10[degrees]C. In vitro isometric force production (N [cm.sup.-2]) was approximately 3-fold lower than measured in vivo force production, caused by, in part, the acceleration of the valves during rapid closure.

MATERIALS AND METHODS

Animals

Thirty Placopecten magellanicus (96.29 mm [+ or -] 4.84 shell height) were obtained from Petoncles 2000 (Cap aux Meules, Iles de la Madeleine, P.Q. Canada) on September 29 (15 animals) and November 1 (15 animals), 2004.

Measurements of In vivo Force Generation During Escape Responses

These responses were measured in the MAPAQ laboratory in the Iles de la Madeleine (P.Q. Canada). A suction clamp was used to immobilize the lower valve of the scallops in the bottom of the water bath, whereas a hook attached to the force gauge was placed under the upper valve (Quantrol by Dillon Advanced Force Gauge, AFG-50 N). The scallops were in circulating sea water during the test (12[degrees]C in October and 8[degrees]C in November). The test stand was adjusted to separate the valves by 15 mm, a distance similar that observed during normal ventilation of resting scallops of this size. Once positioned, the scallops were continually stimulated with an arm of the seastar, Asterias vulgaris, during 600 s. Recordings from the force transducer were taken at 0.1 Hz (Fig. 1). After these measurements, the scallops were individually identified with an indelible marker and flown to Quebec for analysis of their in vitro contractile properties. After their arrival at Universite Laval, scallops were placed in a 1,000 L aquarium containing artificial seawater at 12[degrees]C in October and 8[degrees]C November at the LARSA (Laboratoire Regional des Sciences Aquatiques) for approximately one week before measurement of in vitro contractile properties. The scallops were not red during this period.

Measurements of In Vitro Contractile Properties

The in vitro contractile properties of isolated fiber bundles prepared from each scallop used for the in vivo studies were measured at 5 and 10[degrees]C using equipment and incubation conditions similar to those used by Olson and Marsh (1993). The lower end of the fiber bundle was clamped securely to the bottom of the chamber and the opposite end was attached via a light chain harness to the transducer arm of a Cambridge Technology mode1305 servo-controlled ergometer. Muscles were stimulated via parallel platinum plate electrodes with 0.5 nas pulses from a Grass $48 stimulator amplified by a D.C.-coupled power amplifier. After determining stimulus strength, the muscle was allowed to rest for lh, following Olson and Marsh (1993). Maximal stimulus voltage was rechecked and length adjusted to achieve maximal force during isometric twitch contractions. These twitches and all subsequent recordings were separated by approximately 4 min intervals. For tetanus, stimuli were given for 300 ms at 30 Hz at both measurement temperatures. The preparation was brought to the second temperature over 30 ruin. Peak twitch force (PTF), time to peak force (TPF), and time to 50% relaxation (TR50) were recorded for each isometric twitch. Peak isometric tetanic force (PITF) was recorded for each isometric tetanus. After removal from the chamber, the intact muscle bundle was blotted and weighed to the nearest mg. The cross-sectional area of each bundle was calculated by dividing the volume (mass/density) of the muscle by its length during the measurements of contractile properties. The volume was determined assuming a density for muscle of 1.060 g [cm.sup.-3].

[FIGURE 1 OMITTED]

Experiments with a Model Scallop

To evaluate how the measured force changes with increasing distance from the site of force production, we constructed a model scallop, using two Plexiglas plates (13 cm length, 11 cm wide) connected with metal hinges. A hole in the center of the lower plate (through which a known mass could be dropped) served as a proxy for the adductor muscle. The force meter was used to determine the force at different distances from the outer edge of the upper plate towards the center. One series of measurements examined the impact of distance from the muscle proxy when the upper plate was allowed to fall with the weight (hammer effect) whereas in the second series the impact of distance was measured when the upper place was not free to move.

Analysis of Force Recordings from In Vivo Escape Responses

We evaluated maximum and mean phasic force, the total number of phasic contractions, maximum tonic force and minimal time between phasic contractions (Fig. 1). Phasic force measurements were corrected for the area of the phasic muscle as estimated from photographs of the muscle scar on the inside of the valves (Image J). We did not express tonic force per cross-sectional area of the muscle, given that it was difficult to quantify the area of the tonic muscle scar.

Statistical Analyses

As we had in vivo and in vitro force data for each individual, we used regression analysis to determine the relations between the parameters obtained by each method. To compare in vivo parameters during escape responses between October and November, we used t-tests. To compare the contractile properties of isolated bundles of phasic muscle at the 2 temperatures (5[degrees]C and 10[degrees]C) in a given month, we also used t-tests. T-tests and Anova were used to analyze results obtained with the model scallop. Normality was tested using a Shapiro-Wilk test (Statistica Version 6.0).

RESULTS

In Vivo Performance

The scallops produced an average of 35 phasic contractions during escape responses stimulated, whereas they were attached to the force meter (Table 1). These escape responses all finished with extended tonic contractions (Fig. 1), indicating that the scallops were exhausted by the procedure. Phasic contractions were produced in series, interspersed by tonic contractions, as previously noted in such recordings (Fleury et al. 2005). The maximum phasic force (N cm 2) was approximately 20% greater than the mean phasic force and did not differ between measurements in October at 8[degrees]C and in November at 12[degrees]C. In the force traces, the level of tonic force typically represented at least 50% of that of phasic force (Fig. 1). The minimum time between two claps was approximately 1 s and increased as temperatures fell (Table 1).

In Vitro Performance

Peak twitch force and peak tetanic force measured with isolated fiber bundles (N [cm.sup.-2]) dial not change with temperature and did not differ between scallops measured in October and November (Table 2). Peak twitch force was approximately 25% lower than peak tetanic force at both 5[degrees]C and 10[degrees]C. As expected, force production at the two measurement temperatures was positively correlated (Tetanic force: [R.sup.2] = 0.50, P = 0.00001; Peak twitch force: [R.sup.2] = 0.37, P = 0.006). Measurement temperature influenced the time to peak force as well as the time to 50% relaxation, with average values at 10[degrees]C being lower than those obtained at 5[degrees]C (P = 0.0001). At a given temperature, time to peak force and time to 50% relaxation did not differ between scallops measured in October and November.

Relationship Between In Vivo and In Vitro Performance

Peak twitch force and peak tetanic force (N [cm.sup.-2]) of the isolated fiber bundles were lower than the maximum phasic force measured in vivo. The sum of the time to peak force and the time to 50% relaxation was considerably less than the minimum interval between two contractions measured in vivo, indicating that the natural contraction frequency used during escape responses allows ample time for these processes. Interindividual differences in force production were correlated between measurements in vivo and in vitro. Maximum tetanic force, measured at 5[degrees]C and 10[degrees]C, was positively correlated with the maximum phasic force measured in vivo (Fig. 2). Peak twitch force in vitro and maximum phasic force in vivo were significantly correlated at 10[degrees]C, but not at 5[degrees]C.

Experiments with a Model Scallop

These studies evaluated whether the difference in force production in vivo and in vitro was caused by the biomechanical arrangement of the adductor muscle in the scallop. During the force measurements in vivo, the scallop's upper valve strikes the force transducer with a rapid motion, much like that of a hammer. When the upper plate of the model scallop was free to move and was pulled down by a weight dropped through the central hole, the measured force decreased with distance from the edge (Fig. 3). When the plates of the model scallop were prevented from moving, measured force at the edge was 7.55 [+ or -] 0.58 N whereas it was 13.95 [+ or -] 0.88 at the center of the plate (t-test, P < 0.001). The greater force measured in vivo than in vitro may be partly caused by the increase in angular acceleration with the distance from the muscle. Corroboration for this concept was obtained from observations with scallops during which the sensor for the force gauge was placed at various positions between the edge of the valves and the muscle. Although it was difficult to measure the exact position of the sensor in these in vivo tests, the measured force consistently decreased, as the sensor was placed closer to the muscle (Perez-Cortes, personal observations). We conclude that the difference in the in vivo and in vitro force measurements reflected, in part, the position at which force was measured in vivo.

DISCUSSION

Minimally invasive measurements of force production in vivo reflected interindividual differences in contractile properties of isolated fiber bundles. Although values of force production during contraction of the adductor muscle of Placopecten magellanicus were higher /n vivo than in vitro, scallops that showed higher phasic force production in vivo also showed higher twitch and tetanic force production in vitro. The minimal interval between phasic contractions in vivo decreased as temperature increased, muchas did the time to peak contraction and the time to 50% relaxation of isolated fiber bundles. This relationship between in vivo and in vitro patterns of force production indicates that measurements using a force gauge reflect the contractile capacities of the adductor muscle, although a variety of differences in the techniques lead the absolute values of contractile force to differ.

Several parameters are likely to contribute to differences in force production in vivo and in vitro. The biomechanical arrangement of the muscle in the scallop leads force measurements at the edge of the shell during phasic contractions to be higher than the actual force produced by the muscle. Damage to the muscle fibers during dissection of fiber bundles is likely to have reduced the capacity for force production in vitro (Faulkner et al. 1982). Force measurements at the optimal length along the force: length curve should lead to higher force values in vitro than in vivo. Finally, the physiological condition of the scallops may have declined during transport from the Iles de la Madeleine to Quebec City and during subsequent maintenance in artificial sea water.

[FIGURE 2 OMITTED]

During phasic contractions in vivo, scallops gape widely, separating their valves, and then the adductor muscle rapidly closes the valves. In our in vivo measurements, the sensor of the force gauge is hit by the scallop's upper valve during its downward trajectory. Hence we use the analogy of a hammer hitting the force gauge. The further the head of the hammer from the point at which the force is applied, the greater the force at impact. For a scallop, the greater the gape, the greater the force. It is difficult to ascertain whether this mechanical effect was completely responsible for the approximately 2.5-fold difference in the force values obtained in vivo and in vitro. Our measurements with the model scallop indicate that the force measured at the edge of a plate was approximately twice that measured 4.5 cm from the edge. The adductor muscles in our scallops (shell height 91 mm) were positioned approximately 4.5 cm from the edge of the valves, suggesting that this approximately 2-fold mechanical effect could apply to our scallops, leading the force produced by the muscle to be approximately half that measured at the edge during rapid phasic contractions.

The lower values obtained during measurements of isometric force production in vitro compared with force production in vivo contradict expectations based on the experimental conditions. In vitro measurements of isometric twitch and tetanic force ate done at a constant muscle length, chosen to give maximal force production. In contrast, during in vivo valve adductions, muscle length decreases, suggesting that force production in vivo (at the level of the adductor muscle) should be lower than force production during isometric contractions in vitro. However, as the adductor muscle undergoes cyclic changes in length and force production during repeated phasic contractions (Marsh & Olson 1994), no single parameter measured in vitro fully describes patterns of force production and length change in vivo. Given the difficulty of extrapolating from simple isometric and isotonic measurements, cyclic shortening experiments in vitro better reflect in vivo performance (Marsh & Olson 1994). However, these do not take into account the acceleration provided to the valves by the contraction of the phasic muscle.

[FIGURE 3 OMITTED]

The contractile parameters measured in vitro were similar to values obtained in previous in vitro studies of scallop adductor muscle, except that isometric force was lower than that obtained in previous studies (Rall 1981, Olson & Marsh 1993). Times to peak force and to 50% relaxation agreed well with values in the literature. In vitro force production differed little between 5[degrees]C and 10[degrees]C, but time to peak force and time to 50% relaxation were decreased by an increase in temperature, to a similar extent as in Argopecten irradians adductor muscle (Olson & Marsh 1993). The lower force values in our preparations suggest that a greater proportion of the fiber bundles we used were damaged during dissection than in the experiments presented by Rall (1981) and Olson and Marsh (1993). The positive correlations between in vitro and in vivo force production by the scallop adductor muscle underscores the advantages of the simple, minimally invasive, in vivo method. Although the time resolution of the force gauge prevents in vivo measurements of certain characteristics, such as the time to peak force and time to relaxation, this method reliably demonstrated phasic and tonic contractions as well as changes in phasic and tonic force throughout escape response activity. The sensitivity of in vivo force production to the energetic status of the animals (Guderley et al. 2008, Perez et al. 2008) underscores the potential of this technique for monitoring the characteristics of scallop muscle in differing physiological states.

ACKNOWLEDGMENTS

The authors thank the staff of the MAPAQ and in particular Madeleine Nadeau and Bruno Myrand, for facilitating our work. The collaboration of Sylvain Vigneault and Melanie Bourgeois was central for the success of our experiments. This research was supported by funds from the RAQ and NSERC to HG. HPC was a recipient of a scholarship from the Organization of American States.

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HERNAAN MAURICIO PEREZ, (1), * XAVIER JANSSOONE, (1) CLAUDE COTE (2) AND HELGA GUDERLEYI

(1) Departement de Biologie, Universite Laval, Quebec, Quebec G1K 7P4, Canada; (2) Centre de recherche du CHUQ pavillon CHUL, Universite Laval, Quebec, Quebec, Canada

* Corresponding author. E-mail: hernan-mauricio.perez-cortes.1@ulaval.ca
TABLE 1.
Phasic and tonic force production in vivo during escape responses
of Placopecten magellanicus at 8-12[degrees]C. The minimum time
between two contractions differed significantly between months
(and measurement temperatures) (t-test, P = 0.026).

                              October                  November
                          (12[degrees]C)             (8[degrees]C)

Maximum phasic
  force
  (N/[cm.sup.2])      5.03 [+ or -] 0.71         5.06 [+ or -] 0.66
Mean phasic
  force
  (N/[cm.sup.2])      3.64 [+ or -] 0.73         3.94 [+ or -] 0.63
Maximum tonic
  force (N)          13.53 [+ or -] 2.18        10.72 [+ or -] 1.68
Minimum time
  between two
  contractions (s)    0.90 [+ or -] 0.37 (a)     1.37 [+ or -] 0.69
Total number of
  phasic
  contractions       37.73 [+ or -] 8.46        31.67 [+ or -] 6.16

TABLE 2.
Contractile properties at 5[degrees]C and 10[degrees]C of isolated
bundles of phasic adductor muscle from Placopecten magellanicus. If
values at the two temperatures in a given month are followed by
different letters, they differed significantly (t-test, P < 0.05).

                                          In vitro

                                           October

Parameters                5[degrees]C                10[degrees]C

Peak twitch
  force (PTF)
  (N/[cm.sup.2])     1.53 [+ or -] 0.39         1.46 [+ or -] 0.55
Peak tetanic
  force (PITF)
  (N/[cm.sup.2])     1.98 [+ or -] 0.78         1.98 [+ or -] 0.68
Time to peak
  force (TPF)
  (ms)             122.80 [+ or -] 26.22 (a)   88.53 [+ or -] 14.41 (b)
Time to 50%
  relaxation
  (TR50) (ms)      105.73 [+ or -] 19.29 (a)   66.07 [+ or -] 16.21 (b)

                                          In vitro

                                          November

Parameters                5[degrees]C                10[degrees]C

Peak twitch
  force (PTF)
  (N/[cm.sup.2])     1.48 [+ or -] 0.61         1.26 [+ or -] 0.47
Peak tetanic
  force (PITF)
  (N/[cm.sup.2])     2.36 [+ or -] 0.60         2.10 [+ or -] 0.55
Time to peak
  force (TPF)
  (ms)             132.58 [+ or -] 9.71 (a)    88.83 [+ or -] 9.04 (b)
Time to 50%
  relaxation
  (TR50) (ms)      104.25 [+ or -] 10.70 (a)   64.58 [+ or -] 12.80 (b)
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