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Consequences of Forced Silking Christine S. Ortlepp and John M. Gosline* Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z4 Received July 31, 2003; Revised Manuscript Received February 3, 2004
The forced silking of a spider to obtain major ampullate (MA) silk for experiments is a standard practice; however, this method may have profound effects on the resulting silk’s properties. Experiments were performed to determine the magnitude of the difference in the forces required to draw silk from the MA gland between unrestrained spiders descending on their draglines and restrained spiders from which MA silk was drawn with a motor. The results show that freely falling spiders can spool silk with as little as 0.1 body weights of force, which generates a stress that is about 2% of the silk’s tensile strength. In contrast, forcibly silked spiders apply as much as 4 body weights of force with an internal braking mechanism, and this force creates silk stresses in excess of 50% of the silk’s tensile strength. The large forces observed in forced silking should strongly affect the draw alignment of the polymer network in the newly spun fibers, and this may account for the differences in material properties observed between naturally spun and forcibly spun MA silks. In addition, the heat produced by the internal friction brake during forced silking may set the upper limit of forced silking speed. Introduction Spiders constantly produce major ampullate (MA) silk, reeling out their dragline as they move, spooling it as they fall or descend, and producing it to build the frame and radii of their webs. It is a ubiquitous part of spider biology, and it is important to understand it as such. From the spider’s point of view, what is important is that the properties of the silk must match its intended function. Indeed, we know that spiders can rapidly adjust the material properties and thread diameter of MA silks to achieve this matching of properties to function.1 Several estimates of dragline’s strength relative to the spider’s weight have been made,2,3 and data from adult Nephila claVata and Araneus diadematus suggest that the diameters of draglines from these animals allow the draglines to support 6 and 4-8 body weights, respectively. In addition, a number of studies have shown that the material properties of MA silks vary widely,4-9 particularly with regard to tensile strength, extensibility, and energy to break. Perhaps the clearest example of the ability of a spider to adjust its silk diameter and materials properties is provided by Garrido et al.,10 who show that spiders can create a distinctly different dragline thread when walking up a vertical surface as opposed to walking on a horizontal surface. Draglines formed on a vertical surface have more consistent material properties and are formed with larger thread diameters, producing a dragline with consistently superior properties for its function as a safety line. While it is likely that spiders are able to adjust the spinning duct to control the diameter of their MA threads,1 it is not known how spiders adjust the material properties of their silks. Silk has to be drawn out of a spider; it is generally not * Corresponding author. E-mail:
[email protected].
extruded. To draw silk under natural spinning conditions, spiders attach their dragline to a substrate with glue from the piriform glands, and then they draw the silk out by moving away or by descending and using their weight to draw the silk. Another method is to reach around with a rear leg to grab the silk and draw it out. It is common practice to take advantage of the drawing process to collect silk for experiments by the forced silking of captive animals. Spiders are either encouraged to drop on their dragline, which is then reeled up and collected, or they are strapped down so that the dragline can be pulled out, often by attaching the silk to a motorized cylinder with speeds of 0.004-0.30 m s-1 depending on the spider species and experiment.11-14 It has generally been assumed that MA silk obtained by forced silking is mechanically identical to naturally spun silk, but this may not be correct. If it is not, it is crucial for us to understand the differences between the silk-forming conditions in forcibly and naturally spun silk. Work’s analysis of the difference between naturally spun and forcibly spun MA silk15 provided some hints of differences in material properties, with data for A. diadematus silk suggesting that forcibly spun silk might have a higher initial modulus and lower extensibility than naturally spun threads obtained from webs. Recently, Pe´rez-Rigueiro et al.16 showed clear differences between the material properties of MA silks obtained from Argiope trifasciata. Forcibly silked samples were found to be stiffer, stronger, and less extensible than naturally spun threads, and they demonstrated that the waterinduced supercontraction of forcibly spun threads by 7.5% to 25%, followed by drying in air, produced silks with material properties that matched the range of properties observed for naturally spun threads. They suggested that the controlled supercontraction of artificial silk fibers could
10.1021/bm034269x CCC: $27.50 © 2004 American Chemical Society Published on Web 03/16/2004
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Figure 1. typical vertical fall by a spider filmed at 250 frames/s. Panel A is a composite of the x-y coordinates of the spider’s center of gravity superimposed over the spider hanging from its dragline. The silk attachment is marked by a small black cross at the top of the photo, and the arrow indicates when the friction brake was engaged. Panel B shows the velocity (O) against time. Acceleration was calculated from least squares regressions (solid line) on the linear portions of the velocity-time data and is reported beside the regressions. Velocity is equivalent to the silk spooling speed, so the range of speeds at which A. diadematus can be forcibly silked successfully is indicated by the shaded region in panel B.
provide a mechanism for obtaining silk with tailored material properties, but it remains to be established which feature of the spider’s spinning process account for the difference in properties between forcibly spun and naturally spun silks. We hypothesize that spiders are able to control the tension applied to silks as they are drawn from their spinnerets and that variation in this tension provides a mechanism to control the material properties of the silk. We test this hypothesis by measuring the silk tension required to forcibly draw silk from restrained spiders and by estimating the silk tension developed during natural spinning through a kinematic analysis of spiders falling freely on a dragline in vertical descents. Our results show that spiders can actively control the tension applied to their silk as it is drawn from the spinneret and that this tension can vary more than 20-fold in natural spinning behaviors. Restrained spiders strongly resist forced silking at all times, often generating silk tensions that exceed the maximal values observed when a falling spider applies its friction brake. Our results suggest that large differences in silk tension account for the difference in material properties between forcibly spun and naturally spun silk. Materials & Methods Freely Spooling During Vertical Descents. To document the normal behavior of spiders using dragline as a safety line, 11 adult female A. diadematus spiders were filmed with a high-speed video camera (MotionScope model 1108-002) connected to a personal computer. The experimental arena was set up in a dark room, with a light source placed behind the camera, and a ruler was suspended along one edge of the view field as a reference and calibration aid. A thick glass rod was mounted horizontally at the top of the field of view, and spiders were placed on the rod and filmed at 250 frames/s until they fell or descended (see Figure 1A). Only sequences where the spider fell in the plane of focus were kept and analyzed.
Between 2 and 5 falls were recorded and analyzed for each spider. Digital video sequences were separated into individual bitmaps with AVI Constructor 3.0 and analyzed using Scion Image (beta 3b). The approximate center of mass and the attachment point of the silk to the rod (when visible) were selected for each frame of a series and given x-y coordinates. Two passes with a second-order, zero phase shift Butterworth digital filter17 were used to mathematically smooth the x-y coordinates. Cutoff frequencies of 35 to 50 Hz were carefully selected to prevent oversmoothing. The smoothed positional data were used to calculate the instantaneous velocity between two frames. Pixels per centimeter were calculated for every film section and were used to convert pixel dimensions to length in SI units. For spiders falling vertically with little or no horizontal swinging, the silk spooling rate is equivalent to vertical velocity. Average accelerations were calculated from the slopes of the linear portions of vertical velocity versus time plots. For such vertical descents, a simple relationship exists between acceleration and applied friction force; maximum acceleration is 9.8 m s-2 when only the force of gravity is acting on the falling spider, while 1.0 body weight of friction force results in a spider falling at a constant velocity with zero acceleration. This can also be expressed as FBW )
g-A g
(1)
where FBW is the applied force in body weights, g is the acceleration due to gravity, and A is the spider’s acceleration. Forced Silking. The friction forces applied by a spider to its dragline while it is being forcibly silked can be determined by attaching a spider to a force transducer and measuring the force to draw out the silk. We used a custom-built, semiconductor strain gauge force transducer connected through a preamplifier to a personal computer with LabView 5.0 for Windows. A small platform was screwed to the front of the transducer so that a spider could be tied firmly to the
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FBW )
F Mg
(2)
where force (F) is in Newtons and Mg is the spider’s weight. Preliminary experiments indicated that the maximum speed at which A. diadematus could reliably be silked was 0.30 m s-1. Fifteen spiders were silked at one of three speeds (0.01 m s-1, 0.10 m s-1, or 0.30 m s-1) until the silk broke or ran out. In addition to expressing silking force in spider body weights, the percent of breaking load (%L) was calculated as %L )
Figure 2. Adult A. diadematus tied to a strain gauge force transducer with its dragline leading to an overhead motor. In this photo, one rear leg is holding onto the silk, but the legs would be blocked during forced silking by the cunning placement of small bits of tape.
transducer beam (see Figure 2). Because the spider dimension varied, the distance between the spinneret and the base of the transducer were not constant. Therefore, the transducer beam was calibrated by hanging small objects of known weight at various distances from the base of the transducer beam. A typical calibration was on the order of 30 V N-1. Spiders were placed in CO2 for 10 min to anaesthetise them and were then tied to the platform with the spinnerets exposed and accessible and the rear legs blocked from grabbing the silk. It was crucial that the spider was positioned so that the silk left the spinnerets at a natural angle. To determine this angle, spiders were filmed descending on draglines with a high-speed video camera (MotionScope model 1108-002) connected to a personal computer. Sequences in which rear legs were not in contact with the dragline were analyzed with Scion Image. The angle averaged 160° from the ventral surface of the abdomen or slightly ventral to straight out behind the spider. Thirty minutes18 after removal from CO2, dragline was pulled from the spider and taped to a motor-mounted cylinder directly above the spider. The force transducer was prone to drifting in response to light and temperature fluctuations. Drift could be successfully corrected for in tests lasting less than 1 min by assuming that the rate and direction of drift were constant. In those cases, a regression was fitted through the zero-force baseline before and after silking and used to correct the raw data. If drift exceeded 0.0005 V s-1 (ca. 0.1 g force/min), the test was discarded. Owing to transducer drift in response to ambient light and temperature fluctuations, data could not be corrected accurately when silking lasted for more than 1 min because the rate and direction of drift were not constant, so tests lasting longer than 3 min should be considered to have an uncertainty of about (0.3 body weights. Because the mass of the animals used varied over a large range (0.07-1.2 g), the measured silking force was normalized by expressing it in spider body weights (FBW):
FBW FMAX,BW
(3)
where the estimated silk breaking force (FMAX,BW) is expressed in body weights and depends on the weight of the spider.2 Results Freely Spooling During Vertical Descents. Vertical descents were analyzed in 19 falls by 11 spiders. The maximum silk spooling speed ranged from 0.05 to 1.30 m s-1 with an average of 0.59 ( 0.40 (standard deviation, SD) m s-1. Figure 1 is a sample fall showing the velocity against time during a vertical descent. The spider falls from a point that is very close to the attachment point of its dragline, which is indicated by the black cross just to the left of the first body position in panel A. Thus, the preexisting dragline is only about 1-cm long in this example, and the animal began spooling new silk shortly after it fell from the rod. Initially, the animal accelerates at 9.9 m s-1, indicating that the spider can be applying little, if any, friction force to the silk that is being produced. After a freefall of about 8 cm (see the horizontal arrow in panel A) and the production of about 7 cm of new silk, the spider activates its friction brake at a vertical velocity of about 1.30 m s-1. The friction brake is activated quickly, within about 10 ms, and approximately 1.9 body weights of force are applied to the dragline. This force is maintained at a constant level until the spider comes to a stop about 0.15 s later. Falling behaviors seen in other spiders included freefall, slow vertical descents, pendulum-like swings, and combinations of these behaviors. Friction brakes were applied intermittently or steadily depending on the behavior of the spider. However, freefalls frequently preceded the application of the friction brake, as shown in Figure 1, and in these cases the freefall acceleration indicated that the minimal drawing force required to pull new silk from the spinneret is likely less than 0.1 body weights. The maximal friction force observed when a spider decelerated to a halt in a vertical fall was 2.2 body weights. Thus, an unrestrained spider is capable of modulating the tension on its silk during silk production over more than a 20-fold range. Forced Silking. All spiders responded to forced silking by actively resisting; there was no case of silk being freely spooled without active resistance. While the variation during silking and between individual spiders was large, the general pattern shown in Table 1 is that initial resistance is high but
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Table 1. Forced Silking Tension Summarya silk tension (body weights)
silking speed (m s-1)
0-20 s
0.01 0.1 0.3
2.91 ( 0.73 2.91 ( 0.93 (6)2 2.31 ( 0.63 (5)
total data set (4)1
1.14 ( 0.03 (4)1,2 2.18 ( 0.91 (6) 2.38 ( 0.43 (5)
a Summary of average tension expressed in body weights in forcibly silked spiders reported as mean ( SD (sample size). Average values for the first 20 s are reported in addition to the entire set to illustrate the common pattern of higher initial forces. Statistically different pairs of average tensions are indicated with superscripts as identified with a Tukey Test (P < 0.05).
Figure 3. Example of a forcibly silked 0.42-g adult A. diadematus with force expressed in spider body weights. Percent estimated breaking load is based on a predicted breaking load of 6.1 body weights.2 The top panel shows the entire 4.8-h data set, while the initial and third peaks are expanded in the bottom panel. Note that the scale of the time axis changes between the expanded sections.
often decreases to a steady value within several minutes. Overall, the behavior was very similar to that of the spiders actively decelerating on their draglines, but the maximal forces observed in forced silking commonly exceeded the largest friction force seen in any of the vertical falls by 50% or more. Figure 3 shows the data set for a spider being forcibly silked at 0.01 m s-1. Note that over the full 4.8 h of the test the friction force averaged just above 1 body weight, but short bursts of higher friction force occurred at regular intervals of 16.9 ( 2.8 (SD) min. These intermittent peaks were very common in data sets in which the spiders were silked for more than 20 min. The lower panel in Figure 3
Ortlepp and Gosline
shows an expanded time scale to reveal details of the initial peak in force that is a common response to forced silking and the rapid change in force that characterizes the intermittent peaks. In this instance, the spider resisted with more than 3.5 body weights of force over the first 20 s of the test, and the friction force then dropped to about 1 body weight after 3-4 min. A similar pattern can be seen for the third peak in force at about 50 min into the test. Discussion To test the hypothesis that spiders are able to control the tension applied to their silks as they are drawn from the spinnerets and that this tension differs between natural spinning and forced silking, we need to measure the tension developed when unrestrained spiders form their silk. Ideally, this would include measurements on spiders walking freely and trailing their dragline, as well as spiders spinning webs, but it would be extremely difficult to make these measurements. Fortunately, our analysis of free-falling spiders reveals what we believe is the full spectrum of behaviors available to the animal. At the start of a fall, spiders frequently spool new silk with minimal tension, as indicated by vertical accelerations that approach 1 g. This indicates that the resistance to the fall arising from air resistance plus the tension on the newly formed silk threads is about 0.1 body weight during the freefall phase, and, hence, the minimal silk drawing force is likely smaller than 0.1 body weight. Because the breaking force for dragline from adult A. diadematus averages about 6 body weights,2 this would put silk tension at less than 2% of breaking load during freefalls. While we cannot measure the minimum tension directly, this is the closest approximation of the minimum force needed to draw silk from the spider’s ducts when the friction brakes are disengaged. It should be pointed out that spiders likely do not engage their friction brakes when they are walking or making webs, so the dragline and web frame is likely also spooled with equivalently small tensions. At the other end of the behavioral range, when falling spiders turn on their friction brakes, they can apply up to 2.2 body weights of tension or ∼40% of the silk’s breaking load. Notably, this is only during the short period of deceleration so the duration is on the order of seconds rather than minutes or hours. In comparison, the forces applied by a restrained spider that is being forcibly silked are very high. As Figure 3 shows, the silk is loaded to 60% of its estimated breaking load, a load that spiders do not reach when they are actively decelerating on their dragline in a fall. Another interesting difference between freefall behavior and forced silking is that spiders can produce silk at speeds in excess of 1 m s-1 during unrestrained vertical descents, but forcibly silked spiders normally did not produce silk at speeds in excess of 0.3 m s-1. This is significant because friction brakes work by dissipating energy as heat, and the longer the brake is active, the more heat is produced. A falling spider stops in a fraction of a second and may descend at a constant velocity for only a few seconds, but a forcibly silked spider may spool silk for minutes or hours. The rate of heat production (W) is determined by the product of the
Forced Silking
silk tension (N) and silking speed (m s-1), and it is likely that the upper limit to the forced silking speed is determined by the temperature rise in the friction brake and surrounding tissues. Even at a very low speed, the spiders seem to apply a minimum of 1 body weight of force, so an increase in temperature is inevitable during the extended periods of forced silking. At higher silking speeds, heat production may cause local temperatures to rise to the point where the spider simply abandons silk production. That the localized increase in temperature may affect the properties of the silk itself is a possibility that remains to be analyzed. While the heating effect of friction brakes is one issue, the increase in drawing force may also affect the structure and mechanical properties of the dragline silk. When the silk exits the spigot in the final stages of silk production, it remains hydrated for a few seconds, and this provides an opportunity for the drawing force to enhance the formation of β-sheet crystals and to increase the axial alignment of the molecular chains between these crystals. When the newly spun silk dries a few seconds later, this molecular alignment becomes permanent and will determine the properties of the dry silk. Thus, forcibly silked fibers likely exhibit enhanced strength and stiffness but reduced extensibility as a result of the added draw alignment created by the extra tension applied by the friction brake. Vollrath et al.8 documented the effects of silking speed on material properties of forcibly spun silk, but they did not report on the state of the friction brake at the time the silk was formed. What is needed now is a careful study of the relationship between the material properties and the drawing force at the time of silk formation. Such draw processing in fiber formation may also affect the supercontraction properties of dragline silk. Indeed, supercontraction stresses observed in forcibly silked Nephila claVipes dragline19 approach 400 MPa, whereas the supercontraction stress for naturally formed dragline from the same species is about 50 MPa.20 Clearly, the practice of forced silking needs to be carefully considered in any analysis of silks produced by living spiders, and the draw-processing forces in the manufacture of synthetic silks will need to be carefully controlled because they likely will provide a useful mechanism to modulate the material properties of these silks. Acknowledgment. We thank Margo Lillie for advice and guidance in the writing of this paper. This research was supported by a grant to J.M.G. from National Sciences and Research Council of Canada.
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References and Notes (1) Vollrath, F. Biology of spider silk. Int. J. Biol. Macromol. 1999, 24, 81. (2) Ortlepp, C. S. Scaling and Function of Spider Safety Line. Master Thesis, University of British Columbia, Vancouver, B.C., Canada, 2000. (3) Osaki, S. Spider Silk as Mechanical Lifeline. Nature 1996, 384, 419. (4) Work, W. R. Dimensions, birefringences, and force-elongation behavior of major and minor ampullate silk fibres from orb-webspinning spiders - the effects of wetting on these properties. Text. Res. J. 1977, 47, 650. (5) Cunniff, P. M.; Fossey, S. A.; Auerbach, M. A.; Song, J. W. Mechanical properties of major ampullate gland silk fibers extracted from Nephila claVipes spiders. In Silk Polymers. Materials Science and Biotechnology; Kaplan, D., Adams, W. W., Farmer, B., Viney, C., Eds.; ACS Symposium Series 544; American Chemical Society: Washington, D.C., 1994; pp 234-251. (6) Madsen, B.; Shao, Z. Z.; Vollrath, F. Variability in the mechanical properties of spider silks on three levels: interspecific, intraspecific and intraindividual. Int. J. Biol. Macromol. 1999, 24, 301. (7) Pe´rez-Rigueiro, J.; Elices, M.; Llorca, J.; Viney, C. Tensile properties of Argiope trifasciata dragline silk obtained from the spider’s web. J. Appl. Polym. Sci. 2001, 82, 2245. (8) Vollrath, F.; Madsen, B.; Shao, Z. The Effect of Spinning Conditions on the Mechanics of the Spider’s Dragline Silk. Proc. R. Soc. London, Ser. A 2001, 268, 2339-2346. (9) Garrido, M. A.; Elices, M.; Viney, C.; Pe´rez-Rigueiro, J. The variability and interdependence of spider drag line tensile properties. Polymer 2002, 43, 4495. (10) Garrido, M. A.; Elices, M.; Viney, C.; Pe´rez-Rigueiro, J. Active control of spider silk strength: comparison of dragline spun on vertical and horizontal surfaces. Polymer 2002, 43, 1537. (11) Work, R. W.; Emerson, P. D. An apparatus and technique for the forcible silking of spiders. J. Arachnol. 1982, 10, 1. (12) Shao, Z.; Vollrath, F. The Effects of Solvents on the Contraction and Mechanical Properties of Spider Silk. Polymer 1999, 40, 17991806. (13) Carmichael, S.; Barghout, J. Y. J.; Viney, C. The effect of post-spin drawing on spider silk microstructure: a birefringence model. Int. J. Biol. Macromol. 1999, 24, 219-226. (14) van Beek, J. D.; Ku¨mmerlein, J.; Vollrath, F.; Meier, B. H. Supercontracted spider dragline silk: a Solid-state NMR Study of the Local Structure. Int. J. Biol. Macromol. 1999, 24, 173-178. (15) Work, R. W. The force-elongation behavior of web fibers and silks forcibly obtained from orb-web-spinning spiders. Text. Res. J. 1976, 46, 485. (16) Pe´rez-Rigueiro, J.; Elices, M.; Guinea, G. V. Supercontraction tailors the tensile properties of spider silk. Polymer 2003, 44, 3733. (17) Winter, D. A. Biomechanics and the control of human moVement, 2nd ed.; Wiley-Interscience Publications: New York, 1990. (18) Madsen, B.; Vollrath, F. Mechanics and Morphology of Silk Drawn from Anesthetized Spiders. Naturwissenschaften 2000, 87, 148-153. (19) Bell, F. I.; McEwen, I. J.; Viney, C. Fibre science: Supercontraction stress in wet spider dragline. Nature 2002, 416, 37. (20) Savage, K.; Guerette, P. A.; Gosline, J. M. Supercontraction stress in spider webs. Biomacromolecules 2004, this issue.
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