Article pubs.acs.org/Biomac
Forced Reeling of Bombyx mori Silk: Separating Behavior and Processing Conditions Beth Mortimer,† Chris Holland,*,†,‡ and Fritz Vollrath† †
Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, United Kingdom Department of Materials Science and Engineering, Sheffield University, Mappin Street, Sheffield S1 3JD, United Kingdom
‡
S Supporting Information *
ABSTRACT: Controlled reeling is a powerful tool to investigate the details of silk processing. However, consistent forced reeling of silkworms is hindered by the significant degree of behaviorally induced variation caused by the animal. This paper proposes silkworm paralysis as a novel method to control the animal and thus in vivo spinning conditions. Using these methods, we achieve low and consistent reeling forces during the collection of over 500 m of individual silk fiber while monitoring filament variability, morphology, and properties. Novel techniques to measure the irregular silk crosssectional areas lead to the more accurate calculation of the true engineering values and mechanical property variation of individual silk fibers. Combining controlled reeling and accurate thread measurement techniques allows us to present the relative contributions of processing and behavior in the performance envelope of Bombyx mori silk. resisting silkworms are able to break the thread.4 This is a problem for the industrial use of such forced reeled fibers, as long consistent fibers (with high strength) would be most desirable. The key to addressing these issues is the separation of behavioral influence from the effect of processing conditions. In spiders, the behavioral control of load during reeling has been shown to influence predictably variability and properties of drawn fibers.18,19 However, until now, reeling force in silkworms has not been controlled or quantified. A silkworm can apply load on to the nascent thread through internal or external means. External loading could occur using either or both of two pairs of facial palps (maxilla and labial) or the ‘true’ (thoracic) legs positioned around the spinneret. From observation and scanning electron microscope (SEM) images, we propose that the first pair of legs is best positioned to hook onto the silk. In support of this assertion, we note that the claw on the leg has a nook of a size comparable to a naturally spun silk fiber (Figure 1a). The exact method of load application is presently unknown but is not important for the interpretation of our experiments. Moreover, it is well possible that total loading history on a thread is likely to differ between individual worms and between multiple reelings due to the speed and angle of reeling and the status/experience of the worm. This multiplicity of contributing factors may explain the high variation in forced reeled silks between different silkworms.20 Internally, silkworms possess a silk press which, when activated, increases the diameter of the silk fibers.21 Although
1. INTRODUCTION Silks are fibrous proteins spun by a wide range of insects and spiders, even some crustaceans.1,2 Within the insects, silks from the Chinese mulberry silkmoth Bombyx mori are the most extensively studied because of their huge commercial value derived from a long history of human domestication. An individual strand of silkworm silk is composed of two fibroin fibers and a glue-like sericin coating, which is used by the animal to construct a nonwoven layered cocoon protecting the worm prior to metamorphosis.3−6 Because of the natural range of processing conditions and the need to unravel the threads from the cocoons, naturally spun silks tend to be variable in properties and fiber morphology. Forced reeling allows us to control the processing and environmental conditions with the aim to manipulate the animal to make more consistent fibers that, perhaps equally importantly, do not go through the cocoon layering and unravelling process. Using forced reeling, we can even tailor the mechanical properties of a thread7 by pulling silk directly from the spinnerets of the animal under conditions controlled by the experiment.8 In contrast with the forced reeling of spiders, the forced reeling of silkworms is a relatively new endeavor.9,10 The potential for detailed hypothesis testing is significant because this technique allows the investigation of a wide range of variables such as spinning speed, body temperature, postdraw medium, animal physiology (e.g., anesthesia blood pH), and air humidity.11−18 Despite being more consistent than naturally spun silks,10 forced reeled silks still have high interfiber variability. This complicates quantifying the exact effect of processing conditions on the properties of silks. Additionally, alert and © 2013 American Chemical Society
Received: July 11, 2013 Revised: August 23, 2013 Published: September 4, 2013 3653
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Figure 1. (a) SEM false color image of the claw at the end of the first thoracic leg of a silkworm. Black scale bar denotes 50 μm. Note the notch on the inner side of the claw that corresponds to c. 20 μm, the diameter of a naturally spun silk fiber. (b) Drawing of the silk press inside a silkworm before the spinneret (green, a). The two ducts (blue, b) run from the two silk glands and then merge. Ducts from the Filippi’s gland (purple, c) join before the silk press. At the press, there is a smaller lumen (yellow, d) and a thickening of the tube wall (turquoise, e). There is a long ventral plate that meets the lumen and a smaller plate imbedded in the wall (orange, f). Ventral (red, g) and dorsal (brown, h) muscles control the press to increase lumen size when they are contracted. Elastic recoil of the cuticle-lined wall enables lumen contraction. The inset image gives the transverse section through the press.
unravelling a fiber from a cocoon under wet heat in a chemical environment will inevitably affect the inherent properties of the thread. This is avoided when forced reeling is used. To assess the effect of processing conditions, such as reeling speed, it is important to control the worm’s behavior (i.e., possible effects of it clamping the thread) as well as minimize errors of measuring silk properties (i.e., correct thread dimensions, carefully apply tensile testing, and avoid premeasurement treatments). It is important to be able to directly assess the effects of processing conditions if we want to understand the full silk production system. For example, reeling speed affects the mechanical properties of forced reeled spider and silkworm silks.7,10,11,24 It is assumed that the reeling speed affects the shearing of the silk dope while in the duct,11 and thus a faster reeling speed would result in an increase in β-sheet content while at the same time lead to a decrease in fiber diameter.11,24 In spiders, it has been shown that there is an optimum speed for breaking energy and stress, which matches the average natural spinning rate.11 However with silkworms, increasing reeling rate does not affect breaking energy, and maximum stress increases to an impressive 1 GPa.7,24
it has been a long observed feature of silkworm anatomy,22 so far the mechanics of the press have only been inferred from superficial structural analysis. Using data from silkworm dissection, as well as recent histological and micro-CT observations,21−23 Figure 1b aims to summarize the known anatomy of the silk press. Both the dorsal and ventral muscles act to increase the silk duct lumen when contracted (Figure 1b inset). Lumen size narrows when the muscles are relaxed due to elastic recoil of the cuticle-lined wall. Detail of the musculature may be found elsewhere.21,23 Previous to the application of the technology presented here it was never possible to sufficiently reduce a silkworm’s behavioral control to allow reeling for more than a few meters, with a published current record of 15 m.24 Short fibers with unpredictable properties are undesirable for both research and industry because they increase the costs of manufacturing and reduce the strength of wound threads. Here we present a method that induces natural silkworm paralysis by using the animals’ own ‘play-dead’ antipredator defense, which is induced hormonally by a specific paralytic peptide.25 By thus preventing the animal’s active load application, we were able to prevent fiber breaking, enabling us to reel long consistent fibers (over 500 m), which are desirable for both scientific research and industrial fiber processing.25 Accurate ‘diameter’ measurement to calculate accurate crosssectional area is key to correct calculations of a fiber’s strength, toughness and other mechanical measurements. Unfortunately, area is not easy to measure accurately for silkworm silks because they are not circular (like spider silks). The current literature varies considerably in the methods used − or indeed does not disclose how cross-sectional thread areas were measured or calculated.7,10,24,26 Connected to the issue of calculating accurate mechanical data relates to the material itself. Silk has a strong viscoelastic component and thus will show stress and strain history, and
2. MATERIALS AND METHODS 2.1. Silkworms. Final instar Bombyx mori silkworms were reared on a mulberry diet. Worms were stored in lab conditions (20 °C, 40% relative humidity) until they started spinning. To permit forced reeling, the silkworms were immobilized after they started spinning. Nonparalyzed worms were restrained either by hand around the head or suspended from a pole using tape around their body (Figure 2). Similar worm head movement was still possible with both methods. Paralyzed worms were injected with 0.1 mL of 10 ng μL−1 paralytic peptide in water solution into the first pseudopod base into the hemolymph. The paralytic peptide was custom synthesized by Activotec, Cambridge. The peptide sequence is ENFVGGCATGFKRTADGRCKPTF, modified so that the two Cys amino acids at positions 7 and 19 have an intradisulfide bond.25 Descriptions of the action of the peptide naturally are described elsewhere.27−30 Following 3654
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Figure 2. Experimental setup for silkworm forced reeling: (a) post holding silkworm and (b) tape restraining worm between true legs and prolegs. Reeling device viewed from the side with (c) spool, (d) reeling motor, and (e) horizontal translation motor. injection, all silkworms ceased movement, becoming rigidly straight, and would stop spinning even if silk was protruding from their spinnerets (as for the silk inlaid in Figure 3b). After ∼30 min of complete paralysis they would recover their ability to spin silk which agrees with observations of anesthetized spiders.12 Therefore, worms were left for 30 min after injection under lab conditions (20 °C, 40% relative humidity) until they started spinning again. Worms were then reeled in a semiparalyzed state by suspending them, as described above (Figure 2). Silkworm movement appears normal, but they were less likely to break the silk thread and can be reeled for long periods of time (up to 6 h). During reeling, the appearance of unparalyzed and paralyzed silkworms is identical, and only the head and body movement differs. 2.2. Silk Reeling and Storage. Silk was reeled onto a spool that rotated by a calibrated motor (Figure 2). Reeling speeds were between 6 and 30 mm s−1, spanning most of the natural range for spinning.7 For each speed, samples from the start of a reeling were avoided. Naturally spun silk samples were taken from a single B. mori cocoon, unwound onto a spool. Such samples were taken over a length of 500 m to ensure consistent mechanical properties and area.31 All silk was stored under tension on a spool under lab conditions (20 °C, 40% RH) to minimize variation due to extrinsic factors.31 2.3. Load during Forced Reeling. A silk thread from a stationary silkworm was fixed to a load cell that was then moved to measure the force exerted by the worm during reeling, similar to other published works.18,32 Spinning silkworms were held carefully around their head during such reeling, which in our case was done with a Zwick tensile tester (500 N, Z0.5, Zwick, Germany). The silk was reeled at a constant speed of 10 mm s−1 for up to 70 cm. Load and displacement were recorded over the reeling period. All data were processed in Microsoft Excel by removing slack from the initial stages of reeling then zeroing the initial load. Background levels of load over displacement without silk attachment were also measured to provide a control. 2.4. Cross-Sectional Area. Cross-sectional areas of silkworm silks tend to be highly irregular and variable in dimensions (Figure 4), and cross-sectional areas of all silk fibers must be measured accurately. For this, silk was carefully transferred from the spool onto dividers.12 Still under tension, the silk fibers are glued with cyanoacrylate along their length onto a section of solder wire, as straight as possible. The solder wire was then either glued into rigid plastic tubing and transversely sectioned into discs or sectioned directly using razor blades. The thin discs were then treated with 200 μL mL−1 protease solution in a 38 °C water bath for ∼24 h. This digested some of the silk fiber, leaving an outline in the glue equal to the silk’s original cross section. The digestion helped to make the silk more visible during imaging. These discs were then mounted onto a SEM stub and sputter coated for 150 s, 18 mA, giving a 12.5 nm coating of gold/palladium (Quorum Technologies SC7620). Pictures of the cross sections were taken in a Scanning Electron Microscope (SEM; Neoscope 2000,
Figure 3. Silkworm force over reeling. Zeroed load over cross head extension for: (a) unparalyzed worms (6 worms, 30 reelings): blue line gives maximum load curve, red the median, pink the minimum, and black a representative background curve (out of 12); inlaid SEM image of thread, black bar denotes 20 μm. (b) Paralyzed worms (8 worms, 27 reelings): inlaid is an SEM image of a flattened silkworm thread following paralysis where white bar denotes 50 μm. (c) Average maximum load for the moving cross head for background (no worm attached) and paralyzed and unparalyzed worms, where errors bars give the standard error of the mean between worms. Unparalyzed silkworms show a maximum load significantly higher than the background level (i.e., no worm attached; Moods sign test, p < 0.01**), as do paralyzed worms (Mann−Whitney p = 0.0197*). 3655
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Table 1. Mean Area and Feret’s Shape Coefficient Data for Paralyzed and Unparalyzed Forced Reeled Silks at 6 and 30 mm s−1 and Naturally Spun Silk Feret’s shape coefficienta
area silk type (speed in mm s−1)
n
naturally spun unparalyzed (6) paralyzed (6) unparalyzed (30) paralyzed (30)
145, 67b 69 67 44 67
mean (× 10 mm2) 37.90 19.75 15.71 7.60 7.21
−5
standard deviation (× 10−5 mm2)
coefficient of variation (%)
mean
standard deviation
coefficient of variation (%)
6.45 11.50 5.89 4.34 1.85
17 58 37 57 26
2.25 1.52 1.66 1.48 1.77
0.574 0.327 0.421 0.240 0.630
25 22 25 16 36
a
Feret’s shape coefficient is worked out by dividing the maximum diameter by the minimum diameter for the complex cross-sectional area shape. Values closer to 1 denote a more circular shape. bTwo values of n are given for naturally spun silk: the first for the area samples, the second for the Feret’s shape coefficient.
Nikon Instruments U.K.) at high vacuum, 10 kV and at x2200 magnification. Pictures were analyzed using ImageJ software (NIH). 2.5. Area Allocation. Data in the Supporting Information (Figure S1a,b) give the difference in stress strain curves spread for one data set by assigning either the area of the nearest sample to the sample tested or the average area. As average area gives less spread for the curves; the average area for each speed of a worm is used for area allocation. 2.6. Tensile Tests and Analysis. Fibers were mounted under tension into 10 mm gauge length cardboard frames for tensile testing (5 N load cell, model 5512, Instron, U.K.). Samples were pulled apart at a rate of 40% min−1 until broken, only using samples that broke in the middle. Load-extension data were analyzed using a Microsoft Excel macro and Figures are drawn using Origin Software (OriginPro8). Nonparametric statistics, as suitable tests for low sample numbers, were performed using Minitab 13 software. Paired tests used Mann− Whitney two-tailed tests when the assumption of equal variance is met. In other cases, a Moods sign test was used, which is less powerful but does not assume equal variance.
The significant difference between the background and paralyzed reeling load represents the force taken to pull the fiber out of the worm. It can be explained by the inherent viscosity of the silk dope as it flows.33 Additionally, the silk press will be acting as a friction break on the silk during reeling, as the press is applied when the muscles are relaxed.21 Immediately after paralysis, the silk shows a ribbon shape (Figure 3b inlaid), consistent with a compressive action of the press on the silk. We assume therefore that relaxation of the silk press following paralysis injection constrains fiber spinning for the 30 min that they cannot spin. After reeling restarts, the press continues to constrain the thread without cutting it or preventing spinning. 3.2. Fiber Analysis. Because the action of the silk press during forced reeling appears to influence fiber morphology, we analyzed the cross-sectional images to further quantify the differences between forced reeled (paralyzed and unparalyzed) and naturally spun silkworm silks. Forced reeled silks are thinner than their naturally spun counterparts (Figure 4, Table 1), which is partially due to a more even sericin coating (Figure 4a,b). For naturally spun silks, the interlayer bonding that was present in the cocoon is not completely removed during cocoon unravelling. Furthermore, cross sections show that the brins themselves are smaller in forced reeled silks when compared with naturally
3. RESULTS AND DISCUSSION 3.1. Behavior: Silkworm Load over Reeling. Our results indicate that silkworms can effect load during forced reeling (Figure 3). Paralysis causes this force to be smaller and more consistent (Figure 3a,b). Both paralyzed and unparalyzed silkworms showed a maximum load that was higher than the background (Mann−Whitney two tailed test, p = 0.0197 and Moods sign test p < 0.01 respectively). Importantly, unparalyzed worms applied loads during spinning that were sufficient to break the fiber (Figure 3a). Force calculations give loads of around 0.08 N, equivalent to over 700 MPa based on an average area for a forced reeled silkworm fiber at 10 mm s−1 (the speed of reeling for these tests: 1.05 × 10−4 mm2, n = 23). Such a load would exceed the breaking stress for these silks, which is ∼500 MPa. In contrast, the maximum force that spiders have been observed to apply during forced reeling (using their internal friction breaking mechanism32) never seems to exceed 60% of the breaking load.18,19,32 In contrast, partially paralyzed silkworms were unable to apply a high force during reeling. These worms were able to move and spin silk, but there appeared to be a lasting effect of the paralytic peptide in the muscles which exert load onto the silk. Here we note that as yet a full understanding of the details of the silkworm’s press system is missing. Regardless, the method of paralysis spinning prevents fiber breakage during reeling, so unlike unparalyzed worms, allows reeling for over seven hours (our record was >500 m). Genetic engineering approaches to use the paralysis peptide to allow long reeling will be an interesting next step.25
Figure 4. SEM images of silkworm silks: (a) naturally spun and (b) forced reeled silk where white bars denote 20 μm. SEM images of the protease-digested cross sections of: (c) naturally spun, (d) unparalyzed forced reeled silkworm silk, and (e) paralyzed forced reeled silkworm silk, where black bars denote 10 μm. NB, the dark hole in the glue represents the shape and area of the fiber in the cross sections rather than the white brins, which have been digested in a protease before imaging. Forced reeled silks are reeled at 6 mm s−1. Colored boxes show type of fiber; naturally spun (orange), unparalyzed forced reeled (purple), and paralyzed forced reeled (green). 3656
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Figure 5. (a) Average area over distance for naturally spun (orange) and paralyzed forced reeled silkworm silks (green) reeled at 20 mm s−1, error bars can be seen as variation on the millimeter scale, variation across the x axis as variation on the meter scale. (b) Probability distributions of measured data (bars) and random normal distributions with equal mean and standard deviations of measured data (lines) of paralyzed forced reeled (green; n = 75, area = 8.97 × 10−5 mm2 ± 2.26) and naturally spun silk (orange; data given in Table 1).
Figure 6. Effect of reeling speed on the mechanical properties of paralyzed (green) and unparalyzed forced reeled silkworm silks (purple; which include data where the silkworm broke the thread): (a) the break strain, (b) maximum stress, (c) breaking energy, and (d) mean cross-sectional area. Stars give a comparison to average values for naturally spun silk (not shown in d). Error bars give the standard error of the mean.
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which show higher strength and lower extensibility compared with paralyzed silks at 15 mm s−1, suggesting higher reeling force. Importantly, if the samples surrounding a break are excluded from comparison, then the paralyzed and unparalyzed worms show more similar properties, suggesting more comparable reeling loads. Paralyzed worms give the best indication of the relationship between forced reeling speed and mechanical properties (as their reeling load is consistent; Figure 3b). This observation is underpinned by the consistency of silk-area data for paralyzed worms (Figure 6d), which shows a nonlinear relationship and is consistent with findings in spiders.34 Comparable to the spider silk data, the mechanical data from silks taken from paralyzed worms showed an ‘optimum’ speed for maximum stress and breaking energy.11 Moreover, silkworms can also be reeled ‘too fast’, resulting in a deterioration in properties above a certain speed (100 mm s−1 for spiders,11 20 mm s−1 for silkworms [this paper]). For silkworms, the maximum thread toughness was seen at 10 mm s−1, at the average natural spinning speed.7 Toughness is likely to be the mechanical property on which natural selection acts as the cocoon structure will be selected for high energy absorption ability, for protection against predators.5,35 Both the composite structure of the cocoon and the fiber properties will contribute to this energy absorption.36 Individual fibers will therefore be selected to have high toughness at biologically relevant reeling speeds. Therefore, we can infer that the relatively poor breaking energy above 20 mm s−1 has a limited effect on fitness, as these reeling speeds will not be used by the worm. The patterns of mechanical silk data observed by us differ from previously published literature data on the effect of reeling speed on silkworm silk properties.7,24 We suggest that this is due to differing methods in area measurement and insufficient control of worm ‘behavior’ in the previous experiments. We believe that the methods presented in this paper provide more robust data as the silkworms were more controlled with consistently low fiber loading during reeling (Figure 3c), giving a much more reliable relationship between mechanical properties and reeling speed. The highest breaking stress was found to limit at 540 MPa at a reeling speed of 20 mm s−1. We believe this represents a limit in the strength of silkworm silks and is supported by modeling insights into its protein structure and its diameter.37 This suggests limitations for the current ambitious work using genetic engineering approaches to make stronger silkworm silks.26 It is interesting, however, that for the majority of forced reeled silks the properties appear to be inferior to the naturally spun silks (Figure 6), which show a typical maximum stress of 390 MPa and breaking energy of 67 J cm−3 (data taken from ref 35). Further work looking at the structural basis of mechanical property changes in terms of the processing conditions applied will be an important next step. The removal of behavioral aspects also leaves room for the experimental manipulation of ionic environments in vivo during the spinning process to further understand where variability in silk structure and properties may stem from.38
spun cocoon silks (taking into account protease digestion). We hypothesize that this is due to the silk press contributing a load that decreases fiber diameter. Paralyzed worms appear to have the press consistently applied, leading to the thinner and less circular cross sections (Table 1). Unparalyzed worms have control of their silk press, enabling them to increase silk duct lumen diameter, making fibers more circular on average.21 The implication of this finding is that unparalyzed worms are not only able to apply load during reeling using external mechanisms (Figure 3a), they can also decrease load by removing their silk press. Combined, this increases the variation in reeling load. These relationships hold across the range of reeling speeds (Table 1). Given the morphological differences in the fiber crosssectional area between forced reeled and naturally spun silks, variability on different scales along the fiber was investigated (Figure 5). This ensures the accurate allocation of crosssectional area for stress calculation, another source of variation in mechanical properties. Forced reeled silks are more consistent than naturally spun silks (Figure 5), which fits in with previous literature.10 This is primarily due to their decreased variability in cross-sectional area dimensions as well as less variability in sericin coating (Figure 4). For naturally spun silk, the variation in spinning speed due to the natural figure of eight head movement may also contribute.7 We also observed that area measurements cannot be modeled with a normal distribution, which was unexpected (Figure 5, Anderson−Darling normality test, p < 0.05). There is a slight right skew in both distributions. This appears to be an artifact of the method where overestimation is more likely than underestimation, giving rare high area data. This will therefore slightly underestimate the stress-linked mechanical properties of the silks. Further analysis of these data revealed the number of samples required to give an area with good chance of being representative (compared to mean from over 100 samples; data in Supporting Information, Figure S1c). For cocoon silk, an average of at least 4 samples is required to achieve a comparable area and suitable error (quantified by repeating the method 10 times). For forced reeled silk, the minimum was slightly higher, at 5 samples. Due to the random fluctuation in area for both silks, the benefit of averaging more samples only gradually decreases the chance of deviating from a mean taken with over 100 samples. The samples should be collected randomly across the entire reeled/spooled sample length, to keep it representative. 3.3. Effect of Processing. We have demonstrated that paralysis appears to be a suitable method for decreasing fiber variation by reducing behavioral control of reeling load. The interaction of paralysis with reeling speed will give further insights into the effect of applied load on mechanical properties. Using the methods for cross-sectional area measurement already outlined, the data in Figure 6 support the hypothesis that silkworm behavior influences fiber performance. In samples close to where unparalyzed silkworms broke the thread (most commonly before 15 mm s−1), it appears that the animals were applying load to the fiber. This is clearest when looking at paralyzed and unparalyzed silkworms reeled at the same speed. Research on spider silks has shown that reeling force affects mechanical properties and variability, where higher force increases modulus and breaking stress and decreases breaking strain.18,19 Our unparalyzed data agree with this,
4. CONCLUSIONS Our research demonstrated for Bombyx mori silks how the behavioral aspects of spinning can be separated from the purely physiological processing. Paralyzed silkworms showed consistently low load during reeling, meaning the worms could be reeled for much longer than previously published, at ∼500 m. 3658
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The control of silkworm behavior produced consistent fibers, which were thinner than the naturally spun silks. In contrast, unparalyzed worms behaviorally controlled the load they exert on the fiber by applying load externally, which can break the thread, or by removing load internally. The fibers of forced reeled worms and naturally spun silks were then analyzed to understand fiber variation. Analysis of paralyzed versus naturally spun silk cross-sectional areas suggests that mean areas of at least five or four sections, respectively, across the length of silk are required for accurate stress calculations. Using these techniques for worm immobilization and area allocation we present a solid platform from which the effect of processing conditions can be explored further, not only in mulberry silkworms but also in the silks of other Lepidoptera. Consistent with trends observed in spiders, reeling speed gave an optimum breaking energy at the average natural reeling speed for paralyzed silkworms (10 mm s−1), with a deterioration in properties above a certain speed (20 mm s−1). Last but not least, our data suggest a limit for naturally produced B. mori silk strength of ∼600 MPa.
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ASSOCIATED CONTENT
S Supporting Information *
Figure S1a,b shows the stress strain curves of an unparalyzed worm reeled at 10 mm s−1 using (a) area nearest sample and (b) average area. Figure S1c gives the average deviation from the mean (bars) for 10 repeats using a different number of samples per average (x axis). Error bars give the standard deviation, where there is ∼70% chance that an average using a different N lies within this value. Black axis and green bars give data for paralyzed forced reeled silks, whereas the red axis and orange bars give naturally spun data. The dashed lines correspond to 2, 10, and 20% change in calculated stress when the different area is used. A supplementary video is also available. This is a 20 s clip from a paralyzed silkworm undergoing forced reeling. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: christopher.holland@sheffield.ac.uk. Tel +44 114 222 5477. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Alex Woods for his insights into the reeling of silkworms and for demonstrating the paralysis technique to us. We thank Sophie Scott for help with reeling the silkworms and Dr. David Porter for insightful comments and suggestions. For funding, we thank The Leverhulme Trust (F/08705/D), the U.S. Air Force Office of Scientific Research (FA9550-12-10294), the European Research Council (SP2-GA-2008233409), EPSRC (EP/K005693/1), and Magdalen College, Oxford.
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REFERENCES
(1) Craig, C. Ann. Rev. Entomol. 1997, 42, 231−267. (2) Kronenberger, K.; Dicko, C.; Vollrath, F. Naturwissenschaften 2012, 99, 3−10. (3) Mortimer, B.; Drodge, D. R.; Dragnevski, K. I.; Siviour, C. R.; Holland, C. J. Mater. Sci. 2013, 48, 5055−5062. 3659
dx.doi.org/10.1021/bm401013k | Biomacromolecules 2013, 14, 3653−3659
