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First published online December 14, 2006
Journal of Experimental Biology 210, 12-26 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02613
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Reversibly labile, sclerotization-induced elastic properties in a keratin analog from marine snails: whelk egg capsule biopolymer (WECB)

H. Scott Rapoport{dagger},* and Robert E. Shadwick{ddagger}

Marine Biology Research Division, Scripps Institution of Oceanography, La Jolla, CA 92093, USA


Figure 1
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  		Fig. 1. 15 cycles of extension for a specimen of B. canaliculatum (see Rapoport and Shadwick, 2002Go). The bimodal behavior is delineated by the broken line at approximately 3% strain. Region `A' is the Hookean region and possesses an elastic modulus of 87.9 MPa, an order of magnitude greater than the 3.9 MPa modulus of region `B' (the yield region). The transition between the Hookean and the yield regions presents as apparent failure in the material, but is fully recoverable and repeatable. Arrows indicate the direction of strain during the extension cycles.

 

Figure 2
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  		Fig. 2. The longitudinal stress-strain behavior of a wool fiber in water at 20°C at 0.1% strain min-1; six cycles at a maximum strain of 20% (modelled after fig. 3.19a in Feughelman, 1997Go). The area denoted with an `A' exists to about 2% strain and is termed the Hookean region due to its linear elastic behavior. Area `B' exists from about 3% strain to as much as 30% strain and is termed the `yield' region because it follows an apparent yield point where the Hookean region and its respective modulus undergoes a reversible order-of-magnitude decrease in elastic modulus. For mechanical parameters, see Introduction.

 

Figure 3
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  		Fig. 3. The anatomy of a prosobranch. Adapted from (Fretter and Graham, 1994Go). The capsule gland (nidamental gland, NG) is a prominent feature prior to the creation and deposition of egg capsules. The ventral pedal gland located on the foot is not visible in this schematic.

 

Figure 4
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  		Fig. 4. A female Kelletia kelletii in ventral view. The foot is clinging to an aquarium glass wall as egg capsules are being laid. Red arrow indicates the ventral pedal gland (VPG) where final processing of egg capsules occurs. The shape, size (with respect to overall foot dimensions) and coloration of the VPG were consistent among the prosobranchs in this study.

 

Figure 5
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  		Fig. 5. Comparison of finished capsules with capsules intercepted before passage into the ventral pedal gland (VPG) for three species of prosobranch whelks. In each case, the unfinished or `immature' capsule is located on the left. (A) Kelletia kelletii, (B) Chorus giganteus (courtesy of J. Ram, Wayne State University) and (C) Busycon canaliculatum. Finished capsules have the characteristic mechanical properties as well as a yellow tint. `Immature' capsules are rough approximations of the final shape of the capsule. `Immature' capsules are soluble in common protein denaturants and lack any significant cohesion when strained. Scale bars, 0.5 cm.

 

Figure 6
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  		Fig. 6. Scanning electron micrographs of cross sections of Busycon canaliculatum egg capsules. (A,C) An extract-induced `immature capsule' at low magnification (B; scale bar, 100 µm) and high magnification (C; scale bar, 20 µm). (B,D) A native capsule at low magnification (B; scale bar, 100 µm) and high magnification (D; scale bar, 20 µm). Common to both is a structured layer of sheets that upon closer examination yield distinct ordered fibrous content.

 

Figure 7
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  		Fig. 7. Development of mechanical properties in WECB. Capsule maturation is divided into three distinct phases: (I) pre-pedal manipulation, (II) pedal manipulation and (III) post-pedal manipulation. Pre-pedal manipulation consists of formation of the nascent egg capsule in the nidamental gland including its transport to the pedal gland. Capsules tested in this phase lack material cohesiveness on a scale detectable by our testing apparatuses. These capsules are also very soluble. The pedal manipulation phase involves solely the treatment and manipulation of the capsule while it is in the ventral pedal gland. Capsules from this phase are beginning to show elasticity, due presumably to crosslinking. Post-pedal manipulation includes the deposition of the capsule either on substrate, or to a growing strand. Capsules in this phase show the fully developed mechanical properties with a Hookean and yield region. Additional curing probably occurs over a period of time. (Note, force scales are different in II and III.) Both figures are composed of raw data directly from the MTS tensometer. Multiple curves represent different specimens. Thus, the development of final mechanical and chemical properties is a time-dependent maturation catalyzed by a sclerotizing mechanism applied in the VPG.

 

Figure 8
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  		Fig. 8. Comparison of B. canaliculatum WECB equilibrated in varying concentrations of formic acid (FA) for 1 h prior to testing compared to a native specimen. The tenth cycle of each trial is presented. Higher concentrations of acid at incubation times of 1 h result in a transitional disappearance of the Hookean region of the stress-strain curve. At 88% FA, a 30 min incubation possessed a present, but greatly reduced Hookean region compared to the 1 h incubation pictured above.

 

Figure 9
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  		Fig. 9. The effect of pH on the load curve for WECB from B. canaliculatum. The specimen was allowed to equilibrate in 0.2 mol l-1 citrate/phosphate buffer for 30 min prior to mechanical testing. The pH=2 treatment was the only treatment that appeared to cause a change in the average yield stress and Hookean behavior of the specimen. Pictured are the curves from the tenth cycle of loading.

 

Figure 10
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  		Fig. 10. Recovery of formic acid (FA) treated specimens. Both graphs compare a formic acid treated specimen with the same specimen returned to water. Recovery traces represent 10 stress-strain cycles of B. canaliculatum WECB specimens formerly tested following 1 h incubation in 44% (A) and 88% formic acid (B), respectively, were now returned to deionized water and allowed to equilibrate for 1 h before testing. The specimen from 44% FA appears to have recovered its native behavior. The 88% FA treated specimen recovers somewhat (bimodal behavior appears to be present), but still deviates from native properties. Incubation times approaching 30 min were apparently fully recoverable at 88% FA.

 

Figure 11
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  		Fig. 11. Graphs of WECB resilience based on the three treatments: formic acid, citrate/phosphate buffer changes and temperature exposure. Resilience appears to increase with increasing temperature, and increasing formic acid concentration, but not with decreasing pH. Values are means ± s.e.m. (N=2-5).

 

Figure 12
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  		Fig. 12. Graphs of WECB yield stress based on the three treatments: formic acid, citrate/phosphate buffer changes and temperature exposure. Yield stress decreased with increasing temperature, increasing formic acid concentration and decreasing pH. Values are means ± s.e.m. (N=2-5).

 

Figure 13
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  		Fig. 13. Graphs of WECB elastic modulus based on the three treatments: formic acid, citrate/phosphate buffer changes, and temperature exposure. Open symbols represent the yield region and filled symbols represent the Hookean region. Elastic modulus of the yield region appears to remain fairly constant throughout the treatments. Hookean elastic modulus either remains fairly consistent or decreases. At the extreme of each of the treatments [i.e. high temperature, strong formic acid concentration at longer incubation times (~1 h) and low pH] the Hookean region disappears completely so no modulus can be determined. Values are means ± s.e.m. (N=2-5).

 

Figure 14
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  		Fig. 14. Representative temperature extension cycle sequence for WECB from Busycon canaliculatum. Increasing the temperature of the water bath in which the specimen is being quasi-statically cycled results in a lessening of the yield stress and a recession of the Hookean region. At temperatures approaching 100°C, the Hookean region appears to be absent.

 

Figure 15
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  		Fig. 15. Results from dynamic mechanical testing of native WECB. (A) The storage modulus as a function of frequency as recorded for the three treatments: Hookean, yield and vibe-ramp. (B) Tan{delta} as a function of excitation frequency for the three treatments.

 

Figure 16
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  		Fig. 16. Results from the dynamic testing of 88% formic acid treated WECB (1 h incubation time). (A) Storage modulus as a function of frequency. (B) Tan{delta} as a function of frequency.

 

Figure 17
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  		Fig. 17. Load to failure curves of five dehydrated specimens of WECB from B. canaliculatum. There is an order of magnitude increase in yield stress, and little to no change in yield strain among hydrated and dehydrated specimens. See text for values of toughness, yield strain, yield stress and initial modulus. Red dotted curve represents cyclic loading of native specimen seen in Fig. 1.

 

Figure 18
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  		Fig. 18. Model of WECB mechanics through maturation. Blue arrows denote sequential movement from generalized structure to modified structure as described: (A) generalized structure, (B) pre-ventral pedal gland (VPG; organ located in the foot where a final stabilization process renders WECB insoluble), (C) during VPG, and (D) post-VPG For detailed description, see text.

 

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© The Company of Biologists Ltd 2007