We investigated the hypothesis that calcium salts are used to modify the material properties of baleen α-keratin in a way that partly compensates for the shortcomings of stiffening in a wet environment. This hypothesis predicts that baleen with greater calcification should exhibit a higher modulus and yield stress than less-calcified baleen, and our results were mostly consistent with this prediction. Furthermore, decalcification caused the greatest decrease in the tensile modulus and yield stress of sei baleen, the smallest decrease for minke baleen and an intermediate decrease for humpback baleen, which exhibits an intermediate level of calcification. Decalcification had no effect on the yield strain, suggesting that it is governed entirely by the mechanical behaviour of the coiled coils within intermediate filaments (Hearle 2000
). It is interesting that the material properties of sei and humpback baleen are so similar given that sei baleen is considerably more calcified. This result suggests that there may be other mechanisms of stiffening at work in humpback baleen.
The PIXE results suggest that the calcium salt in all three baleen types is hydroxyapatite, the dominant calcium salt in bone (Pautard 1962
). A major difference between bone and calcified keratin is that calcification in bone is an extracellular process, whereas in α-keratins, calcium salt crystals form within cells (Pautard 1981
). From the AAS data, we can estimate the volume fraction of hydroxyapatite in sei baleen to be about 4.5 per cent, assuming a mass fraction for calcium of 0.399, and densities of hydroxyapatite and α-keratin of 3.156 and 1.3 g cm−3
, respectively. Using the rule of mixtures (Wainwright et al. 1976
), we can then calculate Young's modulus for the calcium salts in sei baleen:
is Young's modulus of the composite (1.19 GPa), and E1V1
refers to the product of the keratin modulus (0.637 GPa) and volume fraction (0.954), and E2V2
refers to the modulus of hydroxyapatite and its volume fraction (0.046). From this equation, we calculate a Young's modulus for the calcium salts in sei baleen of about 13 GPa. Published values for the modulus of hydroxyapatite are typically considerably higher (about 150 GPa for hydroxyapatite single crystals) (Saber-Samandari & Gross 2009
), which suggests that one of the assumptions of the above calculation does not hold. The rule of mixtures equation assumes perfect coupling (i.e. equal strain) between the two phases, which probably does not hold, especially at higher composite strains.
While we were unable to directly measure the effects of calcification on the flexural modulus (Eb), we were able to use the natural variability in calcification among the three baleen types to explore this further. We found the ratio of Eb in highly calcified bristles (sei) to less-calcified (minke) bristles was higher than the same ratio for the tensile modulus (Es) (2.3 versus 1.8). This suggests that calcium salts are laid down in a way that preferentially boosts Eb compared with Es. This result is consistent with the notion that in vivo, bristles are more likely to be loaded in bending than in tension as a result of hydrodynamic forces, collisions with prey and abrasion from the tongue. Adaptation to flexural loading of baleen bristles is also consistent with their tubular morphology, which has greater flexural rigidity than a solid cylinder of equal cross-sectional area.
We have demonstrated that the presence of calcium salts in sei and humpback baleen increases their resistance to bending, but there is a far more straightforward way to increase flexural rigidity, and that is to increase bristle diameter. Flexural rigidity is the product of Young's modulus and the second moment of area, which is proportional to the fourth power of the radius. Large increases in flexural rigidity can therefore be effected by small increases in diameter. This raises the question of why bristle flexural rigidity is boosted via calcification rather than a simple increase in diameter. One possibility is that there are limits on how thick bristles can get before the prey-capturing function of the baleen filter is compromised. This may be particularly relevant in sei whales, which is the only rorqual species that specializes in small mesoplankton like copepods (Pivorunas 1979
), and also possesses the most highly calcified baleen (Pautard 1963
). In sei whales, filter porosity must be low to capture such small prey (Pivorunas 1976
), and this requires fine baleen bristles. However, there is probably a limit to how fine the bristles can be before they are so floppy that they no longer form a competent filter mat between the baleen plates. In this way, calcification may have allowed for the evolution of finer bristles that were still functionally competent, which ultimately decreased filter porosity and allowed for the exploitation of smaller prey. Interestingly, bristles from right and bowhead whale baleen are known to be as fine as sei bristles (Nemoto 1959
), but are relatively uncalcified (Pautard 1963
; St Aubin et al. 1984
). This may be related to the fact that these whales employ a low-speed skim-feeding behaviour that generates far lower hydrodynamic forces than more energetic lunge feeding bouts in sei whales.
If the primary function of calcification in baleen bristles is to increase their resistance to bending, one might predict that the stiffening calcium salts would be preferentially laid down at the bristle periphery, where they would have the greatest effect on the flexural rigidity. However, greater amounts of calcium salts were found near the centre of the bristles in all three baleen types (figures and ). This may be related to the fact that the baleen bristles wear over time, and if all the calcium were at the periphery, there would be a dramatic change in the flexural rigidity at the point where all of the calcium salts were completely worn away. This might create an area that is likely to kink when the bristle is loaded in bending, and would also mean that distal parts of the bristle (where the calcified regions were worn away) would not enjoy any benefits of calcification. A nested ring pattern of calcification ensures calcification of the bristle along its entire length and may minimize drastic changes in flexural rigidity that could lead to kinking. Furthermore, higher calcification near the centre of a bristle may reflect a greater need for reinforcement of the finer distal portion. This notion is supported by the fact that the smallest baleen tubules we observed are typically highly calcified in a similar manner to the central portion of the largest tubules (figures and ).
Our PIXE and von Kossa data demonstrate for the first time, to our knowledge, that there are clear interspecies differences in baleen calcium salt distribution among the three rorqual species examined. The most striking difference we observed was that the intertubular horn of minke baleen is calcified, whereas in humpback and sei baleen, it is uncalcified. Minke whales specialize in larger prey items like fish and krill and therefore have little need for fine baleen bristles. It is perhaps not surprising then that minke tubular horn is relatively uncalcified, except for a few thin rings. The function of intertubular horn calcification is not clear, but we suspect that it may facilitate separation of the bristles from the intertubular horn by creating an abrupt change in material stiffness that leads to stress concentrations and cracks at the interface. In sei baleen, although the pattern is reversed, a similar mechanism could be at work, with the lack of intertubular calcification creating an abrupt interface that facilitates bristle separation.
We have clearly demonstrated that calcification of sei and humpback baleen increases the stiffness of these materials, which may be related to a lack of opportunities for air-drying. However, if one considers that decalcified and uncalcified (i.e. minke) baleen have a tensile modulus that is almost ×100 higher than fully hydrated pure intermediate filaments (Fudge et al. 2003
), this suggests that some other mechanism of stiffening is at work in baleen besides calcification. One possible mechanism is covalent cross-linking within the intermediate filaments, which could raise the modulus to something approaching that of coiled coils, or about 2 GPa (Howard 2001
). Future work should test the hypothesis that baleen is stiffened via these cross-links and investigate the nature of the keratin matrix in this unusual biomaterial.