Selective biodegradation of keratin matrix in feather rachis reveals classic bioengineering

Abstract

Flight necessitates that the feather rachis is extremely tough and light. Yet, the crucial filamentous hierarchy of the rachis is unknown-study hindered by the tight chemical bonding between the filaments and matrix. We used novel microbial biodegradation to delineate the fibres of the rachidial cortex in situ. It revealed the thickest keratin filaments known to date (factor >10), approximately 6 microm thick, extending predominantly axially but with a small outer circumferential component. Near-periodic thickened nodes of the fibres are staggered with those in adjacent fibres in two- and three-dimensional planes, creating a fibre-matrix texture with high attributes for crack stopping and resistance to transverse cutting. Close association of the fibre layer with the underlying 'spongy' medulloid pith indicates the potential for higher buckling loads and greater elastic recoil. Strikingly, the fibres are similar in dimensions and form to the free filaments of the feather vane and plumulaceous and embryonic down, the syncitial barbules, but, identified for the first time in 140+ years of study in a new location-as a major structural component of the rachis. Early in feather evolution, syncitial barbules were consolidated in a robust central rachis, definitively characterizing the avian lineage of keratin.

Figure 1.

SEM of fibres (syncitial barbules) in the cortex of feather rachis of Gallus gallus exposed after fungal biodegradation of matrix (resin embedded and etched). All fibres show regularly spaced syncitial nodes that extend in the proximo-distal direction of the rachis (vertical arrow on the right). The syncitial nodes show variations in morphology, terminating in hooks (arrow; details in figure 2a) or a ring (arrowhead), while others are intermediate between the two. Fibres are densely packed through the cortex (curved arrow) and indicate that the nodes are staggered in arrangement on two- and three-dimensional planes. Inset, detail. Also see the electronic supplementary material, figures S1 and S2. Scale bar, 10 µm.

Figure 2.

SEM of feather rachidial (cortex) fibres (syncitial barbules). (a,b) Biodegraded fibres of Gallus gallus (resin embedded and etched). (a) A detail showing fibres, syncitial nodes and macrofibrils; arrows and arrowheads show hooked and ringed terminations of nodes, respectively. (b) Group of fibres seen in cross section; the thicker cross sections indicate proximity to the syncitial nodes; arrows show macrofibrils. (c)–(e) Non-biodegraded rachidial fibres. (c) Gallus gallus, showing ringed nodes (arrow). (d) Fibres of Falco tinnunculus showing two morphologies of syncitial nodes, hook (arrows) and ring (arrowhead), and component macrofibrils. (e) Fibres of Falco peregrinus. The fibre surface is partially stripped, showing the component macrofibrils (diameter approx. 400–500 nm); part of the syncitial node remains (double-headed arrow). (f) Fibres of taxidermy specimen of Falco biarmicus (Durban Museum, dated 1966, accession no. 479) with matrix degraded and somewhat superficially degraded but intact fibres. (a,c,d) Scale bar, 5 µm; (b) 1 µm; (e,f) 2 µm.

Figure 3.

SEM of biodegraded feather rachis of Gallus gallus (resin embedded and etched). Circumferential fibres (syncitial barbules), identical to the longitudinal fibres (see below in the figure), wound round the outer ‘layers’ of the rachidial cortex. The matrix is partially degraded and shows the honeycomb-like structure in which the fibres are embedded in life (small arrows). Detail shows fibres, comparable to steel rebars in concrete (see text). Arrow shows long axis of rachis. Scale bar, 10 µm.

Figure 4.

SEM of fibres (syncitial barbules). (a) Gallus gallus. Rachidial (cortex) fibres and fungal hyphae and spores (detail in the electronic supplementary material, figure S1). (b) Free syncitial barbules (similar to rachidial cortex fibres) from the downy part of a pennaceous feather of Falco peregrinus. Arrows show both ringed and hooked terminations of the syncitial nodes. Inset shows the megafibrils of the fibres (cf. figure 4a and electronic supplementary material, figure S2). (a) Scale bar, 2 µm and (b) 10 µm.

Figure 5.

A schematic view of the three major structural components of the feather rachis. (a) (i) superficial layers of *fibres, the ultimate size-class in the hierarchy of feather keratin filaments (approx. 6 µm diameter), wound circumferentially round the rachis. (ii) The majority of the fibres extending parallel to the rachidial axis and through the depth of the cortex. Part of the section is peeled back to show why the fibres and even megafibrils are not usually recognized in histological sectioning, but rather only fibrils lower down the hierarchy (based on the electronic supplementary material, figure S2c). Any longitudinal section along the line of the arrows or at any point along the height of the fibre other than at the fibre surface (arrowheads) will fail to show the fibre. (iii) It shows the medulloid pith comprising gas-filled polyhedral structures (based on SEM images, electronic supplementary material, figures S5 and S6). Inset, part of a steel rebar with nodes, used in engineering technology to reinforce high-rise structures, analogous to rachidial fibres. (b) Schematic cross section of fibres and biodegraded ‘matrix’: (i) fibres; (ii) residual cytosol of keratinocytes presumably housing effete organelles and perhaps cytoskeletal elements—all degraded along with corneous envelope; (iii) interdigitating plasma membrane of the original keratinocytes with associated corneous envelope proteins. (c) A schematic three-dimensional cross section of the rachis showing approximate thickness (based on SEMs) of the three keratin layers comprising, (i) circumferential and (ii) longitudinal fibres of the cortex and (iii) polyhedra of medulloid pith. Asterisk denotes homologous with syncitial barbules.