Nanostructural self-assembly of iridescent feather barbules through depletion attraction of melanosomes during keratinization

Abstract

Avian plumage colours are model traits in understanding the evolution of sexually selected ornamental traits. Paradoxically, iridescent structural colours, probably the most dazzling of these traits, remain the most poorly understood. Though some data suggest that expression of bright iridescent plumage colours produced by highly ordered arrays of melanosomes and keratin is condition-dependent, almost nothing is known of their ontogeny and thus of any developmental mechanisms that may be susceptible to perturbation. Here, we use light and electron microscopy to compare the ontogeny of iridescent male and non-iridescent female feathers in blue-black grassquits. Feather barbules of males contain a single layer of melanosomes bounded by a thin layer of keratin-producing blue iridescent colour, while those of females contain disorganized melanosomes and no outer layer. We found that nanostructural organization of male barbules occurs late in development, following death of the barbule cell, and is thus unlikely to be under direct cellular control, contrary to previous suggestions. Rather, organization appears to be caused by entropically driven self-assembly through depletion attraction forces that pin melanosomes to the edge of barbule cells and to one another. These forces are probably stronger in developing barbules of males than of females because their melanosomes are (i) larger, (ii) more densely packed, and (iii) more homogeneously distributed owing to the more consistent shape of barbules during keratinization. These data provide the first proposed developmental pathway for iridescent plumage colours, and suggest that any condition dependence of iridescent barbules is likely driven by factors other than direct metabolic cost.

Figure 1.

Blue-black grassquit feathers early in development (0 mm, ramogenic collar). (a) Developing male barbules within the barb ridge (scale bar, 10 µm). Keratinocytes are undifferentiated and not organized in rows (barbule plates); melanocytes and melanocyte expansions (m) can be seen. (b) Female keratinocyte showing melanosomes (dark spots) and the large nucleus (n) (scale bar, 2 µm). (c) Female melanocyte with developing melanin granules; endoplasmatic reticulum (er) and mitochondria (mt) can be seen at the nucleus (n) edge (scale bar, 1 µm). (d) Female melanocyte expansion (m) with a fused membrane (arrow) to a barbule keratinocyte (k), for transfer of melanosomes (scale bar, 1 µm).

Figure 2.

Male (a–c) and (d–f) female blue-black grassquit feather barbules at intermediate stages of development. (a) Male barbules at 1 mm from the base of the developing feather. Melanosomes (dark spots) fill up most of the barbule volume that is not occupied by the nucleus. (b) Male barbule at 2 mm from the base of the developing feather. Barbule can be seen to elongate laterally, and some heterochromatin (arrow) can be seen at the nucleus edge. (c) Close-up: electron-dense keratin filaments can be seen being formed and phase-separating from the electron-pale cytoplasm. (d) Female barbule at 1 mm from the base of the developing feather, with similar pattern to male barbule, but with considerably fewer and smaller melanosomes. (e) Female barbule at 2 mm from the base of the developing feather, also with a heterochromatic nucleus (arrow). (f) Close-up: keratin filaments can also be seen, as well as large electron-pale vacuoles (exemplified by an asterisk), possibly of lipidic content. Scale bars (a,b,d,e), 2 µm; (c,f), 500 nm.

Figure 3.

Late-development (a–c) male and (d–f) female blue-black grassquit feather barbules. (a) Male barbule at 3 mm from the base of the developing feather. Large electron-dense bundles of keratin can be seen, and melanosomes (dark spots) are concentrated around the edges of the barbule. (b) Close-up: electron-pale areas can be seen in between melanosomes, as a result of their exclusion areas. Nucleus is small and heavily heterochromatic (arrows). (c) Male barbule at 4 mm from the base of the developing feather, mostly keratinized and with a small, electron-dense nucleus. Supportive cells (s) around barbules are also degenerated. (d) Female barbule at 3 mm from the base of the developing feather. As in males, large electron-dense bundles of keratin can be seen. However, the ‘arrowhead’ shape of the barbule and unorganized melanosomes contrast with the ovoid shape and organized melanosomes of the male. (e) Close-up of (d), illustrating as in males the electron lucent areas between melanosomes and the heterochromatin at the edge of the nucleus (arrows). (f) Female barbule at 4 mm from the base of the developing feather; fully keratinized, nucleus no longer visible. Scale bars (a,c,d,f), 2 µm; (b,e), 500 nm.

Figure 4.

Light microscopy of developing barb ridges of male (a,b) and female (c,d) blue-black grassquits during early (a,c; 1 mm from the base of the developing feather) and late (b,d; 3 mm from the base) development (B: barb; bp: barbule plates). During early development, there are contrasting patterns of melanization of barbules, with (a) male barbules being consistently heavily melanized, whereas (c) female barbules are more melanized in the outer barbule plate (bp; left) than the inner, and in both plates being less melanized than male barbules. During late development, the difference in shape of barbules is clearly visible, with (b) male barbules being elliptical in cross section, and (d) female barbules displaying an ‘arrowhead’ shape. Scale bars, 10 µm.

Figure 5.

Schematic of depletion attraction forces during keratinization of iridescent feather barbules. (a) keratin units (grey circles) cannot occupy the excluded volume (dashed lines) around melanosomes (black circles). When they are close to one another or to the barbule edge, these exclusion areas overlap (grey areas), resulting in a greater total volume that can be occupied by keratin, and therefore in less free energy in the system. Because difference in size between melanosomes and keratin units is very large, keratin can occupy gaps between melanosomes and the barbule edge. Inset: since keratin cannot occupy the area between melanocytes, the difference in concentration of the area between melanosomes and the rest of the system results in osmotic pressure that draws solute from that area and back into the system, resulting in an attractive force between particles. (b) As keratin polymerizes and cross-links during keratinization, the range and the strength of these forces increase, resulting in even greater free energy loss from the packing of melanosomes. Keratin units that were ‘trapped’ between melanosomes and the barbule cell membrane also increase in molecular weight, causing these regions to connect and form the keratin thin layer, which will produce iridescence. (c) The product of these interactions results in the formation of a thin film of keratin overlaying a layer of melanosomes when the feather is fully keratinized (as represented by the homogeneous keratin grey area), characteristic of iridescent feather barbules.