Nature’s Palette: Characterization of Shared Pigments in Colorful Avian and Mollusk Shells

Abstract

Pigment-based coloration is a common trait found in a variety of organisms across the tree of life. For example, calcareous avian eggs are natural structures that vary greatly in color, yet just a handful of tetrapyrrole pigment compounds are responsible for generating this myriad of colors. To fully understand the diversity and constraints shaping nature’s palette, it is imperative to characterize the similarities and differences in the types of compounds involved in color production across diverse lineages. Pigment composition was investigated in eggshells of eleven paleognath bird taxa, covering several extinct and extant lineages, and shells of four extant species of mollusks. Birds and mollusks are two distantly related, calcareous shell-building groups, thus characterization of pigments in their calcareous structures would provide insights to whether similar compounds are found in different phyla (Chordata and Mollusca). An ethylenediaminetetraacetic acid (EDTA) extraction protocol was used to analyze the presence and concentration of biliverdin and protoporphyrin, two known and ubiquitous tetrapyrrole avian eggshell pigments, in all avian and molluscan samples. Biliverdin was solely detected in birds, including the colorful eggshells of four tinamou species. In contrast, protoporphyrin was detected in both the eggshells of several avian species and in the shells of all mollusks. These findings support previous hypotheses about the ubiquitous deposition of tetrapyrroles in the eggshells of various bird lineages and provide evidence for its presence also across distantly related animal taxa.

Speckled and plain regions of avian eggshells differ in maternal deposition of calcium and metals: A hitherto overlooked chemical aspect of egg maculation

“…It is therefore assumed that reservoirs of melanin pigments serve as a sink for potentially harmful transition metal ions, adsorbing and harboring the very minerals that helped produce them and thereby offering chemoprotection to adjacent cells and tissues (Larsson 1993, McGraw 2003. A recent investigation into the composition of pigments present in the shells of molluscs and birds' eggs revealed that protoporphyrin pigments occurred in both these evolutionarily distant groups of animals but that biliverdin was present only in birds' eggshells (Verdes et al 2015). Another recent investigation of molluscs showed that the more intensive dark pigmentation due to the presence of protoporphyrin increased the content of some trace elements (measured only by scanning electron microscope) like Cu (Williams et al 2016).…”

Eggshells as hosts of bacterial communities: An experimental test of the antimicrobial egg coloration hypothesis

“…This effect has been shown in a careful set of lab experiments (Ishikawa et al., 2010), using cultures of four commercially obtained bacterial strains. The experiments tested for antibacterial properties of eggshells from domestic chickens ( Gallus gallus domesticus ), comparing eggs that were solid brown, solid blue–green, and white, which contain, respectively, primarily protoporphyrin, biliverdin, and no pigments at all (Verdes et al., 2015). Exposure of bacteria to brown eggshells reduced the survival of Gram‐positive species ( Staphylococcus aureus , Bacillus cereus ) by more than two orders of magnitude, but no effect was seen on Gram‐negative bacteria ( Escherichia coli , Salmonella enteritidis ).…”

Colour in bivalve shells: Using resonance Raman spectroscopy to compare pigments at different phylogenetic levels

“…In previous Raman studies of molluscan shells, black and brown pigments have been attributed to polyenes, but other studies using high‐performance liquid chromatography, mass spectrometry, or spectral analysis have shown that these shell colours can also be due to melanins or tetrapyrroles . The brown tetrapyrrole protoporphyrin IX has been found in the pteriomorph Argopecten sp ., and Raman studies showed that it exhibits Raman peaks attributable to C═C bonds at 1,619, 1,585, and 1,339 cm −1 ; C–C deformation at 1,255 cm −1 ; and C–C and C–H 3 rocking at 970 cm −1 . Raman spectroscopy of black eumelanin isolated from Sepia cuttlefish ink found two intense, broad peaks at 1,380 and 1,580 cm −1 .…”

“…There have been a growing number of studies in the last decade investigating the pigments responsible for molluscan shell colour and the genes and pathways responsible for their production . Techniques used to distinguish pigments in shells have included high‐performance liquid chromatography, mass spectrometry, and UV‐visible spectrophotometry, but difficulties in some studies have arisen from the low concentrations of pigments . Raman spectroscopy overcomes this problem and is particularly effective for detection of polyene pigments, as well as being nondestructive and noninvasive .…”