Identification of Shell Colour Pigments in Marine Snails Clanculus pharaonius and C. margaritarius (Trochoidea; Gastropoda)

Abstract

Colour and pattern are key traits with important roles in camouflage, warning and attraction. Ideally, in order to begin to understand the evolution and ecology of colour in nature, it is important to identify and, where possible, fully characterise pigments using biochemical methods. The phylum Mollusca includes some of the most beautiful exemplars of biological pigmentation, with the vivid colours of sea shells particularly prized by collectors and scientists alike. Biochemical studies of molluscan shell colour were fairly common in the last century, but few of these studies have been confirmed using modern methods and very few shell pigments have been fully characterised. Here, we use modern chemical and multi-modal spectroscopic techniques to identify two porphyrin pigments and eumelanin in the shell of marine snails Clanculus pharaonius and C margaritarius. The same porphyrins were also identified in coloured foot tissue of both species. We use high performance liquid chromatography (HPLC) to show definitively that these porphyrins are uroporphyrin I and uroporphyrin III. Evidence from confocal microscopy analyses shows that the distribution of porphyrin pigments corresponds to the striking pink-red of C. pharaonius shells, as well as pink-red dots and lines on the early whorls of C. margaritarius and yellow-brown colour of later whorls. Additional HPLC results suggest that eumelanin is likely responsible for black spots. We refer to the two differently coloured porphyrin pigments as trochopuniceus (pink-red) and trochoxouthos (yellow-brown) in order to distinguish between them. Trochopuniceus and trochoxouthos were not found in the shell of a third species of the same superfamily, Calliostoma zizyphinum, despite its superficially similar colouration, suggesting that this species has different shell pigments. These findings have important implications for the study of colour and pattern in molluscs specifically, but in other taxa more generally, since this study shows that homology of visible colour cannot be assumed without identification of pigments.

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

“…Protoporphyrin (a cyclic tetrapyrrole) is an immediate precursor of the heme molecule, whereas biliverdin (a linear tetrapyrrole) is a product of hemoglobin breakdown. Protoporphyrin and biliverdin pigments can bind ions of trace metals, primarily iron (Fe) or zinc (Zn), but almost all other metals can be incorporated into their molecules (Ostfeld and Tsutsui 1974, Mikšík et al 1996, Casini et al 2003, Williams et al 2016. Protoporphyrin and biliverdin occur at varying levels in the shells of most birds' eggs, irrespective of their coloration (Mikšík et al 1996, Cassey et al 2011, Wang et al 2011, Brulez et al 2016, and their distribution varies between the speckled and plain areas of the shells .…”

“…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). We therefore hypothesize that an analogous relationship between the presence of protoporphyrin pigments and the content of macrominerals and microminerals (trace elements) should also apply to the maculation of birds' eggs, as suggested earlier by Mikšík et al (1996).…”

Colorful seashells: Identification of haem pathway genes associated with the synthesis of porphyrin shell color in marine snails

“… Specimen number, sampling locality, analyses undertaken in this study (red font) and in (Williams et al., ; black font). …”

“…Total RNA was extracted from mantle edge tissue from specimens of two C. margaritarius, one C. pharaonius and one Ca. zizyphinum, and from colored tissue from the side of the foot of one specimen of C. margaritarius , which is also colored by porphyrins (Williams et al., ; Table ). RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (Qiagen) according to the manufacturer's instructions.…”

“…Conversely, only traces of uroporphyrin were found in the shell of a third species, Calliostoma zizyphinum (Calliostomatidae:Trochoidea) , despite the fact that it is from the same superfamily and has superficially similar coloration, suggesting that shell color in this species is due to different shell pigments (Williams et al., ). The congruence of colors arising from different pigments suggests that there may be selective pressures leading to convergent evolution in these taxa (Williams et al., ). Apart from Clanculus , uroporphyrin pigments are also responsible for coloration of soft tissues and shells of other (mostly marine) mollusks (reviewed in Williams, ), the integument of some annelids (Fox, ), and turaco bird feathers (Nicholas & Rimington, ).…”