DOI: 10.5061/dryad.h70rxwddx
title: Eumelanin and pheomelanin pigmentation in mollusc shells may be less common than expected: insights from mass spectrometry

description: Background: The geometric patterns that adorn the shells of many
 phylogenetically disparate molluscan species are comprised of pigments
 that span the visible spectrum. Although early chemical studies implicated
 melanin as a commonly employed pigment, surprisingly little evidence
 generated with more recent and sensitive techniques exists to support
 these observations. Results: Here we present the first mass
 spectrometric investigations for the presence of eumelanin and pheomelanin
 in 13 different molluscan species from three conchiferan classes:
 Bivalvia, Cephalopoda and Gastropoda. In the bivalve Mytilus
 edulis we demonstrate that eumelanin mainly occurs in the
 outermost, non-mineralised and highly pigmented layer of the shell (often
 referred to as the periostracum). We also identified eumelanin in the
 shells of the cephalopod Nautilus pompilius and the
 marine gastropods Clanculus
 pharaonius and Steromphala adriatica. In the terrestrial
 gastropod Cepaea nemoralis we verify the presence of
 pheomelanin in a mollusc shell for the first time. Surprisingly, in a
 large number of brown/black coloured shells we did not find any evidence
 for either type of melanin. Conclusions: We recommend methods
 such as high-performance liquid chromatography with mass spectrometric
 detection for the analysis of complex biological samples to avoid
 potential false-positive identification of melanin. Our results imply that
 many molluscan species employ as yet unidentified pigments to pattern
 their shells. This has implications for our understanding of how molluscs
 evolved the ability to pigment and pattern their shells, and for the
 identification of the molecular mechanisms that regulate these processes.
description: <strong>Samples and standards</strong>
 Shells from 13 different mollusc species were obtained either
 commercially or by donation from the Natural History Museum Vienna or
 private collectors for ana- lysis (see Fig. 1 for images of samples used
 and Table 1 for previous literature and sample sources). For species
 previously reported to contain eumelanin in their shells (Crassostrea
 gigas, Mizuhopecten yessoensis, Clanculus pharaonius [21, 30, 33]) three
 replicates were analysed. For Mizuhopecten yessoensis the brown coloured
 left valve and for Cepaea nemoralis a morph with yellow background and
 multiple brown bands was analysed. For Mytilus edulis the periostracum was
 removed by scrub- bing the shell with sand for one shell valve, while the
 other valve remained intact. As Steromphala adriatica are very small,
 seven shells were combined into one sample. Samples contained 0.9 to 2.2 g
 of shell material each. For Lioconcha ornata 0.5g of shell material was
 available. Note that shells displaying multiple colours were not
 fragmented or sorted into colour groups. For the Crassostrea gigas sample
 material was taken from the internal shell surface in the region of the
 adductor scar. Care was taken to exclude pigmented material from the outer
 shell layers in this case. For comparison, stan- dards of the melanin
 oxidation products PDCA, PTCA, TDCA and TTCA kindly provided by Prof. Ito
 were used. <strong>Sample preparation, melanin
 oxidation and HPLC–UV–MS analysis</strong>
 Samples were processed as previously described [44]. In brief,
 shells were cleaned in deionized water, dried and weighted, and then
 dissolved in 6 M HCl. Residues were washed with water and were treated
 with proteinase K in 1 M Tris-HCl buffer at 37 °C for 2 h. Pigmented resi-
 dues were treated with alkaline oxidation via H2O2 [41]: Oxidation
 reactions for each sample were carried out for 20 h at 25 °C under
 vigorous shaking using 100 μL H2O, 375 μL 1 M K2CO3 and 25 μL 30% H2O2 as
 reactants. The remaining H2O2 was decomposed by the addition of 50μL 10%
 Na2SO3 and the mixture was acidified with 140μL 6M HCl. The solutions were
 then centrifuged and supernatants were transferred to fresh
 tubes. Samples were treated by solid-phase extraction
 (Phenom- enex Strata-X Polymeric Reversed Phase columns, 33 μm). Columns
 were conditioned with methanol (MeOH) followed by H2O. Shell extracts were
 loaded onto the col- umns and washed with 0.3% formic acid. Columns were
 dried and elution was carried out with MeOH followed by ethyl acetate.
 Solvents were removed under constant nitrogen stream at 40 °C and samples
 were dissolved in 200 μL H2O. Unless otherwise indicated samples were
 directly analysed following solid-phase extraction.
 Measurements were carried out on a Thermo Fisher Scientific
 HPLC–MS system consisting of an Accela HPLC with a Finnigan Surveyor PDA
 Detector coupled to an LTQ Orbitrap XL mass spectrometer equipped with an
 electrospray ionisation (ESI) source. Separation was performed on a
 Phenomenex Gemini C18 column (250 × 2 mm, 5 μm). The mobile phase was 0.3%
 formic acid in H2O:MeOH (80:20). Analyses were performed at 45 °C at a
 flow rate of 0.2 ml/min. UV data were recorded in the range of 200–400 nm.
 Mass spectra were acquired in negative-ion mode over an m/z range of
 120–220. Identification of melanin oxidation products were based on exact
 mass data and retention times. Quantitation was carried out by HPLC–UV in
 the range of 250–290 nm using external calibration with melanin oxidation
 product standards. Evaluation of HPLC–UV–MS data was performed using
 Thermo Xcalibur version 2.2.