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.

Samples and standards

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.

Sample preparation, melanin oxidation and HPLC–UV–MS analysis

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.