The common grove snail Cepaea nemoralis displays a stable pigmentation polymorphism in its shell that has held the attention of scientists for decades. While the details of the molecular mechanisms that generate and maintain this diversity remain elusive, it has long been employed as a model system to address questions related to ecology, population genetics and evolution. In order to contribute to the ongoing efforts to identify the genes that generate this polymorphism we have tested the long-standing assumption that melanin is the pigment that comprises the dark-brown bands. Surprisingly, using a newly established analytical chemical method, we find no evidence that eumelanin is differentially distributed within the shells of C. nemoralis. Furthermore, genes known to be responsible for melanin deposition in other metazoans are not differentially expressed within the shell-forming mantle tissue of C. nemoralis. These results have implications for the continuing search for the supergene that generates the various pigmentation morphotypes.

Cepaea nemoralis

Six living animals and approximately 100 empty shells of C. nemoralis were collected at University of Göttingen, Germany (51°33'24.0"N 9°57'27.3"E). Empty shells were cleaned and dried, then crushed. Shell pieces were sorted according to replicate group and colour fraction. Tissue samples were taken from fresh material by careful dissection of mantle and foot tissue.

Sample Preparation, Melanin Oxidation and LC-UV-MS Analyses

Two major morphs of C. nemoralis (pink banded and yellow banded) were investigated. Their corresponding colour-sorted shell fragments were as follows: pink background; brown band on pink background; yellow background; brown band on yellow background (see Figure 1A). Three technical replicates of each colour group were performed, with each replicate comprised of up to 8 shells.

Analysis of eumelanin and pheomelanin oxidation products was carried out as previously described ¹⁴ (see main text): In brief, shells were cleaned in deionized water and weighted. Cleaned shell pieces were dissolved in 6 M HCl and centrifuged at 13000 rpm for 15 min. Residues were washed with HPLC grade water. Samples were treated with proteinase K in 1 M Tris-HCl buffer at 37 °C for 2 h. Treatment was stopped by acidification with 6 M HCl, and samples were centrifuged and washed as described above.

Oxidation reactions and solid-phase extractions were performed as previously described ¹⁴. For each sample, oxidation was performed for 20 h at 25 °C under vigorous shaking using 100 μL H₂O, 375 μL 1 M K₂CO₃ and 25 μL 30% H₂O₂. Remaining H₂O₂ was decomposed by the addition of 50 μL 10% Na₂SO₃ and the mixture was acidified with 140 μL 6 M HCl. Solutions were then centrifuged at 13,000 rpm for 40 min and the supernatants transferred to fresh tubes. Samples were then treated by solid-phase extraction on Phenomenex Strata-X 33 μm polymeric reversed phase columns under vacuum. Columns were first conditioned with 5 mL methanol (MeOH) followed by 5 mL H₂O. Shell extracts were loaded onto the columns which were then washed with 5 mL 0.3% formic acid. The columns were then dried for 30 min and elution was carried out with 3 mL MeOH followed by 3 mL ethyl acetate. Solvents were removed under constant nitrogen stream at 40 °C and samples were dissolved in 200 μL H₂O.

Measurements were also performed as previously described ¹⁴. Briefly, a Thermo Fisher Scientific LC-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 was employed. Separation was performed on a Phenomenex Gemini C18 column (250 × 2 mm, 5 μm) at 45 °C using a flow rate of 0.2 mL/min. The mobile phase was 0.3% formic acid in H₂O:MeOH (80:20). UV data were recorded between 200 and 400 nm. Mass spectra were acquired in negative ion mode over an m/z range of 120–220.

Reverse transcription quantitative PCR (qPCR) of melanin pathway genes in C. nemoralis

Four genes known to be involved in melanin synthesis and dark pigmentation were chosen for qPCR testing: tyrosinase(Tyr), tyrosinase related protein (TyrRP), yellow-like gene (Yellow) and laccase 2 (Lacc2). These sequences were identified from within a C. nemoralis mantle tissue transcriptome data set using tBLASTx. Primers for qPCR were designed with Primer3 (Untergasser et al., 2012). Primer sequences and Genbank accession codes are listed in Table 2.

The experimental design for RT-qPCRs followed the protocol described in Affenzeller et al. ¹⁵: Six sub-adult individuals of C. nemoralis were collected at the University of Göttingen. Total RNA from pigmented mantle (producing the band in the shell), unpigmented mantle (producing background coloured shell) and foot tissue was extracted from each individual using Qiazol (Qiagen) according to the manufacturer’s instructions resulting in a total of twelve RNA extractions. These underwent a DNase treatment (RQ1 RNase-free DNase, Promega) according to the manufacturer’s instructions. Nanodrop and agarose gel electrophoresis were employed to verify quality and integrity of RNA. Synthesis of cDNA was carried out with 1 μg of total extracted RNA per sample using Promega M-MLV reverse transcriptase and oligo dTs. Reaction was run at 42 °C for 75 min, followed by 15 min at 70 °C to inactivate reverse transcriptase. The cDNA was stored at -20 °C until further use.

All qPCR runs followed a maximum sample layout, comply with the MIQE guidelines ²⁹ and included no template controls (NTC) for each primer pair and three inter run calibrators (IRC) EF1α, RNAP and UBI. Samples were run in triplicate, NTC and IRCs were run in duplicate. Amplification reactions contained 5 μL 2x Rotor-Gene SYBR Green PCR Master Mix, 0.4 μL cDNA, 1 μM final Primer concentration and 4.4 μL ddH₂O to a final volume of 10 μL. Reactions were run on a Rotor-Gene Q (Qiagen) using Rotor-Gene Q software (version 2.0.2) with the following temperature profile: 5 min initial activation and denaturation at 95 °C; 45 cycles of 5 sec denaturation at 95 °C, 10 sec annealing and extension at 60 °C (data collection at this step); a final melt curve analysis from 60 °C to 95 °C at a rate of 5 sec/1 °C.

Quantification and Statistical Analyses

LC-UV-MS of melanin oxidation products

Quantitation of melanin oxidation products was carried out by external calibration with standard mixtures (obtained from S. Ito). External calibration was set with 9-point calibration curves. Limit of quantitation (LOQ) was set at 10:1 signal-to-noise ratio. All manual peak integrations of chromatograms and analyses of mass spectra were done in Xcalibur 2.2 Qual Browser (Thermo Scientific, Waltham, USA). Quantitation was based on areas gained from peak integrations of UV chromatograms in a range of 250-290 nm. Each colour fraction was run for three replicates (comprised of up to eight shells each). Statistical analyses (mean and standard deviation calculations) were carried out in Microsoft Excel for Office 365 MSO (16.0.11629.20192).

qPCR

Raw fluorescence data were baseline and amplification efficiency corrected in LinRegPCR (Ruijter et al., 2009). Inter run correction was performed using Factor-qPCR ³⁰. So gained corrected cycle threshold (Cq) values were used to calculate the geometric means of technical replicates. Normalisation and relative expression were calculated based on the Pfaffl method ³¹ with beta-actin and elongation factor 1 alpha serving as reference genes as previously tested for mantle tissue in C. nemoralis ¹⁵.

Descriptive statistical analyses (mean and standard deviation calculations) of six biological replicates for each sample set (pigmented mantle, unpigmented mantle, foot) were carried out in Microsoft Excel for Office 365 MSO (16.0.11629.20192). Statistical comparisons between pigmented mantle and unpigmented mantle, as well as between all mantle samples and foot tissue, were run in PAST 3.15 as t-tests using Mann-Whitney as a significance measure (* p≤ 0.05, ** p≤ 0.01).

Data were generated with XCalibur, and one would need a license of that software in order to open them.