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

Williams ST, Ito S, Wakamatsu K, Goral T, Edwards NP, Wogelius RA, Henkel T, de Oliveira LF, Maia LF, Strekopytov S, Jeffries T, Speiser DI, Marsden JT - PLoS ONE (2016)

Bottom Line: Additional HPLC results suggest that eumelanin is likely responsible for black spots.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.

View Article: PubMed Central - PubMed

Affiliation: Natural History Museum, Department of Life Sciences, London, United Kingdom.

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.

No MeSH data available.


Related in: MedlinePlus

Confocal images showing uroporphyrin distribution in areas of pink-red and yellow-brown pigmentation on Clanculus margaritarius shell.Shell images on each line are of a single shell and are in approximately the same orientation and magnification. (A-C) Top of shell, Clanculus margaritarius A (specimen #2). (A) Visible pink-red pigmentation. (B) Shaded volume 3D projection of a spectrally mixed image of the same shell showing fluorescent red areas congruent with visible pink-red pigment. (C) Maximum intensity projection of a spectrally unmixed image showing distribution of uroporphyrin I and III. (D-F) Clanculus margaritarius B (specimen #10). (D) Visible pink-red pigmentation. (E) Maximum intensity projection of a spectrally mixed image showing fluorescent red areas under UV light congruent with pink-red pigment. (F) Maximum intensity projection of a spectrally unmixed image showing distribution of uroporphyrin I and III. (G-L) Two different areas with yellow-brown, black and white colouration on shell of C. margaritarius A (specimen #2). (M-O) Yellow brown areas on shell of Clanculus margaritarius B (specimen #10). (G, J, M) Visible pigmentation for approximately the same areas as used in analyses. (H, K, N) Maximum intensity projection of spectrally mixed images showing red fluorescent areas under UV light congruent with yellow-brown pigmentation. Panel N is shown in 3D. (I, L, O) Maximum intensity projection of spectrally unmixed images showing distribution of uroporphyrin I and III. Panel O is shown in 3D. Note that in unmixed images green corresponds to uroporphyrin I and red to uroporphyrin III and yellow to areas where both co-occur. Note also that fluorescence is quenched in black spots and nearby white areas. Visible pigmentation is marked on each image: pr–pink-red, bl–black spot, w–nearby white area, yb–yellow-brown. Panels (G-I) include a small strip of blu-tack used to locate coloured areas.
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pone.0156664.g003: Confocal images showing uroporphyrin distribution in areas of pink-red and yellow-brown pigmentation on Clanculus margaritarius shell.Shell images on each line are of a single shell and are in approximately the same orientation and magnification. (A-C) Top of shell, Clanculus margaritarius A (specimen #2). (A) Visible pink-red pigmentation. (B) Shaded volume 3D projection of a spectrally mixed image of the same shell showing fluorescent red areas congruent with visible pink-red pigment. (C) Maximum intensity projection of a spectrally unmixed image showing distribution of uroporphyrin I and III. (D-F) Clanculus margaritarius B (specimen #10). (D) Visible pink-red pigmentation. (E) Maximum intensity projection of a spectrally mixed image showing fluorescent red areas under UV light congruent with pink-red pigment. (F) Maximum intensity projection of a spectrally unmixed image showing distribution of uroporphyrin I and III. (G-L) Two different areas with yellow-brown, black and white colouration on shell of C. margaritarius A (specimen #2). (M-O) Yellow brown areas on shell of Clanculus margaritarius B (specimen #10). (G, J, M) Visible pigmentation for approximately the same areas as used in analyses. (H, K, N) Maximum intensity projection of spectrally mixed images showing red fluorescent areas under UV light congruent with yellow-brown pigmentation. Panel N is shown in 3D. (I, L, O) Maximum intensity projection of spectrally unmixed images showing distribution of uroporphyrin I and III. Panel O is shown in 3D. Note that in unmixed images green corresponds to uroporphyrin I and red to uroporphyrin III and yellow to areas where both co-occur. Note also that fluorescence is quenched in black spots and nearby white areas. Visible pigmentation is marked on each image: pr–pink-red, bl–black spot, w–nearby white area, yb–yellow-brown. Panels (G-I) include a small strip of blu-tack used to locate coloured areas.

Mentions: Red fluorescence under UV light suggested the presence of porphyrins in the shell of C. margaritarius (Figs 2 and 3) and C. pharaonius (Fig 2, Figure B in S1 File), which was confirmed by HPLC analyses. Two large peaks in the HPLC traces corresponding to uroporphyrin I and uroporphyrin III were observed, although the peak area ratio differed between the two species (Fig 4). Retention times of the porphyrins from the shell extracts showed very slight drift during HPLC analysis when compared with standards. A recognised explanation for this is that the analysis was run overnight and the ambient temperature of the laboratory fluctuates during the evening. Temperature fluctuation can affect evaporation of the organic phase affecting the retention times of the porphyrins eluting from the column. The use of internal quality controls run on all assays at the beginning and the end of each run ensures that peaks were identified correctly, even in the presence of slight drift.


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

Williams ST, Ito S, Wakamatsu K, Goral T, Edwards NP, Wogelius RA, Henkel T, de Oliveira LF, Maia LF, Strekopytov S, Jeffries T, Speiser DI, Marsden JT - PLoS ONE (2016)

Confocal images showing uroporphyrin distribution in areas of pink-red and yellow-brown pigmentation on Clanculus margaritarius shell.Shell images on each line are of a single shell and are in approximately the same orientation and magnification. (A-C) Top of shell, Clanculus margaritarius A (specimen #2). (A) Visible pink-red pigmentation. (B) Shaded volume 3D projection of a spectrally mixed image of the same shell showing fluorescent red areas congruent with visible pink-red pigment. (C) Maximum intensity projection of a spectrally unmixed image showing distribution of uroporphyrin I and III. (D-F) Clanculus margaritarius B (specimen #10). (D) Visible pink-red pigmentation. (E) Maximum intensity projection of a spectrally mixed image showing fluorescent red areas under UV light congruent with pink-red pigment. (F) Maximum intensity projection of a spectrally unmixed image showing distribution of uroporphyrin I and III. (G-L) Two different areas with yellow-brown, black and white colouration on shell of C. margaritarius A (specimen #2). (M-O) Yellow brown areas on shell of Clanculus margaritarius B (specimen #10). (G, J, M) Visible pigmentation for approximately the same areas as used in analyses. (H, K, N) Maximum intensity projection of spectrally mixed images showing red fluorescent areas under UV light congruent with yellow-brown pigmentation. Panel N is shown in 3D. (I, L, O) Maximum intensity projection of spectrally unmixed images showing distribution of uroporphyrin I and III. Panel O is shown in 3D. Note that in unmixed images green corresponds to uroporphyrin I and red to uroporphyrin III and yellow to areas where both co-occur. Note also that fluorescence is quenched in black spots and nearby white areas. Visible pigmentation is marked on each image: pr–pink-red, bl–black spot, w–nearby white area, yb–yellow-brown. Panels (G-I) include a small strip of blu-tack used to locate coloured areas.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4930200&req=5

pone.0156664.g003: Confocal images showing uroporphyrin distribution in areas of pink-red and yellow-brown pigmentation on Clanculus margaritarius shell.Shell images on each line are of a single shell and are in approximately the same orientation and magnification. (A-C) Top of shell, Clanculus margaritarius A (specimen #2). (A) Visible pink-red pigmentation. (B) Shaded volume 3D projection of a spectrally mixed image of the same shell showing fluorescent red areas congruent with visible pink-red pigment. (C) Maximum intensity projection of a spectrally unmixed image showing distribution of uroporphyrin I and III. (D-F) Clanculus margaritarius B (specimen #10). (D) Visible pink-red pigmentation. (E) Maximum intensity projection of a spectrally mixed image showing fluorescent red areas under UV light congruent with pink-red pigment. (F) Maximum intensity projection of a spectrally unmixed image showing distribution of uroporphyrin I and III. (G-L) Two different areas with yellow-brown, black and white colouration on shell of C. margaritarius A (specimen #2). (M-O) Yellow brown areas on shell of Clanculus margaritarius B (specimen #10). (G, J, M) Visible pigmentation for approximately the same areas as used in analyses. (H, K, N) Maximum intensity projection of spectrally mixed images showing red fluorescent areas under UV light congruent with yellow-brown pigmentation. Panel N is shown in 3D. (I, L, O) Maximum intensity projection of spectrally unmixed images showing distribution of uroporphyrin I and III. Panel O is shown in 3D. Note that in unmixed images green corresponds to uroporphyrin I and red to uroporphyrin III and yellow to areas where both co-occur. Note also that fluorescence is quenched in black spots and nearby white areas. Visible pigmentation is marked on each image: pr–pink-red, bl–black spot, w–nearby white area, yb–yellow-brown. Panels (G-I) include a small strip of blu-tack used to locate coloured areas.
Mentions: Red fluorescence under UV light suggested the presence of porphyrins in the shell of C. margaritarius (Figs 2 and 3) and C. pharaonius (Fig 2, Figure B in S1 File), which was confirmed by HPLC analyses. Two large peaks in the HPLC traces corresponding to uroporphyrin I and uroporphyrin III were observed, although the peak area ratio differed between the two species (Fig 4). Retention times of the porphyrins from the shell extracts showed very slight drift during HPLC analysis when compared with standards. A recognised explanation for this is that the analysis was run overnight and the ambient temperature of the laboratory fluctuates during the evening. Temperature fluctuation can affect evaporation of the organic phase affecting the retention times of the porphyrins eluting from the column. The use of internal quality controls run on all assays at the beginning and the end of each run ensures that peaks were identified correctly, even in the presence of slight drift.

Bottom Line: Additional HPLC results suggest that eumelanin is likely responsible for black spots.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.

View Article: PubMed Central - PubMed

Affiliation: Natural History Museum, Department of Life Sciences, London, United Kingdom.

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.

No MeSH data available.


Related in: MedlinePlus