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Modifications of great scallop (Pecten maximus) shells have been noticed in many sites of scallop fisheries in Brittany, especially in shallow waters.
Aquaculture International 4, 2 3 7 - 2 5 2 (1996)

Abnormal melanization and microstructural distortions of the shell of great scallop living in shallow water H. L a r v o r 1'*, J.P. C u i f 2 a n d N. D e v a u c h e l l e 1 IlFREMER, DRV/A, BP 70, 29280 Plouzane, France 2Universit# de Paris XI, Laboratoire de Pal#ontologie, BAT 504, F-91405 Orsay C#dex, France

Modifications of great scallop (Pecten maximus) shells have been noticed in many sites of scallop fisheries in Brittany, especially in shallow waters. These calcification abnormalities are linked to the a p p e a r a n c e of a brown colouration of the internal calcified shell layer, due to the p r e s e n c e of a eumelanin a s s o c i a t e d with the insoluble organic matrix of the biocrystals. The a p p e a r a n c e of this pigment generates m a n y d i s t u r b a n c e s of the calcified foliated m i c r o s t r u c t u r e of the scallop internal shell layer. The mantle structure is not modified in brown shells as c o m p a r e d with white ones. No pathogenic signs such as h y p e r p l a s i a or h a e m o c y t i c infiltration have been o b s e r v e d . According to this observation, we h y p o t h e s i z e that the brown colour p h e n o m e n o n is more a result of environmental d i s t u r b a n c e r a t h e r than a s y m p t o m of a pathogenic disease. The colour abnormalities of the internal shell layer can be d e t e c t e d b y a spectral analysis of its reflectance before it can be d e t e c t e d with the naked eye. This method, correlated to m i c r o s t r u c t u r a l o b s e r v a t i o n s , gives a rapid and precise analysis of the a p p e a r a n c e of the pigmentation on adult or juvenile scallops. It m a y be a useful m e t h o d for the evaluation of the influence of environmental p a r a m e t e r s , for example, on calcification and its abnormalities. KEYWORDS: Calcification, Environmental disturbances, Eumelanin, Great scallop (Pecten

maximus), Shell colour, m i c r o s t r u c t u r e and melanization

INTRODUCTION A l t e r a t i o n s in t h e e x o s k e l e t o n of m a r i n e i n v e r t e b r a t e s a r e o f t e n a s i g n of n o t i c e a b l e c h a n g e s in t h e i r l i v i n g e n v i r o n m e n t . P o l l u t a n t s a n d p a t h o g e n s c a n d i s t u r b t h e n o r m a l b i o m i n e r a l i z a t i o n p r o c e s s a n d t h e n l e a d t o g r o s s d i s t o r t i o n s of t h e s h e l l s t r u c t u r e . A l z i e u et al. (1982) h a v e e s t a b l i s h e d a c o r r e l a t i o n b e t w e e n t h e u s e of TBT ( t r i b u t y l t i n ) a n t i f o u l i n g p a i n t s a n d o y s t e r (Crassostrea gigas) c h a m b e r i n g . T h e TBT a c e t a t e a l s o a f f e c t s d e v e l o p m e n t of C. gigas l a r v a l s h e l l s (His a n d R o b e r t , 1980). A t r a z i n - s i m a z i n e , a h e r b i c i d e w i d e l y u s e d in c o r n c u l t u r e , p r o d u c e s t h e s a m e l a r v a l s h e l l a b n o r m a l i t i e s ( R o b e r t et al., 1986). M a c h a d o et al. (1990) d e m o n s t r a t e d t h a t *Author to whom correspondence should be addressed. 0967-6120 © 1996 Chapman & Hall

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diflubenzuron, an inhibitor of chitin synthesis in insect cuticule, generates microstructural distortions of the nacreous layer of Anodonta cygnea shells. Bacterial diseases (Dungan and Elston, 1988; Goulletquer et al., 1989; Getchell, 1991; Paillard 1992), parasitic infestations ~ l a k e and Evans, 1973; Bartoli, 1976; Bower et al., 1992; Sato-Okoshi and Okoshi, 1992) or ecological disturbances of as yet unknown origin could induce the formation of organic sheets in place of normal shell material (Mori, 1975; Palmer, 1980; Isaji, 1993; Ford and Paillard, 1994; Farley and Lewis, 1994), modifications of their microstructure (Epifanio, 1976; Marin and Dauphin, 1992; SatoOkoshi and Okoshi, 1993; Dunca et al., in press) and/or of their colour (Johannessen, 1973; Palmer, 1980; Prezant and Chalermwat, pers. comm.; Pass et al., 1987; Medakovic et al., pers. comm.). These modifications, which often lead to mortality, represent a serious threat to shellfish farming, affecting the production quality and commercial viability. Such an alteration has been noted in great scallop (Pecten m a x i m u s ) shells in certain fishery grounds and experimental rearing sites in Brittany, France, following the sowing of scallops (Dao, pets. comm.). A deep brown colouration is observed on the inner surface of the scallop valves, but, contrary to the observations of Baird and Gibson (1956) and Minchin (1991), no change is evident on the outer surface of the shell. This phenomenon, which was until recently observed only in old scallops, is now known to be widespread in younger animals (2 or 3 years old) in shallow waters and in ecologically unstable places, which might be subjected to pollution and reduced salinity, and is often associated with serious winter mortalities. The brown colour, which can adversely affect consumer approval, does not affect the commercial value of the shellfish (muscle and gonad quality) unless it is very evident (Larvor et al., 1995). However, at the ultimate stage of development, when the brown colour covers all the shell, the animal presents many signs of weakening; the adductor muscle is weakened and poorly attached to the shell, and brown-shelled scallops have a higher water content than normal white-shelled scallops (Larvor et al., 1995). The presence of opportunistic boring parasites such as algae, sponges, worms or fungi is sometimes noted (Devauchelle et al., 1994) in very brown shells, but the parasites do not seem to have carried browning of the shell; they rather seem to take advantage of the poor shell condition. Such shell defects and pigmentation have been noted in Ireland and Scotland, in wild and ranched great scallops (Paice, 1974). A study was carried out to investigate whether this brown colour of shells is a response to a disturbance of the scallops and to examine whether these calcification abnormalities could be considered as a result of physiological stress or as an expression of the quality of the immediate environment. The shell composition was investigated to characterize the calcification problems associated with the development of the brown colour in the internal layer of the valves. This analysis of the biomineral composition did not reveal any strong modification of the calcite of brown shells compared with white ones (Larvor et al., in press), no special accumulation of organic material was detected in brown shells and the amino-acid composition of the biomineral organic matrices was not modified, but the crystal structure was disorganized. The decalcification technique showed that the brown colour was generated by an insoluble compound associated with the insoluble organic envelopes of the crystals.

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This paper presents the characterization of the brown compound. The features of the pigment were established using histochemical studies. Its spectral characteristics were analysed with reflectance and spectroscopic techniques in the visible and the infrared ranges. A histological study of the mantle and the shell structure and microstructure was also undertaken to check whether the appearance of the brown colour could be related to modification of the shell-secreting epithelia similar to that induced in the brown ring disease of Manila clams (Tapes philippinarum) (Paillard, 1992), the juvenile oyster disease of Crassostrea virginica (Ford and Paillard, 1994), or disturbances in the microstructure of the shell as observed in diseased pearl oysters (Pinctada margaritfera) from French Polynesia (Marin and Dauphin, 1992).

MATERIAL AND METHODS Great scallops were sampled in winter 1993 and 1994 in the Bay of Brest (France) and in the English Channel (Bay of Seine). Scallops from the English Channel were dredged near the Greenwich buoy, at about 40 m, on a sandy bottom. Scallops from the Bay of Brest were dredged at different depths ranging from 5 to 35 m, and substrata ranging from gravel-sandy bottoms, to sandy and light muddy-sandy bottoms. Different age classes from 1 to 5 years old were selected, depending on the proposed studies.

Microstructural analyses Twenty valves with white inner shell surface fished from the Bay of Seine and from the Bay of Brest, 10 valves with very brown inner shell surface, and 20 valves showing just light brown spots on the inner shell surface provided from different sites in the Bay of Brest were selected for microstructural observations. Shells of 4-or-5-year-old scallops were used. Small pieces approximately 25 × 10 mm were cut from the pallial area of the valves, between the pallial line and the shell margin, with a high-speed hand drill fitted with a cutting disc. They were cleaned with deionized water using an ultrasonic technique, and then dried, mounted on stubs and coated with gold to observe the internal shell layer of the valves with a scanning electron microscope.

Histological observations of the mantle and histochemical study of the shell matrix Thirty white and 30 deep-brown scallops were observed. Scallops were anaesthetized in 1% MgC1 (seawater solution) for 24 h, to prevent contraction of the adductor muscle during the manipulation and strong retraction of the mantle during the fixation processes. The adductor muscle was then cut carefully prior to preservation of the mantle epithelia. Scallops with their whole valves were fixed in Bouin's solution (Gabe, 1968). This solution was changed periodically (every 2 days), using freshly added acetic acid until decalcification of the valves was complete. This took about t5 days. Small pieces of the marginal shell border and the adjacent mantle were preserved together, dehydrated with graduated ethanol,

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cleared in toluene and embedded in paraffin. Sections 4 ~m thick were cut and mounted on slides, using a Eukitt medium. Different staining techniques were selected: Masson trichrome and Giemsa staining for topographic observations, Alcian blue used to detect acidic secretions and PAS (periodic acid-Schiff) to detect glycoproteins. The shell pigment was characterized using different histochemical techniques: hydrogen peroxide discolouration (H~O2 10%, 24 h), the Lillie reaction (Lillie, 1957) specific for melanin pigments, hydrogen peroxide treatment coupled to the Lillie reaction (Paillard, 1992), the Hueck reaction and the Sudan black reaction (Ganter and Joll~s, 1969) specific to lipoid pigments. The solubility of the pigment was studied after acetic acid extraction of the insoluble organic matrix (IOM) of the shell ~ e v a u c h e l l e et al, 1994; Larvor et al., in press). Ten brown IOMs were compared with 10 white IOMs. Insolubility in acid solutions, acetic acid 6M (24 h) and boiling HC! 6M (24 h), were demonstrated elsewhere (Larvor et al., in press). The resistance to NaOH treatments (0.1M, 0.5M and 1M, for 24 h) and the solubility in organic solvents (acetone, methanol, ethanol, petroleum spirit, xylene, toluene) were investigated.

Studies of the spectral characteristics The spectral characteristics of the pigment were studied both on small untreated pieces of shells and on the insoluble acid-extracted organic matrices which contained the brown pigment. Studies in the visible range (400-800 nm) were performed by a reflectance technique. Different degrees of brown and white valves were compared. The valves of 4-or 5-year-old scallops showing different development of brown colour were selected to establish a reflectance scale corresponding to the colour observed with the naked eye. About 100 white valves from 2-5-year-old scallops were examined for their reflectance characteristics to try to detect the appearance of the brown colour sooner than with the naked eye. Small pieces of shell approximately 25 × 10 mm were cut from the shell edge using the same technique as for microstructural study, cleaned and dried. Pieces of shell were observed under a photonic microscope coupled to a spectral analyser ('Spectro 100', optical spectrum analyser, Instrument systems, Optische Messtechnik). The percentage reflectance of each shell piece was established by comparison with a pure white standard (magnesium oxide) at 2 nm intervals of wavelength from 400 to 750 nm. A spectroscopic study was also performed on the insoluble organic matrix of brown and white shells, dissolved in NaOH 1M (24 h). Absorbency was measured over the range 200-700 nm. NaOH 1M was used as a control. For the infrared (IR) study, mixed insoluble organic matrix (about 1%) and KBr pellets were crushed together to obtain a homogeneous powder. A small amount of this dry powder was pressed in a small dish and analysed for its IR absorbency characteristics. Ten white and 10 brown insoluble organic matrices were analysed. The correlation between the different spectra were established, relative to the position of the absorbency bands and their intensity.

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RESULTS The brown colour begins in the hinge region and can extend to the ventral shell margin and to the pallial line (Fig. 1). The inner surface of the valve progressively turns entirely brown, but the external surface remains unaffected. Microstructural observations White valves from the Bay of Brest and from the Bay of Seine or light brown valves with white areas from the Bay of Brest present a normal foliated microstructure, a characteristic of pectinids (Fig. 2 A-B). Changes from the normal foliated microstructure can be observed on very brown valves and in the brown patches of light brown shells; the growing tips of the crystals become eroded and etched (Fig. 2C) and the lateral margins can be distorted (Figure 2D). Abnormal quadrangular mineral deposits may also be observed at the growing edge of the crystals (Fig. 2D) and the foliated microstructure can become completely disorganized (Fig. 2E-F). No consistent relationship between the intensity of the brown colour and the intensity of degradation of the foliated microstructure was observed. Histological observations of the mantle The mantle of brown shells shows no morphological modification compared with the mantle of white shells. The external shell-secreting and marginal region of the epithelium consists of a monolayer of elongated cells (about 25 i~m x 8 ~m). These cells become more cuboidal (about 13 i~m × 10 I~m) with progression towards the attachments of the pallial muscles. They have an apical tip composed of microvilli,

FIG. 1. Upper valves of scallops showing the development of the brown colour in their inner shell layer. Scale bar: 5 cm.

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a basal or central nucleus which occupies most of the cell volume, and a clearstaining cytoplasm with granules. Mucous cells are common throughout this epithelium. Many collagen fibres and fascicules of muscle fibres cross the connective tissue. Few haemocytes are observed. In brown and white shells, the periostracal lamina consists of a thin (1-4 v~m), wrinkled, uncoloured monolayer, emerging between the middle and the outer fold of the mantle (Clark, 1974). It is secreted by the external epithelium of the middle fold and the internal epithelium of the outer fold. The cells that secrete this lamina appear the same in both colour forms. They have cuboidal cells with microvilli, a basal nucleus and a clear cytoplasm in the outer fold, and tall elongated cells with a basal nucleus and very long microvilli in the middle fold. Some exocytosis vacuoles can be observed around the periostracal lamina or at the tip of these elongated cells. The internal epithelium of the outer fold is scattered with mucocytes and a few cells with strongly PAS-stained glucidic granules at the apical part. The mantle showed no signs of hyperplasia or of haemocytic infiltration. Melanin pigments were not observed in the cells of the outer fold epithelia or at the surface of this fold.

Histochemical studies of the shell pigment The principal histochemical results are summarized in Table 1. The brown shell pigment is highly insoluble in acids and in organic solvents. The resistance to the basic solvent NaOH is variable depending on its concentration. Complete solubility of brown IOMs (insoluble organic matrices) was best achieved using NaOH 1M for 24 h at ambient temperature. White IOMs were soluble in any NaOH treatment tested. The results of the Hueck reaction were strongly positive for the whole shell matrix of brown and white shells. This technique, regularly used to detect lipoid pigment, seems to interfere with the acidity of the shell matrices, so it has not been taken into account for further interpretation of the characterization of the pigment. The strong insolubility in acids and in organic solvents, and the positive reaction to Lillie staining and to H202 discolouration, are characteristic of a eumelanin pigment. Lillie staining was positive in the matrix of the internal shell layer of brown shells, in the extremely thin external layer of white shells and in the epithelial cells of the inner and middle folds OF and MF) of the mantle. The pigment is not present in the cells of the periostracal groove and in the external shell-secreting epithelium of the mantle. Nor is it present in the periostracal lamina. This melanin pigment is confined to small granules at the apical tips of the cells of the IF and ME In the shell, it is

FIG. 2. Microstructure of the calcified inner shell of white and brown scallops. (A-B) Normal foliated microstructure of white calcite material; (C-F) altered microstructure of brown calcite material. See text for further details.

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TABLE 1 Tested characteristics and histochemical reactions of the brown pigment present in brown shells (0, negative response; +, positive response) Properties and histochemical reactions

Results

Solubility in acidic solvent (HCI 6N, 110 °C, 24 h) Solubility in basic solvent (NaOH 0.1, 0.5, 1M) Solubility on organic solvents Hydrogen peroxide discolouration (H202 10%, 24 h) Lillie reaction (ferrous ion capture) H202 treatment and Lillie reaction Diacetin-Sudan Black reaction Hueck reaction

0 0,0,+ 0

Conclusion

Eumelanin pigment

+ +

0 0 + for the whole shell matrix (brown or white)

w i d e s p r e a d and a s s o c i a t e d with the matrix; we could not d e t e c t any preferential a c c u m u l a t i o n s in p a t c h e s or in lamina along the shell matrix.

Studies of the spectral characteristics

Reflectance study The c o l o u r i m e t r i c s t u d y of different b r o w n and white shells shows that the brown c o l o u r a t i o n is a s s o c i a t e d with a d e c r e a s e of the reflectance o v e r a wide s p e c t r u m from 400 to 600 nm (Fig. 3A). This d e c r e a s e is c o r r e l a t e d to the intensity of the b r o w n colour: the d e e p e r the b r o w n colour, the m o r e the reflectance decreases. Some of the white shells from different sites in the Bay of Brest a p p e a r e d to p r e s e n t a small d e c r e a s e in reflectance even w h e n the beginning of the b r o w n c o l o u r a t i o n could not b e distinguished with the naked eye. Fig. 3(B) p r e s e n t s an example of this d e c r e a s e in reflectance for scallops d r e d g e d in the Roscanvel site of the Bay of Brest.

Spectrometric study Two of the a b s o r b e n c y s p e c t r a of b r o w n and white acid-insoluble organic matrices (BIOM and WlOM) dissolved in NaOH 1M are p r e s e n t e d in Fig. 4, c o m p a r e d with the NaOH 1M spectra. T h e p r e s e n c e of the pigment in the BIOM is c h a r a c t e r i z e d by a progressive increase of the a b s o r b e n c y o v e r the wavelength range 600-300 nm. A small increase of the a b s o r b e n c y of the WlOM is also n o t e d from 400 to 240 nm. The NaOH solvent has a strong a b s o r b e n c y at 223 nm which is also p r e s e n t on the BIOM and WlOM s p e c t r a and which might c o n c e a l the a b s o r b e n c y c h a r a c t e r i s t i c s of the pigment in the UV. T h e a p p e a r a n c e of the b r o w n c o m p o u n d in scallop shells is c h a r a c t e r i z e d by the s a m e reflectance and a b s o r b e n c y p r o p e r t i e s in the visible range: a m o n o t o n i c increase of the a b s o r b e n c y with d e c r e a s i n g wavelength which was r e c o r d e d b y Crippa et al. (1989) as "the typical s p e c t r a of b o t h eu- and phaeomelanin". T h e

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extension of the spectral range to the UV in the second method shows that the presence of the organic shell matrix induces interferences linked to the absorption of its proteic component.

2.50

White

2.00

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1.50

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450

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550

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w

600

650

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Wavelength (nm) 2.15 2.10 2.05

'6 2.00 0 0 t-

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1.90 1.85 1.80

B

1.75 4OO



s

450

500

'i ...........

550

i

.

i

600

650

700

Wmt~JeJ)aJb.(prnl, FIG. 3. Reflectance study of scallop shells. (A) Reflectance of white and brown shells. The graph presents an example of some individual measurements. (B) Reflectance of white shells from the Bay of Brest (individual measurements). The level of whiteness established with the naked eye can be measured by the reflectance analysis: the upper set of curves relate to pure white shells, and the lower set to shells that appear white to the naked eye but are revealed to be starting to become brown when tested instrumentally.

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246 .5

.

.

.

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.

.

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°0.~

.....

200

250

300

350

, ...............

400

450

500

550

600

Wavelength (nm) FIG. 4. Absorbency spectra of brown and white insoluble organic matrices (BIOM and WIOM) dissolved in NaOH 1M solution compared with the control spectrum (NaOH 1M).

Infrared study The vibrational bands of brown and white IOM are presented in Table 2. The vibrational bands o b s e r v e d for BIOM and WIOM are the same, and coincide with many of th o s e of natural and synthetic melanins noted by Crippa et al. (1989). Some of the bands are not specific to melanins, but rather to glycoproteins (amine and amide bands, COOH-carboxylate, sugars). The aromatic C-H bonding, which could be representative of melanin products, is not recorded in all the analysed specimens. At this stage, the difference between BIOM and WlOM could not be established and the melanic pigment could not be recognized. The correlation coefficients calculated between the different spectra of BIOM and WlOM are all over 0.9, except for one BIOM and one WIOM. Then the differences between melanized and white insoluble organic matrices of the shell could not easily be established even when considering the levels of the absorption coefficients for each vibrational band.

DISCUSSION The brown c o m p o u n d abnormally deposited in the internal calcified layer of brown valves is a eume|anin pigment, associated with the insoluble shell matrix. Melanin pigments are normal components of the mantle and shell of the great scallop, as of many molluscs (Comfort, 1951), but were not clearly demonstrated in the shell because of their high insolubility. In scallops with a white internal shell layer, th ey are responsible for the external colour of the upper valve, where it is present in the thin external calcified layer. This layer is synthesized by the marginal cells of the external epithelium of the mantle. These are unpigmented cells, as are all

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TABLE 2 VibrationN bands and c4haracteristic tR absorption regions of the brown and white ~nsoluble organic matdces (BIOM and W]OM). The last column gives the bands for different natural and synthetic meiaNns (from Crippa et aL, 1989). +, Vibrational band was observed; -, vibrational band was not observed; +/-, vibrational band was observed on only a few specimens; um, unmeasured vibrational band

Vibrational band

v (cm -~)

BIOM

WlOM

Amide A Amide B Aliphatic CM4 stretching

3300 3000-3100 2930 2860 17t 6 - I 735 1680-1636 1573 1530-1550 1420 1050-1100 900-730 600

+ + _ + + + + + + +/um

+ + _ + + + 4+ + +/um

COOHAmide I COO- asymmetrical Amide tt COO- symmetrical Sugars Aromatic C-H bonding OH liberations (H20)

Synthetic and natural melanins + + +~ +i

+/+ + um + + + +

~Except for Sepia melanin.

the ceils of the external epithelium of the mantle. They produce uncoIoured melanin precursors which are polymerized in the extrapallial fluid. In the mantle of white and brown shells, melanin is restricted to the melanocytes of the epithelia of the inner fold and of the :middle sensory fold, where it is confined to small granules at the tip of the cells. This form of cellular melanin, called phaeomelanin, is soluble. No such melanin patches have been recorded in the external epithelium of the mantle of brown shells. According to this observation and their different solubility (Crippa et al., 1989), the two melanin pigments, phaeomelm nin in the mantle and eumelanin in the shell, are considered to have different origins and to be unrelated. The presence of the pigmentation in the external shell layer of the upper valve of white shells demonstrates that the melanin, or rather melanin precursors, are normally synthethized at the margin of the outer fold of the mantle which secretes this shell layer (Clark, I974). Then we could consider that the appearance of the Ngment in the internal shetl layer might be due to a disorder in the shell-secreting epithelia, which could get the properties of the cells of the tip of the fold. tn the process of shell repair (Watabe, 1983), the cells of the shell-secreting epithelium change their morphology to form successively the required layer to rebuild the damaged region. The synthesis of melanin in the internal shell layer could be generated by a similar change in cell physiology. The presence of melanin has already been recorded in exoskeleton alterations of crustaceans and insects (Smolowitz et al., 1992; S6derhfill and Cerenius, 1992) and many observations of cuticular diseases underline the presence of brown spots which might be melanic (Sindermann, 1979), These metanization processes were demonstrated as being associated with the non-specific immune systems of

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a r t h r o p o d s (Pye, 1974; S6derh~Ul, 1982, 1992; S6derh~ill et al., 1994). Otherwise, squid's melanin has an anti-inflammatory action on ulceration of gastric mucosa of rats (Mimura et al., 1987) and its precursors are cytotoxic (Graham et al., 1978). The presence of melanin in diseased shells of molluscs was first noted by Paillard (1992) in Manila clams Ruditapes philippinarum suffering from a bacterial disease, the brown-ring disease (BRD). This pigment is included in the periostracal lamina and in the brown organic deposit covering the inner side of the shell. We have previously detected melanin in such organic deposits in Polydora chambers (Larvor et al., in press) and in BRD-like layers of some great scallop shells (personal observation). In these deposits it is associated with an organic matrix which has an amino acid composition clearly different from the normal calcified layer organic matrices (Devauchelle et al., 1994). These brown deposits have been observed in many species of bivalves (Blake and Evans, 1973; Bartoli, 1976; Mann and Taylor, 1981; Goulletquer et al., 1989; Getchell, 1991; Gould and Fowler, 1991). In the case of the brown calcified material, no clear modification of the proteic composition of the shell matrices was evident in brown valves compared with white ones (Larvor et al., in press). Furthermore, the calcium content was not modified and no special metal accumulation was noted. Thus the conditions required for normal biomineralization processes were considered unchanged, except for the problem of the synthesis of the melanin component. Nonetheless, the foliated microstructure of the inner shell layer of the scallop is clearly distorted. However, it could be the appearance of the melanin in the insoluble envelopes of the crystals which may disturb the normal sclerotization of the organic matrix and the growing of normal foliated crystals. Melanin and sclerotized proteins have two precursors in common, tyrosine and DOPA @Vaite, 1992), and implicate two enzymes, monophenol monooxygenase (which converts tyrosine to DOPA and is therefore also called tyrosinase) and catechol oxidase (which converts catechol to o-quinone). Robb (1984) noted that these reactions o c cu r at different phases of biosynthesis. In DOPA-sclerotization other enzymes which convert the DOPA to dopamine and N-acetyl dopamine lead to the formation of N-acetyldopaquinone, which cannot undergo intramolecular addition as in melanin biosynthesis. We could hypothesize that modifications of these enzyme levels or activities, or modifications of the availability of the substrata, could induce synthesis of melanin as a deviation from the normal sclerotization process. The fact that the crystal structure is distorted, even if the composition of the shell matrix did not change, urges us to think that the normal arrangement of the proteic substratum for calcium carbonate chelation is modified. The presence of eumelanin chelated to the insoluble matrix may be one reason for this abnormal organization. However, the details of the DOPA-sclerotization process, tg-sclerotization or quinone tanning, remain unclear and the function of pigmentation by melanin formation is still debated (Hill, 1992). Histological study of the scallop mantle revealed no alterations of the shellsecreting epithelium or of the cells of the periostracal groove cells. This is quite different from other shell diseases. In juvenile oyster disease of Crassostrea virginica (Ford and Paillard, 1994; Farley and Lewis, 1994) and in brown ring disease of Manila clams (Paillard, 1992), inflammation of the mantle epithelia underlying the brown deposits was observed. The modifications consisted of a deep hyperplasia of the

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shell and periostracum-secreting ceils, an intense haemocytic infiltration of the connective tissue and lesions in the mantle epithelium. This was o b s e r v e d on only a few scallops developing brown ring diseaseAike symptoms on their shells (personal observations). No pathological symptoms of the mantle were evident in this study, It appears therefore that the colouration of the inner shell layer of great scallop and the modification of its normal foliated microstructure may be a response to environmental disturbances rather than the result of infectious disease. Contrasts in pigmentation have already been o b s e r v e d on the outer surface of the shell of the great scallop. Minchin (1991) studied the variability of shell colour and pattern of young great scallops from different places in Ireland: he recorded five colour morphs and four of them demonstrated geographic variations. In Ireland and Cornwall, Baird and Gibson (t956) showed that scallop populations from adjacent places could differ in shell colour and size, depending on depth and substratum: large, nearly unpigmented scallops were dredged in deep water and on a muddy bottom and, in an area within 200 m, small dark-pigmented scallops were dredged in shallow water and on a gravel bottom. The pigmentation of the inner surface was not mentioned, Medakovic et al. (pers. comm.) reported an occurrence of mussels with a pink inner shell layer in polluted areas of the northern Adriatic Sea. Robb (1984) noted that tyrosinase activation could b e accomplished b y detergent, ageing, pH changes or proteolysis. Gadd et aL (1990) and Gadd and de Rome (I988) demonstrated the role of melanin in fungal biosorption of copper and TBT. Otherwise, microstructuraI distortions, whether linked to colour changes or not, have been o b s e r v e d in different species of bivalves. Marin and Dauphin (1992) noted an alteration of the nacreous layer of the shell of diseased pearl oysters Pinctada margaritifera held in bad ecological conditions (overcrowding, underfeeding). Machado and Coimbra (in press) demonstrated that tributyl-tin oxide (TBTO), diflubenzuron and pH changes could induce modifications of the nacreous microstructure of Anodonta cygnea, and Prezant and Chalermwat (pers. comm.) showed that variation of water temperature and of trophic level could have an influence on the shell of Corbicula fluminea, Such observations of microstructural changes were made on starving scallops in experimental conditions 0)evauchelle et aL, 1994),

CONCLUSIONS 1. Further experiments need to be made to discriminate the nature of the disturbances that could induce colour and shell crystal modifications. 2. The reflectance technique presented in this paper, coupled with microstructural observations, will have interesting applications in the monitoring of the appearance of the colouration in white shells of juvenile and adult scallops. 3. It is a more convenient method than the classical spectrometric study in the liquid phase b e c a u s e it does not need much preparation of the material and studies the properties of the brown melanic pigment in place, with no influence of interference a b s o r b a n c e of solvents.

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ACKNOWLEDGEMENTS We t h a n k C. Pailtard for h e r helpful a d v i c e c o n c e r n i n g t h e pigment discrimination study, and Y. Dauphin for reviewing the infrared results. This w o r k was s u p p o r t e d b y IFREMER, station of Brest, France, the University of Paris XI, Orsay, France, and the Brest Urban Community,

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