The Yellow Pigment Cells of Hydrobia Ulvae (Pennant) (Mollusca : Prosobranchia) J.D

J. moll. Stud. (1979), 45, 345-352

THE YELLOW PIGMENT CELLS OF HYDROBIA ULVAE (PENNANT) (MOLLUSCA : PROSOBRANCHIA) J.D. FISH Downloaded from https://academic.oup.com/mollus/article/45/3/345/1003004 by guest on 29 September 2021 Department of Zoology, University College of Wales, Penglais, Aberystwyth, SY23 3DA. (Received 3 January 1979)

ABSTRACT The distribution of yellow pigment cells in the veliger larvae and post-metamorphic snails of Hydrobia ulvae has been recorded. The number of cells increases with the size of the snail and the pigment is characteristic of the foot of the veliger larva, and the foot, mantle, tentacles and penis of adult snails. The cells contain numerous, double membrane-bound vesicles and have a highly granular cytoplasm. The absorption spectra of acetone and methanolic HC1 extracts show single peaks at 337 and 392 nm respectively, while chloroform extracts show peaks at 249, 274 and 283 nm with an inflexion at 293 nm. The pigment has a pale green fluorescence in ultra- violet light. The results of feeding and starvation experiments using larval and post-metamorphic snails lead to the hypothesis that the pigment is a waste product of metabolism which is stored in the vesicles of the cells.

INTRODUCTION One of the characteristic features of the veliger larva of Hydrobia ulvae is a number of conspicuous pigment cells arranged in a V-shaped area on the mesopodium. These distinct cells appear black under transmitted light and yellow-green under reflected light and were first recorded by Henking (1894). It has subsequently been shown that they are an important aid in identification and are one of the characters by which the veligers of Hydrobia can be readily separated from those of the edible periwinkle, Littorina littorea (L.) (Fish & Fish, 1977) with which they are often found in inshore waters. Observations on adult snails show that similar cells are present in dense concentrations in specific areas of the body to which they impart a distinct yellow colouration. Studies on the pigmentation of Hydrobia have been largely concerned with the black pigmentation of the tentacles and snout, a feature which is used in species separation (Muus, 1962) and only passing reference has been made to the yellow pigment. Clay (1960) noted yellow pigment spots on the snout and foot of H. ulvae and more recently Fretter & Graham (1978) have referred to "sulphur-yellow speckles" on the foot, snout, tentacles and penis. In view of the occurrence of the pigment cells in both veliger larvae and adults, and their importance as diagnostic features in the former, the present investigation was designed to study their distribution, structure and possible function.

MATERIAL AND METHODS Adult snails were collected from the Dovey estuary (SN 613943) and dissected under filtered sea water. Tissues rich in yellow cells (mantle and penis) were removed and fixed according to the nature of the histochemical test to be applied. Stains for mucopolysaccharides were alcian blue, toluidine blue and the periodic acid Schiff technique. The possible presence of lipofuscins was investigated by the chrome alum haematoxylin and indophenol methods. All staining procedures were carried out according to Pearse (1968). Cryostat sectioned material was used in the investigation of glycogen and lipid deposits. The ultrastructure of the pigment cells in both veliger larvae and adult snails was studied using a AEI EM6B electron microscope. Tissues which had been fixed for 2 h in 4% glutaraldehyde in cacodylate buffer followed by 4 h in 1% osmium tetroxide were block stained in 3% aqueous uranyl acetate and sectioned on an LKB ultramicrotome. The sections were mounted on colloidin-coated grids and double stained in 5% aqueous uranyl acetate for 5 min followed by lead citrate for 5 min (Reynolds, 1963). Pigment extracts were made from adult snails by the laborious and time-consuming procedure of dissecting away tissues rich in pigment cells and homogenising with a suitable solvent. Absorption spectra were obtained by means of a Unicam SP 800 spectrophotometer. Thin layer chromatography was carried out on 0.2 mm thick silica gel plates using the following solvent systems: hexane/ether (70:30 v/v); chloroform/acetone/methanol (70:25:5 v/v); chloroform/meth'anol/ acetic acid (30:30: 1 v/v). 346 THE JOURNAL OF MQLLUSCAN STUDIES Downloaded from https://academic.oup.com/mollus/article/45/3/345/1003004 by guest on 29 September 2021

Fig. 1. i he distribution of pigment cells in A) the foot B) the tentacle C) the penis and D) the veliger larva (squashed preparation) of H. ulvae. Pigment cells arrowed. Line indicates 500/jm (A,C); lOOjjm (B,D). FISH: PIGMENT CELLS OF HYDROBIA 347 Newly hatched veliger larvae of Hydrobia were obtained by incubating egg masses at I5°C in millipore filtered sea water (pore size 0.2^m). The veligers were removed within 1 h of hatching and used in either feeding or starvation experiments. In feeding experiments the veligers were maintained at 15°C in filtered sea water containing the haptophycean, Isochrysis galbana Parke at a concentration of about 35x10' cells per ml. Veligers were starved in millipore filtered sea water which was changed every second day. The effects of starvation and feeding on the density of the pigment cells in post-metamorphic snails were investigated by recording changes in the number of cells in the tentacles of juvenile snails during periods of starvation and feeding. Snails were fed on detritus rich in Enteromorpha and when under starvation individual snails were kept in separate dishes containing millipore filtered sea water which was changed every second day. The tentacles were chosen for study as they are almost transparent and the cells are sufficiently dispersed to allow accurate counts to be made. This is particularly true of recently metamorphosed specimens. Downloaded from https://academic.oup.com/mollus/article/45/3/345/1003004 by guest on 29 September 2021

RESULTS Distribution of pigment cells in the veliger larva Veliger larvae taken in inshore plankton samples show the characteristic V-shaped area of pigment cells on the mesopodium (Fig. ID). These cells are so conspicuous that they can be clearly seen through the semitransparent operculum when the larva is withdrawn into the shell and are thus an important aid in identification. The number of cells increases with the age of the larva and just before metamorphosis there may be one hundred or more restricted to the mesopodium. Yellow pigment cells are absent from veliger larvae hatched in the laboratory and they failed to develop in larvae kept under starvation for up to 24 days. On the other hand, veligers fed on Isochrysis galbana showed conspicuous yellow pigment cells on the mesopodium after six days.

200-,

= 100-

E 3

2 3-4 Shell height (mm)

Fig. 2. Variation in the number of yellow pigment cells in the tentacles of H. ulvae with shell height. Each point is the mean of the number of cells in right and left tentacles. Y = —23.071 + 26.857X; r = 0.892 348 THE JOURNAL OF MOLLUSCAN STUDIES

— -o

20- Downloaded from https://academic.oup.com/mollus/article/45/3/345/1003004 by guest on 29 September 2021

November 1977 December January 1978

Fig. 3. The effects of starvation and feeding on the number of yellow cells in the tentacles of two juvenile H. ulvae. O O right tentacle: • • left tentacle.

Distribution of pigment cells in the post-metamorphic snail After metamorphosis the distribution of the pigment cells widens as the animal grows and they become conspicuous on the snout, mantle edge, tentacles and.in the male specimens, the penis. In the larger snails the cells are so numerous that they impart a distinct yellow colouration to these areas. In the foot the pigment is concentrated around the margin and its distribution is best observed while the snail is suspended from the surface film by a mucous raft (Fig. 1 A). In the tentacles the cells are concentrated in the mid-line giving a prominent yellow band along the length of each tentacle (Fig. IB). The penis shows a concentration of pigment at the tip and around the lateral margins (Fig. 1C). Both the snout and mantle edge bear large numbers of pigment cells and there is a distinct crescent of pigment on either side of the mouth. In large specimens a small patch of pigment overlies the viscera but in many cases this is obscured by an extensive development of black pigment in this area. FISH: PIGMENT CELLS OF HYDROBIA 349 Downloaded from https://academic.oup.com/mollus/article/45/3/345/1003004 by guest on 29 September 2021

Fig. 4. A) Penis tip of H. utvae viewed under ultra-violet light using a standard fluorescence microscope Line indicates 25^m. B) Transmission electron micrograph showing the cytoplasm of a pigment cell Line indicates 2jjm. C) Transmission electron micrograph of a group of pigment cells from the penis Line indicates 2um. • r~

350 THE JOURNAL OF MOLLUSCAN STUDIES The observation that the density of yellow pigment cells appears to increase with the size of the snail has been confirmed by counting the number of separate cells in the tentacles and expressing these values against the shell height of the snail (Fig. 2). The effect of starvation and feeding on the number of cells in the tentacles of juvenile specimens is shown in Fig. 3. During periods of starvation there is no increase in density, whereas a steady increase is maintained once feeding has commenced. There was no increase in the number of yellow cells in control specimens kept under starvation.

Structure of the pigment cells Electron microscope studies show that the pigment cells of larval and adult snails have the same

structure. They lie beneath the epidermis and vary from about 10 to 20 pm in maximum dimension. Downloaded from https://academic.oup.com/mollus/article/45/3/345/1003004 by guest on 29 September 2021 There is a well defined nucleus, two or three mitochondria and a large number of double membrane- bound vesicles which give the cells their characteristic vacuolated appearance (Fig. 4C). The cytoplasm of the cell is granular, the granules being of two types, a densely-staining rosette, and a smaller, less dense but more numerous granule (Fig. 4B). Histochemical tests for mucopolysaccharides, glycogen, lipid and lipofuscins all gave negative results.

Pigment extractions The absorption spectra of the pigment in acetone, chloroform and methanol containing 5% concentrated HC1 are shown in Fig. 5. The acetone and methanol extracts show single peaks at 337 and 392 nm respectively, while the chloroform extract shows peaks at 249, 274 and 283 nm with an inflexion at 293 nm. The pigment was not soluble in ether. Pigment extracts show a pale green fluorescence under ultra-violet light, a property also shown by intact cells in situ (Fig. 4A).

Thin layer chromatography Thin layer chromatography showed a single major component with Rf-values of 0.04 (hexane, ether), 0.25 (chloroform, acetone, methanol) and 0.55 (chloroform, methanol, acetic acid). In the chloroform-methanol systems there was a faint suggestion of a second component.

DISCUSSION \ Veliger larvae of prosobranch molluscs are frequently pigmented and the colour and distribution of pigment associated with the mesopodium and velar lobes can be useful in identification. The colour of the digestive gland is often a reflection of the plant food on which the veliger has been feeding and this character has also been used in identification (Thorson, 1946; Fretter & Montgomery, 1968) even though in many veligers the colour of the gland is lost during short periods of starvation (Fretter & Montgomery, 1968). The veligers of Hydrobia develop yellow cells only after feeding and these are not lost during starvation, a feature which enhances their value in identification. In Mangelia nebula (Montagu) the veliger is characterized by yellow-orange pigment on the velar lobes which at metamorphosis passes into the cephalic haemocoel and becomes the first accumulation of the characteristic pigment of the adult (Fretter, 1972). Other species of prosobranch veliger are known to have pigment spots on the velar lobes and mesopodium but little is known about these and their relationship with the pigmentation of the adult snail. In Hydrobia the ultrastructure of the pigment cells is the same in both larval and post-metamorphic snails and it would appear that the pigment is held in the spherical vacuoles which are characteristic of the cells. Molluscs exhibit a wide diversity of colours and in many cases these are related to the food ingested by the snail. Bannister, Bannister & Micallef (1968) have reported a bilatriene in the foot of Monodonta turbinata (Born) which they suggested was derived from algal precursors in the food. The yellow pigment isolated from Hydrobia may be a mixture of more than one component, and is characterized by the absorption spectra in acetone, chloroform and methanolic HC1 (Fig. 5) and a green fluorescence in ultra-violet light. Together with the Rf-values from thin layer chromatography the data provide basic information on the nature of the pigment but do not permit positive identification. Further biochemical studies are hampered by the small amounts of material available and are beyond the scope of the present work. However, in Hydrobia the increase in density of the pigment cells and the widening distribution with increase in size of the snail, together with the results of feeding and starvation experiments all lead to the hypothesis that these cells are related«to feeding activity and contain waste products of metabolism. The pigment might be derived directly from the FISH: PIGMENT CELLS OF HYDROBIA 351 Downloaded from https://academic.oup.com/mollus/article/45/3/345/1003004 by guest on 29 September 2021

250 300 350 Wavelength nm

Fig. 5. Absorption spectra of the yellow pigment in A) chloroform B) acetone and C) methanolic HC1. Each extract was scanned from 200-800 nm; the portion shown is that for which peaks were obtained. food source or be the result of structural modifications to dietary compounds. It is interesting to note that H. ventrosa (Montagu), which is often found with H. ulvae, shows yellow pigment spots (Clay, 1960; Fretter & Graham, 1978) while the freshwater Potamopyrgus jenkinsi (Smith) has white pigment cells. Because of the low density of these cells in Potamopyrgus a suitable extract has not been made, but they have the same ultrastructure as the yellow pigment cells of H. ulvae and the difference in colour might be a reflection of different food. Any suggestions as to the importance of these cells to the snail must be speculative but it is possible that the accumulation of waste products in this way is a means of relieving the kidney of some of its excretory function. This has been suggested by Fretter & Graham (1962) for prosobranch molluscs in which the digestive gland takes up waste products of metabolism from the blood. This may be of particular significance to animals living in waters of reduced salinity.

SUMMARY Veliger larvae of H. ulvae are characterized by yellow pigment cells on the mesopodium. In post- metamorphic snails the distribution of the pigment widens as the snail grows and it is found on the mantle, foot, tentacles and penis. The pigment cells have a well-defined nucleus and numerous, double membrane-bound vesicles. The cytoplasm is highly granular and the cells have a pale green fluorescence in ultra-violet light. The absorption spectra of the pigment in acetone and methanolic HC1 show single peaks at 337 and 392 nm respectively, while chloroform extracts have peaks at 249, 274 and 283 nm with an inflexion at 293 nm.-Thin layer chromatography showed a single, major component. These results of feeding and starvation experiments on the presence and distribution of the pigment in veliger larvae and post-metamorphic snails, lead to the hypothesis that the pigment is a waste product of metabolism which is stored in the vesicles of the cells.

ACKNOWLEDGMENTS I wish to thank Dr. J. Barrett and Dr. B. D. Davies of the Departments of Zoology and Biochemistry respectively, for helpful advice and assistance. 352 THE JOURNAL OF MOLLUSCAN STUDIES REFERENCES BANNISTER, W. H., BANNISTER, J. V. & MICALLEF, H. 1968. The green pigment in the foot of Monodonta (Mollusca) species. Comparative Biochemistry and Physiology, 24, 839-46. CLAY, E. 1960. Hydrobia ulvae Pennant, H. ventrosa Montagu and Potamopyrgus jenkinsi Smith. Literature Survey of the Common Fauna of Estuaries, No. 8, 41pp. I.C.I. Paints Division, •Brixham Res. Memorandum PVM45/A/381. FISH, J. D. & FISH, S. 1977. The veliger larva of Hydrobia ulvae with observations on the veliger of Littorina littorea (Mollusca: Prosobranchia). Journal of Zoology, London. 18, 495-503. FRETTER, V. 1972. Metamorphic changes in the velar musculature, head and shell of some prosobranch veligers. Journal of the Marine Biological Association of the U.K., 52, 161-77. FRETTER, V. & GRAHAM, A. 1962. British Prosobranch Molluscs. London, Ray Society. FRETTER, V. & GRAHAM, A. 1978. The prosobranch molluscs of Britain and Denmark, Part 3 — Neritacea, Downloaded from https://academic.oup.com/mollus/article/45/3/345/1003004 by guest on 29 September 2021 Viviparacea, Valvatacea, terrestrial and freshwater Littorinacea and Rissoacea. Journal of Molluscan Studies, Suppl. 5. FRETTER, V. & MONTGOMERY, M. C. 1968. The treatment of food by prosobranch veligers. Journal of the Marine Biological Association of the U.K., 48, 499-520. HENKING, H. 1894. Beitrage zur Kenntniss von Hydrobia ulvae Penn. und deren Brutpflege. Berichte der Naturforschenden Gesellschaft zu Freiburg i.B., 8, 89-110. MUUS, B. J. 1962. Some Danish Hydrobiidae with the description of a new species, Hydrobia neglecta. Proceedings of the Malacological Society, London, 35, 131-38. PEARSE, A. G. E. 1968. Histochemistry. Theoretical and Applied. London, Churchill Livingstone. REYNOLDS, E, S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology, 17, 208-17. THORSON, G. 1946. Reproduction and larval development of Danish marine bottom invertebrates, with special reference to the planktonic larvae of the Sound (0resund). Meddelelser Fra Kommissionen For Danmarks Fiskeri- og Havundersfgelser, Serie Plankton, 4, No. 1, 1-523.