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.