4°5 ON THE STRUCTURE AND FUNCTION OF THE WANDERING CELLS IN THE WALL OF THE ALIMENTARY CANAL OF NUDI- BRANCHIATE MOLLUSCA BY NORMAN MILLOTT, M.Sc. Demonstrator in Zoology, the Victoria University of Manchester (Received 17 December 1936) (With Five Text-figures) INTRODUCTION IN the lining epithelium and subjacent tissues of the gut of nudibranch molluscs are certain wandering cells. These differ from the lymphocytes, which are also present in the epithelium, submucosa, and underlying blood spaces of the gut, in being somewhat larger; though both types of cell are very variable in size. Unlike the lymphocytes, these cells possess a very definite, almost corpuscular, form after fixation. They are rare in the general vascular system, being largely confined to the wall and lumen of the gut. In the majority of animals examined, they appeared most numerous in the hindgut. In the gut epithelium of all the forms examined, the wandering cells often occupied spacious cavities. Figs. 2-5 indicate the uniformity of appearance of the wandering cells, and the characteristic manner in which they occur in intra-epithelial spaces (int.ep.c). The following investigation aims at discovering whether these cells have any significance in the excretory processes of nudibranchs. The term "excretion" is used in its widest sense. No attempt is made to dis- tinguish between water excretion, nitrogen excretion, or the elimination of other, by- or end-products of metabolism. I wish to express my gratitude to Prof. H. Graham Cannon, for the great help he has given me in the preparation of this paper. My thanks are also due to Dr C. F. A. Pantin for a great deal of kind criticism and advice. Finally I wish to thank Prof. E. A. Spaul for permission to use the Leeds University Laboratories at Robin Hood's Bay wjhere a portion of the work was carried out. 406 NORMAN MILLOTT MATERIAL AND TECHNIQUE The wandering cells were examined in the alimentary canal of the following seven species chosen from different families of Nudibranchiata. Aeolidiidae Aeolidia papillosa (Linnaeus) Okeniidae Ancula cristata (Alder) Goniodoris nodosa (Montagu) Onchidoridae Acanthodoris pilosa (Abildgaard) Euphuridae Polycera quadrilineata (O. F. Miillef) Doridigitatidae Jorunna tormentosa (Cuvier) Archidoris brittanica (Johnston) Sections of material fixed in Flemming without acetic or Susa (made up in sea water) were used. The former gave better fixation, but on account of its lack of penetration it necessitated removal of the tissues surrounding the gut before fixation. The latter fixative, while not preserving cytological detail so well, proved useful in virtue of its power of penetration, which rendered the removal of any tissues unnecessary. Material was embedded in wax, sections were cut 5 and io/x thick, and stained in Delafield's haematoxylin and eosin, safranin and light green, and Mallory's triple stain. Structure (Fig. 1) The wandering cells showed a perfectly uniform structure and the following account applies to all the species examined. cl.z. l.xt.U. bl.b. b.pos.a. Fig. 1. Structure of a typical wandering cell, from the midgut of Goniodoris nodosa. Fixed Susa (in sea water), stained Delafield's haematoxylin and eosin. x 5820. b.eos.a. boundary of eosinophile area (seen by transparency); bl.b. blackish granules in wandering cell; cl.z. clear zone of cytoplasm; d.st.b. deeply stained body in wandering cell; n. nucleus; ncl. nucleolus; vac. vacuole. The cells are regularly spherical, though they occasionally present evidence of being amoeboid. It is highly probable that the living cells are amoeboid, as the tendency of such cells to assume a spherical form on fixation is well known. Alimentary Canal of Nudibranchiate Mollusca 407 Their size is very variable after fixation, some being only 4/u. in diameter while occasional giant forms with a diameter of over IO/A were observed. The average size lies between 5 and 6/1. A single nucleus (n.) is present which may appear spherical or ovoid, but which is often characteristically crescentic in form. It stains very deeply in haematoxylin and it is often difficult to see the contained chromatin. A distinct nucleolus {ncl.) can occasionally be observed. Frequently the nucleus is separated by a clear zone of cytoplasm (cl.z.) from a well-marked crescentic area (eos.a., Figs. 2 and 4), which is devoid of inclusions and is strongly eosinophile. Sometimes, as in Figs. 2, Fig. 2. Portion of a transverse section of the epithelium and underlying tissues of the hindgut of Ancula cristata, showing wandering cells. Fixed Susa (in sea water), stained Delafield's haematoxylin and eosin. x 2560. b.c. blood cells; eos.a, eosinophile area. 3 and 5, the eosinophile area may be absent or obscured by the dense staining of the surrounding cytoplasm. The remaining cytoplasm varies greatly in appearance. In some forms it is highly vacuolated, in others it appears loaded with solid inclusions which may appear deeply stained (d.st.b.) or blackish (bl.b.). Sometimes the vacuoles and granules co-exist in the cytoplasm, in other cells both may appear to be absent. The reaction of the cytoplasm to stains is also varied. After osmic fixation the cytoplasm may fail to take up stain and appears greyish. In other cases the cyto- plasm may stain very deeply in stains such as safranin, orange G, and haematoxylin, and consequently the cells appear as deeply stained corpuscles devoid of any obvious structure (Fig. 3, d.w.c). Sometimes the cytoplasm may stain differentially with safranin and light green or Mallory. 408 NORMAN MILLOTT Function Technique. In order to discover whether the wandering cells have an excretory significance, a suspension of iron-saccharate was injected into the haemocoele of several individuals of Archidoris brittanica. The injection was performed in the foot, in a position slightly anterior, and to the right-hand side, where the haemocoele is occupied largely by the reproductive system. By this means, the possibility of introducing saccharate into the digestive gland, or the so-called "stomach", both of which lie close against the foot, was avoided as far as possible. d.w.c. int.ep.c. d.w.c. Fig. 3. Portion of transverse section of the epithelium and underlying tissues of the hindgut of Goniodoris nodoia showing wandering cells. Fixed Susa (in sea water), stained Delafield's haema- toxylin and eosin. x 2560. b.c. blood cells; d.w.c. deeply staining wandering cell (see text); int.ep.c. intraepithelial space; After injection, the animals were washed thoroughly in sea water, thus removing from the integument any traces of iron saccharate which might have entered the gut. Most of the specimens injected recovered within 30 min. and moved about quite normally; a few, however, remained in a state of torpidity in which the branchiae and rhinophores were only slightly extended, and movement was very limited. None of the individuals died. Specimens were fixed by Yonge's modification of the older method for iron saccharate (Yonge, 1926), using equal parts of 5 per cent ammonium sulphide in 95 per cent alcohol, and Bouin's fluid, at ii, 4, 7, 9 and 19.V hours after the time of injection. Sections were cut 10/x thick, and subsequently treated with a 10 per cent solution of potassium ferrocyanide and dilute hydrochloric acid; by this means the iron in the tissue was converted into Prussian blue. The sections were counter- stained in eosin. Alimentary Canal of Nudibranchiate Mollusca 409 A portion of the epithelium of the hindgut of a specimen fixed if hours after injection is shown in Fig. 4. The blood spaces (b.sp.) and blood cells (b.c.) in the submucosa are seen heavily laden with iron. Very little iron is present in the lumen of the gut, though traces are present in the wandering cells (w.c. 1), or more commonly in the mucus secreted from the mucus glands (mc.g.). w.c. w.c. 2 mc.g. eos.a. w.c. 4 w.c. 3 eos.a b.c. Fig. 4. Portion of a transverse section of epithelium and submucosa of hindgut of ArcYndoris brittanica (from a specimen fixed i J hours after injection with a suspension of iron saccharate). Fixed, and stained, as in text, x 2560. b.c. blood cells; b.tp. blood space in sub-mucosa stained deep blue (see text); eos.a. eosinophile area; mc.g. mucus gland; tacc. particles of iron saccharate; w.c. 1-4 wan- dering cells. The wandering cells (ic.c. 1, w.c. 3, w.c. 4) throughout the epithelium and subjacent tissues are seen to contain particles of ingested iron (sacc). Blood cells (b.c.) are common in the epithelium and submucosa. They are laden with iron, which some of them appear to be actively excreting into the gut. In sections of specimens fixed after a longer interval, there was an increase in the number of wandering cells that were apparently discharging particles of iron into the lumen of the gut, and there was a much smaller number of wandering cells in the epithelium and subjacent tissues. In the specimens fixed after 19J hours, there was still a large amount of saccharate in the haemocoele, and the wandering cells in the lumen of the gut still appeared to 410 NORMAN MILLOTT be actively discharging iron saccharate. Thus specimens treated in the above manner indicate that iron saccharate begins to appear in the gut after 11 hours, and is still being discharged by the wandering cells in the lumen of the gut after 19^ hours. In view of the varying manner in which the injected animals responded to their treatment, as shown by their varying degrees of recovery, it was not thought advisable to attach any significance to the relative quantities of iron saccharate appearing in the gut at the various stages between i.\ and 19J hours.