Semper's organ, a cephalic gland in certain gastropods

By NANCY J. LANE

(From the Cytological Laboratory, Department of Zoology, University Museum, Oxford.Present address: Department of Pathology, Albert Einstein College of Medicine, Yeshiva

University, New York 61, N.Y., U.S.A.)

With z plates (figs. 4 and 5)

SummarySemper's organ has been studied and compared in two genera, Limax and Helix. Inboth cases the organ, which lies on each side of, and beneath, the buccal mass, iscomposed of glandular elements surrounding ganglia; the latter originate from twonerves arising from each side of the cerebral ganglia. In both, the organ produces theexternal swellings that form the mouth lobes on each side of the head. The chiefdifference between the two is that Semper's organ is much larger in Limax. In Helixit is reduced to strands of secretory and nervous tissue, dispersed among abundantmuscle-fibres.

In H. aspersa the glandular area surrounding the ganglionic part of the organ iscomposed of three cell-types. The cytoplasm of the chief cellular component,'Semper's' cells, contains a number of lipochondria, some of which possess the cortexof concentric lamellae typical of phospholipid globules. The cytoplasm of the secondcell-type, or 'mucus' cell, is filled with electron-lucent globules between which arescattered electron-dense bodies that contain some polysaccharide and lipid. The thirdcell-type, the reticulate cell, is rather similar to the mucus cell, except that its cytoplasmcontains a reticulum formed by strands which lie within, or applied to, the electron-lucent globules; these cells contain acid mucopolysaccharides. The structure of themucus and reticulate cells bears certain resemblances to secretory cells in the sheathof the optic tentacles in the same species.

The function of Semper's organ is discussed. Certain evidence suggests that someof its secretory cells may have an endocrine activity, and the significance of lipochon-dria in such actively secreting invertebrate cells is considered.

IntroductionI N 1856 Semper described glandular lobes in the cephalic region of certainpulmonates, since then referred to as 'Semper's organ'. The componentparts of these lobes, lying beside the buccal mass in close association withthe tentacles, have subsequently been reappraised and assigned to differentcategories, nervous and glandular (Eckardt, 19*14). Semper's organ variesin size in different pulmonates, being largest in the slug Limax (Semper,1856).

Investigators have studied the arrangement and anatomical details ofSemper's organ in different genera, and many have put forward suggestionsas to its possible function (Flemming, 1869; Leydig, 1876; Simroth, 1876,1891; Sochaczewer, 1881; Sarasin, 1883; Hanitsch, 1888; Babor, 1895;Tauber, 1900; Beutler, 1901; Keller, 1902; Hackel, 1911; Meisenheimer,1912; Eckardt, 1914; Hoffman, 1925; Bronn, 1928; Rotarides, 1929; Baecker,1932). In a preliminary examination I found that both in Semper's organ[Quart. J. micr. Sci., Vol. 105, pt. 3, pp. 331-342, 1964.]

332 Lane—Semper's organ

and in the tentacles of the Stylommatophora, secretory cells are arrangedaround ganglia; further, certain cells in Semper's organ resemble some ofthe secretory cells in the optic tentacles (Lane, 1962). This similarity tocells in the tentacular sheaths has also been mentioned by several earlierworkers (Eckardt, 1914; Bronn, 1928; Baecker, 1932). Since the cells in theoptic tentacles have been found to produce a hormonal effect on the ovotestis(Pelluet and Lane, 1961), it seemed worth while to examine Semper's organin greater detail, to determine to what extent the structure of its componentcells paralleled that of the tentacular secretory cells and whether its cellswere also responsible for the production of secretions which might behormonally active.

No previous histochemical or detailed morphological investigation appearsto have been made of the cells that compose the glandular portion of Semper'sorgan.

Material and methodsThe animal chiefly used in this study was the common garden snail, H.

aspersa Miiller, which was employed throughout all the histochemical andelectron microscopical investigations. The slug Limax flavus Linnaeus wasalso used to make a comparison between the structure of Semper's organ inthe two genera. Serial paraffin sections were made of the entire head regionof H. aspersa, which had been fixed with the tentacles extended; thesesections were stained with haematal 8/Biebrich scarlet (Baker, 1962) in orderto ascertain the organization and localization of Semper's organ. For thehistochemical studies, either Semper's organ or the whole cephalic area wasremoved and fixed in Helly's solution or formaldehyde-calcium followed bypost-chroming (Baker, 1944); the tissue was then embedded in paraffin orgelatine respectively. The histochemical tests performed, with the detailsof preparation, are summarized in the appendix (p. 342).

Tissues for the electron microscopical study were fixed in Palade's bufferedosmium solution (1952), dehydrated in an ascending series of alcohols, treatedwith propylene oxide, and embedded in araldite according to Luft's specifica-tions (1961). Sections showing silver or gold interference colours were cuton the Huxley ultramicrotome and mounted on uncoated or formvar-filmedgrids. In some cases the sections were stained with potassium permanganatefor 10 min before inspection, to increase the contrast (Lawn, i960). Thespecimens were examined in an Akashi electron microscope (model TRS50EI).

Araldite sections were also cut at 0-25 /x on the ultramicrotome, placed onpieces of glass coverslips, and dried. After being stained for 1 min in 1%toluidine blue borax solution and differentiated with 50% ethanol, they weremounted on slides under liquid paraffin (Meek, 1963). These preparationswere examined by light microscopy in order to establish the presence of thecells comprising Semper's organ, and to determine their localization in thesection, before the thinner serial sections were examined under the electron

Lane—Semper's organ 333

microscope. This precaution is especially necessary in H. aspersa, becausethe cells of the gland are dispersed amongst muscle-fibres and often do notappear in sections.

ResultsStructure of Semper's organ

The description of Semper's organ by Eckardt (1914) has been acceptedby most subsequent workers as definitive. In a study of Limax and Agrio-limax he concluded that Semper's organ consisted of diverse component

labial ganglia lying beneath buccal mass

inferior tentacle ^ buccal mas:

Semper's organ

area oftentacul,ganglion

oesophagus

optic \ cerebral commissu

cerebral ganglion

optic tentacle retracted

to both Semper's orga,\nd inferior tentacle

bands of muscle

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FIG. I. Diagrams of dissections, viewed dorsally, made on one side of the cephalicregion in A, Limax flavus and B, H. aspersa, illustrating the arrangement of Semper'sorgan in relation to the inferior and optic tentacles, brain, and buccal mass. Thecephalic area of the snail in B contains the same organs as those labelled for the slugin A, with the exceptions noted. None of the other nerves arising from the cerebral

ganglion are included in this diagram.

parts: 'lip ganglia' and 'mouth lobe ganglia', surrounded by packets andlayers of unicellular glands which form the 'mouth lobe glands'. Eckardtconsidered that the mouth lobe ganglia were innervated by thick nervesthat arose from each side of the cerebral ganglia and ran to the inferiortentacles. He also found that certain superficial cells in the glandular packetsdischarged their products externally, and he considered that they therebyfunctionally supported the lateral belt of the foot glands; he was, however,not able to discover for the other, non-superficial, cells, any ducts to theexterior or to the digestive tract.

My findings are on the whole in agreement with those of Eckardt. Forexample! I find that some, but not all, of the cells in the glandular portion ofSemper's organ have ducts to the external surface, and none have ducts to

334 Lane—Semper's organ

the digestive tract. In Limax, however, in the animals I have examined, Ihave found 2 nerves arising side by side from each cerebral ganglion, ofwhich one runs to the inferior tentacle, and the other to the lobes ofSemper's organ (fig. 1, A), albeit there is a branch from the latter nerveacross to the inferior tentacle.

mouthlobe

FIG. 2. Diagram to illustrate an anterior external view of the cephalic region ofH. aspersa.

I have also found 2 other smaller nerves arising from each side of thecerebral ganglia just beside those innervating the tentacles and the lobes ofSemper's organ; each of these runs ventrally under the buccal mass toterminate in a small labial ganglion (fig. 1, A), around which lie cords ofsecretory tissue. These no doubt correspond to Eckardt's Lippenganglien,and the 2 main lobes on each side of the buccal mass (fig. 1, A) may beidentified with his mouth lobe ganglia and glands. The mouth lobe gangliaand glands would appear to produce the external swellings of the mouthlobes, one on each side of the head between the inferior tentacles and the foot(similar to the arrangement in fig. 2).

In Limax, Semper's organ can easily be seen and dissected out as a whiteor white-grey mass. In Helix it is not so immediately evident, for it is muchsmaller. It lies in the same site, but the organ is restricted to a small area at

Lane—Semper's organ 335

the end of a number of muscular strands, situated just beside the point wherethe sheath of the inferior tentacles arises (figs. 1, B ; 4, A). The gland, therefore,is not readily dissected out, and the cells can most easily be located in serialsections of the whole cephalic area. Externally, however, the mouth lobes,within which the gland cells and ganglia are situated, are quite extensiveswellings (fig. 2).

llpid granules

FIG. 3. Diagram to illustrate the differences between the three secretory cell-typesthat make up Semper's organ in H. aspersa, as they appear after staining withchrome-haematoxylin-phloxine. A, Semper's cell; B, mucus cell; c, reticulate cell.

The structure of Semper's organ in Helix differs from that in Limax, then,as shown in fig. 1. Not only is the organ smaller, but the muscular strandsbetween which its secretory cells lie continue in a posterior direction to forma muscle band that joins up with the retractor muscle of each optic tentacle(fig. 1, B). This does not occur in Limax. Further, the innervation of theorgan differs, in that in Helix only one nerve arises from the cerebral ganglionto supply both Semper's organ and the inferior tentacle (fig. r, B).

In addition to the numerous nerve-fibres from the ganglia running amongthe secretory cells of Semper's organ, there is an extensive vascular supply. Inone section from H. aspersa, the duct of a cell from the organ appeared torun between the epithelial cells directly into a blood-vessel (fig. 4, c).

At the ultrastructural level, the surface of the mouth lobe area in H.aspersa is covered with a cuticle composed of microvilli, similar to the cuticleon the optic tentacles and the dorsal body surface (Lane, 19636). In themouth lobe and cephalic area generally, unicellular calcium glands are presentbeneath the surface epithelium, like those in the optic tentacles (Lane,1963c). Ultrastructurally these appear to correspond to cells composed ofvast quantities of spheroids and ellipsoids of varying size, having a concentriclayered structure (probably the form taken by the calcium salt of which theyare composed).

Types of cell in Semper's organ

In H. aspersa, three different cell-types are found in the glandular area ofSemper's organ; these cells lie around the ganglia of the organ and thenerve-fibres arising from them. It is difficult to delimit the glandular portionproper, as many of the cells are drawn out into long processes and lie between

336 Lane—Semper's organ

the nerve and muscle-fibres in cords or packets (fig. 4, A). The cells which Ipropose to describe form what could be considered the mouth lobe gland area,in that they lie around the ganglionic regions and nerve-fibres on each sideof the buccal mass. Other secretory cells, lying beneath the mouth cavityand forming the glands of the lip ganglia, appear to be similar to the cell-types to be described, except that the cells corresponding to the third cell-type occur only infrequently.

The chief cell-type, which comprises the bulk of the glandular area, will bereferred to as 'Semper's' cells. They are ellipsoidal and measure up to 30 fxin length and 15 ju. in width. Their cytoplasm contains a great many spheroi-dal or ellipsoidal granules from 1 to 2 /A in diameter (figs. 3, A; 4, B). In somecases vacuoles, 1 to 2 /x. in diameter, are also present. The nuclei of Semper'scells are spheroidal, up to 9 ju, in diameter; often, however, they are indis-tinguishable, completely obscured by the quantities of granules in the cell.The duct that entered a blood-vessel directly, as described earlier (fig. 4, c),contained granules similar to those in Semper's cells; a number of these cellslay around the blood-vessel and presumably the duct arose from one of them.A few of Semper's cells have ducts running to the external surface, but mostof them do not appear to do so.

The second cell-type is frequently found lying between Semper's cells,but, in serial sections, it can also be seen in abundance in the area of the footgland. I shall refer to these cells provisionally as 'mucus' cells, since theirstructure is rather similar to that of some vertebrate mucus cells. They areabout 33 fx in length and 16/x in width, with a spheroidal nucleus of about8 JU in diameter. These mucus cells contain large quantities of spheroidal orellipsoidal globules, 1 to 2 fx in diameter, which are refractile and, in contrastto the inclusions in Semper's cells, take up few dyes (figs. 3, B; 4, D). Insome preparations a number of smaller granules can be seen lying betweenthese globules.

The cells of the third type, in or associated with the glandular area ofSemper's organ, I shall term reticulate cells, because they contain a reticularmaterial that can only be with difficulty resolved into globular inclusions

FIG. 4 (plate). All the micrographs shown in this figure are from sections of Semper'sorgan in H. aspersa.

A, a section through Semper's organ, showing how its secretory cells (sc), lying amongmuscle-fibres (mf), are found close by the sheath of the inferior tentacle (inf). Note thenerve (ne) from the cerebral ganglion running into the organ, as well as blood-vessels (bv).Helly/haematal 8/Biebrich scarlet.

B, a Semper's cell (s), the chief cellular constituent of Semper's organ. Note the spheroi-dal and ellipsoidal granules (g) which almost fill the cytoplasm. Brasil/chrome-haematoxylin-phloxine.

c, a cellular process (p), apparently from a Semper's cell, running directly into the lumen (/)of a blood-vessel (br). Helly/haematal 8/Biebrich scarlet.

D, mucus cells (me) located alongside Semper's cells (s). n, nucleus; p, process; gl, globules;gr, granules lying between the globules. Brasil/chrome-haematoxylin-phloxine.

E, a reticulate cell (re) situated on the periphery of Semper's organ, Note the globules (g/)with strands (st) lying upon them or inside them, gr, smaller granules scattered among theglobules; p, cellular process. Helly/haematal 8/Biebrich scarlet.

FIG. 5

N. J. LANE

Lane—Semper's organ 337

(from less than 1 to 3 or more fj. in diameter) and strands that either lie in, orare superficially applied to, the globules (figs. 3, c; 4, E). Smaller granularbodies lie between the globules and strands, but they cannot always beresolved as separate from the other two components. The reticulate cellsthemselves tend to be ellipsoidal and measure approximately 40 /x in length.They have spheroidal nuclei that are usually obscured by the cytoplasmicinclusions. These cells often lie at the periphery of the area in which theSemper's and mucus cells are found. In addition, they are most extensivelylocated in the region near the foot and in some cases have long ducts thatrun to the surface of the sole, opening between the cilia. These reticulatecells, therefore, may not form part of Semper's organ proper, but are foundsufficiently often between and near the other two cell-types to warrantinclusion in a description of the glandular area.

Histochemistry and fine structure of the secretory inclusions

The results of the histochemical tests applied to the cells which composethe glandular area of Semper's organ are to be found in the appendix (p. 342).

The granules in the cytoplasm of Semper's cells respond positively to testsfor carbohydrate and for lipids in general, and also, in most cases, to a testfor phospholipids; this suggests that they may be partially composed ofcerebroside. These secretory granules, or another cytoplasmic componentwith a similar distribution, react positively to tests for 'neurosecretory' sub-stances. Ultrastructurally, the granules appear to correspond to spheroids ofvarying electron-density and diameter (fig. 5, A, C) that contain electron-dense granules and lamellae which are in some cases arranged concentricallyin the cortex (fig. 5, B). Although the granules are fairly numerous withineach cell, there is a good deal of cytoplasm between them which containsmembranes, mitochondria, vesicles, and smaller granules (fig. 5, A, C).

The globules of the mucus cells are very non-reactive histochemically, butmay contain slight amounts of carbohydrate. Under the electron microscope,they are electron-lucent, and are either empty or contain only a faint granularsubstance; very little cytoplasm lies between them (fig. 5, E). Occasionally,

FIG. S (plate). All the electron micrographs shown in this figure are from Semper's organof H. aspersa; the tissues have been fixed in Palade's buffered osmium and embedded inaraldite.

A, a cell from the region of Semper's organ that appears to be a Semper's cell. Note themuscle-fibres (mf) which lie adjacent to it and the lipochondria (Ip) within its cytoplasm.gr, smaller granules; v, vesicles; m, mitochondria; mb, membranes; n, nucleus of a muscle-cell.

B, a lamellated granule, or phospholipid globule (pip), from a Semper's cell, whose lamellaehave been somewhat disrupted from their concentric arrangement. Ip, lipochondrion; gr.granule.

C, lipochondria (Ip), showing different degrees of electron-density, in the cytoplasm of aSemper's cell, tr, triglyceride droplets; n, nucleus; v, vesicles.

D, an electron-dense body (edb) from the cytoplasm of a mucus cell, gl, globule.E, an area of cytoplasm from a mucus cell in Semper's organ, containing electron-lucent

globules (gl). cy, ground cytoplasm.F, a reticulate cell from Semper's organ containing electron-lucent globules (gl) in its

cytoplasm (cy). Note the electron-dense granules and strands (st) lying in the globules.

338 Lane—Semper's organ

amorphous, electron-dense bodies are found dispersed between the globules(fig. 5, D); these may represent the smaller granules seen scattered amongthe globules under the light microscope. The smaller granules contain, inaddition to some lipid, rather more polysaccharide than the globules, and theytake up the 'neurosecretory' stains. However, it is difficult to determine theirtrue nature, for they are not visible by light microscopy in all preparations,and their size and shape vary a great deal in electron micrographs.

In the reticulate cells, sulphated mucopolysaccharides are present, andboth the globules and the strands contain acid mucopolysaccharides; somelipid is present in the strands and scattered granular bodies. The fine struc-ture of these cells seems to differ only slightly from that of the mucus cells,though the proximity of the two cell-types makes it impossible to distinguishthem with certainty under the electron microscope. However, one cell-typecontains, in addition to electron-lucent globules and scattered amorphouselectron-dense bodies, a number of granular lumps and strands of highelectron density that lie within or upon the electron-lucent globules (fig. 5, F).These strands, in my opinion, correspond to those seen in the reticulate cellsunder the light microscope.

DiscussionOver the years there has been confusion in the terminology of Semper's

organ. The external lobes beneath which the organ lies have been consideredto represent a third pair of Fiihler (tentacles) (Leydig, 1876). They have beentermed Mundlappen by some investigators (among them Semper, 1856;Eckardt, 1914; Hoffman, 1925) and Lippen by others (Simroth, 1876). Thelobes of the organ have often been confused with the papillae of skin aroundthe entrance to the mouth. However, to avoid the possibility of confusingthe other tentacles or the lips round the entrance to the mouth for Semper'sorgan, it seems best to use the term 'mouth lobe' for the external swellingsformed by it. As described earlier, Eckardt's (1914) division of the organinto mouth, lobe ganglia, mouth lobe glands, and labial ganglia in Limaxseems to be established as accurate (Bronn, 1928; Baecker, 1932), thoughthere are variations in different genera, as for example in Helix, which I havealready described, in Paralimax (Tauber, 1900), or in Amalia (Bronn, 1928).

There has also been a controversy as to the actual nature and function ofthe cells comprising the glandular portions of Semper's organ. Semperhimself considered the secretory cells to form a Geruchsorgan. Others havevariously described it, like Semper, as a gustatory sense organ, a Gesch-macksorgan (Simroth, 1876), or as a mixed sensory and glandular organ(Hanitsch, 1888), as a purely glandular organ (Sochaczewer, 1881), as a slimegland pouring secretions on to the surface of the head (Rotarides, 1929), asa pharyngeal salivary gland opening into the digestive tract (Babor, 1895;Meisenheimer, 1912), or as a connective tissue element (Tauber, 1900;Bronn, 1928). Eckardt (1914) considered that the glandular cells servedduring gliding as padding and protection for the ganglia.

Lane—Semper's organ 339

Although the ganglionic component of Semper's organ certainly suggestsa sensory function, I have no definite evidence bearing on this. Some of thesuperficial cells of the glandular component have an exocrine secretoryfunction, particularly the reticulate cells that lie on the side of the organ nearthe foot. It is improbable that the hypothesis of Tauber (1900) and Bronn(1928) is correct, for not only do some of the gland cells have obvious ducts,but they do not lie in a matrix that is demonstrably composed of mucopoly-saccharides, like the ground substance of connective tissue. In addition, likeother investigators (Semper, 1856; Eckardt, 1914; Bronn, 1928), I have notfound any excretory ducts from Semper's organ entering the digestive tract,so that it cannot be a salivary gland. Since there is an extensive blood supply,it is possible that those cells in Semper's organ that do not have externalducts empty their secretory products into the blood-stream, perhaps directly.If this is so, the cells form an endocrine gland. It is noteworthy that Quattrini(1956; 1957) has found that the amounts of DNA in the nuclei of the cells ofSemper's organ are variable in a cyclic manner that is connected with a cyto-plasmic secretory cycle. Such results are also suggestive of endocrine activity.

Certain cells in Semper's organ bear similarities to the cells of the footgland (Eckardt, 1914), to which area sensory (Hanitsch, 1888) and perhapsolfactory (Sochaczewer, 1881) functions have also been ascribed. Campion(1961) described cells in the pedal gland of H. aspersa which had, at least intheir ducts, a 'reticular' structure; these she found similar to those termed the'sole-glands' by other investigators. The cells, as she characterizes them, arevery like the reticulate cells of Semper's organ, which, as I have alreadysuggested, form at least part of the foot gland. Another indication of theidentity of the reticular cells in the foot with those in Semper's organ is thedifficulty Bronn found (1928) in deciding whether the 'upper lip gland'described by Hackel in Chilina corresponds to a mouth lobe gland or to a footgland.

The cytological similarities between the secretory cells in Semper's organand the Spindeizellen in the sheath of the optic tentacles (termed the 'lateral'cells by myself, 1962) have been noted previously by Eckardt (1914) andBaecker (1932). Both groups of cells are characterized by a lack of excretoryducts (Bronn, 1928). The mucus and reticulate cells in Semper's organ bearstrong resemblances to the lateral oval and lateral processed cells of the optictentacles respectively, both by light and electron microscopical methods(Lane, 1962; 1963c), although there are certain differences. Ultrastructuralsimilarities are to be found between both the lateral cells of the tentacles andthe mucus and reticulate cells on one hand, and certain vertebrate mucus cellson the other (Lane, 1963c; Florey, i960; Shearman and Muir, i960; Palay,1958; Rhodin and Dalhamn, 1956). In particular, electron-dense bodies ofsimilar structure (as in fig. 5, D) are found in all the afore-mentioned cell-types.In the mucus and reticulate cells, and the tentacular cells, these bodies con-tain lipid; in the latter, they are also the sites of acid phosphatase activity,which suggests that they may correspond to lysosomes (Lane, 1963d).

340 Lane—Semper''s organ

The granular inclusions in Semper's cells contain carbohydrate and lipid,with a partial phospholipid component. In correspondence, their finestructure is similar to that of lipochondria such as are found, for example, inthe neurones of Helix (Chou and Meek, 1958; Dalton, i960; Meek and Lane,1964), and of Locusta (Ashhurst and Chapman, 1962). In some cases thegranules resemble non-crenated triglyceride droplets or mixed lipid globules(fig. 5, c); in others they contain the concentric cortical lamellae typical ofphospholipid globules (Chou and Meek, 1958; Mercer, 1962; Meek andLane, 1964). In spite of the fact that at the light microscopical level the'neurosecretory' stains are taken up by Semper's cells, there is no evidence ofthe presence of elementary neurosecretory granules.

Semper's cells are arranged around a ganglionic centre in a way verysimilar to the collar cells round the ganglion of the optic tentacle. In spite ofthis resemblance, in a comparison with the collar cells it is evident thatSemper's cells do not correspond structurally or cytochemically to them, norto any of the other tentacular cells. Rather than a neurosecretory endocrinecomplex like that formed by the collar cells, Semper's cells (or at least some ofthem) may form a purely glandular endocrine organ.

The cyclic secretory activity of Semper's cells (Quattrini, 1956; 1957),combined with the fact that their chief inclusions are in the form of lipo-chondria, is noteworthy in view of recent investigations (Lane, 1963a, d\Meek and Lane, 1964), which have shown that lipid globules in invertebratesecretory and neurosecretory cells possess a complex of diverse enzymes, andmay, therefore, play a prominent role in cellular activity. The presence ofmany lipochondria in an active secretory cell such as Semper's cell may befurther evidence of the importance of lipochondria in invertebrate cells.

I am indebted to Dr. J. R. Baker, F.R.S., for his supervision during thecourse of my work on gastropod cytology, and to Professor J. W. S. Pringle,F.R.S., for accommodation in his Department. The Huxley ultramicrotomewas provided by the Royal Society, and the Akashi electron microscope bythe Wellcome Trustees (grants to Dr. J. R. Baker). I wish to thank theNational Research Council of Canada; this research was carried out duringthe tenure of their N.A.T.O. Science Scholarship.

ReferencesAshhurst, D. E., and Chapman, J. A., 1962. Quart. J. micr. Sci., 103, 147.Babor, J. F., 1895. S. B. bohn. Ges. Wiss., 34, 1.Baecker, R., 1932. Z. ges. Anat., 29, 449.Baker, J. R., 1944. Quart. J. micr. Sci., 85, 1.

1946. Ibid., 87, 441.1949. Ibid., 90, 293.1956- Ibid., 97, 621.1962. Ibid., 103, 493.

Beutler, B., 1901. Zool. Jb., Abt. 2, 14, 369.Bronn, H. G., 1928. In Klassen und Ordungen des Tier-Reichs. Bd. Ill , Mollusca, Abt. 2,

ii. Leipzig (Akademische Verlagsgesellschaft).Campion, M., 1961. Quart. J. micr. Sci., 102, 195.

Lane—Semper's organ 341

Chou, J. T. Y., and Meek, G. A., 1958. Ibid., 99, 279.Dalton, A. J., i960. In Cell physiology of neoplasia (M. D. Anderson Hospital and Tumor

Institute). Austin (University of Texas Press).Eckardt, E., 1914. Jena. Z. Naturw., 51, 211.Flemming, W., 1869. Arch. mikr. Anat., 5, 415.Florey, H. W., i960. Quart. J. exp. Physiol., 45, 329.Gomori, G., 1941. Amer. J. Path., 17, 395.Hackel, W., 1911. Zool. Jb., Suppl. 13 (Fauna Chilensis 4), 89.Hanitsch, R.( 1888. Proc. Biol. Soc, Liverpool, 2, 152.Heath, I. D., 1961. Nature, Lond., 191, 1370.

1962. Quart. J. micr. Sci., 103, 457.Herlant, M., 1958. Arch. Anat. micr. Morph. exp., 47, 1.Hoffman, H., 1925. Jena. Z. Naturw., 61, 1.Keller, W., 1902. Zool. Jb., Suppl. 5 (Fauna Chilensis 2), 607.Lane, N. J., 1962. Quart. J. micr. Sci., 103, 211.

1963a. Ibid., 104, 401.19636. Ibid., 104, 495.1963c. Ibid., 105, 35.1963^. Ibid., 105, 49.1963 .̂ Ibid., 105, 61.

Lawn, A. M., i960. J. biophys. biochem. Cytol., 7, 197.Leydig, F., 1876. Arch. Naturg., 42, 209.Luft, J. H., 1961. J. biophys. biochem. Cytol., 9, 409.McGee-Russell, S. M., 1955. As in Pearse, 1954.McManus, J. F. A., 1948. Stain. Tech., 23, 99.Meek, G. A., 1963. J. R. micr. Soc, Proc. Symposium on Cytochemical Progress in Electron

Microscopy, 81 (3 and 4), 184.and Lane, N. J., 1964. J. R. micr. Soc, 82, 193.

Mercer, E. H., 1962. In The interpretation of ultrastructure, Symposium of InternationalSociety for Cell Biology, vol. 1, edited by Harris. London (Academic Press).

Meisenheimer, J., 1912. Monogr. einheim. Tiere, Bd. 4, Leipzig, 1.Palade, G. E., 1952. J. exp. Med., 95, 285.Palay, S. L., 1958. The morphology of secretion. In Frontiers in cytology, edited by Palay.

New Haven (Yale University Press).Pearse, A. G. E., 1954. Histochemistry, theoretical and applied. London (Churchill).Pelluet, D., and Lane, N. J., 1961. Canad. J. Zool., 39, 789.Quattrini, D., 1956. Boll. Zool., 23 (2), 679.

1957. Ibid., 24 (2), 243.Rhodin, J., and Dalhamn, T., 1956. Z. Zellforsch., 44, 345.Rotarides, M., 1929. 10 Intern. Zool. Kongr., Budapest, 2, 952.Sarasin, P. B., 1883. Wiirzburg. Zool. Arb., 6, 91.Semper, C, 1856. Z. wiss. Zool., 8, 366.Shearman, D. J. C, and Muir, A. R., i960. Quart. J. exp. Physiol., 45, 337.Simroth, H., 1876. Z. wiss. Zool., 26, 227.

1891. Zool. Jb., Suppl. 2, Bd. s, 861.Sochaczewer, D., 1881. Z. wiss. Zool., 35, 30.Tauber, H., 1900. Annu. Mus. Zool. Acad. St. Pe'tersb., 5, 373.

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ts o

btai

ned

Tes

t

PAS

..

..

PA

S af

ter

dias

tase

PAS

afte

r hy

alas

eH

eath

's

for

sulp

hate

dm

ucop

olys

acch

arid

esm

etac

hrom

asia

(to

luid

ine

blue

, i %

aq.

) .

chro

me-

haem

atox

ylin

-ph

loxi

ne .

alci

an b

lue

.ha

emat

al 8

/Bie

bric

hsc

arle

tnu

clea

r fa

st

red

for

calc

ium

.

Suda

n bl

ack

B

.

acid

hae

mat

ein

acid

ha

emat

ein

wit

hpy

ridi

ne e

xtra

ctio

n .

He

He

He

He

Bra He

He

He

F/C

a +

PC

F/C

a +

PC

WB

+ P

E

Sem

per'

s ce

ll

Ref

eren

ce

McM

anus

(19

48)

Pea

rse

(195

4)Pe

arse

(19

54)

Hea

th (

1961

, 19

62)

Bak

er (

unpu

blis

hed)

Gom

ori

(194

1)

Her

lant

(19

58)

Bak

er (

1962

)

McG

ee-R

usse

ll (1

955)

Bak

er

(194

4,

1949

,19

56)

Bak

er (

1946

)B

aker

(194

6)

Ret

icul

ate

cell

+ +

red

+ /o

+ /o

03

KE

Y:

Bra

=

Bra

sil;

F/C

a +

PC

= f

orm

alde

hyde

/cal

cium

wit

h po

stch

rom

ing;

G =

ge

lati

ne; H

e =

Hel

ly;

P =

pa

raff

in;

PA

S =

pe

riod

ic a

cid/

Schi

ffte

st;

WB

+ P

E =

wea

k B

ouin

wit

h py

ridi

ne e

xtra

ctio

n .

+ +

+

= s

tron

g re

acti

on;

++

=

m

oder

ate

reac

tion

; +

=

wea

k re

acti

on;

+/

+ +

=

im

pos -

sibl

e to

det

erm

ine

whe

ther

a w

eak

reac

tion

or a

mod

erat

e re

acti

on;

+/o

=

im

poss

ible

to

dete

rmin

e w

heth

er a

wea

k re

acti

on o

r no

rea

ctio

n ; o

=

nore

acti

on;

— =

no

t vi

sibl

e; e

xt.

=

reac

tion

in t

he c

orte

x of

the

inc

lusi

on;

int.

=

reac

tion

in

the

inte

rnum

of

the

incl

usio

n .