271
A study of the replacement mechanism of the
pulmonate radula
By N. W. RUNHAM
(From the Department of Zoology, University College of North Wales,
Bangor, Caernarvonshire)
With i plate (fig. 2)
Summary
Results obtained from a histological, autoradiographic, and operative investigation
of the radulae of two pulmonates have led to the formulation of the following theory
of radula replacement. The radula is secreted continually by the permanent odontoblasts and continually moves anteriorly. The superior epithelium of the radular
gland is produced by division of cells near the odontoblasts and moves forward at the
same rate as the radula; the cells produce materials that harden the radula and then
die. The inferior epithelium is also produced by division of cells near the odontoblasts, but initially moves anteriorly at only a quarter of the rate of the radula. The
epithelium then changes shape from a columnar to a pavement epithelium. It is
suggested that it then moves forward at the same rate as the radula. The pavement
epithelium secretes the subradular membrane which may serve to attach the epithelium to the radula, and the supralateral radula tensor muscles are attached to the
basement membrane of this epithelium. At the extreme anterior end of the radula
the inferior epithelium becomes again columnar and secretes a cuticle continuous
with the buccal cuticle. This process detaches the subradular membrane and the
radula from the epithelium. The epithelial cells then die and disintegrate. The
detached radula and subradular membrane show a change of staining properties
suggestive of reversal of the tanning, and they then break up.
Introduction
T H E R E have been many theories about the replacement mechanism of the
molluscan radula, particularly in the older German literature. This older
work has been excellently summarized by Market (1958), Hubendick (1945),
and more recently by Fretter and Graham (1962), so that no attempt to do
so will be made here. The above-mentioned authors agree that the radula
is probably secreted by the odontoblasts, is altered chemically and physically
by secretions from the superior epithelium of the radular gland, is replaced
continually, moves forward continually into the buccal cavity, and is eliminated in some way. Markel has confirmed previous observations that there
is a subradular membrane beneath the older radula, and he also distinguished
a fibrous layer under that. A diagram of a sagittal section through a typical
pulmonate radula is given in fig. 1.
The belief that the radula moves forward continually has been based on
indirect evidence—Markel's from a study of the histology of the radular
gland, and Hubendick's from a study of the growth of the radula. Recently
the author has produced direct evidence for a continual replacement in the
[Quart. J. micr. Sci., Vol. 104, pt. 2, pp. 271-7, 1963.]
272
Runham—Replacement
of pulmonate radula
radula of Lymnaea stagnalis (Runham, 1962). By cauterizing the tip of the
radula gland to destroy some odontoblasts and a small part of the radula
and then killing the animals at intervals after the operation, it was possible
to show that the radula moves forward, and that it does so at the rate of
approximately 2-9 rows a day. The present study is concerned with the
mechanism of this continual replacement.
FIG. 1. A diagram of a sagittal section through the radula and associated structures of a
typical Pulmonate.
Material and methods
Fully grown L. stagnalis and Helix pomatia were purchased from L. Haig
& Co. Ltd., Surrey. The animals were decapitated and the radulae were
dissected out, fixed in either Zenker's or Clarke's fixative, embedded in ester
wax, and sectioned at 8 //.. Sections were stained in Mallory's triple stain
or Heidenhain's iron haematoxylin.
For autoradiography, fully grown L. stagnalis were each injected with
10 pc of tritiated thymidine (Radiochemical Laboratories, Amersham) and
at various intervals the radulae were dissected out and fixed in Clarke's
fixative and sectioned as above. Kodak AR10 stripping film was applied to
the dewaxed slides, which were then exposed in a light-tight box at 4 0 C
for 6 weeks. The slides were developed and stained by Belanger's (1961)
basic fuchsin technique, with the substitution of Kodak Digb for his diluted
D19 developer.
Runham—Replacement of pulmonate radula
273
The numbering of the rows of teeth in the radula starts in all cases from
the most recently formed row: in other words, the rows are numbered from
behind forwards.
Results
Markel (1958) has given a very accurate description of the histology of the
radula and radular gland of H. pomatia and L. stagnalis, with which I am
in almost complete agreement. He omits, however, to observe the important
changes that occur in the superior epithelium.
In H. pomatia, at the lower posterior end of the radular gland, there is a
group of large cells, the odontoblasts. Beneath the radula there is an epithelium called the inferior epithelium. At the posterior end of the radular
gland its cells are tall and columnar, 70 /J, in height; while under the functional
anterior part of the radula, from about the 115th tooth row, there is a sharp
transition to a pavement epithelium only 8 /x in height. The supralateral
radula tensor muscles (Carriker, 1945) are attached to these cells by the
basement membrane. Under the very oldest part of the radula, at about
the 170th tooth row, the cells again form a columnar epithelium 70 fj. in
height. Finally, they appear to disintegrate at the point where they meet
the epithelium underlying the general buccal cavity cuticle (fig. 2, D). The
epithelium above the radula—the superior epithelium—is a tall columnar
epithelium whose cells are of irregular height as they interdigitate with the
teeth. Between the 22nd and the 42nd row some of the nuclei of this epithelium stain darkly (fig. z, A), and from the 42nd row these nuclei clump
together in groups of about 10 nuclei (fig. 2, B), eventually disintegrating to
form an intensely staining mass (fig. 2, B). About 70% of the cells disintegrate
in this way, while the remainder form a low epithelium which secretes a
cuticular layer clearly disengaging the superior epithelium from the underlying radula (fig. 2, c). At about the 92nd row these remaining cells apparently disintegrate (fig. 2, c) where they abut on to the epithelium which is
continuous with that of the collostylar hood (Carriker, 1945). This epithelium lies above the radula until the 112th row, when it is reflexed from the
radula; it secretes a cuticle which is thin above the radula and then forms
the very thick cuticle typical of the collostylar hood. At the 115th row, where
the inferior epithelium forms a pavement epithelium, a subradular membrane
is formed beneath the radula by the inferior epithelium cells. Markel describes a fibrous layer beneath the subradular membrane; while I find this
layer in wax embedded material I do not find it in cryostat sections of fresh
material, so that I suspect it to be an artifact.
The histology of the radular gland of L. stagnalis is very similar to that
of H. pomatia, but the subradular membrane is proportionately much
thinner and there are important differences in the superior epithelium. The
dying nuclei do not clump as in Helix but the dying cells form a fairly
complete layer above an epithelium formed by the remaining cells, and then
disintegrate, followed by the remainder of the cells of the superior epithelium.
274
Runham—Replacement of pulmonate radula
The epithelium of the collostylar hood does not extend into the radula gland
as in Helix.
As Markel has observed, in both these animals the only cell-divisions are
found in the regions of the inferior and superior epithelia nearest to the
odontoblasts.
The autoradiographic study was undertaken to investigate the movements of the epithelial cells. It has been shown that desoxyribonucleic acid
is metabolically stable so that by administering tritiated thymidine the
nuclei become specifically labelled and remain so for the life of the cell (e.g.
Quastler and Sherman, 1958). In fig. 3 the previously published results on
the movement of the actual radula of L. stagnalis (Runham, 1962) are compared with the autoradiograph results. It can be seen that the cells of the
superior epithelium move forward at the same rate as the radula (the operated
animal takes about 5 days to recover from the operation). The inferior
epithelial cells move forward but only at about a quarter of the speed of the
radula. No labelling of the odontoblasts was ever observed. A similar
experiment was undertaken to label the cells of the radular gland of H.
potnatia, but the animals hibernated soon after the beginning of the experiment and then no growth of the radula occurred.
Discussion
As Markel has emphasized, any theory of radula replacement must account
for the production, forward movement, and loss of the radula.
There is little disagreement that the odontoblasts secrete the substance of
the radula. There are, however, two opposing views on the permanence of
the odontoblasts. The majority view is that they are permanent, forming
the same longitudinal row of teeth throughout the life of the animal (e.g.
Gabe and Prenant, 1952; Markel, 1958); Pruvot-Fol (1926) on the contrary, maintains that there is a continual replacement of the odontoblasts,
Fie. Z (plate). Longitudinal sections through the radulae and radular glands of H. pomatia
and L. stagnalis. Zenker's fixative; 8/i; Heidenhain's iron haematoxylin.
A, H. pomatia. Superior epithelium above tooth rows 31 and 32. Some of the nuclei are
strongly stained and distorted.
B, H. pomatia. Superior epithelium above tooth rows 75 to 77. Behind the tooth of the
75th row some of the nuclei have clumped together, while behind the 76th row they have
broken down to form an intensely staining mass. The remainder of the cells form a low
epithelium.
c, H. pomatia. Superior epithelium above tooth rows 91 and 92. The remaining cells of
the epithelium disintegrate where they abut on to the epithelium and cuticle of the collostylar
hood (left). Note the cell debris.
D, L. stagnalis. Inferior epithelium beneath the n 6th tooth row. This is an enlargement
of E to show the breakdown of the cells of the epithelium where they abut on to the buccal
epithelium and its cuticle (right).
E, L. stagnalis. Anterior end of the radula. The worn teeth and tooth-free membrane can
be seen. The radula and subradular membrane have become detached from the inferior
epithelium. The epithelium and cuticle of the buccal cavity can be seen on the right. The
swelling of the subradular membrane has been exaggerated by fixation and embedding.
^rv*
Fir,. 2
N. \V. RUXHAM
Runham—Replacement of pulmonate radula
275
which become transformed into the superior and inferior epithelia of the
radular gland. The location of cell-divisions support, and the autoradiography results confirm the view, that the odontoblasts are permanent.
10
20
30
40
Days after start of experiment
50
60
FIG. 3. Movement of the radula and cells of the radular gland of L. stagnate, a, a, movement of the radula. The radula and radular gland were cauterised and the rate of formation
of new teeth rows determined, b, b, movement of the superior epithelial cells. The nuclei
of the cells were labelled with tritiated thymidine, and the rate of forward movement of the
cells determined, c, c, movement of the inferior epithelial cells. These cells were similarly
labelled and their rate of forward movement determined.
The inferior and superior epithelial cells of the radular gland are produced
in a region bordering the odontoblast layer, as has been shown by radioactive labelling. The superior epithelium moves forward at the same rate
as the radula, which is not unexpected when one considers the intimate
interdigitation of the cells with the teeth. During forward movement of the
superior epithelium the cells secrete materials which alter the chemical
composition and physical properties of the radula (Gabe and Prenant, 1958;
Runham, 1961). In the older superior epithelium about 70% of the cells
die initially, followed later by the remainder. The basement membrane of
the superior epithelium and the epithelium of the collostylar hood is continuous, but it is not known whether the superior epithelial cells move over
it or whether it is also continually replaced.
In these experiments the forward movement of the inferior epithelium
was followed for only 50 rows of teeth, but the cells radically alter shape at
about the 60th row. At the level of this change there is no evidence of cell
death, so that it is reasonable to suppose that the tall columnar cells, length
5 JU., become transformed into the flat cells of length 12 y,. If this is so, the
cross-sectional area of the cells—assuming that they are circular—is 19-7 [i?
276
Runham—Replacement of pulntonate radula
and 113-1 p.2 respectively, i.e. a difference of about 6 times. Considering
the great inaccuracy of this estimation it is surprisingly near 4—the factor
by which the growth of the radula exceeds that of the inferior epithelium.
If this relationship is correct, the inferior epithelium from this point would
move at the same rate as the radula. Long-term experiments are in progress
to investigate this suggestion.
The radula must, however, slide over the inferior epithelium at the
posterior end of the radular gland. It is not known how firmly the inferior
epithelium is attached to the radula in this region but there are other wellknown examples where a structure moves forward over an epithelium while
remaining firmly attached to it—e.g. the human finger nail. If the functional
part of the radula is firmly attached to the underlying epithelium a simpler
mechanism would be sufficient to transmit the pull of the supralateral radular
tensor muscle through the epithelium. It is also of interest that the subradular membrane is located in this area. Possibly the function of this
layer is to establish a firm attachment of the epithelium to the radula.
This view is different from the current theories of movement of the radula
in relation to the inferior epithelium. Hoffman (1932) believed that the
radula is permanently attached to the inferior epithelium (the sub-radular
membrane having a cementing function) and that the radula only moves
forward through a growth of the whole buccal mass. Markel (1958), on histological evidence, suggested that the radula and the inferior epithelium are
firmly attached to each other but are both continually replaced. He points
out, however, that the changes in shapes of the cells imply that this attachment is not permanent. Hubendick (1945) dismisses Hoffman's suggestion
and puts forward evidence for the view that the radula slides over the subradular membrane, which he believes is continuous with the buccal cuticle.
The subradular membrane is, however, not continuous with the buccal
cuticle (fig. 2, E) and I cannot envisage how the radula could be used for
feeding if the only resistance to the sliding of the radula over the subradular
membrane was friction—as suggested by Hubendick (1945). I do not know
how the radula is attached to the inferior epithelium but it is possible that a
similar mechanism to that shown by Hubendick (1958) for the attachment
of the shell muscle to the shell of molluscs applies, i.e. by means of microvilli
from the epithelial cell surface being inserted into corresponding holes in
the shell.
At the extreme posterior end of the radula the staining properties of the
radula change, reverting to those found in the recently secreted radula
(Runham, 1962). This would imply the presence of an enzyme to reverse
the tanning of the radula, which would then become very soft and easily
abraded. I do not think that a chitinase alone, as suggested by Markel (1958),
would be sufficient to dissolve a tanned structure. Presumably if an enzyme
is secreted it is derived from the epithelium. The detachment of the radula
from the epithelium appears to be brought about by the secretion of a
cuticle under the radula which is continuous with the buccal cavity cuticle,
Runham—Replacement of pulmonate radula
277
or possibly by detachment of the cells as they change shape. If the radula
is firmly attached to the epithelium over the functional zone, then the forward movement of the radula is probably brought about by the movement
of this epithelium. Such a movement of the epithelium implies a change in
the attachment of the muscle: fibres must lose their attachment at the anterior
end and form new connexions at the posterior end, or alternatively there
must be a sliding of the epithelium over the basement membrane.
References
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CARRIKER, M. R., i94S- Trans. Wisconsin Acad. Sci., 38, 1.
FRETTER, V., and GRAHAM, A., 1962. British prosobranch molluscs: their functional anatomy
and ecology. London (Ray Society).
GABE, M., and PRENANT, M., 1952. C.R. Acad. Sci. Paris, 235, 1050.
1958. Ann. d'Histochim., 3, 95.
HOFFMAN, H., 1932. Jen. Ztschr. Naturwiss., 67, 535.
HUBENDICK, B., 1945. Arkiv. Zool., 36, 1.
1958. Ibid., 11, 31.
MARKEL, K., 1958. Z. wiss. Zool., 160, 213.
PRUVOT-FOL, A., 1926. Arch. Zool. exp. g£n., 65, 209.
QUASTLER, H., and SHERMAN, F. G., 1959.
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