311
Presumed photoreceptive cilia in a ctenophore
By G. A. HORRIDGE
(From the Gatty Marine Laboratory and Department of Natural History, the University,
St. Andrews, Fife)
With 6 plates (figs, i to 6)
Summary
Four groups of lamellate bodies are symmetrically arranged inter-radially in the floor
of the apical organ. Each is composed of many streamed-out membranes of a group
of about a dozen cilia, within an invagination of a cell. The rings of ciliary fibrils, of
the 9 + 0 pattern, become disarrayed not far from the base. Their dense membranes
are lined with granules so that the successive lamellae resemble those of the modified
cilia of vertebrate eyes, although flattened in a different plane. On this basis, of ciliary
origin, and their resemblance to photoreceptors in fine detail, these structures are interpreted as photoreceptors.
Introduction
I T is commonly accepted that ctenophores have no special light-sensitive
organ. But in his monograph on the ctenophores of Naples, Chun (1880)
shows 4 crescent-shaped groups of round bodies, each of which is about the
size of a nucleus, symmetrically placed about the floor of the apical organ.
Chun finds these in all ctenophores and suggests (p. 166) that they are primitive photoreceptor organs, but hardly to be called an eye. Responses of these
animals to light are also supposed to be indefinite (Hyman, 1940). No reports
of immediate or rapid responses to illumination or darkening have come to
hand, and tests show none in Pleurobrachia, although it is possible that
ctenophores migrate vertically in the plankton in relation to diurnal light
changes.
However, upon close examination at the electron-microscope level, the
organs mentioned by Chun turn out to have a structure which is remarkably
similar to that of photoreceptors in the eyes of vertebrates (Brown, Gibbons,
and Wald, 1963) and in the distal retina of the clam Pecten (Miller, 1958), in
both of which, in different ways, the folded membranes of the photoreceptors
are derived from the membranes of cilia. Recently Eakin (1963) has
found that the photoreceptors of starfish, coelenterates, and Sagitta are also
complicated elaborations of cilia. The justification for regarding as photoreceptors the structures to be described in ctenophores rests only on this
anatomical similarity. Direct electrophysiological micro-methods would be
necessary to prove the case, because otherwise it is difficult to experiment with
only one group of the diversely differentiated cells of the ctenophore apical
organs.
[Quart. J. micr. Sci., Vol. 105, pt. 3, pp. 311-317, 1964.]
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Horridge—Ctenophore photoreceptor
Methods
Pleurobrachia pilots, collected from the plankton of St. Andrews bay, was
fixed in 2 % osmic acid mixed with an equal volume of sea-water of pH 7-2
to 7-4. As in coelenterates, the tissue is very watery and it is difficult to
achieve good fixation of a variety of components and tissues at the same time,
no matter what combinations of dilution and added sucrose are tried. Apical
organs were embedded in araldite, and sectioned and stained with lead acetate,
according to the alcohol-ether method described in a previous paper (Horridge
and Mackay, 1964). The correct region of the apical organ was identified by
examination of 1 /x sections stained in aqueous toluidine blue (fig. 1, A, B).
Results
As seen with the light microscope in horizontal or vertical sections of the
apical organ there are 4 groups of round basiphilic objects in the rounded
angles at the base of the side wall (fig. 1, A). Each group consists of up to 20
lamellated objects, each of 4 to 8 ju. in diameter. Here and there an individual
of the group is associated with a space or vacuole. Each of these objects lies
separate and distinct from its neighbours within the thickness of the ectodermal epithelium of the apical organ. The epithelium consists of tightly
packed columnar cells up to 100 fi tall, which are of several types distinguished
by their contents. These oval dark objects are quite distinct from the developing grains of the statolith, which lie in a different region of the wall of the
apical organ.
Under the electron microscope each of these objects is resolved as a
lamellate body (figs. 2, 3) of up to 50 coiled membranes which are thicker than
normal plasma membranes and which, when fixed and stained as described,
become electron-dense (figs. 4, 5). The membranes are those of cilia which
have extended sideways, on one side or on both sides of their axis, and the
membranes have coiled around themselves and round each other within the
ambit of a cavity. This cavity originates as an invagination of the plasma
membrane of the cell where its distal end meets the sea-water in the cavity of
the apical organ. Apart from the directions of the fibrils of the cilia, the
structure of one of these lamellate bodies appears fairly similar no matter
FIG. I (plate), A, horizontal (equatorial plane) section through the apical organ of Pleurobrachia, just grazing the underside of the statolith, which is visible in the centre. This section
is transverse to the main axis of symmetry of the animal. The top 4 groups of lamellate bodies
are just beginning to come into view at this level.
B, a section, deeper in the same series, parallel to the above but through the ectodermal
floor of the apical organ, showing the 2 groups of lamellate bodies on one side. This is about
the maximum number found in any one section.
c, a typical cilium of the 9 + 0 type, of a cell of the side wall of the apical organ, in the
region above the lamellate bodies, which are generated from cilia of this type.
D, a cilium of the 9 + 2 type, which is abundant in the apical organ.
b, otolith balancers; c, cilia at the beginning of the ciliated groove; end, endoderm;
o, lamellate bodies; J, sagittal plane (Hyman, 1940); st, statolith.
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Horridge—Ctenophore photoreceptor
313
what plane it is sectioned in, showing that they are spherical and not cylindrical in general form.
The exact pattern of the membrane differs in each lamellate body. However, the same fundamental relationship between the membranes of the cilia
and the plasma membrane of the vacuole occurs in all. A group of between 6
and 12 cilia of the 9 + 0 pattern (fig. 1, c) spring from the wall of the invagination in the cell on its side towards the cavity of the apical organ. As can be
followed more clearly in lamellate bodies which are interpreted as growing
ones (see below), the membranes of the cilia spread progressively out sideways until they have circled round the inside of the vacuole (figs. 3, A, B; 6, c).
This process of membrane extension happens on one side (figs. 3, B; 4, A)
or on both sides (figs. 5, c; 6, c) of the axis of the cilium. The lamellate body
is built up of successive turns as the membranes extend in the spaces available
to them. The overlapping of adjacent layers becomes extremely complicated
because the process happens by growth of structures, topologically the shape
of folded pieces of paper, which come to fit into layers within a sphere.
Frequently the plane of a section includes some cilia which are seen in longitudinal section and others which are cut more transversely (fig. 5, B). Because
they are the extensions of the side wall of a tube, the membranes are paired
and the 2 membranes of a pair meet to form a loop either in the centre of the
lamellate body or, more commonly, against the plasma membrane of the
invagination in which the lamellate body lies (fig. 4, A, B, C, in different
patterns). Finger-like protrusions from the edges of the folded membranes
become sectioned and appear as totally enclosed vesicles with walls which are
not seen as being continuous with the folded ends of adjacent lamellae
(figs. 3, B; 6, B). Brown, Gibbons, and Wald (1963) consider that similar
vesicles at the ends of folded lamellae in vertebrate rods and cones are not
artefacts: it is necessary to say this in view of criticisms of fixation by osmic
acid (Rosenbluth, 1963).
The membranes lie in pairs and, in many examples, the spaces between
them can be traced back to the inside and outside of the cilium which gave
rise to them: the outside surface is the smooth one (fig. 4, A). The inside
surface of each membrane is rough and covered with granules which add to
the thickness of the membrane (fig. 4, A). In situation and appearance the
granules closely resemble those thought to contain the photoreceptor pigments in vertebrate eyes, as for example discussed by Brown, Gibbons, and
Wald (1963) in relation to the lamellar micelles in the cones of the amphibian
Necturus. In the vertebrate photoreceptors the lamellar membranes are
invaginations of the cilium membrane and have the granules on the outside
Fie. z (plate). A group of lamellate bodies in the side wall of the apical organ, cut in a
sagittal plane (parallel to the principal axis at right angles to the line between the tentacles).
The cells containing the lamellate bodies have a distinct complement of vesicles and neurptubules. The lamellate body which is surrounded by loose membranes (centre right) is one
which is degenerating, and all stages to final dissolution have been found. The mesogloeal
side is towards the bottom.
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Horridge—Ctenophore photoreceptor
surfaces of a fold, whereas here they are extensions and have the granules on
the inside. The lamellate bodies of the ctenophore Callianira were reported
as being brownish-red by Kolliker (1853).
The regularity of the spacing between the membranes is outstanding in
some isolated regions of lamellate bodies but in most places the regular
spacing is only between the outsides of 2 cilia. The distance, where it is
regular, between outer ciliary surfaces is consistently 10 to 12 m/x: whereas
between inner surfaces it varies between 15 m/x and 100 m/A. This invites the
interpretation that the constant separation between cilia is maintained by the
regular arrangement of a solid packing which is, however, not seen. Other
cilia in ctenophores, such as those of the comb-plates and the balancers, are
fused closely together in groups with a regular spacing, again suggesting an
adhesion of the outer surfaces.
The cilia which give rise to the lamellate bodies have the 9 + 0 pattern
typical of sensory cilia (Barnes, 1961) including the vertebrate rods and cones,
and the photoreceptors of Pecten. In the apical organ there are several other
types of cilia, some of the 9 + 2 pattern with a specialized root structure which
lies at right angles to the cilium shaft, others with the 9 + 0 pattern. The cilia
of the lamellate body differ from all these in having the granules which suggest photopigments.
The basal structure is different from that of any other cilium which I have
seen described. The basal body of each cilium contains the 9 triple fibrils
which are characteristic of a centriole (fig. 5, A). Certainly 1 and probably 2
of the fibrils of the basal body triplets are continuous with the paired fibrils
of the cilium (figs. 4, B; 5, B; 6, B). The basal body is not at an angle to the
cilium shaft and it lies just inside the meeting of the cilium membrane with
the plasma membrane. A structure resembling the spokes of a cartwheel
projects outwards from the sides of the basal body (fig. 5, A, left). There is
no definite root structure, in contrast to ctenophore comb-plate cilia, which
have branched roots more or less parallel to their axis, and apical organ
balancer cilia, each of which has a striated flattened root at right angles to the
shaft. Occasionally a few fibres lie in the cytoplasm around the basal body
(fig. 4, B). Commonly there are small vesicles which can lie within the hollow
of the cilium shaft (fig. 5, B).
The appearance of the basal body in longitudinal sections suggests that a
fine amorphous substance lies in the centre of the shaft both in growing and
in mature examples. This varies slightly in position in relation to the base
and can form cross bands (fig. 4, B). Distally the filaments of the cilium become disarranged (figs. 3, B; 4, A; 6, c) and eventually they peter out. The
FIG. 3 (plate). Details of a lamellate body which is interpreted as being in process of
development. The invaginated cavity is not yet filled and many of the 9 + 0 type cilia still
have recognizable round outlines. B is a high power picture of the relevant region of the top
portion of A.
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a, amorphous bodies, which are commonly present; I, longitudinal section of basal body;
t, transverse section of basal body; va, vacuole.
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Horridge—Ctenophore photoreceptor
315
paired 'hooks' typical of the filaments of normal cilia (fig. 1, D) are not found
in the 9 + 0 pattern (fig. 1, c). The dissociation of the filaments shows that
they are less closely bound together laterally than are those of the normal
9-)-2 type characteristic of unspecialized ctenophore cells, apical organ
balancers, ciliated grooves, and comb-plates. The size of the ring of 9 is the
same in both types of cilia. Typical cilia of the side wall of the apical organ
are shown in fig. 1, c (9+0 pattern) and fig. 1, D (9+2 pattern).
Other features of the cells which produce the lamellate bodies are those of
sensory nerve-cells elsewhere in coelenterates and ctenophores. The cells
are elongated and their bases terminate among other elongated cells and fibres
at the base of the epithelium where it adjoins the mesogloea. Occasionally
there are typical asymmetrical synapses in this region, with vesicles only in
the cytoplasm which can be traced back in a cell containing a lamellate body
or to a ciliated cell of the wall of the apical organ. The cytoplasm around the
lamellate body contains a great number of vesicles and some neurotubules
(canaliculi) which resemble those in nerve-cells elsewhere in the animal.
Neighbouring cells, without lamellate bodies, contain different types of
vesicles. Unlike other cells, those which bear the lamellate bodies are not
ciliated where they meet the cavity of the apical organ; this is to be expected
because their basal bodies and cilia lie deep below in the invagination.
Instead there is sometimes a concentrated region of orientated endoplasmic
reticulum, in which the membranes lie at right angles to the cell surface and
appear as if they can open to the sea-water. In this region, on other cells,
there are secretory cilia, which are readily identified by their bulky contents
and swollen ends.
Evidence for a continual production and disintegration of lamellate bodies
conies from their size distribution, from the occurrence of degenerate-looking
examples, and from the fact that all stages of their formation occur in mediumsized sexually mature animals. The sequence can be picked out from cells
which are always situated in a definite pattern. The first recognizable stage is
of several cilia of the 9 + 0 type growing inwards from the surface of the
epithelium into a small space which is an invagination of the distal plasma
membrane. In every case examined there have been relatively large irregular
granular particles lying within the invagination, and the extensions of the cilia
membranes sometimes grow around these (fig. 3, A, but best seen in fig. 6, c).
FIG. 4 (plate). Membranes of the lamellate body with intercalated ciliaryfilaments,vesicles,
and the granular lining of the membrane. The folded ends of the paired membranes meet the
plasma membrane which originally surrounded the invaginated space. Note the constancy of
separation between the outer, but not the inner surfaces of the ciliary membranes.
A, transverse section of cilia.
B, longitudinal section of cilia.
C, regular membrane folds arrive at the plasma membrane of the vacuole.
b, transverse bars in the cilium shaft; cm, paired cell membrane of adjoining cells; d,
double membrane formed by the wall of the cilium and the folded end of another cilium.
lamella; /, fibrils of a cilium; pm, plasma membrane of the wall of the invagination which
holds the lamellate body; v, vesicles within ciliary lamellae.
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Horridge—Ctenophore photoreceptor
Near its base, the stem of each cilium usually persists as a round pillar, while
the lateral extension of the membrane starts further along it, as can be seen in
a series (fig. 5). The largest and best-developed lamellate bodies lie further
away from the surface of the epithelium, and degenerate lamellate bodies
are always situated towards the mesogloeal side of the group (fig. 2).
Discussion
Although photoreceptors present a vast array of anatomical diversity
throughout the animal kingdom, 2 features which many have in common
relate to their fundamental mechanism. There is a multiplication of the area
of a membrane, which may be loaded with granules and, in a few investigated
examples, is known to be associated with the photoreceptor mechanism.
There are at least 3 ways in which the convoluted membrane arises, (a) by
successive folding of a membrane, which may be associated with a cilium as
vertebrates, (b) by growth of a mass of crowded villi which, pressed together,
form a rhabdom, and (c) by the coiling of a membrane to form a lamellate
body. Arthropod rhabdoms have not so far been related to cilia, but in the
arrow-worm Sagitta a rhabdom stands upon a projection which is a modified
sensory cilium (Eakin, personal communication). In developing eyes of the
clam Pecten, whirled lamellate bodies are formed from sensory cilia (Miller,
1958), although the relationship between the cilia membranes and the plasma
membrane is not clearly shown. Identification of ctenophore lamellate bodies
as photoreceptors depends on a combination of morphological features. In
a mixed tissue, below the organ level of organization, as here in coelenterates
and ctenophores, there are usually no other criteria available for the recognition of cell types; similar difficulties have been encountered in the recognition of nerve-cells in ctenophores.
The wide distribution of cilia of the few known examples of the 9 + 0
pattern, always in situations where a sensory function is known or suspected,
is reviewed by Barnes (1961). The present example from ctenophores has a
basal body formed from a centriole 'in line' and does not fit the classification
by Barnes, who suggests that 9 + 0 photoreceptor cilia, as in rods and cones,
are likely to have a basal body together with an additional centriole. Also
relevant to Barnes's discussion is the fact that here and elsewhere in ctenophores there are many cilia per cell, and none has an additional centriole.
Onion bodies which are not known to be associated with cilia, and which
are not suspected of being photoreceptor organelles, occur commonly
FIG. S (plate). Details of bases of cilia of a fully developed lamellate body. The 3 sections,
A, B, and C are from the same series, showing regions a few microns apart.
A, section through the basal bodies. Note the radiating structures spread out from the
fibrils, seen especially in the one on the left.
B, 2 stems just above the basal bodies and a cilium in longitudinal section at right angles to
the others. No flattened membranes are continuous with the cilium membranes in this
section, v, vesicles within cilia.
c, a few microns towards the mesogloeal end of the cell, the membranes become continuous with those of the cilia.
c. A
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Horridge—Ctenophore photoreceptor
317
throughout the animal kingdom, especially in nerve-cells. An onion body is
an organelle of concentric membranes of 100 m//. to 5-0/^ in diameter, which
appears to lie wholly within the cytoplasm. Where closely parallel membranes
are curved in a spherical shape the distinction between spiral and concentric
layers depends on the plane of the section. It is necessary to distinguish between lamellate bodies formed by the plasma membrane and onion bodies
formed by intracellular membranes.
Transport of material down the axes of the cilia presumably occurs during
the process of extension of the ciliary membranes, and especially during the
subsequent laying down of the granular substance which lines the extended
membrane. Transport down the visual cilia perhaps occurs also in the narrow
region in the rod and cone cells of vertebrates. There is nothing in the structure of the basal body to suggest mechanisms which could move material
along the shaft. Related structures in other cilia may be relevant here. In the
apical organ of Pleurobrachia there are cilia with swollen ends which contain
numerous small vesicles. The statolith in ctenophores stands upon 4 groups
of cilia, called balancers, although its constituent particles are synthesized in
the cells below. On account of the difficulty of sectioning the hard otolith,
I have not been able to determine whether these cilia distended with vesicles
are concerned in the formation of the otolith, although such a relation is likely.
Even comb-plate cilia sometimes contain vesicles near their basal ends. It is
evident that in the formation of otoliths on sensory cilia, and in the related
topic of the transport of energy-rich material which maintains ciliary activity,
much remains to be discovered. In ctenophore comb-plates the mitochondria
are not closely associated with ciliary rootlets as they are in Amphioxus
(Olsson, 1962). Possibly the resemblance between neurotubules (canaliculi)
and ciliary fibrils is relevant, in that both may in some way act as lines along
which streaming occurs. Ctenophores certainly present many opportunities
for analysis of processes in cilia.
References
Barnes, B. G., 1961. J. Ult. Res., 5, 453Brown, P. K., Gibbons, I. R., and Wald, G., 1963. J. Cell Biol., 19, 79.
Chun, C, 1880. Die Ctenophorendes GolfesvonNeapelundderangrenzendenMeeres-Abschnitte.
Leipzig (Engelmann).
Eakin, R. M., 1963. In General physiology of cell specialization. Ed. Mazia, D., and
Tyler, A. London (McGraw-Hill).
Horridge, G. A., and Mackay, B., 1964. Quart. J. micr. Sci. (In the press.)
Hyman, L., 1940. The invertebrates Protozoa through Ctenophora. London (McGraw-Hill).
Kolliker, A., 1853. Zeit. wiss. Zool., 4, 315.
Miller, W. H., 1958. J. biophys. biochem. Cytol., 4, 117.
Olsson, R., 1962. J. Cell Biol., 15, 596.
Rosenbluth, J., 1963. Ibid., 16, 143.
FIG. 6 (plate), A, the peripheral end of a typical cell containing a lamellate body, reaching
to the sea-water in the cavity of the apical organ.
B, longitudinal section through the base of a cilium showing the close similarity between
neurotubules of the cytoplasm and cilia fibrils.
C, a group of cilia in an immature lamellate body, showing the initiation of the lamellae.
a, amorphous body, round which cilia sometimes wrap; m, mitochondrion; n, neurotubules (canaliculi).