Ultrastructure of the Ciliated Cups of a Synaptid Holothuroid, Leptosynapta galliennei

(Echinodermata)
Author(s): Didier VanderSpiegel, Angélique Geronnez-Lecomte and Michel Jangoux
Reviewed work(s):
Source: Invertebrate Biology, Vol. 117, No. 3 (Summer, 1998), pp. 253-260
Published by: Blackwell Publishing on behalf of American Microscopical Society
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Invertebrate Biology 117(3): 253-260.
C 1998 American Microscopical Society, Inc.

Ultrastructure of the ciliated cups of a synaptid holothuroid,

Leptosynapta galliennei (Echinodermata)

and Michel Jangoux, 2

Didier VandenSpiegella, Angelique Geronnez-Lecomte2,
1Laboratoire de Biologie marine, Universite de Mons Hainaut, 19 ave Maitriau 7000-Mons, Belgium
2 Laboratoire de Biologie marine,Universite Libre de Bruxelles, 1050-Bruxelles,Belgium

Abstract. The medial epidermal surface of the buccal tentacles of the synaptid holothuroid,
Leptosynapta galliennei, bears 5-30 ciliated cups. Each cup consists of an epidermal elevation
with a central invagination forming a small pit. Whereas the epidermis of the outer lining of
the cups is similar to that on the adjacent tentacle, the epidermis lining each pit consists of
ciliated cells gathered together in a central bulb and surrounded by elongated support cells.
The basal parts of ciliated cells are tapered and, together, form a nerve bundle entering the
intradermal nerve plexus of the tentacle. We suggest that the ciliated cells and thus the entire
ciliated cup has chemo-sensory function. It is further suggested that the cups allow the holo-
thuroid to test the properties of the surrounding water and could also provide information on
the palatibility of collected food material.
Additionalkey words: Sensory organs, Synapta

Sensory structures in echinoderms usually consist Methods

of scattered sensory cells that are occasionally organ-
ised into organs. Such organs are well developed in Adult Leptosynapta galliennei (Herapath 1865)
were hand-collected at low tide in the "Anse du Poul-
the synaptid holothuroids where they are said to com-
du" near Roscoff (Brittany, France) in September
pensate for the lack of podia (for review, see Hyman
1995. They were transported to the marine biology
1955; Reese 1966; Smiley 1994). Many synaptid spe-
laboratory of the Mons University and kept alive in a
cies have photo-receptor organs (i.e., the ocelli of
closed-circuit marine aquarium (13?C, 33%o salinity).
Opheodesmosa spectabilis, Yamamoto & Yoshida For light microscopy, buccal tentacles and ciliated
1978), mechano-receptor organs (i.e., the statocysts of
cups were removed from individuals previously an-
Leptosynapta inhaerens, Ehlers 1997) and supposed aesthetized for 1 h in a marine solution of propylene
chemo-receptor organs (i.e., the ciliated cups of Lep- phenoxetol (Nipa Laboratories, UK, see Hill & Reis-
tosynapta inhaerens, Cuenot 1891). The ciliated cups chmidt 1976). They were fixed in Bouin's fluid, em-
have been extensively studied in the species Leptosy- bedded in
Paraplast, and cut into 5-[xm thick sections.
napta inhaerens by classical light microscopy (e.g., Sections were stained with Masson's trichrome and
Cuenot 1891; Clark 1907) but there have been no ul- Mayer's hemalum coupled with phloxine and light
trastructural accounts of these organs reported to be green (Ganter & Jolles 1969-1970). Histochemical ob-
sensory (Cuenot 1891; Clark 1907; Heding 1928; Hy- servations were performed using Alcian Blue (pH 2.6)
man 1955; McKenzie 1988). Cuenot (1891) and Clark and the periodic acid-Schiff (PAS) techniques for the
(1907) reported that the cups are elevations of the ten- detection of mucopolysaccharides (Ganter & Jolles
taclar epidermis surrounding a ciliated pit, and that a 1969-1970).
nerve plexus connects each cup to the main tentaclar For scanning electron microscopy (SEM), cups were
nerve. The aim of the present paper is to describe the fixed in Bouin's fluid for 24 h (Lahaye & Jangoux
fine structure and discuss the function of these cups in 1985). They were dehydrated in a graded ethanol se-
light of ultrastructural detail in the burrowing species ries, dried by the critical point method (using CO2 as
Leptosynapta galliennei. transition fluid), mounted on aluminium stubs, coated
with gold, and observed with a JEOL JSM 6100 scan-
ning electron microscope.
a
Authorfor correspondence. For transmission electron microscopy (TEM),
E-mail: VandenSpiegel @umh.ac.be cups were fixed by immersion in 3% glutaraldehyde
254 VandenSpiegel, Geronnez-Lecomte, & Jangoux

Figs. 1-3. Leptosynaptagalliennei. Inner surface of a buccal tentacle and its ciliated cups. Fig. 1. Inner surface of an
entirebuccal tentacle.Fig. 2. Ciliatedcups. Fig. 3. SEM of a ciliated cup showing the centralbulb (CB). Cilia (C), ciliated
cup (CC), ciliated pit (CP), pinnule (P), outer epidermis(OE), stem (ST).

in cacodylate buffer (0.1 M, pH 7.8) for 3 h at 4?C. Results

They were then rinsed in cacodylate buffer and post-
fixed for 1 h in 1% osmium tetroxide in the same
buffer. After a final buffer wash, they were decalci-
fied according to the method of Dietrich & Fontaine
(1975), dehydrated in a graded ethanol series, and
embedded in Spurr's resin. Sections were cut on an
LKB V ultramicrotome, contrasted with uranyl ac-
etate and lead citrate, and observed with a Zeiss EM
10 transmission electron microscope.
Water currents around sensory cups were investi-
gated in vitro (light microscopy) using single tentacles
immersed in seawater containing suspended carmine
particles (5 mg/ml).
External morphology
The mouth of Leptosynapta galliennei is surrounded
by twelve pinnate buccal tentacles of almost equal
size. Each tentacle measures -0.4 cm in length when
extended, and is comprised of a stem from which 3-
6 pairs of distal lateral branches ("pinnules") and a
single, long, apical filament arise (Figs. 1, 2). On the
inner proximal parts of the tentacle stems (i.e., the sur-
face facing the mouth), 5-30 ciliated cups occur (Fig.
1). The cups measure about 60 |jm in diameter and 70
pLmin height (Fig. 2). Apically, each cup is invagi-
nated, forming a small pit up to 12 asm deep. The
 ::

The ciliated cups of a synaptid holothuroid 255

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Fig. 4. Leptosynapta galliennei. Schematic representation of a longitudinal section through a ciliated cup. Basal lamina
(b), bundle of filaments(bf), cilium (c), centralbulb (CB), ciliated cell (CC), connective tissue (co), cuticle (cu), ciliated
cells processess (ccp), ciliary rootlet (r), junctional complex (jc), invaginatedsupportcell (isc), lateral nerve (ln), mito-
chondrion (m), mucocyte (mu), nucleus (n), outer epidermis (OE), outer support cell (osc). (Not to scale).

lateral wall of the pit is smooth while its bottom is tive tissue underlying the epidermis follows the con-
heavily ciliated; the cilia may protrude slightly beyond tour of the cup (Fig. 4); it is composed of scattered
the edge of the pit (Fig. 3). Observations using sus- collagen fibres embedded in an extracellular matrix, in
pended carmine particles failed to reveal water cur- which no cellular components have yet been identified.
rents at the tip of the ciliated cups; thus, the cilia of The outer epidermal lining and connective tissue of
the pit appear to be non-motile. the cup are continuous between the cups and their
equivalents in the tentacle stem.
Fine structure
The epidermis, measuring 50 tLmat the most, pro-
The ciliated cups consist of epidermal elaborations; gressively changes in cellular composition from the
these are small elevations with a central pit in which tentacle stem to the deepest part of the ciliated cup.
the epidermis is specialised (Figs. 4, 5). The connec- The epidermis of the stem and of the outer lining of
256 VandenSpiegel, Geronnez-Lecomte, & Jangoux

the cup measures -50 pLmthick, although it is much
thinner in some areas. It is made up of support cells
and scattered interspersed mucocytes (Figs. 4-10). At
the point where the epidermis invaginates, the muco-
cytes are lost and the invaginated epidermis is made
entirely of support cells that organise around a cluster
of ciliated cells (Figs. 4-7, 11-13). Epidermal cells are
bound together by apico-lateral junctional complexes
consisting of a zonula adhaerens and subjacent septate
desmosome (Fig. 12). They rest on a basal lamina to
which they are anchored by hemidesmosome-like
structures. A thin cuticle (0.5 [Lm) overlies the entire
epidermis of the cup except the pit aperture. Under-
neath the cuticle is a narrow subcuticular space (1 tRm)
which is crossed by the microvilli of the epithelial cells
(Figs. 10A, 12). Bacteria occur in the subcuticular
space (Figs. 8, 12).
The support cells differ in shape with position on
the cup. In the outer lining of the cup, they have a
classical T-shaped structure: a thin extended apical
area, a neck, and an enlarged nucleus-containing body.
At the edge of the pit, the support cells progressively
change from T-shaped to club-shaped cells; the support
cells in the pit possess an elongated neck which forms
a kind of collar surrounding a central cluster of ciliated
cells (Figs. 4, 6). The support cells of the outer lining
contain bundles of filaments that link the connective
tissue fibers and the apical extensions of the cell by
way of hemidesmosomes (Figs. 4, 10A-B). Except for
these filaments, the cytoplasm of all support cells is
similar. It contains numerous vesicles measuring -0.2
pm in diameter, enclosing an electron-lucent material.
There are also membrane-bound granules of various
sizes and a few mitochondria (Figs. 9, 10).
Mucocytes only occur in the epidermis of the ten-
tacle stem and outer lining of the cup, where they are
scattered between support cells. They are round to egg-
shaped cells, and their cytoplasm is densely packed
with vacuoles (Figs. 7, 8). These are about 0.4 pLmin
diameter and contain Alcian Blue and PAS positive
materials.
Within the cup, ciliated cells are gathered together
in a central bulb surrounded by support cells (Figs. 4,
5, 6). The ciliated cells have a narrow neck, an en-
larged nucleus-containing central part, and an elongate
and tapered basal process (Figs. 4, 11-13). These pro-
cesses converge to form a nerve up to 100 Itm long
and 15 iLm in diameter that enters the lateral nerve of
the tentacle (Figs. 4, 5, 14). Each cell bears a single
apical cilium (-10 pm long) surrounded by a ring of
-10 microvilli. The cilia have a regular 9 x 2 + 2
arrangement of microtubules, and are associated with
a prominent ciliary rootlet extending through the api-
cal two thirds of the cell (Figs. 4, 11, 12). The cyto-
plasm contains numerous spherical membrane-bound
granules; these measure -0.25 pLmin diameter and
contain an electron-dense material. The apical cyto-
plasm also harbours many large mitochondria (Figs.
11, 12). At the place where the ciliated-cup nerve dif-
ferentiates, the basal lamina of the epidermis is in con-
tinuity with the basal lamina of the lateral nerve.
Discussion
The buccal tentacles of Leptosynapta galliennei
possesses cups that consist of a cluster of ciliated
cells surrounded by tall elongate support cells. The
ciliated cells are morphologically similar to other
echinoderm sensory cells described by Holland
(1984); they are columnar and bear a basally directed
process or axon which contacts a lateral nerve of the
tentacle. As shown by McKenzie (1988), this nerve
is connected to the main nerve of the tentacle which
itself contacts the perioesophageal nerve ring. There
is, thus, a clear connection between the ciliated cells
of the cup and the central part of the nervous system
of the individual.
The particular arrangement of the sensory cells
within the cups is reminiscent of the sensory-secretory
epidermal elevations that occur throughout the integ-
ument of synaptid holothuroids (Cuenot 1891; Clark
1907; Hyman 1955). Those elevations consist of a ba-
sal circle of gland cells that surround a slightly
projecting sensory bud. The latter is made up of ciliated
neuroepithelial cells whose basal parts are tapered,
forming together a nerve fibre entering the intradermal
nerve plexus (Cuenot 1891; Clark 1907; Hyman

Figs. 5-10. Leptosynapta galliennei. Cellularstructureof a ciliated cup. Fig. 5. Longitudinalsection throughcup (semithin
section). Fig. 6. Transversesection throughapical part of cup. Fig. 7. Transversesection throughbasal part of cup. Figs.
8, 9. Transversesections throughthe outer epidermis.Fig. 10. Detailed views of the apex of a supportcell (A) and a
bundle of filaments(B). Basal lamina (B), bacterium(BA), bundle of filaments(BF), cilium (C), centralbulb (CB), ciliated
cell (CC), connectivetissue (CO), ciliatedpit (CP), ciliated cell processes (CCP),cuticle (CU), granule(G), hemidesmosome
(H), invaginatedsupportcell (ISC), lateralnerve (LN), mitochondrion(M), mucocyte (MU), microvilli (MV), nucleus (N),
outer epidermis(OE), outer supportcell (OSC), vacuole (V).
The ciliated cups of a synaptid holothuroid 257

_e _ ____I____Y___Y_I?____q_?i-?h?L-?JII

i ' . .H]~~~~~~~~~ - 1-- 111?11 1 1 1 I --I -?""?ILIPLgBs*Is

258 VandenSpiegel, Geronnez-Lecomte, & Jangoux

i
I

I
The ciliated cups of a synaptid holothuroid
1955). According to Clark (1907), these structures are
tactile (mechano-receptor) organs.
Though ciliated cups occur in a number of species
belonging to different genera of apodous holothuroids
(Clark 1907; Hyman 1955), their structure was inves-
tigated only to the light microscope level and only in
Synapta inhaerens. Although there have been no stud-
ies on chemoselectivity in the feeding behaviour of
synaptids, the cups were suggested to test the sur-
rounding medium (Cuenot 1891; Clark 1907). Accord-
ing to Cuenot (1891), the cilia beat continuously and
thus produce a steady water current through the cili-
ated pit where chemicals would be detected. Our ob-
servations failed to reveal water currents at the tip of
the cup. Nonetheless, our ultrastructural observations
clearly show that the cups contain typical neurosen-
sory cells and thus, there is a little doubt that these
cups serve as sensory organs; whether the cups are
mechano-receptor or chemo-receptor organs is, how-
ever, uncertain. According to Cobb (1987), it is not
possible to separate these two modalities of perception
ultrastructurally.
From the feeding behavior of L. galliennei, it is
tempting to suggest that the ciliated cells are chemo-
receptors. L. galliennei is a burrowing holothurioid
which assumes an "L-shaped" posture in the sedi-
ment, with the oral tentacles projecting onto the sub-
stratum with their inner surface facing the water column
(Feral 1985). The ciliated cups are thus exposed to
surrounding water current and could function as che-
mo-receptor organs, perhaps in triggering the feeding
activity of the individual. Leptosynapta galliennei is
known to eat all kinds of detritus that can be removed
from the substratum (Feral 1985). When feeding, L.
galliennei picks up food particles on the sticky outer
surfaces of the tentacles (McKenzie 1988); a loaded
tentacle then bends towards the mouth, and the outer
surface with attached particles comes into close con-
tact with the proximal inner surface of the opposite
tentacle, where ciliated cups occur. Thus, the ciliated
cups could provide information on the palatability of
collected food material. The results of this study em-
phasize the need for studies on sensory abilities in the
feeding behaviour of synaptids, which would contrib-
ute to our understanding of the functional capabilities
4-
Figs. 11-14. Leptosynapta galliennei. Fine structure of a ciliated cup. Fig. 11. Longitudinal section through the apical part
of the central bulb. Fig. 12. Detailed view of the apex of ciliated cells. Fig. 13. Detailed view of the basal part of ciliated
cells. Fig. 14. Longitudinal section through the ciliated cells processes. Basal lamina (B), bacterium (BA), cilium (C),
ciliated cell (CC), connective tissue (CO), ciliated cell processes (CCP), ciliary rootlet (R), granule (G), mitochondrion
(M), microvilli (MV), nucleus (N), septate desmosome (SD).
259
of the ciliated cups and other senory structures in the
tentacles of these holothuroids.
Acknowledgments. We thank Dr. RD. Reynolds for critical
reading of the manuscript; Professor P Lassere for providing
facilities at the Observatoire Oc6anologique de Roscoff
(Brittany, France); Professor J.A. Heuson-Stiennon for the
use of transmission electron microscope and P. Postiau for
technical assistance. Contribution of the "Centre Interuniv-
ersitaire de Biologie Marine".
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