~oo1ogicd3ournalof the Linnean Sock@ (1996), 116: 123-1 38. With 13 figures
Tardigrade biology. Edited (y S. J. McInnes and D. B. Norman
@
On the nature of pharyngeal muscle cells in the
Tardigrada
JETTE EIBYE-JACOBSEN
zoological Museum, UniversiQ of Copenhagen, Uniuersitetsparken 15, DK-2100 Copenhagen
0, Denmark
As part of a transmission electron microscopic study of the embryological development in tardigrades,
the ultrastructure of the pharynx was examined. The intent was to establish whether the pharyngeal
muscle cells constitute an ectodermal myoepithelium (as in many aschelminth pharynges) or whether
they are mesodermal (as in certain other aschelminth groups and among articulates). In the latter case
the cuticle would be produced solely by specialized epithelial cells. The eutardigrade species Halobiotw
crispae Kristensen, 1982, was investigated in four embryological stages, as a newly hatched juvenile, in
the active adult stage, and in the hibernation stage pseudosimplex 1. A comparison was made with the
arthrotardigrade Actinarctus dolyphms Schulz, 1935, in the active adult stage and in the simplex stage.
The results indicate that the tardigrade pharynx is an ectodermal myoepithelium. The muscles appear
to be truly cross-striated and monosarcomerial. The phylogenetic implications of these findings are
discussed briefly.
01996 The Linnean Society of London
ADDITIONAL KEY WORDS:
phylogeny.
~
pharynx - myoepithelium
-
muscle attachment
-
ultrastructure -
CONTENTS
Introduction . . . . . . . . . . . . . . . . . . .
Material and methods . . . . . . . . . . . . . . . .
Halobiotus crispae . . . . . . . . . . . . . . . .
Actinarctus dolyphoms . . . . . . . . . . . . . . .
Abbreviations used in figures . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . .
Halobiotus crkpae . . . . . . . . . . . . . . . .
Actinarctw dolypholur . . . . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . . . . . . . .
Conclusion regarding the nature of the pharyngeal muscle cells .
Interpretation of the observed ultrastructural details of the cuticle
Comparison between somatic and pharyngeal muscle attachments
Phylogenetic implications . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . .
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123
125
125
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126
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135
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137
INTRODUCTION
The tardigrade pharynx is an ovoid, muscular suction pump with a triradiate
cuticle-lined lumen. Marcus (1928, 1929) described the organ as being composed of
002.1-1082/96/010123+16
$18.00/0
123
01996 The Linnean Society of London
124
J. EIBYE-JACOBSEN
two different categories of cells: muscle cells and apical cells. The apical cells, situated
at the bottoms of the luminal clefts (termed “apices of the luminal ventricles” by
Dewel & Clark, 1973), were thought to be epithelial and the only producers of
cuticle. The muscle cells were thus considered mesodermal, at least in position if not
in origin.
Marcus (1929) claimed that the muscle cells withdraw from the cuticle and the
apical cells penetrate to the centre of the pharynx to produce the new cuticle during
moulting. If this is true, one would expect the pharyngeal cells to become
reorganized at the ultrastructural level during cuticular resynthesis.
Most recent authors working on pharyngeal ultrastructure describe the tardigrade
pharynx as a myoepithelium (e.g. Dewel & Clark, 1973; Walz, 1973; Ruppert, 1982).
That is, the cuticle would be synthesized by the muscle cells themselves and the
apical cells would serve a different purpose than the one suggested by Marcus (1929).
According to this interpretation, all the cells constituting the pharynx would be
ectodermal in origin and position.
At the ultrastructural level, Marcus’ interpretation would imply that an apical cell
of ectodermal origin is always interposed between a mesodermal muscle cell and the
cuticle. If this is true, the narrow gap between the apical plasma membrane of the
muscle cell and the cuticle should contain two plasma membranes, some cytoplasm
and a basement membrane. If, on the other hand, the tardigrade pharynx constitutes
an ectodermal myoepithelium, all pharyngeal cells would form one continuous
epithelium with a common basement membrane, and no cellular components would
be present between the apical cell membrane of the muscle cells and the cuticle.
These two possibilities are outlined schematically in Figure 1.
Since thc qucstion of the nature and origin of the pharyngeal muscle cells has
implications in the current debate on the phylogenetic position of the tardigrades
(Ruppert, 1982), this study re-examines the problem as part of an overall
investigation of the development, ultrastructure and function of the tardigrade
pharynx. The organization of pharyngeal cells was investigated in the adult active,
Figure 1 . Two different situations: A, all cells of the pharynx form a continuous epithelium with a
common basement membrane,each cell of the organ being in direct contact with the cuticle; B, the apical
cells forni a continuous epithelium and are separated from the muscle cells by a basement membrane;
note that the muscle cells are not in direct contact with the cuticle, as the apical cells are interposed
between the two.
PHARYNGEAL MUSCLE CELLS IN THE TARDIGRADA
125
pseudosimplex 1 (hibernating stage, see Kristensen, 1982a) and embryological stages
of the eutardigrade Halobiotus crispae Kristensen, 1982, and in the adult active and
simplex (moulting) stages of the arthrotardigrade Actinarctus dolyphorus Schultz,
1935.
The ontogeny of the pharynx of Halobiotus crispae could reveal two things: (1) A
dual origin of the pharyngeal cells would be proof that the pharynx is a bilayered
structure. (2) As the cuticle becomes synthesized while the organ is forming, the
opportunity arises to determine how many cells participate in the process.
The comparison with A. dolyphoms was made to increase the probability that the
findings based on the pharynx of H. crispae are representative of the entire tardigrade
group. Since the arthrotardigrades are considered to possess a greater number of
plesiomorphic characters than eutardigrades, one might expect to find a more
plesiomorphic configuration of the pharynx within that group.
MATERIAL AND METHODS
Halobiotus crispae
Material was collected at Vellerup Vig in the Isefjord, Denmark, during May of
1992, 1993 and 1994. Since the population of H. crispae is limited to a very restricted
zone at this locality, SCUBA divers were necessary to obtain samples containing
specimens. Samples consisted of carefully selected old individuals of Mytilus edulis
taken from boulders or handfuls of coarse sand removed from the bottom, just
beneath the stones from depth of 2.5 to 3.0m.
The samples were freshwater-shocked, filtered through a 33 pm plankton net,
reintroduced into salt water, and sorted out under the microscope to collect adult
specimens and the egg-containing exuvia. The collected specimens and eggs were
fixed in trialdehyde as described by Kalt and Tandler (1971) and modified by
Kristensen (1982a). Postfixation was in a 1YO solution of osmium tetroxide at 5OC for
10 minutes. The material was embedded in Epon 8 12 and cut into ultrathin sections
using an Ultracut E, Reichert-Jung ultramicrotome. Further adult specimens and the
pseudosimplex stage specimen were collected by Dr R. M. Kristensen in 1978-79 in
West Greenland, Disko Island, and subsequently treated for TEM as described
above. Transmission electron microscopic photographs were taken on aJEOL JEMlO0SX microscope except for that presented in Figure 3, which was taken on a JEOL
JEM- 1OOCX microscope at the Botanical Laboratory, University of Copenhagen.
Actinarctus dolyphoms
Material of this species was collected in Frederikshavn, Denmark, from
Amphioxus-sand (leg. R. M. Kristensen). The sample was bulk fixed in trialdehyde,
followed by filtration in a 63 pm plankton net. The collected specimens were treated
by Dr. Kristensen as described above for Halobiotus crisljae.
126
J. EIBYE-JACOBSEN
ABBREVLATIONS USED IN FIGURES
A
at
bm
ch
cs
cLl
dcd
del
di
CC
es
hz
lam
it1
apical cell
muscle attachment site
basement membrane
chorion of egg
columnar structure in cuticle
cuticula
diffuse electron-dense layer
diffuse electron-lucent layer
distinct electron-lucent layer
ectodermal cell
esophagus
H-zone of cross striated muscle fibre
intracellular attachment material
inner trilaminatc laver
lu
M
me
mf
mi
mu
N
nu
Otl
Ph
Pl
SP
tza
lumen
muscle cell
mesodermal cell
muscle filament
microvilli
mucus coat
nerve cell
nucleus
outer trilaminate layer
Pharynx
placoid
supportive structure
'tight' zonula adherens
RESUI.'I'S
Halobiotw cr$m
Actzue stage
The muscle attachment sites at the lumen of the active stage pharynx of Hulobiotus
criSpae (Figs 3 & 4) were examined at a magnification of 75 000 X . At the luminal
face of the cuticle a trilaminate layer is visible. Some flocculent, mucus-like material
is sometimes obscrvcd in the lumen itself (Fig. 3). Thc bulk of the cuticle proximal to
the trilaminate layer appears to be homogeneous, although the electron density
varies from light grey at the external and internal borders to dark grey in the central
part. Overlying the attachment sites, a relatively electron-dense diffuse layer
(approximately 25 nm thick) and an electron-lucent diffuse layer (approximately
11 nm thick) are present (Figs 2-4). 'The electron-lucent layer appears to contain a
number of columnar structures (Fig. 4).
At the attachment face of the diffuse electron-lucent layer, an internal trilaminate
layer (thickness approximately 7 nm) is seen (Fig. 3). Next to this layer, a very distinct
electron-lucent layer (approximately 6 nm thick) is evident, interposed just above the
cell membrane of the muscle cells (Figs 3 & 4). It is not possible to distinguish the
plasma membrane of the muscle cell as a trilaminate structure due to the presence
of extremely electron-dense attachment material.
A striated electron-dense layer, approximately 100nm thick, lies inside the muscle
cells. 'The thin filaments of the contractile apparatus appear to pass through the
electron-dense layer to insert on the plasmalemma. The filaments apical to the dense
material are grouped together to form the striated layer (Figs 2-4).
The interface between the cuticle and the muscle cells is smooth throughout.
'There are no convolutions of the cell membrane as seen in somatic muscle
attachments within the tardigrades (Shaw, 1974; Kristenson, 1978), but in some
specimens the attachment area seems to be broken up into plates that are not totally
in register with one another (Fig. 4). At the basement membrane some small conical
prpjections into the basal parts of the muscle cells are seen, especially at the anterior
PHARYNGEAL MUSCLE CELLS IN THE TARDIGRADA
Figures 2-4. Fig. 2. An overview of the structures involved in muscle attachment sites within the
tardigrade pharynx. Fig. 3. Enlarged view of the luminal cuticle at a muscle attachment site in a
longitudinal section of a newly hatched specimen of Halobktur ckpae. Fig. 4. Enlarged view of the luminal
cuticle at a muscle attachment site in a transverse section of an active adult specimen of Halobwhu
crispae.
127
1‘LH
J . E1BYE;JACOBSEN
and posterior ends of the organ of fully mature, active specimens (unpublished
data).
The zones of transition from a muscle attachment site to one of contact between
the general cell membrane and the cuticle are very difficult to resolve. It is evident
that the cell membrane meets the cuticle at a level closer to the lumen than at the
muscle attachment sites. The diffuse electron-dense layer is present, but it is
narrower than at the attachment sites. The diffuse electron-lucent layer is absent and
the internal trilaminate layer is impossible to follow.
Abutting muscle cells at the centre of the pharynx are held together by ‘tight’
zonulae adherentes in which the cell membranes appear fused or very closely
apposed and are layered with densely staining material (Dewel et al., 1993) (Fig. 3).
The junctions between muscle cells and apical cells are very similar to the ones
between two muscle cells, and no compartmentalization is apparent within the
pharynx. The cuticle covers the apical faces of all pharyngeal cclls, muscle cells as
well as apical cells.
~ s e ~ d Io ~ ~ ~ ~ ~ x
As seen in Figure 5, the muscle attachment sites greatly resemble the ones in the
active stage pharynx. Although the structure of the supportive cuticular components
is totally different from that in the active configuration (macro- and micro-placoids
are absent; see Kristensen, 1982a and Eibye-Jacobsen, in press, a), the attachment
sites appear unaltered ultrastructurally. Furthermore, the interrelationships between
the cells at the centre of the pharynx are unaltered as compared to the active stage
(Fig. 5). All muscle and apical cells are held together by ‘tight’ zonulae adherentes,
thus constituting one continuous cell layer surrounding the lumen in this stage of the
life cycle as well.
Development
In the following, the pharynx of embryos from approximately day 7, day 9, day 1 1
and day 14 after the egg-laying (development at 8”C, see Eibye-Jacobsen, in press,
b) will be described:
Day 7 (Fig. 6). The forming head region of the embryo contains an unorganized
aggregation of cells that surrounds an irregular lumen. A basement membrane is not
detectable and different cell types are indistinguishable.
Day 9 (Fig. 7). A thin but unmistakable basement membrane surrounds the
pharyngeal cells. Outside this membrane a few cells are closely associated with it.
The nuclei of the phaqngeal cells contain one or two large nucleoli. The lumen is
riot visible due to the plane of section.
Day 11 (Fig. 8). The pharyngeal lumen is clearly triradiate and a cuticle is being
synthesized. Serial sections reveal that all cells of the pharynx are in contact with the
cuticle during its formation. The number of cells present, determined by counting
the cell nuclei, is 54 and this remains constant throughout ontogeny and moulting
stages. Each cell is predetermined to fill a specific function (ix. future muscle or nerve
cell) and can be identified by its position within the organ.
Day i1 (Figs 9 & 10).The cuticular structures have almost reached their final size
and configuration. Muscle filaments are seen in the immature muscle cells, which are
in direct contact with the cuticle. Small microvilli are seen at the luminal surface of
the pharyngeal cells.
PHARYNGEAL MUSCLE CELLS IN THE TARDIGRADA
129
Actinarctus do?yphorus
Active stage
The pharynx of this species differs considerably from that of Halobiotus. One of the
most obvious differences is that the supportive structures (placoids) of the luminal
cuticle of Actinarctus are three in number, fused longitudinally, calcified, and situated
at the bottoms of the luminal clefts (Fig. 11). In contrast, the supportive structures of
Halobiotus are six in number (in a cross section), broken up into separate, bead-like
structures longitudinally and situated in pairs alongside the sides of the luminal clefts
(Eibye-Jacobsen, in press, a).
As a result of intense staining of the uncalcified part of the cuticle, it is impossible
to observe the ultrastructural details of the muscle attachment sites within this
structure (Fig. 11). However, the intracellular part of the attachments shows
similarities with the muscle attachments described for H. crkpae (Figs 11 & 12). Apical
cells are easy to identify, since their cytoplasm stains heavily compared to that of
muscle cells. The apical cell surrounds the supportive cuticular structure and meets
the electron dense cuticle at the site of muscle attachment (Fig. 1 l), but it does not
penetrate between the cuticle and the luminal cell membrane of the muscle cell (Fig.
12).
Simplex
The investigation of a specimen of Actinarctus in the simplex stage shows that the
Figure 5. Transverse section of the pharynx of a pseudosimplex specimen of Hul0620h1.r mispae, showing
bottom of luminal cleft.
J. EIBYE-JACOBSEN
6
Fi,pre 6. A, day 7 e m b q o showing a great number of cell nuclei. The developing head is to the left and
contains a loosely organized mass of cells with a lumen. B, sketch showing outline of embryo and cell
nuclri in A. C:. earlier section from the same series as B. D, later section from the samc series as B. Znset
skctch showing the orientation of the sections in this series, lateral vicw of embryo (head to the right).
131
PHARYNGEAL MUSCLE CELLS IN THE TARDIGRADA
8
Figures 7 & 8. Fig. 7. Sketch showing the appearance of the pharynx in a day 9 embryo. Inset: sketch
showing the orientation and position of the section, dorsal view of embryo. Fig. 8. Sketch of transverse
section through the pharynx of a day 11 embryo. Inset: sketch showing the orientation and position of the
section, lateral view of embryo.
positions of the muscle cells are unaltered during cuticular synthesis. The apical
membranes of the muscle cells are in contact with the cuticle at all times (Fig. 13),
but the thick muscle filaments are withdrawn from the centre of the organ. An
electron-lucent band is observed in the middle region of the bundles of detached
thick muscle filament (Fig. 13).
DISCUSSION
Conclusions regarding the nature Ofp h a y g e a l muscle cells
It is concluded from the evidence presented here that the tardigrade pharynx is an
ectodermal myoepithelium (alternative A in Fig. 1). This is in accordance with Walz
(1973) and Dewel & Clark (1973). This conclusion is based on the following:
(1) All pharyngeal cells have a common ectodermal origin during development.
(2)All pharyngeal cells participate in the synthesis of the cuticle during
development as well as during moulting.
(3) All pharyngeal cells are joined together at the luminal cuticle by ‘tight’zonulae
adherentes.
(4)All pharyngeal cells share a common basement membrane and there is no
basement membrane between any of them.
(5)No trace of cellular components are present between the apical cell membranes
of the muscle cells and the cuticle.
J. EIBI’E-JACOBSEN
Fi,qures 9 8r 10. Fig. 9. Horizontal section through a day 1.1 embryo showing the pharnyx with placoids
almost in functiot~alconfiguration. Anterior end in the upper left corner. Ins& Sketch showing orientation
and position of srrtion. lateral \-ie\v of emhr)o. Fig. 10. Enlargpment of the pharynx shown in Fig. 9.
Soticc thc immature inusck filamrnts and the micro\illi at the interface hetwrpn the apical parts of the
cells and the cuticular structures.
PHARYNGEAL MUSCLE CELLS IN THE TARDIGRADA
Figures 11 & 12. Fig. 11. Transverse section through the active pharynx of Actinarctus dovphonrs. Fig. 12.
Enlargement of the section shown in Fig. 11. Notice that the apical cells do not penetrate beneath the
muscle cells at the attachment sites.
133
.J. EIBYE:Jr\COBSEN
Figure 13. Transverse section through a simplex phavnx of .ktinarchCr dovphonu. As the new cuticle is
hring synthesized. thc lumen is occluded. Notice the light zones in the middle regions of the bundles of
thirk musrlr filaments.
PHARYNGEAL MUSCLE CELLS IN THE T A R D I G M A
135
The study of the development of Halobiotus crispae makes it clear that the
pharyngeal cells have a common origin: no distinction exists between the precursor
cells of the pharynx at day 7, and a common basement membrane appears very early
in ontogeny (approximately day 9). Furthermore, the cells on the outside of the
basement membrane at day 9 show that mesodermal cells are actually present at this
stage.
Later developmental stages provide information on the primary synthesis of the
luminal cuticle. It is very dimcult to establish whether or not the muscle cells are in
fact in direct contact with the cuticle, since the area of contact available to each cell
at the centre of the organ is very limited. This situation is a result of (1) the narrow
space at the central region, (2) the relatively large number of cells present and (3) the
fact that the cells at the two ends of the organ are orientated obliquely to its
longitudinal axis. Thus, a considerable fraction of the sections in a transverse section
series will fail to reveal cells extending from the basement membrane to the luminal
cuticle. A few sections in each series do show this, however, and from these it is
concluded that all muscle cells are in direct contact with the developing cuticle at all
times.
Examination of the pseudosimplex 1 specimen of Halobiotus crispae indicates that no
rearrangement of the pharyngeal cells occurs during this hibernation stage. The
positions and relations between cells remain unaltered and the ‘tight’ zonulae
adherentes are present in the pseudosimplex 1 as well as in the active form although
the configuration of the cuticular structures is remarkably different between the two.
The simplex (moulting) stage of Halobiotus crispae has not been examined in this
study.
The comparison with the arthrotardigrade Actinarctus dovphorus shows that
although the structure of the cuticular elements is very different from that of
Halobiotus, a similarity in pattern of cellular interrelationships is evident. The apical
cells are not interposed between the cuticle and the muscle cells in the investigated
specimen. The specimen of Actinarctus dog@?orms in the simplex stage shows that the
muscle cells remain in direct contact with the cuticle during its resynthesis. The
calcified supportive structures in the radial positions are solely produced by the apical
cells, but the cuticle of the attachment areas is laid down by the muscle cells
themselves. Furthermore, this specimen appears to have electron-lucent H-zones in
the middle region of the thick muscle filaments, thereby strongly suggesting that the
pharyngeal muscle cells are true cross-striated monosarcomerial muscles. This
confirms the findings of Walz (1973).
The cells of the tardigrade pharynx thus form one continuous epithelium in which
all the cells have a common ectodermal origin. The function of the apical cells is
different from that of the muscle cells, however, as they do not contain any thick
muscle filaments. This issue will be dealt with in a paper concerning the
ultrastructure, development and function of the pharynx of Halobiotus crispae (EibyeJacobsen, in press, a).
Interpretation
of the obsmed
ultrastructural details of the cuticle
According to Greven (1980) the body cuticle of eutardigrades consists of a mucus
coat, an outer trilaminate layer, an epicuticle of varied complexity (exocuticle
according to Dewel & Dewel, 1979), an inner trilaminate.layer, an intracuticle and
I36
J. EIBYE-JACOBSEN
a procuticle (mesocuticle and endocuticle according to Dewel & Dewel, 1979). The
inner epicuticle is a complex structure in its supposedly plesiomorphic configuration,
being divided into two layers by pillars in, for instance, Macrobiotus dianeae (Kristensen
198213). In Halobiotus crkpae the inner epicuticle of the integument consists of one
homogeneous layer (pers. obs.).
As described above, an inner 7 nm thick trilaminate layer was found just above the
apical membranes of the muscle cells in the pharynx of Halobiotus crispae in the active
form. Since no other signs of cellular components interposed between the cuticle and
the muscle cell borders were observed, the inner trilaminate layer is interpreted as
being part of the cuticle. It could be homologous to the inner trilaminate layer of the
body cuticle, if the reduced thickness (this layer is 16nm in the body cuticle
according to Dewel & Dewel, 1979) and lack of undulation are considered to be
specializations reflecting specific functional demands of the pharyngeal cuticle. The
intracuticle and the procuticle would thus be completely reduced and the two diffuse
layers above the triilaniinate layer would be specializations within the epicuticle.
The distinct electron-lucent layer found just apical to the muscle cell membranes
is seen in the muscle attachments of other myoepithelial pharynges (see figures in e.g.
Ruppert, 1982 (gastrotrichs), Byers & Anderson, 1972 (nematode), Albertson &
Thomson, 1976 (nematode), Endo 1983, 1984 (nematodes), Kristensen 1991
(loriciferans)).This layer is an extracellular space, possibly fluid-filled, that transmits
forces in myoepithelial pharynges. Other myoepithelial muscle attachments in
tardigrades do not show this feature (Dewel & Dewel, 1979: in the oviduct of
Milnesium tardgradum; Kristensen, 1979; in the rosette cells of the gonopore in females
of HakchinZScw sp.)
Inside the muscle cell, the thin muscle filaments (actin filaments) converge on the
plasma membrane through the dense attachment material forming a hemidesmosome. The striation of the l00nm attachment zones probably arises as the actin
filaments gather in bundles before inserting on the cell membrane.
Camparkon between somatic and phavngeal muscle attachments
As a result of this work, an interesting comparison can be made between somatic
and pharyngeal muscle attachments in tardigrades. Somatic attachments are
characterized by the following (Shaw , 1974; Greven & Groht, 1975; Kristensen,
1978; Dewel & Dewel, 1979; Greven, 1984): (1) easily identified epidermal cell
bctween thc cuticle and the muscle cells, (2) rod-like structures within the intracuticle
and the procuticle, (3) conical projections of cuticle into the epidermis cell forming
conical hemidesmosomes, (4) extracellular substance (specialized basement membrane) present between the epidermal cell and the muscle cell(s) (up to five muscle
cell5 may attach at the same epidermal cell), (5) apical plasma membrane of the
muscle cells more or less undulated, and (6) hemidesmosomes in the muscle cell.
In contrast, the pharyngeal muscle attachments in Halobiotus crzspae show: (1)
m>oepithelial muscle cells, (2) no rod-like or fibrous structures within the cuticle, (3)
no conical projections of the cuticle into the cell, and (4)apical plasma membrane is
mooth. The only similarity between the two types of muscle attachments is the
presence of hemidesmosomes. In the somatic attachment there are true hemidesmosome\ at thc apical membrane of the muscle cell and so-called conical hemidesmosomes (Dewel & Dewel, 1979; described as “everse hemidesmosomes” by Kristensen,
PHARYNGEAL MUSCLE CELLS IN THE TARDIGRADA
I37
1978) at the cuticular face of the epidermal cell. The hemidesmosomes of the
myoepithelial muscle cells are probably equivalent to the hemidesmosomes of the
somatic muscle cells. It remains unresolved whether there are any differences in the
ultrastructural details between the two. The specializations within the cuticle of the
two types of attachments are clearly different, irrespective of which layers are present
in the pharyngeal cuticle. The evolution of the somatic muscle attachment and the
pharyngeal muscle attachment in the tardigrades could be two separate events, each
as a response to functional needs.
Phylogenetic implications
The phylogenetic implications of this study are very few. The myoepithelial
condition of epithelial cells represents a plesiomorphic character state within the
animal kingdom. As such, the myoepithelial, triradiate pharynx could have evolved
several times as independent events. Therefore, the presence of such a pharynx is by
itself of little use as a character in discussing large phylogenetic issues, such as the
position of the Tardigrada within Bilateria.
If the pharynx is to be used in phylogenetic discussions, a far more detailed
investigation of the tardigrade pharynx is required. Comparisons should include
knowledge of cuticular specializations, secretory cells, valve systems, the number and
relative positions of muscle and sensory cells, nerve tracts and the innervation of the
organ.
ACKNOWLEDGEMENTS
I wish to thank the technical staff at the Zoological Museum, Copenhagen,
(ZMUC) for their help and support. Ms Stine Elle has produced the drawings and
the lay-out of the figures, Mr Kenn Kristiansen and Mr Geert Brovad have assisted
me in copying the photographs and Ms Eva von Linstow Roloff and Ms Karen
Meilvang have produced the ultrathin sections.
Dr Danny Eibye-Jacobsen and Dr Claus Nielsen have critically read and corrected
the manuscript and Dr Reinhardt M. Kristensen encouraged me to start this project
and provided much of the material used (all ZMUC). Dr Birger Neuhaus (Den
Kongelige Veterinare & Landboh0jskole) is thanked for his contributions to the
discussion and for critical revision of the material.
SCUBA divers: Marianne Thorsen, Lars Nielsen, Charlotte Lundius,Jane Grooss,
Johnny Rostholm, Peter Funch Andersen, Tom Schi~tteand Mikael Larsen and his
‘Amonitterne’ are thanked for their valuable help in collecting the samples.
Dr Lise Bolt Jmgensen, Botanical Laboratory, University of Copenhagen, is
thanked for her help in taking micrographs at very high magnification.
Funds for this project were provided by the Danish Natural Science Research
Council (grant number 1 1-9400).
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