JOURNAL OF MORPHOLOGY 273:1227–1245 (2012)
Neuroanatomy of Halobiotus crispae (Eutardigrada:
Hypsibiidae): Tardigrade Brain Structure Supports
the Clade Panarthropoda
Dennis K. Persson,1,2* Kenneth A. Halberg,2 Aslak Jørgensen,3 Nadja Møbjerg,2
and Reinhardt M. Kristensen1
1
Invertebrate Department, Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15,
DK-2100 Copenhagen Ø, Denmark
2
Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark
3
Laboratory of Molecular Systematics, Natural History Museum of Denmark, University of Copenhagen,
Sølvgade 83, DK-1307 Copenhagen K, Denmark
ABSTRACT The position of Tardigrada in the animal
tree of life is a subject that has received much attention,
but still remains controversial. Whereas some think tardigrades should be categorized as cycloneuralians, most
authors argue in favor of a phylogenetic position
within Panarthropoda as a sister group to Arthropoda or
Arthropoda 1 Onychophora. Thus far, neither molecular
nor morphological investigations have provided conclusive results as to the tardigrade sister group relationships. In this article, we present a detailed description
of the nervous system of the eutardigrade Halobiotus
crispae, using immunostainings, confocal laser scanning
microscopy, and computer-aided three-dimensional
reconstructions supported by transmission electron microscopy. We report details regarding the structure of
the brain as well as the ganglia of the ventral nerve
cord. In contrast to the newest investigation, we find
transverse commissures in the ventral ganglia, and our
data suggest that the brain is partitioned into at least
three lobes. Additionally, we can confirm the existence of
a subpharyngeal ganglion previously called subesophagal
ganglion. According to our results, the original suggestion
of a brain comprised of at least three parts cannot be
rejected, and the data presented supports a sister group
relationship of Tardigrada to 1) Arthropoda or 2) Onychophora or 3) Arthropoda 1 Onychophora. J. Morphol.
273:1227–1245, 2012. Ó 2012 Wiley Periodicals, Inc.
KEY WORDS: neuroanatomy; tardigrade; phylogeny
INTRODUCTION
Tardigrades are small invertebrates predominantly found in mosses and lichens but also
on marine macroalgae and between sand grains
(Bertolani, 1982; McInnes, in press). They exhibit
many autapomorphic features and were categorized as a phylum, Tardigrada, by Ramazzotti and
Maucci (1983). When subjected to adverse environmental conditions, they may endure by means of i)
active regulating processes or ii) entering diapause
or a stress tolerant state called cryptobiosis
(Møbjerg et al., 2011). Cryptobiosis enables tardigrades to survive severe physical stress, and conÓ 2012 WILEY PERIODICALS, INC.
sequently, they occupy some of the most inhospitable habitats (Renaud-Mornant, 1975; Dastych and
Kristensen, 1995; Pugh and McInnes, 1998;
Wright, 2001; Møbjerg et al., 2007; Halberg et al.,
2009b; Persson et al., 2011).
The position of Tardigrada in the animal tree of
life is a subject that has received much attention
but remains controversial. Ever since their discovery in the middle of the 18th century, their position
in the tree of life has been debated, and affiliations
to Rotifera, Pentastomida, Onychophora, Nematoda, and Arthropoda have been suggested
(Dujardin, 1851; Plate, 1889; Marcus, 1929; Crowe
et al., 1970; Dewel and Clark, 1973; Ramsköld and
Hou, 1991). Most evidence suggests one of the two
large, species-rich and economically important
groups Nematoda or Arthropoda as the closest
relatives to Tardigrada. Therefore, the position of
tardigrades has been at the front of the ongoing discussion on metazoan systematics. The two most
supported theories are that tardigrades either
belong to Cycloneuralia, as a close relative to nematodes (Crowe et al., 1970, Dewel and Clark, 1973;
Ruppert and Barnes, 1994), or to Panarthropoda,
along with arthropods and onychophorans (Baccetti
and Rosati, 1971; Bussers and Jeuniaux, 1973;
Contract grant sponsor: Danish Carlsberg Foundation; Contract
grant sponsor: Danish Natural Science Research Council; Contract
grant sponsor: National Science Foundation under the AToL program.
*Correspondence to: Dennis Krog Persson, Invertebrate Department, Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark.
E-mail:
[email protected]
Received 16 November 2011; Revised 26 April 2012;
Accepted 27 May 2012
Published online 18 July 2012 in
Wiley Online Library (wileyonlinelibrary.com)
DOI: 10.1002/jmor.20054
1228
D.K. PERSSON ET AL.
Greven, 1982; Kristensen, 1976, 1981; Møbjerg and
Dahl, 1996; Nielsen, 2001). Molecular investigations seem to support both views (Garey et al.,
1996; Giribet et al., 1996; Aguinaldo et al., 1997;
Garey, 2001; Mallatt et al., 2004; Dunn et al., 2008;
Rota-Stabelli et al., 2010; Campbell et al., 2011),
whereas morphological data more often support the
view that tardigrades are related to onychophorans/
arthropods (Schmidt-Rhaesa et al., 1998; Budd,
2001). Importantly, recent papers emphasize that
the molecular data are not conclusive as to whether
tardigrades are more closely related to arthropods
and onychophorans or to the nematodes and nematomorphs (Dunn et al., 2008; Edgecombe, 2010;
Edgecombe et al., 2011; Campbell et al., 2011). Phylogenomic analyses have yielded different results
depending on which substitution model that was
used to analyze the data (Dunn et al., 2008). In
addition, morphological investigations have generated contradictory results, inferring an inconsistency in the phylogenetic analyses (Dewel and
Clark 1973; Kristensen and Higgins, 1984a,b;
Dewel et al., 1993; Dewel and Dewel, 1996; Zantke
et al., 2008). This is in part due to the fact that tardigrades possess morphological characters relating
to both nematodes and arthropods. As an example,
they possess a muscular myoepithelial triradiate
pharynx (Ruppert, 1982; Ruppert and Barnes,
1994), which is also seen in nematodes and loriciferans (Kristensen, 2003), and conversely have lobed
cerebral ganglia connected with a ladder-type chain
of ventral trunk ganglia, much like an arthropod
nervous system. For review of some of the inconsistencies, see Greven (1982). The architecture of the
nervous system, and in particular, the brain of Tardigrada, has often been emphasized as important
for phylogenetic analysis attempting to elucidate
the relation of the group to other phyla, however, a
general consensus has yet to be reached (Marcus,
1929; Dewel and Dewel, 1996; Zantke et al., 2008).
Even the most recent investigation on the tardigrade nervous system, using confocal laser scanning microscopy, immunocytochemical staining,
and three-dimensional (3D) reconstruction, did not
provide explicit conclusion toward the phylogenetic
relationship between tardigrades and arthropods or
nematodes (Zantke et al., 2008).
Earlier investigations of tardigrade cephalic
sense organs and their innervations have led to
suggestions of a three-lobed brain (Kristensen and
Higgins, 1984a,b; Dewel and Dewel, 1996; Wiederhöft and Greven, 1996). Furthermore, the ventral
nerve cord has been described to comprise paired
segmental ganglia interconnected by longitudinal
connectives, and connected intrasegmentally by
transverse commissures giving the appearance of a
rope-ladder like organization (Kristensen, 1982).
This structuring of the central nervous system
(CNS) is similar to that observed in arthropods
(Scholtz, 2002; Müller, 2006; Scholtz and
Journal of Morphology
Edgecombe, 2006) and would therefore strengthen
the hypothesis that Tardigrada is closely related to
Arthropoda. However, the results obtained by
Zantke et al. (2008) on the eutardigrade Macrobiotus hufelandi C.A.S. Schultze (1833) showed a
three lobed brain, but did not support the existence of three segments in the head. Their argument against three brain segments is founded on a
suggested hypothetical model based on their data,
in which it seems all brain commissures are
located in the hypothetical deutocerebrum. Also,
they find no connective from the hypothetical tritocerebrum to the first ventral trunk ganglion and
transverse commissures of the ventral trunk ganglia were not observed.
Studies on tardigrade development support the
sister group relationship of Tardigrada and Arthropoda 1 Onychophora (Hejnol and Schnabel,
2006), although it was not determined whether or
not a third brain lobe is present. In addition, the nature of the subpharyngeal ganglion was questioned
as it seemed to be an outgrowth of the brain.
Due to these differences between earlier morphological investigations, we found it is necessary
to reinvestigate tardigrade neuroanatomy. A clarification of whether or not certain structures exist
is needed, and a detailed neuroanatomical investigation can furthermore provide possible evidence
for the sister group relationship of Tardigrada.
Here, we provide a detailed description of
the nervous system of the marine eutardigrade
Halobiotus crispae Kristensen (1982) based on
immunocytochemical staining, confocal laser
scanning microscopy, and computer-aided 3D
reconstructions, supported by transmission electron microscopy. Specifically, the brain and the
ventral ganglia receive much attention, as these
structures are of phylogenetic importance. Our
investigation expands on the current knowledge of
tardigrade neuroanatomy, and our data are interpreted according to existing data and theories.
MATERIALS AND METHODS
Specimens of the tardigrade Halobiotus crispae were sampled
at Vellerup Vig, in the Isefjord. Animals were extracted according to methods previously described (see Kristensen, 1982;
Eibye-Jacobsen, 1997; Møbjerg and Dahl, 1996, Møbjerg et al.,
2007; Halberg et al., 2009a,b; Halberg and Møbjerg, 2012). Live
tardigrades were stored at 48C in seawater (20%) from the locality and supplied with substrate. The animal examined with
transmission electron microscopy was collected intertidally at
the type locality Nipissat Bay, Disko Island, West Greenland.
Relaxation and Fixation
Specimens of H. crispae were stretched in freshwater and subsequently relaxed using CO2-enriched water to prevent muscle
contractions during fixation. The CO2-enriched water was
applied drop by drop until the animals were completely passive.
Immediately after relaxation, the specimens were fixed in 4%
paraformaldehyde in 0.1 mol l21 phosphate buffered saline (PBS)
NEUROANATOMY OF Halobiotus crispae
21
(for 500 ml of a 53 concentrated stock solution of 0.5 mol l
PBS: 33.48 g Na2HPO42H2O, 7.93 g NaH2PO4H2O, pH 7.2–7.4)
for 60 min at room temperature, and washed at least 83 10–15
min in washing buffer (0.1 mol l21 PBS with 0.1% NaN3).
Permeabilization and Blocking
After fixation, the animals were perforated using a very fine
needle to facilitate penetration of the antibodies, followed by
90 s of sonication in a sonication bath at 30 W (Branson,
B-1200 E1). Subsequently, the tardigrades were transferred to a
blocking and permeabilizing solution consisting of 0.1 mol l21
PBS with 1% Triton X-100, 0.1% NaN3 and normal goat serum
(NGS, 6% working concentration; Sigma cat. no. G9023), and
incubated over night at 48C.
Staining
For immunolabeling, the primary antibody used was antiacetylated a-tubulin at a concentration of 1:250 (antimouse, monoclonal; Sigma, cat. no. T6793). Specimens were incubated with
primary antibody on a shaker (celloshaker variospeed) at 48C
for 48 h, and subsequently rinsed with washing buffer for at
least 63 20–30 min and finally over night. Specimens were
then incubated with secondary antibody (Alexa flour 488 goatantimouse, Invitrogen cat. no. A11001) at a concentration of
1:200 for 48 h on a shaker (celloshaker variospeed) at 48C.
Antibodies were diluted with PBS with 0.1% NaN3 and NGS.
During the last half of the incubation period with secondary
antibody, 40 ,6-diamidino-2-phenylindole (5 lg/ml, DAPI; Invitrogen cat. no. D21490) was added for nuclear staining. After incubation, the animals were rinsed thoroughly in washing buffer
for at least 63 20–30 min followed by an overnight wash. A
total of 58 animals were used. A complementary immunolabeling with serotonin was performed following the same protocol
as described above, using antiserotonin 5-HT 1:200 (antirabbit,
polyclonal; Sigma, cat. no. S1561).
Negative controls were performed during the initial experiments. The controls were performed by omitting the primary
antibody from the staining procedure. The controls showed no fluorescence other than the autofluorescence of cuticular structures.
Mounting and Image Acquisition
Prior to mounting, specimens were treated with a glycerol series. This was done to prevent any deformation of the animals,
due to the density and viscosity of the mounting media. The
glycerol was applied at the start of the third buffer rinsing (see
above), one drop at a time, starting with 5% glycerol. A few
drops were applied and a small amount of liquid was simultaneously removed, until all the rinsing liquid had been replaced
with 5% glycerol. This process was repeated with 10% followed
by 25–100% glycerol. Then, the specimens were mounted on
coverslips in Flouromount mounting medium (Southern Biotechnology Associates, Birmingham, AL). Using an Erwin loop,
specimens were placed in droplets of the mounting medium and
carefully manipulated with a fine needle into the desired position and the coverslips were sealed. Image acquisition was performed on a Leica DM RXE 6 TL microscope equipped with a
Leica TCS SP2 AOBS confocal laser scanning unit, using the
488-nm line of an argon/crypton laser for antibody detection
and UV laser for DAPI. A maximum projection of the image series was used and processed in the 3D image program IMARIS
(Bitplane, Zurich, Switzerland).
Transmission Electron Microscopy
8 specimens were used for transmission electron microscopy.
The specimens were collected in Nipissat Bay, Disko Island,
West Greenland (N 698 25.9340 , E 548 10.7680 ) in April 1979
1229
(type material: Kristensen, 1982), and fixed with a trialdehyde
solution followed by postfixation with 1% OsO4. Ultrathin sections The specimens were cut with a diamond knife. For details
on the preparation of transmission electron microscopy sections
see Kristensen (1976).
Regarding the nomenclature, we follow that used by Marcus
(1929), also implemented by Zantke et al. (2008), and to some
extent Kristensen (1982). Common neuroanatomical terms are
in accordance with Richter et al. (2010).
RESULTS
H. crispae showed pronounced immunoreactivity
against acetylated a-tubulin, particularly in the brain
as well as the longitudinal nerve cords and ventral
ganglia (Fig. 1). The acetylated a-tubulin immunoreactions combined with nuclear labeling with DAPI
produced detailed images of the nervous system. In
the following description, the CNS, comprising the
brain, ventral longitudinal nerve cords, and ventral
ganglia, will be described separately from the peripheral nervous system (PNS), which is comprised of
every nervous structure outside the CNS. The sensory areas described as papilla cephalica (Figs. 1 and
2; pc) and temporalia (Fig. 2A,B; t. see Kristensen,
1982) are very closely associated with the brain and
will be treated as part of the CNS.
The nervous system of H. crispae has a clear
segmental organization, with four paired ventral
trunk ganglia each connected with two leg ganglia
and two lateral neurons. Each of the lateral neurons in turn communicates with a dorsal neuron.
The brain appears to be composed of three paired
lobes each containing a commissure. The brain is
connected with a subpharyngeal ganglion (g0) by a
pair of connectives from the ventrolateral lobes.
The subpharyngeal ganglion is in turn connected
by a ventral longitudinal nerve cord to the first
ventral trunk ganglion.
The Central Nervous System
The brain. The gross structure of the brain
of H. crispae consists of 11 nerve cell clusters
containing the soma of the nerve cells, and an
elaborate network of nerve fibers and fiber bundles
connecting the clusters (Figs. 2–6). The number of
nerve cells and cells associated with these in the
head region accounts for approximately one-quarter to one-third of all the cells in the animal (see
review by Møbjerg et al., 2011). The interpretation
of the number of brain clusters depends on which
structures are considered to be part of the brain as
well as on the interpretation of what comprises a
cluster. Here, we define a brain cluster as a distinct group of cells or cells forming part of a lobe.
The overall brain structure of H. crispae comprises
two lateral outer lobes, two inner lobes, a median
ganglion, and two ventrolateral lobes (Fig. 2). The
laterally located outer brain lobes are comprised of
two clusters, which is also true for the two inner
brain lobes. The median ganglion, situated
Journal of Morphology
1230
D.K. PERSSON ET AL.
Fig. 1. Overview of the nervous system of Halobiotus crispae showing immunoreactivity against antiacetylated a-tubulin, maximum projection. (A) Double labeling with antiacetylated a-tubulin and DAPI. Lateral view, showing the strong innervation of the
papilla cephalica (pc) from the brain, as well as the arrangement of the PNS. (B) Dorsal and (C) ventral view, respectively, giving
an overview of the CNS with brain and ventral ganglia as well as the arrangement of the PNS. cl, cloacal neurons; gI-IV, ventral
ganglion I-IV; dne, dorsal neuron; dn, dorsal nerve; lgg, leg ganglion; lne, lateral neuron; lln, lateral longitudinal nerve; ln, lateral
nerve; pc, nerves of papilla cephalica.
Journal of Morphology
NEUROANATOMY OF Halobiotus crispae
1231
Fig. 2. Halobiotus crispae, immunoreactivity against antiacetylated a-tubulin in the brain viewed from four different angles.
(A) Dorsal view showing the five nerves of the papilla cephalica (n7, n8, n9, n10a, n10b) as well as the head nerves n12 and n13.
(B) Frontal view revealing the preoral commissure (prcm) just ventral to the dorsal commissure. Maximum projection. White
arrowheads indicate conspicuous immunoreactive nerves of the inner lobes. (C) Lateral and (D) anterolateral view, both showing
the 3D arrangement of the outer and inner lobes. Normal shading, Imaris. (E, F) Frontal view showing immunoreaction in the
area of the ventrolateral lobe and the ventral commissure, maximum projection and iso-surface rendering, respectively. dc, dorsal
commissure; ic, inner connective; il.acl, inner lobe anterior cluster; il, inner lobe; il.pcl, inner lobe posterior cluster; mg, median
ganglion; oc, outer connective; ol, outer lobe; pc, nerves of papilla cephalica; prcm, preoral commissure; t, temporalia; vc, ventral
commissure; vll, ventrolateral lobe. Double arrows indicate orientation of the image. Anterior and posterior is indicated with a and
p, respectively, whereas d and v indicate dorsal and ventral, respectively.
Journal of Morphology
1232
D.K. PERSSON ET AL.
Fig. 3. Halobiotus crispae, close-up of the brain, with individual stack images showing specific details. (A) Dorsal view of a maximum-projection. Here, the connection of the brain to the nervous system of the trunk is revealed as well as the stylet commissure
(stcm) which is connected with the stylets. (B–E) Individual sections from dorsal to ventral. (B) Notice the innervations of the temporalia (t), as well as the conspicuous immunoreactive areas of the inner lobes, lateral to the dorsal commissure (white arrowheads). The n13 nerve is clearly visible in the anteriodorsal part of the head. (C) Going further ventral reveals the large nerve bundle from the outer lobes, which become part of the dorsal commissure (white arrowheads). (D) This section shows the postoral commissure (pocm) and the connectives (co) to the ventral part of the brain. Also, notice the buccal neurons (bn) dorsal to the mouth
opening. (E) The mouth opening (mo) is innervated by several nerves connected with the ventral part of the brain (white arrowheads). In the same focal plane as the mouth opening, the ventral commissure (vc) is clearly discernible. bn, buccal neurons; co,
connective; dc, dorsal connective; ey, eye; gI, first ventral ganglion, ic, inner connective; ln, lateral nerve; mo, mouth opening; oc,
outer connective; pc, nerves of papilla cephalica; pocm, post oral commissure; t, temporalia; vc, ventral commissure.
Journal of Morphology
NEUROANATOMY OF Halobiotus crispae
Fig. 4. Halobiotus crispae, close-up of the mouth opening,
showing the external and internal structures of the buccal
lamella. (A) Scanning electron micrograph revealing the external morphology of the six buccal lamella (asterisk) surrounding
the mouth opening. (B) Transmission electron micrograph of a
section through the buccal lamella shows the internal structure
of the buccal lamella and the surrounding tissue. (C) Antiacetylated a-tubulin immunoreaction in the mouth opening. ci, cilia.
Scale bars are 15 lm.
between the inner lobes, consists of a single cluster
of cells and innervates the forehead via the nerve
n13, whereas the ventrolateral lobe comprises one
cluster on each side of the buccal tube (Figs. 2 and
3). The mouth is innervated by nerves from both
ventrolateral and inner lobes (Figs. 3E and 4; se
1233
below). The brain contains a commissure between
each of the paired brain lobes and a smaller commissure in connection with the innervations of the
stylets. In total, we identified four commissures:
the large dorsal commissure connects the inner
lobes, the preoral commissure connects the outer
lobes, the ventral commissure connects the ventrolateral lobes and the stylet commissure innervates
the stylets and stylet supports (Figs. 2,3, 5 and 6).
A pair of connectives extending from the ventrolateral lobes, connects the brain to the central
nervous system of the trunk through a subpharyngeal ganglion (Fig. 7-8). The subpharyngeal ganglion communicates with the first ventral trunk
ganglion through the ventral longitudinal nerve
cord. In addition, so-called outer and inner connectives extend from respectively the outer and ventrolateral lobes, connecting the brain to the first
ventral trunk ganglion (see below).
The lateral outer brain lobes constitute the largest and most conspicuous part of the brain. These
large lobes are elongated and positioned dorsolaterally to the buccal tube and extend caudally; they
are referred to as the outer lobes (Figs. 2, 3, 6, 9,
and 10; ol) and they contain the eye spots
(Fig. 3A1D; ey). The outer lobes can be divided
into an anterior and a posterior cluster (Figs. 2C
and 6; ol.acl, ol.pcl). The second pair of lobes is
closer to the median plane of the animal and is
referred to as the inner lobes (Figs. 2, 5, 6, and 10;
il). The inner lobes also seem to be composed of
two nerve cell clusters, a posterior cluster and an
anterior cluster (Figs. 2A1C and 5; il.pcl, il.acl).
Both pairs of brain lobes are positioned dorsal to
the buccal tube, and the two ‘‘hemispheres’’ are
interconnected by a massive bundle of nerve fibers
containing the preoral and predorsal commissures
(Fig. 2A1B; prcm, dc). On each side of the dorsal
commissure small, highly immunoreactive areas of
the inner lobes are clearly visible (Fig. 2A,B; white
arrowheads), which seem to be connected with
nerves running through the dorsal commissure. A
very pronounced nerve runs between the anterior
cluster of the inner lobe and the dorsal commissure (Fig. 5; n4, see also Zantke et al., 2008). The
anterior clusters of the inner lobes receive nerve
extensions from the sensory area of the anterior
part of the head, known as the papilla cephalica
(Figs. 1–315). A total of five nerves can be distinguished in the papilla cephalica named n7, n8, n9,
n10a, and n10b (Figs. 2A1C and 5), of these only
n9 originates from the outer lobe, whereas the
others originate from the inner lobe.
From the posterior region of the outer lobes, in
the vicinity of the eyes, extensive dorsal innervations of the cuticle are clearly visible (Figs. 2A–D,
3A1B, and 5; t). These innervations are more or
less similar to the innervations seen in the cuticle
of the papilla cephalica and are termed temporalia
by Kristensen (1982); the exact number of nerves
Journal of Morphology
1234
D.K. PERSSON ET AL.
Fig. 5. Halobiotus crispae, antiacetylated a-tubulin immunoreactions of the brain. (A) Maximum projection revealing the n4
nerve. Also, the head nerves n12 and n13 are clearly visible. (B) Normal shading rendering, using the software Imaris, reveal the
n13 nerve to originate from the median ganglion (mg). Furthermore, the individual nerves, n7-n10a1b, of the papilla cephalica can
be distinguished. dc, dorsal commissure; il.acl, inner lobe anterior cluster; mg, median ganglion; pc, nerves of papilla cephalica; t,
temporalia.
innervating the area cannot be distinguished.
Between the inner lobes, an unpaired triangularshaped median ganglion is located (Figs. 2A1C
and 5; mg). From this ganglion, a double nerve
cord extends anteriorly above the dorsal commisJournal of Morphology
sure, ending in a small circular nerve structure in
the cuticle of the forehead. This nerve corresponds
with the n13 nerve described in Zantke et al.
(2008), and it follows that the very fine paired
nerve lateral to n13 is n12 (Figs. 1B,C, 2A–D, 3A–
NEUROANATOMY OF Halobiotus crispae
1235
Fig. 6. Halobiotus crispae, individual sections from an image stack, showing immunoreaction to antiacetylated a-tubulin and
corresponding DAPI labeling. (A) A large part of the dorsal commissure (dc) originates from the outer lobes (arrows). (B) The DAPI
labeling in the same focal plane as A shows the posterior and anterior clusters of the outer lobes, and the posterior cluster of the
inner lobes. (C and D) The position of the stylet commissure coincides with the stylet supports (ss). (E and F) In the ventral part
of the animal, a ventral commissure is clearly visible (E, vc), which corresponds very well with the position of two ventrolateral
lobes revealed with DAPI labeling (F, vll). (G) In the most ventral part of the animal, the antiacetylated a-tubulin staining show
that the outer connectives connects to the outer lobes and the inner connectives connects to ventrolateral lobes, which is connected
with the subpharyngeal ganglion (g0). bt, buccal tube; dc, dorsal commisure; g0, ventral ganglion; ic, inner connective; il, inner
lobe; oc, outer connective; ol, outer lobe; ol.acl, outer lobe anterior cluster; il.pcl, inner lobe posterior cluster; ol.pcl, outer lobe posterior cluster; ss, stylet support; stcm, stylet commissures; stn, stylet nerves; vll, ventrolateral lobe.
C, and 5; n12, n13). The outer lobes, inner lobes,
the median ganglion, and associated commissures
constitute the dorsal part of the brain.
Ventral to the buccal tube another commissure
is located, the ventral commissure (Figs. 2E1F,
3E, and 6E; vc). Posterior to the ventral commisJournal of Morphology
1236
D.K. PERSSON ET AL.
Fig. 7. Halobiotus crispae, lateral view of the brain and ventral ganglia. (A) Immunoreactivity against antiacetylated a-tubulin
in the brain showing the longitudinal nerve cord connecting the first ventral trunk ganglion (gI) to the subpharyngeal ganglion
(g0). (B) DAPI staining showing the nuclei of gI and g0. (C) Lateral view of the ventral trunk ganglia and the subpharyngeal ganglion (g0). Asterisks indicate possible perikarya. bt, buccal tube; clg, claw gland; g0, subpharyngeal ganglion; gI, first ventral trunk
ganglion; ic, inner connective; oc, outer connective; lgg, leg ganglion; lnc, longitudinal nerve cord.
sure and slightly dorsal to the buccal tube, a
fourth commissure is visible in the area of the
stylets (Fig. 3D; stcm). From this stylet commissure, stylet nerves (Fig. 3D; stn) extend anterolaterally into the ventrolateral brain lobes. These
lobes consist of a cell cluster on each side of the
buccal tube (Figs. 2E1F and 6C–H). From the ventrolateral lobes, several nerves extend anteriorly
toward the mouth (Fig. 3E; white arrowheads). In
addition, the mouth receives nerves from several
nerve cells dorsal to the mouth opening (Fig. 3E;
bn) which are connected with the inner lobes.
These nerves are all connected with the six buccal
lamellae surrounding the mouth opening (Fig. 4;
asterisks).
Sections of the image stack in the area of the
ventral commissure reveal cell clusters around the
buccal tube and immediately ventral to it, as well
Journal of Morphology
as a ganglion-like structure posterior to these
clusters, these are termed the ventrolateral lobes
(vll) and the subpharyngeal ganglion (g0), respectively (Fig. 6F1H; stippled circles/oval). The ventrolateral lobes are also clearly visible in Figure
2E and F, interconnected by the ventral commissure. Additionally, a transmission electron microscopic image of the same area support the existence of a third pair of brain lobes and also shows
the connective extending ventrally from these
lobes (Fig. 9).
The ventral ganglia and longitudinal nerve
cords. From the posterior region of the outer
lobes, a double nerve tract extends ventrocaudally,
with one lateral nerve (Fig. 3A; ln) branching off
into the lateral nervous system and the other connecting to the first ventral trunk ganglion. The latter is referred to as the outer connective (Fig. 3A;
NEUROANATOMY OF Halobiotus crispae
1237
ally, serotonin immunoreactivity is shown in these
regions as well, which could indicate the presence
of perikarya (Fig. 7, asterisks).
In addition, we find a fifth ventral ganglion, the
subpharyngeal ganglion (g0), which is connected
with the ventrolateral brain lobes and the first
ventral trunk ganglion (gI) via the longitudinal
nerve cord (Fig. 7).
The Peripheral Nervous System
Fig. 8. Halobiotus crispae, the nervous system of the trunk.
Insets show close-up of the ventral ganglia I–IV and arrows
indicate the number of commissures. cl, cloacal nerves/neurons;
cm, commissure.
oc). From the ventrolateral part of the brain
extends the inner connective (Fig. 3A; ic); the
inner connective is also connected with the first
ventral trunk ganglia. These nerve tracts connect
the brain with the CNS of the trunk comprised of
paired ventral trunk ganglia and longitudinal
nerve cords. There are four paired ventral trunk
ganglia, each composed of approximately 40 cells
that are associated with a corresponding leg pair
(Figs. 1 and 8; gI–gIV). The ventral trunk ganglia
are intrasegmentally connected by transverse commissures and intersegmentally connected by ventral longitudinal connectives, giving the appearance
of
a
rope-ladder-like
arrangement
(Figs. 118). In the first three ganglia, we observe
two commissures, whereas in the fourth, only one
can be observed (Fig. 8; inserts). The longitudinal
connectives are collectively referred to as the longitudinal nerve cords (Fig. 1; lnc), and extend
through most of the length of the animal, terminating in the fourth ventral trunk ganglion (Fig.
1; gIV). It is not unambiguous whether the longitudinal nerve cords contain perikarya or not; the
a-tubulin stainings show some thickenings of the
longitudinal nerve cords in the regions between
the ventral trunk ganglia (Figs. 1 and 8). Addition-
The PNS is mainly comprised of four dorsal and
four lateral neurons on each side of the animal
(Fig. 1; dne and lne). The dorsal and lateral neurons are connected by dorsal and lateral nerves
(Fig. 1A1B; dn, ln). The lateral nerves connect the
lateral neurons with the ventral trunk ganglia. In
addition, there is a lateral nerve connecting the
first lateral neuron to the brain (Fig. 3A; ln). This
lateral nerve originates from the outer lobe, propagating in parallel with the outer connective, and
connects to the first lateral neuron (Fig. 1A; lne).
From the first lateral neuron, nerve projections
extend both in an anterior–posterior as well as in
a dorsal direction, and are referred to as lateral
longitudinal nerves (Fig. 1A–C; lln) and dorsal
nerves (Fig. 1A1B; dn), respectively. The lateral
nerve extends through most of the length of the
animal, connecting the four lateral neurons. The
lateral neurons are connected with dorsal neurons
(Fig. 1; dne) via dorsal nerves (Fig. 1; dn). From
all the dorsal ganglia/neurons sensory cilia extends
further toward the dorsal side. These modified cilia
could very well correspond to the lateral cirri of
heterotardigrades.
At the fourth ventral trunk ganglion, nerves
extend dorsocaudally to the dorsal neuron (Fig. 1;
dne) and nerves branch off into the hind legs. The
nerves in the hind legs connect to leg ganglia in
the distal part of the legs (Fig. 1; lgg), and two
nerves extend from these ganglia terminating in a
cilia in the proximal part of the legs. In addition, a
pair of nerves extends from the fourth ventral
trunk ganglion and terminates near the cloaca
(Fig. 1A; cl).
Leg ganglia are connected with nerves originating from the ventral trunk ganglia. From the second and third ventral trunk ganglion, three nerves
n1, n2, and n3 originates (Fig. 1C). The n3 and n2
nerves connect with the anterior and posterior
region of the associated leg, with the posterior
nerve attaching at the distal part of the leg, and
the middle nerve connect to a ganglion in the proximal part of the leg—a leg ganglion (Fig. 1C; lgg).
The n1 nerve connect to the lateral neuron. For
the first ventral trunk ganglion, it follows that the
outer connectives correspond to the n1 nerves of
the second and third ventral ganglia, whereas the
n2 and n3 nerves exhibit the same pattern in ventral trunk ganglia I–III. For complete overview of
Journal of Morphology
1238
D.K. PERSSON ET AL.
Fig. 9. Halobiotus crispae, transmission electron micrograph merging of five cross-sections of
the brain of H. crispae. Image merging performed in CorelDraw. Notice the ventrolateral lobes
(vll) and their connectives to the ventral region. dc, dorsal commissure; bt, buccal tube; co, connectives; ol, outer lobe; stn, stylet nerve; vll, ventrolateral lobe.
the described nervous structures and nerves in the
present study, as well as comparison to the nervous structures previously described in the literature see table 1.
DISCUSSION
Marcus (1929) described the nervous system of
Macrobiotus hufelandi with impressive detail,
exemplified by the fact that most of our findings
(using contemporary techniques) in H. crispae confirm what he described. Moreover, our results support most of the descriptions of the tardigrade
nervous system by Zantke et al. (2008), albeit we
report some new findings together with increased
details. In particular, we have obtained greater
details on brain structure, and important structures in the ventral ganglia. Noticeably, several of
the details uncovered in our study pertain to structures that were declared missing in tardigrades or
possibly misinterpreted by Zantke et al. (2008).
Journal of Morphology
In the nervous system investigation of M. hufelandi by Zantke et al. (2008) a preoral commissure
is described in the brain to be positioned just
below the dorsal commissure. In addition, they
show that a postoral commissure is connected with
the first ventral ganglion via the inner connectives, and to the dorsal commissure via the circumbuccal connectives, forming a circumbuccal ring.
Our data show a similar preoral commissure positioned just ventral to the dorsal commissure
(Fig. 2B, prcm). In addition, we find a commissure
ventral to the buccal tube which could be equivalent to the postoral commissure described by
Zantke et al. (2008, Fig. 2F, pocm). We choose to
term this structure the ventral commissure
(Fig. 3E, vc) as it appears more similar to the ventral ring commissure found in the heterotardigrade Echiniscus viridissimus by Dewel and Dewel
(1996). In H. crispae, the ventral commissure is
not connected with the first ventral trunk ganglia
via the inner connectives; more precisely, the ventral commissure connects the ventrolateral lobes
NEUROANATOMY OF Halobiotus crispae
1239
Fig. 10. Conceptual drawing constructed on the basis of the data in this study, showing our interpretation of the brain structure
of H. crispae. (A) Lateral view. (B) Frontal view. clg, claw gland; co, connective; dc, dorsal commissure; ey, eye; g0, subpharyngeal
ganglion; gI, first ventral trunk ganglion; ic, inner connective; il, inner lobe; lgg, leg ganglion; mg, median ganglion; mo, mouth
opening; oc, outer connective; ol, outer lobe; pc, papilla cephalica; pb, pharyngeal bulb; st, stylet; t, temporalia; vll, ventrolateral
lobe. Arrows indicate the approximate area of the transmission electron microscopical section in figure 9.
Journal of Morphology
1240
Journal of Morphology
TABLE 1. Table listing the structures of the nervous system of H. crispae described in this study and compared with previous studies of H. crispae or other tardigrades
Identified structures
and named nerves
CNS, head
PNS
M. hufelandi
M. hufelandi
H. crispae
Constructed
ancestral tardigrade
Persson et al. (this study)
Marcus (1929)
Zantke et al. (2008)
Kristensen (1982)
Nielsen (2001)
Inner lobes (il) with anterior
and posterior clusters (acl, pcl)
Dorsal commissure (dc)
Outer lobes (ol) with anterior
and posterior clusters (acl, pcl)
Preoral commissure (prcm)
Median ganglion (mg)
n12, n13
n4
Buccal neurons (bn)
Ventrolateral lobes (vll)
Ventral commissure (vc)
Subpharyngeal ganglion (g0)
Stylet commissure (stcm)
Stylet nerve (stn)
Outer connective (oc)
Inner connective (ic)
Innervation of papilla cephalica
(pc, n7, n8, n9, n10a, n10b)
Innervation of temporalia (t)
Innervation of buccal lamella
Four ventral trunk ganglia
(gI-IV)
Transverse commissures (cm)
Longitudinal nerve cords (lnc)
Leg ganglia (lgg)
Leg nerves (n2+n3)
Lateral nerves (n1, ln)
Lateral neurons (lne)
Dorsal nerves (dn)
Dorsal neurons (dne)
Lateral longitudinal nerves (lln)
Cloacal nerves (cl)
Inner lobes
Inner lobes (posterior cluster)
Protocerebrum
Protocerebrum
Outer lobes
Dorsal commissure
Outer lobes (dorsal cluster)
Deutocerebrum
Deutocerebrum
Tritocerebrum
Tritocerebrum
Subesophageal ganglion
Subesophageal ganglion
Stylet nerve (s.ne)
Outer connective
Stylet nerve (s.ne)
Outer connective
Innervation of papilla
cephalica
Innervation of temporalia
Innervation of papilla
cephalica
Innervation of temporalia
Innervation of buccal
lamella
Four ventral trunk
ganglia
Transverse commissures
Longitudinal nerve cords
Preoral commissure
Median ganglion
n11, n12n, n13
n4, n5, n6
Postoral commissure?
Subesophageal ganglion (I)
Postoral commissure?
Outer connective
Innervation of papilla
cephalica
Innervation of temporalia
Innervation of buccal
lamella
Four ventral trunk
ganglia (IIV)
Transverse commissures
Longitudinal nerve cords
Leg ganglia
Leg nerves (n3)
Lateral nerves (n1, n2)
Lateral neurons
Dorsal nerves
Dorsal neurons
Cloacal nerves
Outer connective
Inner connective
Innervation of papilla
cephalica (n7, n8, n9, n10)
Four ventral trunk
ganglia (gI-IV)
Longitudinal nerve cords
Leg ganglia
n1, n2, n3
Neurophils of the hind legs
Four ventral trunk ganglia
Transverse commissures
Longitudinal nerve cords
Leg nerves
Sensilla
D.K. PERSSON ET AL.
CNS, trunk
H. crispae
NEUROANATOMY OF Halobiotus crispae
flanking the buccal tube. It is these lobes that are
connected with the first ventral trunk ganglia via
the inner connective.
When looking at a DAPI staining of the area of
the stylet commissure, there is not an actual ganglion (compare Fig. 6C,D) and the commissure is
positioned exactly at the stylet supports. Also,
nerves extend laterally from the commissure
(Figs. 3A1D and 6C; co) maybe connecting to the
stylet muscles. So, this seems to be a stylet commissure in connection with the innervations of the
stylets. Therefore, we chose the name stylet commissure. In addition, the stylet commissure is not
directly connected with the dorsal commissure, but
it is actually connected with the ganglion-like
structure associated with the ventral commissure.
Furthermore, through closer observations of the
brain, it is obvious that the stylet commissure is
positioned dorsal to the buccal tube, so it should
not be confused with the postoral commissure
described by Zantke et al. (2008), which is positioned ventral to the buccal tube.
The transmission electron microscopical image
in Figure 9 and the image stack of the a-tubulin
and DAPI staining on Figure 6 supports the
immunoreactions seen in Figure 2F, which suggests that the ganglion of the ventral commissure
is a paired, lobed structure situated lateral to the
buccal tube. More ventral, we find a cell cluster
much like the first ventral trunk ganglion, in very
close proximity to the lobes of the ventral commissure (Fig. 6H). The DAPI staining shows a cell
structure very similar to the first ventral trunk
ganglion, and it is positioned in the same level.
Combined with the immunoreaction and complementary DAPI staining in Figure 7, it seems to be
a ganglion in the ventral part of the head connected with the first ventral trunk ganglion.
Although we cannot determine if these nerve cells
have a paired cluster arrangement as is the case
for the ventral trunk ganglia, we cautiously
hypothesize that it is a subpharyngeal ganglion.
The presence of the subpharyngeal ganglion is also
supported by a developmental study, in which a
subesophageal ganglion (the subpharyngeal ganglion) is observed and suggested to be an outgrowth of the brain (Hejnol and Schnabel, 2005).
From this suggestion, it is argued that the subpharyngeal ganglion is part of the brain, which
then forms a circumbuccal ring resembling cycloneuralian conditions (Zantke et al., 2008). This is
still at a hypothetical stage and actual evidence
is needed to clarify if the subpharyngeal ganglion
is part of the brain. Nevertheless, if we consider
the idea of cephalization of several anterior segments to be true, it would not be surprising if the
subpharyngeal ganglion seemed to originate from
the same region as the three paired brain lobes. In
order to fully understand the nature and origin of
the subpharyngeal ganglion, it will be important
1241
to investigate the brain of arthrotardigrades as
they are considered to have many plesiomorphic
characters. Therefore, they may be closer to the
ancestral state in appearance compared to the
more specialized eutardigrades.
Furthermore, it seems plausible that the lobes of
the ventral commissure may be a third paired
brain lobe, and it is clearly connected with the
subpharyngeal ganglion via two connectives
(Fig. 9). The fact that we find two connectives, one
from each ventrolateral lobe, suggests that the
subpharyngeal ganglion is or may have been a
paired cluster like that found in the ventral trunk
ganglia. The ventrolateral lobes are also connected
with the stylet nerves (Fig. 3A). If we accept the
hypothesis of the stylets being internalized claws,
that is, internalized leg, this supports the idea of
segmentation in the head. Hence, we suggest a
brain of at least three parts, and the existence of a
subpharyngeal ganglion in tardigrades.
The third paired lobe has previously been
described in H. crispae by Kristensen (1982) and
could be homologs to the arthropod tritocerebrum.
In addition, if there is homology between the
outer and inner lobes and the protocerebrum and
deutocerebrum of arthropods, then, the brain configuration of tardigrades could be interpreted as
arthropod-like, as suggested in earlier descriptions
(Kristensen and Higgins, 1984a,b; Dewel and
Dewel, 1996; Nielsen, 2001, 2011), though further
studies on tardigrade development is needed to
verify a possible homology. In Figure 6, the subpharyngeal ganglion is indicated by DAPI labeling,
but we do not see a distinct immunoreaction for
a-tubulin. However, on Figure 7A, which shows
antiacetylated a-tubulin staining from the lateral
side, we find clear immunoreaction in the subpharyngeal ganglion, as well as in the nerve cord connecting it to the first ventral trunk ganglion. This
is also supported by serotonergic immunoreaction
in the ventral trunk ganglia as well as g0 (Fig.
7C). One explanation for the lack of immunoreaction in Figure 6 could be failure of antibody recognition, which we have previously encountered with
tardigrades. An example is the inner lobes which
show immunoreaction in Figure 2C1D, but in
Figure 2A no immunoreaction can be observed in
the posterior clusters of the inner lobes.
The n13 nerve running dorsally from the median
lobe (Fig. 5B) terminating in a circular shape in
the forehead is interpreted as a rudiment of the
median cirrus of Heterotardigrada (see also Zantke
et al., 2008). Generally, the median cirrus is present in all Arthrotardigrada (though reduced in
some Archechiniscus species), and missing from all
Eutardigrada and Echiniscoidea. Assuming that
the marine Arthrotardigrada represents the ancestral condition (Renaud-Mornant, 1982; Jørgensen
et al., 2010), the presence of a median cirrus
within Tardigrada represents the plesiomorphic
Journal of Morphology
1242
D.K. PERSSON ET AL.
condition. Additionally, connected with the dorsal
neurons, we found modified cilia that likely are
reduced sensory structures, homologs to the lateral
cirri of Heterotardigrada. Consequently, these
structures could be important in an intra-phylum
phylogenetic perspective.
When looking at the ventral longitudinal nerve
cords and the ventral trunk ganglia, a distinct segmentation is readily recognizable. Apart from the
head, each of the four segments bears a leg and
contains a ventral ganglion with peripheral
nerves, which innervate the associated leg as well
as the lateral and dorsal sides. The ventral trunk
ganglia are paired and intersegmentally connected
by transverse commissures, as described in the
Results section. These commissures were originally
described by Marcus (1929); however, their presence in tardigrades was recently questioned
(Zantke et al., 2008). Our investigation shows that
tardigrades possess intersegmental transverse
commissures. Their presence is important to the
phylogenetic debate as they are a critical component in the rope-ladder type nervous system. This
type of nervous system is not encountered in any
cycloneuralians, but is seen in most arthropods
(Bullock and Horridge, 1965; Brusca and Brusca,
2002).
The PNS primarily comprises the paired nerves
n1, n2, and n3, which are forming a repeated pattern in association with the first three ventral
ganglia. Only the n1 nerves of the first ventral
ganglia differ from the pattern by connecting to
the outer lobes of the brain. Also, the nerves
associated with the fourth ventral trunk ganglion
are arranged in a slightly different pattern.
Although the difference in nerve arrangement in
the fourth ventral ganglion is linked to the orientation and function of the hind legs, the difference in the n1 nerve in the first ventral trunk
ganglion is due to its connection to the brain. We
hypothesize that this deviation could be linked to
an evolutionary scenario involving cephalization
of at least three anterior segments of a tardigrade
ancestor.
In the light of our results, we propose the following hypothetical model to explain the organization
of the tardigrade brain as three segments: The
area of the outer lobes, with the eyes and innervations of the temporalia, which we interpret as
homologs to the primary clavae and lateral cirri of
the heterotardigrades, could be interpreted as the
first segment or protocerebrum. This would then
correspond to the first segment in arthropods bearing the compound eye, though this structure is not
homologs to the tardigrade eye. Also, it would correspond to the first segment in the onychophorans
which bears the antenna and the eyes. Although
again these structures are not considered homologs to any of the structures found on the first segment in tardigrades.
Journal of Morphology
The next segment holds the inner lobes (possibly
deutocerebrum), which innervates the papilla
cephalica, homologs to the secondary clavae and
the internal cirri of heterotardigrades (for homology on primary and secondary clava, see also
Zantke et al. 2008). Consequently, this would be
homologs to the second segment in the head of
both arthropods and onychophorans, though again
it is not readily possible to homologize between
tardigrade sense organs and the modified limps of
arthropods and onychophorans.
The third segment contains the ventrolateral
lobes (possibly tritocerebrum), which innervate the
buccal lamella as well as the stylets and stylet
supports. As it has been hypothesized that the
stylet apparatus may have been formed by internalization of a leg it could be homologs to the second antenna/pedipalp of crustaceans/chelicerates.
According to some authors, the tritocerebrum is
not present in onychophorans (see Erikson and
Budd, 2000; Mayer et al., 2010), if this is true it is
difficult to homologize between tardigrades and
onychophorans with respect to the third brain
region. However, if we hypothesize that the cephalization in the Onychophora lineage excluded the
ganglion which in tardigrades became the third
brain lobe, then, the slime papilla could be homologs to the stylet apparatus.
Of course, in order to accept this hypothesis,
this needs to be supported by more data—for
example, molecular data. Indeed, answers to some
of the questions regarding homology may be found
in future comparative developmental studies, like
those performed by Jager et al. (2006) on Hox gene
expression and by Gabriel and Goldstein (2007) on
expression patterns of Pax 3/7 and Engrailed
homologs.
Whether or not tardigrades possess a tritocerebrum is important with regards to their phylogenetic position in relation to Onychophora and Arthropoda. As mentioned above, a tritocerebrum
has not been found in the onychophorans. This is
because the ganglion innervating the third segment is not part of the brain, but is part of the
ventral nerve cord. Consequently, it was hypothesized that the last common ancestor of onychophorans and arthropods possessed a bipartite
brain comprised of a protocerebrum and a deutocerebrum (Mayer et al., 2010). As H. crispae possess a third brain lobe, this could then support a
clade with Tardigrada and Arthropoda as sister
groups within Panarthropoda.
The presence of a third brain lobe in tardigrades
generates three possible phylogenetic configurations for Panarthropoda, assuming true homology
between the brain lobes of Tardigrada and Arthropoda. In one scenario, the ancestor to Panarthropoda could have had three brain lobes, and
consequently, the third lobe was lost in Onychophora. If this is true, brain morphology will not
NEUROANATOMY OF Halobiotus crispae
aid in resolving the phylogeny within Panarthropoda, as all placements of Onychophora are
equally parsimonious. In contrast, if the last common ancestor of Panarthropoda had a bipartite
brain, the outcome would be that Onychophora
could be sister group to Tardigrada 1 Arthropoda,
or grouped together with one of them in which
case the development of the third brain lobe would
be convergent. Hence, the most parsimonious hypothesis, when only considering brain morphology,
is that the last common ancestor to Panarthropoda
had a bipartite brain and that tritocerebrum was
developed in a common ancestor to Tardigrada and
Arthropoda. This is of course speculative, and even
though molecular data support the inclusion of
Tardigrada into Panarthropoda, it does not support a sister group relationship between Tardigrada and Arthropoda (Campbell et al., 2011). As
noted above more investigations, especially on tardigrade development, are needed in order to substantiate our suggestions.
The stylet apparatus may be internalized claws,
as earlier transmission electron microscopical
investigations have shown that stylets and claws
are formed in the same way (Kristensen, 1976;
Nielsen, 2001, 2011; Halberg et al., 2009a) and are
similar to the jaws of Onychophora (Storch and
Ruhberg, 1993; Mayer and Harzsch, 2007; Mayer
et al., 2010). It is also a possibility that the stylets
and the supports could be greatly modified mouth
appendages.
The nervous system is distinctly metameric, consisting of the three-lobed brain, the subpharyngeal
ganglion, and the four ventral trunk ganglia.
Characteristic to all tardigrades is the large paired
outer connective with a small ganglion that connects the outer lobe (protocerebrum) to the first
ventral ganglion (Marcus, 1929). However, we also
found a thin connective between the ventrolateral
lobe (tritocerebrum) and the first ventral trunk
ganglion. Our interpretation of the brain structure
is summarized in a concept drawing in Figure 10
and represents an integration of all our immunoreactive data from the head region as well as transmission electron microscopy.
On the basis of this investigation, we find that
tardigrades possess a brain of at least three parts.
We hypothesize that the three paired lobes could
originate from three head segments and that the
subpharyngeal ganglion originated from a fourth
segment. Along with the commissures of the ventral trunk ganglia and the segmentation of the
body, this leads us to suggest that the tardigrade
nervous system structure supports the clade Panarthropoda. The similarities of the tardigrade
nervous system toward Cycloneuralia (loriciferans
and kinorhynchs, Kristensen, 2003), especially the
kinorhynchs, which have segmentally arranged
ventral ganglia (Kristensen and Higgins, 1991),
have been pointed out by morphologists as being
1243
surprising analogies. However, tardigrades may
have some plesiomorphic characters in the nervous
system as well as a myoepithelial triradiate pharyngeal bulb, so the mentioned similarities
between Tardigrada and Cycloneuralia could be
true homologies.
ACKNOWLEDGMENT
The authors thank Stine Elle for preparing the
line art illustrations.
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