Neuroanatomy of

Neuroanatomy of Halobiotus crispae (Eutardigrada:Hypsibiidae): Tardigrade Brain Structure Supportsthe Clade Panarthropoda

Dennis K. Persson,1,2* Kenneth A. Halberg,2 Aslak Jørgensen,3 Nadja Møbjerg,2

and Reinhardt M. Kristensen1

1Invertebrate Department, Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15,DK-2100 Copenhagen Ø, Denmark2Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark3Laboratory 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 animaltree of life is a subject that has received much attention,but still remains controversial. Whereas some think tar-digrades should be categorized as cycloneuralians, mostauthors argue in favor of a phylogenetic positionwithin Panarthropoda as a sister group to Arthropoda orArthropoda 1 Onychophora. Thus far, neither molecularnor morphological investigations have provided conclu-sive results as to the tardigrade sister group relation-ships. In this article, we present a detailed descriptionof the nervous system of the eutardigrade Halobiotuscrispae, using immunostainings, confocal laser scanningmicroscopy, and computer-aided three-dimensionalreconstructions supported by transmission electron mi-croscopy. We report details regarding the structure ofthe brain as well as the ganglia of the ventral nervecord. In contrast to the newest investigation, we findtransverse commissures in the ventral ganglia, and ourdata suggest that the brain is partitioned into at leastthree lobes. Additionally, we can confirm the existence ofa subpharyngeal ganglion previously called subesophagalganglion. According to our results, the original suggestionof a brain comprised of at least three parts cannot berejected, and the data presented supports a sister grouprelationship of Tardigrada to 1) Arthropoda or 2) Onycho-phora or 3) Arthropoda 1 Onychophora. J. Morphol.273:1227–1245, 2012. � 2012 Wiley Periodicals, Inc.

KEY WORDS: neuroanatomy; tardigrade; phylogeny

INTRODUCTION

Tardigrades are small invertebrates predomi-nantly found in mosses and lichens but alsoon marine macroalgae and between sand grains(Bertolani, 1982; McInnes, in press). They exhibitmany autapomorphic features and were catego-rized as a phylum, Tardigrada, by Ramazzotti andMaucci (1983). When subjected to adverse environ-mental conditions, they may endure by means of i)active regulating processes or ii) entering diapauseor a stress tolerant state called cryptobiosis(Møbjerg et al., 2011). Cryptobiosis enables tardi-grades to survive severe physical stress, and con-

sequently, they occupy some of the most inhospita-ble habitats (Renaud-Mornant, 1975; Dastych andKristensen, 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 oflife is a subject that has received much attentionbut remains controversial. Ever since their discov-ery in the middle of the 18th century, their positionin the tree of life has been debated, and affiliationsto Rotifera, Pentastomida, Onychophora, Nema-toda, and Arthropoda have been suggested(Dujardin, 1851; Plate, 1889; Marcus, 1929; Croweet al., 1970; Dewel and Clark, 1973; Ramskold andHou, 1991). Most evidence suggests one of the twolarge, species-rich and economically importantgroups Nematoda or Arthropoda as the closestrelatives to Tardigrada. Therefore, the position oftardigrades has been at the front of the ongoing dis-cussion on metazoan systematics. The two mostsupported theories are that tardigrades eitherbelong to Cycloneuralia, as a close relative to nem-atodes (Crowe et al., 1970, Dewel and Clark, 1973;Ruppert and Barnes, 1994), or to Panarthropoda,along with arthropods and onychophorans (Baccettiand Rosati, 1971; Bussers and Jeuniaux, 1973;

Contract grant sponsor: Danish Carlsberg Foundation; Contractgrant sponsor: Danish Natural Science Research Council; Contractgrant sponsor: National Science Foundation under the AToL pro-gram.

*Correspondence to: Dennis Krog Persson, Invertebrate Depart-ment, Natural History Museum of Denmark, University of Copen-hagen, 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 inWiley Online Library (wileyonlinelibrary.com)DOI: 10.1002/jmor.20054

JOURNAL OF MORPHOLOGY 273:1227–1245 (2012)

� 2012 WILEY PERIODICALS, INC.

Greven, 1982; Kristensen, 1976, 1981; Møbjerg andDahl, 1996; Nielsen, 2001). Molecular investiga-tions 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 theview that tardigrades are related to onychophorans/arthropods (Schmidt-Rhaesa et al., 1998; Budd,2001). Importantly, recent papers emphasize thatthe molecular data are not conclusive as to whethertardigrades are more closely related to arthropodsand onychophorans or to the nematodes and nema-tomorphs (Dunn et al., 2008; Edgecombe, 2010;Edgecombe et al., 2011; Campbell et al., 2011). Phy-logenomic analyses have yielded different resultsdepending on which substitution model that wasused to analyze the data (Dunn et al., 2008). Inaddition, morphological investigations have gener-ated contradictory results, inferring an inconsis-tency in the phylogenetic analyses (Dewel andClark 1973; Kristensen and Higgins, 1984a,b;Dewel et al., 1993; Dewel and Dewel, 1996; Zantkeet al., 2008). This is in part due to the fact that tar-digrades possess morphological characters relatingto both nematodes and arthropods. As an example,they possess a muscular myoepithelial triradiatepharynx (Ruppert, 1982; Ruppert and Barnes,1994), which is also seen in nematodes and loricifer-ans (Kristensen, 2003), and conversely have lobedcerebral ganglia connected with a ladder-type chainof ventral trunk ganglia, much like an arthropodnervous system. For review of some of the inconsis-tencies, see Greven (1982). The architecture of thenervous system, and in particular, the brain of Tar-digrada, has often been emphasized as importantfor phylogenetic analysis attempting to elucidatethe relation of the group to other phyla, however, ageneral consensus has yet to be reached (Marcus,1929; Dewel and Dewel, 1996; Zantke et al., 2008).Even the most recent investigation on the tardi-grade nervous system, using confocal laser scan-ning microscopy, immunocytochemical staining,and three-dimensional (3D) reconstruction, did notprovide explicit conclusion toward the phylogeneticrelationship between tardigrades and arthropods ornematodes (Zantke et al., 2008).

Earlier investigations of tardigrade cephalicsense organs and their innervations have led tosuggestions of a three-lobed brain (Kristensen andHiggins, 1984a,b; Dewel and Dewel, 1996; Wieder-hoft and Greven, 1996). Furthermore, the ventralnerve cord has been described to comprise pairedsegmental ganglia interconnected by longitudinalconnectives, and connected intrasegmentally bytransverse commissures giving the appearance of arope-ladder like organization (Kristensen, 1982).This structuring of the central nervous system(CNS) is similar to that observed in arthropods(Scholtz, 2002; Muller, 2006; Scholtz and

Edgecombe, 2006) and would therefore strengthenthe hypothesis that Tardigrada is closely related toArthropoda. However, the results obtained byZantke et al. (2008) on the eutardigrade Macrobio-tus hufelandi C.A.S. Schultze (1833) showed athree lobed brain, but did not support the exis-tence of three segments in the head. Their argu-ment against three brain segments is founded on asuggested hypothetical model based on their data,in which it seems all brain commissures arelocated in the hypothetical deutocerebrum. Also,they find no connective from the hypothetical trito-cerebrum to the first ventral trunk ganglion andtransverse commissures of the ventral trunk gan-glia were not observed.

Studies on tardigrade development support thesister group relationship of Tardigrada and Ar-thropoda 1 Onychophora (Hejnol and Schnabel,2006), although it was not determined whether ornot a third brain lobe is present. In addition, the na-ture of the subpharyngeal ganglion was questionedas it seemed to be an outgrowth of the brain.

Due to these differences between earlier mor-phological investigations, we found it is necessaryto reinvestigate tardigrade neuroanatomy. A clari-fication of whether or not certain structures existis needed, and a detailed neuroanatomical investi-gation can furthermore provide possible evidencefor the sister group relationship of Tardigrada.

Here, we provide a detailed description ofthe nervous system of the marine eutardigradeHalobiotus crispae Kristensen (1982) based onimmunocytochemical staining, confocal laserscanning microscopy, and computer-aided 3Dreconstructions, supported by transmission elec-tron microscopy. Specifically, the brain and theventral ganglia receive much attention, as thesestructures are of phylogenetic importance. Ourinvestigation expands on the current knowledge oftardigrade neuroanatomy, and our data are inter-preted according to existing data and theories.

MATERIALS AND METHODS

Specimens of the tardigrade Halobiotus crispae were sampledat Vellerup Vig, in the Isefjord. Animals were extracted accord-ing 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). Livetardigrades were stored at 48C in seawater (20%) from the lo-cality and supplied with substrate. The animal examined withtransmission electron microscopy was collected intertidally atthe type locality Nipissat Bay, Disko Island, West Greenland.

Relaxation and Fixation

Specimens of H. crispae were stretched in freshwater and sub-sequently relaxed using CO2-enriched water to prevent musclecontractions during fixation. The CO2-enriched water wasapplied 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)

1228 D.K. PERSSON ET AL.

Journal of Morphology

(for 500 ml of a 53 concentrated stock solution of 0.5 mol l21

PBS: 33.48 g Na2HPO4�2H2O, 7.93 g NaH2PO4�H2O, pH 7.2–7.4)for 60 min at room temperature, and washed at least 83 10–15min in washing buffer (0.1 mol l21 PBS with 0.1% NaN3).

Permeabilization and Blocking

After fixation, the animals were perforated using a very fineneedle to facilitate penetration of the antibodies, followed by90 s of sonication in a sonication bath at 30 W (Branson,B-1200 E1). Subsequently, the tardigrades were transferred to ablocking 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), andincubated over night at 48C.

Staining

For immunolabeling, the primary antibody used was antiace-tylated a-tubulin at a concentration of 1:250 (antimouse, mono-clonal; Sigma, cat. no. T6793). Specimens were incubated withprimary antibody on a shaker (celloshaker variospeed) at 48Cfor 48 h, and subsequently rinsed with washing buffer for atleast 63 20–30 min and finally over night. Specimens werethen incubated with secondary antibody (Alexa flour 488 goat-antimouse, Invitrogen cat. no. A11001) at a concentration of1:200 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 secondaryantibody, 40,6-diamidino-2-phenylindole (5 lg/ml, DAPI; Invitro-gen cat. no. D21490) was added for nuclear staining. After incu-bation, the animals were rinsed thoroughly in washing bufferfor at least 63 20–30 min followed by an overnight wash. Atotal of 58 animals were used. A complementary immunolabel-ing with serotonin was performed following the same protocolas described above, using antiserotonin 5-HT 1:200 (antirabbit,polyclonal; Sigma, cat. no. S1561).Negative controls were performed during the initial experi-

ments. The controls were performed by omitting the primaryantibody from the staining procedure. The controls showed no flu-orescence other than the autofluorescence of cuticular structures.

Mounting and Image Acquisition

Prior to mounting, specimens were treated with a glycerol se-ries. This was done to prevent any deformation of the animals,due to the density and viscosity of the mounting media. Theglycerol was applied at the start of the third buffer rinsing (seeabove), one drop at a time, starting with 5% glycerol. A fewdrops were applied and a small amount of liquid was simultane-ously removed, until all the rinsing liquid had been replacedwith 5% glycerol. This process was repeated with 10% followedby 25–100% glycerol. Then, the specimens were mounted oncoverslips in Flouromount mounting medium (Southern Bio-technology Associates, Birmingham, AL). Using an Erwin loop,specimens were placed in droplets of the mounting medium andcarefully manipulated with a fine needle into the desired posi-tion and the coverslips were sealed. Image acquisition was per-formed on a Leica DM RXE 6 TL microscope equipped with aLeica TCS SP2 AOBS confocal laser scanning unit, using the488-nm line of an argon/crypton laser for antibody detectionand UV laser for DAPI. A maximum projection of the image se-ries 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

(type material: Kristensen, 1982), and fixed with a trialdehydesolution followed by postfixation with 1% OsO4. Ultrathin sec-tions The specimens were cut with a diamond knife. For detailson the preparation of transmission electron microscopy sectionssee Kristensen (1976).

Regarding the nomenclature, we follow that used by Marcus(1929), also implemented by Zantke et al. (2008), and to someextent Kristensen (1982). Common neuroanatomical terms arein accordance with Richter et al. (2010).

RESULTS

H. crispae showed pronounced immunoreactivityagainst acetylated a-tubulin, particularly in the brainas well as the longitudinal nerve cords and ventralganglia (Fig. 1). The acetylated a-tubulin immunore-actions combined with nuclear labeling with DAPIproduced detailed images of the nervous system. Inthe following description, the CNS, comprising thebrain, ventral longitudinal nerve cords, and ventralganglia, will be described separately from the periph-eral nervous system (PNS), which is comprised ofevery nervous structure outside the CNS. The sen-sory areas described as papilla cephalica (Figs. 1 and2; pc) and temporalia (Fig. 2A,B; t. see Kristensen,1982) are very closely associated with the brain andwill be treated as part of the CNS.

The nervous system of H. crispae has a clearsegmental organization, with four paired ventraltrunk ganglia each connected with two leg gangliaand two lateral neurons. Each of the lateral neu-rons in turn communicates with a dorsal neuron.The brain appears to be composed of three pairedlobes each containing a commissure. The brain isconnected with a subpharyngeal ganglion (g0) by apair of connectives from the ventrolateral lobes.The subpharyngeal ganglion is in turn connectedby a ventral longitudinal nerve cord to the firstventral trunk ganglion.

The Central Nervous SystemThe brain. The gross structure of the brain

of H. crispae consists of 11 nerve cell clusterscontaining the soma of the nerve cells, and anelaborate network of nerve fibers and fiber bundlesconnecting the clusters (Figs. 2–6). The number ofnerve cells and cells associated with these in thehead region accounts for approximately one-quar-ter to one-third of all the cells in the animal (seereview by Møbjerg et al., 2011). The interpretationof the number of brain clusters depends on whichstructures are considered to be part of the brain aswell as on the interpretation of what comprises acluster. Here, we define a brain cluster as a dis-tinct group of cells or cells forming part of a lobe.The overall brain structure of H. crispae comprisestwo lateral outer lobes, two inner lobes, a medianganglion, and two ventrolateral lobes (Fig. 2). Thelaterally located outer brain lobes are comprised oftwo clusters, which is also true for the two innerbrain lobes. The median ganglion, situated

NEUROANATOMY OF Halobiotus crispae 1229


Fig. 1. Overview of the nervous system of Halobiotus crispae showing immunoreactivity against antiacetylated a-tubulin, maxi-mum projection. (A) Double labeling with antiacetylated a-tubulin and DAPI. Lateral view, showing the strong innervation of thepapilla cephalica (pc) from the brain, as well as the arrangement of the PNS. (B) Dorsal and (C) ventral view, respectively, givingan overview of the CNS with brain and ventral ganglia as well as the arrangement of the PNS. cl, cloacal neurons; gI-IV, ventralganglion I-IV; dne, dorsal neuron; dn, dorsal nerve; lgg, leg ganglion; lne, lateral neuron; lln, lateral longitudinal nerve; ln, lateralnerve; pc, nerves of papilla cephalica.



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. Whitearrowheads indicate conspicuous immunoreactive nerves of the inner lobes. (C) Lateral and (D) anterolateral view, both showingthe 3D arrangement of the outer and inner lobes. Normal shading, Imaris. (E, F) Frontal view showing immunoreaction in thearea of the ventrolateral lobe and the ventral commissure, maximum projection and iso-surface rendering, respectively. dc, dorsalcommissure; ic, inner connective; il.acl, inner lobe anterior cluster; il, inner lobe; il.pcl, inner lobe posterior cluster; mg, medianganglion; oc, outer connective; ol, outer lobe; pc, nerves of papilla cephalica; prcm, preoral commissure; t, temporalia; vc, ventralcommissure; vll, ventrolateral lobe. Double arrows indicate orientation of the image. Anterior and posterior is indicated with a andp, respectively, whereas d and v indicate dorsal and ventral, respectively.



Fig. 3. Halobiotus crispae, close-up of the brain, with individual stack images showing specific details. (A) Dorsal view of a max-imum-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 tem-poralia (t), as well as the conspicuous immunoreactive areas of the inner lobes, lateral to the dorsal commissure (white arrow-heads). The n13 nerve is clearly visible in the anteriodorsal part of the head. (C) Going further ventral reveals the large nerve bun-dle from the outer lobes, which become part of the dorsal commissure (white arrowheads). (D) This section shows the postoral com-missure (pocm) and the connectives (co) to the ventral part of the brain. Also, notice the buccal neurons (bn) dorsal to the mouthopening. (E) The mouth opening (mo) is innervated by several nerves connected with the ventral part of the brain (white arrow-heads). 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.



between the inner lobes, consists of a single clusterof cells and innervates the forehead via the nerven13, whereas the ventrolateral lobe comprises onecluster on each side of the buccal tube (Figs. 2 and3). The mouth is innervated by nerves from bothventrolateral and inner lobes (Figs. 3E and 4; se

below). The brain contains a commissure betweeneach of the paired brain lobes and a smaller com-missure in connection with the innervations of thestylets. In total, we identified four commissures:the large dorsal commissure connects the innerlobes, the preoral commissure connects the outerlobes, the ventral commissure connects the ventro-lateral lobes and the stylet commissure innervatesthe stylets and stylet supports (Figs. 2,3, 5 and 6).

A pair of connectives extending from the ventro-lateral lobes, connects the brain to the centralnervous system of the trunk through a subphar-yngeal ganglion (Fig. 7-8). The subpharyngeal gan-glion communicates with the first ventral trunkganglion through the ventral longitudinal nervecord. In addition, so-called outer and inner connec-tives extend from respectively the outer and ven-trolateral lobes, connecting the brain to the firstventral trunk ganglion (see below).

The lateral outer brain lobes constitute the larg-est and most conspicuous part of the brain. Theselarge lobes are elongated and positioned dorsolat-erally to the buccal tube and extend caudally; theyare 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 dividedinto an anterior and a posterior cluster (Figs. 2Cand 6; ol.acl, ol.pcl). The second pair of lobes iscloser to the median plane of the animal and isreferred to as the inner lobes (Figs. 2, 5, 6, and 10;il). The inner lobes also seem to be composed oftwo nerve cell clusters, a posterior cluster and ananterior cluster (Figs. 2A1C and 5; il.pcl, il.acl).Both pairs of brain lobes are positioned dorsal tothe buccal tube, and the two ‘‘hemispheres’’ areinterconnected by a massive bundle of nerve fiberscontaining the preoral and predorsal commissures(Fig. 2A1B; prcm, dc). On each side of the dorsalcommissure small, highly immunoreactive areas ofthe inner lobes are clearly visible (Fig. 2A,B; whitearrowheads), which seem to be connected withnerves running through the dorsal commissure. Avery pronounced nerve runs between the anteriorcluster of the inner lobe and the dorsal commis-sure (Fig. 5; n4, see also Zantke et al., 2008). Theanterior clusters of the inner lobes receive nerveextensions from the sensory area of the anteriorpart of the head, known as the papilla cephalica(Figs. 1–315). A total of five nerves can be distin-guished in the papilla cephalica named n7, n8, n9,n10a, and n10b (Figs. 2A1C and 5), of these onlyn9 originates from the outer lobe, whereas theothers originate from the inner lobe.

From the posterior region of the outer lobes, inthe vicinity of the eyes, extensive dorsal innerva-tions of the cuticle are clearly visible (Figs. 2A–D,3A1B, and 5; t). These innervations are more orless similar to the innervations seen in the cuticleof the papilla cephalica and are termed temporaliaby Kristensen (1982); the exact number of nerves

Fig. 4. Halobiotus crispae, close-up of the mouth opening,showing the external and internal structures of the buccallamella. (A) Scanning electron micrograph revealing the exter-nal morphology of the six buccal lamella (asterisk) surroundingthe mouth opening. (B) Transmission electron micrograph of asection through the buccal lamella shows the internal structureof the buccal lamella and the surrounding tissue. (C) Antiacety-lated a-tubulin immunoreaction in the mouth opening. ci, cilia.Scale bars are 15 lm.



innervating the area cannot be distinguished.Between the inner lobes, an unpaired triangular-shaped median ganglion is located (Figs. 2A1Cand 5; mg). From this ganglion, a double nervecord extends anteriorly above the dorsal commis-

sure, ending in a small circular nerve structure inthe cuticle of the forehead. This nerve correspondswith the n13 nerve described in Zantke et al.(2008), and it follows that the very fine pairednerve lateral to n13 is n12 (Figs. 1B,C, 2A–D, 3A–

Fig. 5. Halobiotus crispae, antiacetylated a-tubulin immunoreactions of the brain. (A) Maximum projection revealing the n4nerve. Also, the head nerves n12 and n13 are clearly visible. (B) Normal shading rendering, using the software Imaris, reveal then13 nerve to originate from the median ganglion (mg). Furthermore, the individual nerves, n7-n10a1b, of the papilla cephalica canbe distinguished. dc, dorsal commissure; il.acl, inner lobe anterior cluster; mg, median ganglion; pc, nerves of papilla cephalica; t,temporalia.



C, and 5; n12, n13). The outer lobes, inner lobes,the median ganglion, and associated commissuresconstitute the dorsal part of the brain.

Ventral to the buccal tube another commissureis located, the ventral commissure (Figs. 2E1F,3E, and 6E; vc). Posterior to the ventral commis-

Fig. 6. Halobiotus crispae, individual sections from an image stack, showing immunoreaction to antiacetylated a-tubulin andcorresponding DAPI labeling. (A) A large part of the dorsal commissure (dc) originates from the outer lobes (arrows). (B) The DAPIlabeling in the same focal plane as A shows the posterior and anterior clusters of the outer lobes, and the posterior cluster of theinner lobes. (C and D) The position of the stylet commissure coincides with the stylet supports (ss). (E and F) In the ventral partof the animal, a ventral commissure is clearly visible (E, vc), which corresponds very well with the position of two ventrolaterallobes revealed with DAPI labeling (F, vll). (G) In the most ventral part of the animal, the antiacetylated a-tubulin staining showthat the outer connectives connects to the outer lobes and the inner connectives connects to ventrolateral lobes, which is connectedwith the subpharyngeal ganglion (g0). bt, buccal tube; dc, dorsal commisure; g0, ventral ganglion; ic, inner connective; il, innerlobe; oc, outer connective; ol, outer lobe; ol.acl, outer lobe anterior cluster; il.pcl, inner lobe posterior cluster; ol.pcl, outer lobe poste-rior cluster; ss, stylet support; stcm, stylet commissures; stn, stylet nerves; vll, ventrolateral lobe.



sure and slightly dorsal to the buccal tube, afourth commissure is visible in the area of thestylets (Fig. 3D; stcm). From this stylet commis-sure, stylet nerves (Fig. 3D; stn) extend anterolat-erally into the ventrolateral brain lobes. Theselobes consist of a cell cluster on each side of thebuccal tube (Figs. 2E1F and 6C–H). From the ven-trolateral lobes, several nerves extend anteriorlytoward the mouth (Fig. 3E; white arrowheads). Inaddition, the mouth receives nerves from severalnerve cells dorsal to the mouth opening (Fig. 3E;bn) which are connected with the inner lobes.These nerves are all connected with the six buccallamellae surrounding the mouth opening (Fig. 4;asterisks).

Sections of the image stack in the area of theventral commissure reveal cell clusters around thebuccal tube and immediately ventral to it, as well

as a ganglion-like structure posterior to theseclusters, these are termed the ventrolateral lobes(vll) and the subpharyngeal ganglion (g0), respec-tively (Fig. 6F1H; stippled circles/oval). The ven-trolateral lobes are also clearly visible in Figure2E and F, interconnected by the ventral commis-sure. Additionally, a transmission electron micro-scopic image of the same area support the exis-tence of a third pair of brain lobes and also showsthe connective extending ventrally from theselobes (Fig. 9).

The ventral ganglia and longitudinal nervecords. From the posterior region of the outerlobes, a double nerve tract extends ventrocaudally,with one lateral nerve (Fig. 3A; ln) branching offinto the lateral nervous system and the other con-necting to the first ventral trunk ganglion. The lat-ter is referred to as the outer connective (Fig. 3A;

Fig. 7. Halobiotus crispae, lateral view of the brain and ventral ganglia. (A) Immunoreactivity against antiacetylated a-tubulinin 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 gan-glion (g0). Asterisks indicate possible perikarya. bt, buccal tube; clg, claw gland; g0, subpharyngeal ganglion; gI, first ventral trunkganglion; ic, inner connective; oc, outer connective; lgg, leg ganglion; lnc, longitudinal nerve cord.



oc). From the ventrolateral part of the brainextends the inner connective (Fig. 3A; ic); theinner connective is also connected with the firstventral trunk ganglia. These nerve tracts connectthe brain with the CNS of the trunk comprised ofpaired ventral trunk ganglia and longitudinalnerve cords. There are four paired ventral trunkganglia, each composed of approximately 40 cellsthat are associated with a corresponding leg pair(Figs. 1 and 8; gI–gIV). The ventral trunk gangliaare intrasegmentally connected by transverse com-missures and intersegmentally connected by ven-tral longitudinal connectives, giving the appear-ance of a rope-ladder-like arrangement(Figs. 118). In the first three ganglia, we observetwo commissures, whereas in the fourth, only onecan be observed (Fig. 8; inserts). The longitudinalconnectives are collectively referred to as the lon-gitudinal nerve cords (Fig. 1; lnc), and extendthrough most of the length of the animal, termi-nating in the fourth ventral trunk ganglion (Fig.1; gIV). It is not unambiguous whether the longi-tudinal nerve cords contain perikarya or not; thea-tubulin stainings show some thickenings of thelongitudinal nerve cords in the regions betweenthe ventral trunk ganglia (Figs. 1 and 8). Addition-

ally, serotonin immunoreactivity is shown in theseregions as well, which could indicate the presenceof perikarya (Fig. 7, asterisks).

In addition, we find a fifth ventral ganglion, thesubpharyngeal ganglion (g0), which is connectedwith the ventrolateral brain lobes and the firstventral trunk ganglion (gI) via the longitudinalnerve cord (Fig. 7).

The Peripheral Nervous System

The PNS is mainly comprised of four dorsal andfour lateral neurons on each side of the animal(Fig. 1; dne and lne). The dorsal and lateral neu-rons are connected by dorsal and lateral nerves(Fig. 1A1B; dn, ln). The lateral nerves connect thelateral neurons with the ventral trunk ganglia. Inaddition, there is a lateral nerve connecting thefirst lateral neuron to the brain (Fig. 3A; ln). Thislateral nerve originates from the outer lobe, propa-gating in parallel with the outer connective, andconnects to the first lateral neuron (Fig. 1A; lne).From the first lateral neuron, nerve projectionsextend both in an anterior–posterior as well as ina dorsal direction, and are referred to as laterallongitudinal nerves (Fig. 1A–C; lln) and dorsalnerves (Fig. 1A1B; dn), respectively. The lateralnerve extends through most of the length of theanimal, connecting the four lateral neurons. Thelateral neurons are connected with dorsal neurons(Fig. 1; dne) via dorsal nerves (Fig. 1; dn). Fromall the dorsal ganglia/neurons sensory cilia extendsfurther toward the dorsal side. These modified ciliacould very well correspond to the lateral cirri ofheterotardigrades.

At the fourth ventral trunk ganglion, nervesextend dorsocaudally to the dorsal neuron (Fig. 1;dne) and nerves branch off into the hind legs. Thenerves in the hind legs connect to leg ganglia inthe distal part of the legs (Fig. 1; lgg), and twonerves extend from these ganglia terminating in acilia in the proximal part of the legs. In addition, apair of nerves extends from the fourth ventraltrunk ganglion and terminates near the cloaca(Fig. 1A; cl).

Leg ganglia are connected with nerves originat-ing from the ventral trunk ganglia. From the sec-ond and third ventral trunk ganglion, three nervesn1, n2, and n3 originates (Fig. 1C). The n3 and n2nerves connect with the anterior and posteriorregion of the associated leg, with the posteriornerve attaching at the distal part of the leg, andthe middle nerve connect to a ganglion in the prox-imal part of the leg—a leg ganglion (Fig. 1C; lgg).The n1 nerve connect to the lateral neuron. Forthe first ventral trunk ganglion, it follows that theouter connectives correspond to the n1 nerves ofthe second and third ventral ganglia, whereas then2 and n3 nerves exhibit the same pattern in ven-tral trunk ganglia I–III. For complete overview of

Fig. 8. Halobiotus crispae, the nervous system of the trunk.Insets show close-up of the ventral ganglia I–IV and arrowsindicate the number of commissures. cl, cloacal nerves/neurons;cm, commissure.



the described nervous structures and nerves in thepresent study, as well as comparison to the nerv-ous structures previously described in the litera-ture see table 1.

DISCUSSION

Marcus (1929) described the nervous system ofMacrobiotus hufelandi with impressive detail,exemplified by the fact that most of our findings(using contemporary techniques) in H. crispae con-firm what he described. Moreover, our results sup-port most of the descriptions of the tardigradenervous system by Zantke et al. (2008), albeit wereport some new findings together with increaseddetails. In particular, we have obtained greaterdetails on brain structure, and important struc-tures in the ventral ganglia. Noticeably, several ofthe details uncovered in our study pertain to struc-tures that were declared missing in tardigrades orpossibly misinterpreted by Zantke et al. (2008).

In the nervous system investigation of M. hufe-landi by Zantke et al. (2008) a preoral commissureis described in the brain to be positioned justbelow the dorsal commissure. In addition, theyshow that a postoral commissure is connected withthe first ventral ganglion via the inner connec-tives, and to the dorsal commissure via the circum-buccal connectives, forming a circumbuccal ring.Our data show a similar preoral commissure posi-tioned just ventral to the dorsal commissure(Fig. 2B, prcm). In addition, we find a commissureventral to the buccal tube which could be equiva-lent to the postoral commissure described byZantke et al. (2008, Fig. 2F, pocm). We choose toterm this structure the ventral commissure(Fig. 3E, vc) as it appears more similar to the ven-tral ring commissure found in the heterotardi-grade Echiniscus viridissimus by Dewel and Dewel(1996). In H. crispae, the ventral commissure isnot connected with the first ventral trunk gangliavia the inner connectives; more precisely, the ven-tral commissure connects the ventrolateral lobes

Fig. 9. Halobiotus crispae, transmission electron micrograph merging of five cross-sections ofthe 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, con-nectives; ol, outer lobe; stn, stylet nerve; vll, ventrolateral lobe.



Fig. 10. Conceptual drawing constructed on the basis of the data in this study, showing our interpretation of the brain structureof H. crispae. (A) Lateral view. (B) Frontal view. clg, claw gland; co, connective; dc, dorsal commissure; ey, eye; g0, subpharyngealganglion; gI, first ventral trunk ganglion; ic, inner connective; il, inner lobe; lgg, leg ganglion; mg, median ganglion; mo, mouthopening; oc, outer connective; ol, outer lobe; pc, papilla cephalica; pb, pharyngeal bulb; st, stylet; t, temporalia; vll, ventrolaterallobe. Arrows indicate the approximate area of the transmission electron microscopical section in figure 9.



TABLE

1.Table

listingthestru

cturesof

thenervou

ssystem

ofH.crispaedescribed

inthis

studyandcomparedwithpreviousstudiesof

H.crispaeor

other

tard

igra

des

Iden

tified

stru

ctures

andnamed

nerves

H.crispae

M.hufelandi

M.hufelandi

H.crispae

Con

stru

cted

ancestraltard

igrade

Persson

etal.(this

study)

Marcus(1929)

Zantkeet

al.(2008)

Kristen

sen(1982)

Nielsen

(2001)

CNS,hea

dIn

ner

lobes

(il)withanterior

andposterior

clusters(acl,pcl)

Inner

lobes

Inner

lobes

(posterior

cluster)

Protocerebru

mProtocerebru

m

Dorsa

lcommissu

re(dc)

Dorsa

lcommissu

reOuterlobes

(ol)withanterior

andposterior

clusters(acl,pcl)

Outerlobes

Outerlobes

(dorsa

lcluster)

Deu

tocerebru

mDeu

tocerebru

m

Preoralcommissu

re(prcm)

Preoralcommissu

reMed

ianganglion

(mg)

Med

ianganglion

n12,n13

n11

,n12n,n13

n4

n4,n5,n6

Buccalneu

rons(bn)

Ven

trolaterallobes

(vll)

Postoralcommissu

re?

Tritocerebru

mTritocerebru

mVen

tralcommissu

re(vc)

Subpharyngea

lganglion

(g0)

Subesop

hagea

lganglion

(I)

Subesop

hagea

lganglion

Subesop

hagea

lganglion

Styletcommissu

re(stcm)

Postoralcommissu

re?

Styletnerve(stn)

Styletnerve(s.ne)

Styletnerve(s.ne)

Outerconnective(oc)

Outerconnective

Outerconnective

Outerconnective

Outerconnective

Inner

connective(ic)

Inner

connective

Innervation

ofpapilla

cephalica

(pc,

n7,n8,n9,n10a,n10b)

Innervation

ofpapilla

cephalica

Innervation

ofpapilla

cephalica

(n7,n8,n9,n10)

Innervation

ofpapilla

cephalica

Innervation

ofpapilla

cephalica

Innervation

oftemporalia(t)

Innervation

oftemporalia

Innervation

oftemporalia

Innervation

oftemporalia

Innervation

ofbuccallamella

Innervation

ofbuccal

lamella

Innervation

ofbuccal

lamella

CNS,trunk

Fou

rven

traltrunkganglia

(gI-IV

)Fou

rven

traltrunk

ganglia(IIV

)Fou

rven

traltrunk

ganglia(gI-IV

)Fou

rven

traltrunkganglia

Fou

rven

traltrunk

ganglia

Transverse

commissu

res(cm)

Transverse

commissu

res

Transverse

commissu

res

Transverse

commissu

res

Lon

gitudinalnervecord

s(lnc)

Lon

gitudinalnervecord

sLon

gitudinalnervecord

sLon

gitudinalnervecord

sLon

gitudinalnervecord

sPNS

Leg

ganglia(lgg)

Leg

ganglia

Leg

ganglia

Leg

nerves

(n2+n3)

Leg

nerves

(n3)

n1,n2,n3

Leg

nerves

Lateralnerves

(n1,ln)

Lateralnerves

(n1,n2)

Neu

rophilsof

thehindlegs

Lateralneu

rons(lne)

Lateralneu

rons

Sen

silla

Dorsa

lnerves

(dn)

Dorsa

lnerves

Dorsa

lneu

rons(dne)

Dorsa

lneu

rons

Laterallongitudinalnerves

(lln)

Cloacalnerves

(cl)

Cloacalnerves



flanking the buccal tube. It is these lobes that areconnected with the first ventral trunk ganglia viathe inner connective.

When looking at a DAPI staining of the area ofthe stylet commissure, there is not an actual gan-glion (compare Fig. 6C,D) and the commissure ispositioned exactly at the stylet supports. Also,nerves extend laterally from the commissure(Figs. 3A1D and 6C; co) maybe connecting to thestylet muscles. So, this seems to be a stylet com-missure in connection with the innervations of thestylets. Therefore, we chose the name stylet com-missure. In addition, the stylet commissure is notdirectly connected with the dorsal commissure, butit is actually connected with the ganglion-likestructure associated with the ventral commissure.Furthermore, through closer observations of thebrain, it is obvious that the stylet commissure ispositioned dorsal to the buccal tube, so it shouldnot be confused with the postoral commissuredescribed by Zantke et al. (2008), which is posi-tioned ventral to the buccal tube.

The transmission electron microscopical imagein Figure 9 and the image stack of the a-tubulinand DAPI staining on Figure 6 supports theimmunoreactions seen in Figure 2F, which sug-gests that the ganglion of the ventral commissureis a paired, lobed structure situated lateral to thebuccal tube. More ventral, we find a cell clustermuch like the first ventral trunk ganglion, in veryclose proximity to the lobes of the ventral commis-sure (Fig. 6H). The DAPI staining shows a cellstructure very similar to the first ventral trunkganglion, and it is positioned in the same level.Combined with the immunoreaction and comple-mentary DAPI staining in Figure 7, it seems to bea ganglion in the ventral part of the head con-nected with the first ventral trunk ganglion.Although we cannot determine if these nerve cellshave a paired cluster arrangement as is the casefor the ventral trunk ganglia, we cautiouslyhypothesize that it is a subpharyngeal ganglion.The presence of the subpharyngeal ganglion is alsosupported by a developmental study, in which asubesophageal ganglion (the subpharyngeal gan-glion) is observed and suggested to be an out-growth of the brain (Hejnol and Schnabel, 2005).From this suggestion, it is argued that the sub-pharyngeal ganglion is part of the brain, whichthen forms a circumbuccal ring resembling cyclo-neuralian conditions (Zantke et al., 2008). This isstill at a hypothetical stage and actual evidenceis needed to clarify if the subpharyngeal ganglionis part of the brain. Nevertheless, if we considerthe idea of cephalization of several anterior seg-ments to be true, it would not be surprising if thesubpharyngeal ganglion seemed to originate fromthe same region as the three paired brain lobes. Inorder to fully understand the nature and origin ofthe subpharyngeal ganglion, it will be important

to investigate the brain of arthrotardigrades asthey are considered to have many plesiomorphiccharacters. Therefore, they may be closer to theancestral state in appearance compared to themore specialized eutardigrades.

Furthermore, it seems plausible that the lobes ofthe ventral commissure may be a third pairedbrain lobe, and it is clearly connected with thesubpharyngeal ganglion via two connectives(Fig. 9). The fact that we find two connectives, onefrom each ventrolateral lobe, suggests that thesubpharyngeal ganglion is or may have been apaired cluster like that found in the ventral trunkganglia. The ventrolateral lobes are also connectedwith the stylet nerves (Fig. 3A). If we accept thehypothesis of the stylets being internalized claws,that is, internalized leg, this supports the idea ofsegmentation in the head. Hence, we suggest abrain of at least three parts, and the existence of asubpharyngeal ganglion in tardigrades.

The third paired lobe has previously beendescribed in H. crispae by Kristensen (1982) andcould be homologs to the arthropod tritocerebrum.In addition, if there is homology between theouter and inner lobes and the protocerebrum anddeutocerebrum of arthropods, then, the brain con-figuration of tardigrades could be interpreted asarthropod-like, as suggested in earlier descriptions(Kristensen and Higgins, 1984a,b; Dewel andDewel, 1996; Nielsen, 2001, 2011), though furtherstudies on tardigrade development is needed toverify a possible homology. In Figure 6, the sub-pharyngeal ganglion is indicated by DAPI labeling,but we do not see a distinct immunoreaction fora-tubulin. However, on Figure 7A, which showsantiacetylated a-tubulin staining from the lateralside, we find clear immunoreaction in the subphar-yngeal ganglion, as well as in the nerve cord con-necting it to the first ventral trunk ganglion. Thisis also supported by serotonergic immunoreactionin the ventral trunk ganglia as well as g0 (Fig.7C). One explanation for the lack of immunoreac-tion in Figure 6 could be failure of antibody recog-nition, which we have previously encountered withtardigrades. An example is the inner lobes whichshow immunoreaction in Figure 2C1D, but inFigure 2A no immunoreaction can be observed inthe posterior clusters of the inner lobes.

The n13 nerve running dorsally from the medianlobe (Fig. 5B) terminating in a circular shape inthe forehead is interpreted as a rudiment of themedian cirrus of Heterotardigrada (see also Zantkeet al., 2008). Generally, the median cirrus is pres-ent in all Arthrotardigrada (though reduced insome Archechiniscus species), and missing from allEutardigrada and Echiniscoidea. Assuming thatthe marine Arthrotardigrada represents the ances-tral condition (Renaud-Mornant, 1982; Jørgensenet al., 2010), the presence of a median cirruswithin Tardigrada represents the plesiomorphic



condition. Additionally, connected with the dorsalneurons, we found modified cilia that likely arereduced sensory structures, homologs to the lateralcirri of Heterotardigrada. Consequently, thesestructures could be important in an intra-phylumphylogenetic perspective.

When looking at the ventral longitudinal nervecords and the ventral trunk ganglia, a distinct seg-mentation is readily recognizable. Apart from thehead, each of the four segments bears a leg andcontains a ventral ganglion with peripheralnerves, which innervate the associated leg as wellas the lateral and dorsal sides. The ventral trunkganglia are paired and intersegmentally connectedby transverse commissures, as described in theResults section. These commissures were originallydescribed by Marcus (1929); however, their pres-ence in tardigrades was recently questioned(Zantke et al., 2008). Our investigation shows thattardigrades possess intersegmental transversecommissures. Their presence is important to thephylogenetic debate as they are a critical compo-nent in the rope-ladder type nervous system. Thistype of nervous system is not encountered in anycycloneuralians, but is seen in most arthropods(Bullock and Horridge, 1965; Brusca and Brusca,2002).

The PNS primarily comprises the paired nervesn1, n2, and n3, which are forming a repeated pat-tern in association with the first three ventralganglia. Only the n1 nerves of the first ventralganglia differ from the pattern by connecting tothe outer lobes of the brain. Also, the nervesassociated with the fourth ventral trunk ganglionare arranged in a slightly different pattern.Although the difference in nerve arrangement inthe fourth ventral ganglion is linked to the orien-tation and function of the hind legs, the differ-ence in the n1 nerve in the first ventral trunkganglion is due to its connection to the brain. Wehypothesize that this deviation could be linked toan evolutionary scenario involving cephalizationof at least three anterior segments of a tardigradeancestor.

In the light of our results, we propose the follow-ing hypothetical model to explain the organizationof the tardigrade brain as three segments: Thearea of the outer lobes, with the eyes and innerva-tions of the temporalia, which we interpret ashomologs to the primary clavae and lateral cirri ofthe heterotardigrades, could be interpreted as thefirst segment or protocerebrum. This would thencorrespond to the first segment in arthropods bear-ing the compound eye, though this structure is nothomologs to the tardigrade eye. Also, it would cor-respond to the first segment in the onychophoranswhich bears the antenna and the eyes. Althoughagain these structures are not considered homo-logs to any of the structures found on the first seg-ment in tardigrades.

The next segment holds the inner lobes (possiblydeutocerebrum), which innervates the papillacephalica, homologs to the secondary clavae andthe internal cirri of heterotardigrades (for homol-ogy on primary and secondary clava, see alsoZantke et al. 2008). Consequently, this would behomologs to the second segment in the head ofboth arthropods and onychophorans, though againit is not readily possible to homologize betweentardigrade sense organs and the modified limps ofarthropods and onychophorans.

The third segment contains the ventrolaterallobes (possibly tritocerebrum), which innervate thebuccal lamella as well as the stylets and styletsupports. As it has been hypothesized that thestylet apparatus may have been formed by inter-nalization of a leg it could be homologs to the sec-ond antenna/pedipalp of crustaceans/chelicerates.According to some authors, the tritocerebrum isnot present in onychophorans (see Erikson andBudd, 2000; Mayer et al., 2010), if this is true it isdifficult to homologize between tardigrades andonychophorans with respect to the third brainregion. However, if we hypothesize that the cepha-lization in the Onychophora lineage excluded theganglion which in tardigrades became the thirdbrain lobe, then, the slime papilla could be homo-logs to the stylet apparatus.

Of course, in order to accept this hypothesis,this needs to be supported by more data—forexample, molecular data. Indeed, answers to someof the questions regarding homology may be foundin future comparative developmental studies, likethose performed by Jager et al. (2006) on Hox geneexpression and by Gabriel and Goldstein (2007) onexpression patterns of Pax 3/7 and Engrailedhomologs.

Whether or not tardigrades possess a tritocere-brum is important with regards to their phyloge-netic position in relation to Onychophora and Ar-thropoda. As mentioned above, a tritocerebrumhas not been found in the onychophorans. This isbecause the ganglion innervating the third seg-ment is not part of the brain, but is part of theventral nerve cord. Consequently, it was hypothe-sized that the last common ancestor of onycho-phorans and arthropods possessed a bipartitebrain comprised of a protocerebrum and a deuto-cerebrum (Mayer et al., 2010). As H. crispae pos-sess a third brain lobe, this could then support aclade with Tardigrada and Arthropoda as sistergroups within Panarthropoda.

The presence of a third brain lobe in tardigradesgenerates three possible phylogenetic configura-tions for Panarthropoda, assuming true homologybetween the brain lobes of Tardigrada and Ar-thropoda. In one scenario, the ancestor to Panar-thropoda could have had three brain lobes, andconsequently, the third lobe was lost in Onycho-phora. If this is true, brain morphology will not



aid in resolving the phylogeny within Panarthro-poda, as all placements of Onychophora areequally parsimonious. In contrast, if the last com-mon ancestor of Panarthropoda had a bipartitebrain, the outcome would be that Onychophoracould be sister group to Tardigrada 1 Arthropoda,or grouped together with one of them in whichcase the development of the third brain lobe wouldbe convergent. Hence, the most parsimonious hy-pothesis, when only considering brain morphology,is that the last common ancestor to Panarthropodahad a bipartite brain and that tritocerebrum wasdeveloped in a common ancestor to Tardigrada andArthropoda. This is of course speculative, and eventhough molecular data support the inclusion ofTardigrada into Panarthropoda, it does not sup-port a sister group relationship between Tardi-grada and Arthropoda (Campbell et al., 2011). Asnoted above more investigations, especially on tar-digrade development, are needed in order to sub-stantiate our suggestions.

The stylet apparatus may be internalized claws,as earlier transmission electron microscopicalinvestigations have shown that stylets and clawsare formed in the same way (Kristensen, 1976;Nielsen, 2001, 2011; Halberg et al., 2009a) and aresimilar to the jaws of Onychophora (Storch andRuhberg, 1993; Mayer and Harzsch, 2007; Mayeret al., 2010). It is also a possibility that the styletsand the supports could be greatly modified mouthappendages.

The nervous system is distinctly metameric, con-sisting of the three-lobed brain, the subpharyngealganglion, and the four ventral trunk ganglia.Characteristic to all tardigrades is the large pairedouter connective with a small ganglion that con-nects the outer lobe (protocerebrum) to the firstventral ganglion (Marcus, 1929). However, we alsofound a thin connective between the ventrolaterallobe (tritocerebrum) and the first ventral trunkganglion. Our interpretation of the brain structureis summarized in a concept drawing in Figure 10and represents an integration of all our immunore-active data from the head region as well as trans-mission electron microscopy.

On the basis of this investigation, we find thattardigrades possess a brain of at least three parts.We hypothesize that the three paired lobes couldoriginate from three head segments and that thesubpharyngeal ganglion originated from a fourthsegment. Along with the commissures of the ven-tral trunk ganglia and the segmentation of thebody, this leads us to suggest that the tardigradenervous system structure supports the clade Pan-arthropoda. The similarities of the tardigradenervous system toward Cycloneuralia (loriciferansand kinorhynchs, Kristensen, 2003), especially thekinorhynchs, which have segmentally arrangedventral ganglia (Kristensen and Higgins, 1991),have been pointed out by morphologists as being

surprising analogies. However, tardigrades mayhave some plesiomorphic characters in the nervoussystem as well as a myoepithelial triradiate pha-ryngeal bulb, so the mentioned similaritiesbetween Tardigrada and Cycloneuralia could betrue homologies.

ACKNOWLEDGMENT

The authors thank Stine Elle for preparing theline art illustrations.

LITERATURE CITED

Aguinaldo AMA, Turbeville JM, Linford LS, Rivera MC, GareyJR, Raff RA, Lake JA. 1997. Evidence for a clade of nemato-des, arthropods and other moulting animals. Nature 387:489–493.

Baccetti B, Rosati F. 1971. Electron microscopy on tardigrades.III. The integument. J Ultrastruct Res 34:214–243.

Bertolani R. 1982. Tardigradi (Tardigrada). Guide per il ricono-scimento delle specie animali delle acque interne Italiane.Verona, Italy: Consiglio Naz. Delle Richerche 15. pp 1–114.

Brusca R, Brusca GJ. 2002. Invertebrates. Sunderland, Massa-chusetts: Sinauer Associates. pp 1–936.

Budd GE. 2001. Tardigrades as ‘Stem-Group Arthropods’: TheEvidence from the Cambrian Fauna. Zool Anz 240:265–279.

Bussers JC, Jeuniaux CH. 1973. Chitinous cuticle and system-atic position of Tardigrada. Biochem Syst 1:77–78.

Bullock TH, Horridge GA. 1965. Structure and function in thenervous system of invertebrates. San Franscisco: WH Free-mand.

Campbell LI, Rota-Stabelli O, Edgecombe GD, Marchioro T,Longhorn SJ, Telford MJ, Philippe H, Rebecchi L, PetersonKJ, Pisani D. 2011. MicroRNAs and suggest that velvetworms are the sister group of Arthropoda. PNAS 108:15920–15924.

Crowe JH, Newell IM, Thomson WW. 1970. Echiniscus viridis(Tardigrada): Fine structure of the cuticle. Trans Am MicroscSoc 89:316–325.

Dujardin F. 1851. Sur les Tardigrades et sur une espece a longspieds vivant dans l’eau de mer. Ann Sci Nat Zool Ser 3. T 15,160–166.

Dastych H, Kristensen RM. 1995. Echiniscus ehrenbergi sp. n.,a new water bear from the Himalayas (Tardigrada). EntomolMitt zool Mus Hamburg 11:221–230.

Dewel RA, Clark WH. 1973. Studies on tardigrades. I: Finestructure of the anterior foregut of Milnesium tardigradumDoyere. Tissue Cell 5:133–146.

Dewel RA, Dewel WC. 1996. The brain of Echiniscus viridissi-mus Peterfi, 1956 (Heterotardigrada): A key to understandingthe phylogenetic position of tardigrades and the evolution ofthe arthropod head. Zool J Linn Soc 116:35–49.

Dewel RA, Nelson DR, Dewel WC. 1993. Tardigrada. In: Harri-son FW, Rice ME, editors. Microscopic Anatomy of Inverte-brates, Volume 12: Onychophora, Chilopoda and Lesser Proto-stomata. New York: Wiley-Liss, Inc. pp 143–183; Chapter 5.

Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, SmithSA, Seaver E, Rouse GW, Obst M, Edgecombe GD, SørensenMV, Haddock SHD, Schmidt-Rhaesa A, Okusu A, KristensenRM, Wheeler WC, Martindale MQ, Giribet G. 2008. Broadphylogenomic sampling improves resolution of the animaltree of life. Nature 452:665–780.

Edgecombe GD. 2010. Arthropod phylogeny: An overview fromthe perspectives of morphology, molecular data and the fossilrecord. Arthropod Struct Dev 39:74–87.

Edgecombe GD, Giribet G, Dunn CW, Hejnol A, Kristensen RM,Neves,RC, Rouse GW, Worsaae K, Sørensen MV. 2011.Higher-level metazoan relationships: Recent progress andremaining questions. Org Divers Evol 11:151–172.



Eibye-Jacobsen J. 1997. Development, ultrastructure and func-tion of the pharynx of Halobiotus crispae Kristensen, 1982(Eutardigrada). Acta Zool (Stock) 78:329–347.

Erikson BJ, Budd GE. 2000. Onychophoran cephalic nerves andtheir bearing on our understanding of head segmentation andstem-group evolution of Arthropoda. Arth Struct Dev 29:197–209.

Gabriel WN, Goldstein B. 2007. Segmental expression of Pax3/7and Engrailed homologs in tardigrade development. DevGenes Evol 217:421–433.

Garey JR, Krotec M, Nelson DR, Brooks J. 1996. Molecularanalysis supports a Tardigrade-Arthropod association. Inver-tebr Biol 115:79–88.

Garey JR. 2001. Ecdysozoa: The relationship between Cyclo-neuralia and Panarthropoda. Zool Anz 240:321–330.

Giribet G, Carranza S, Baguna J, Riutort M, Ribera C. 1996.First molecular evidence for the existence of a Tardigrada 1Arthropoda clade. Mol Biol Evol 13:76–84.

Greven H. 1982. Homologues or analogues? A survey of somestructural patterns in Tardigrada. In: Nelson DR, editor. Pro-ceedings of the Third International Symposium on the Tardi-grada, August 3–6, 1980, Johnson City, Tennessee. JohnsonCity: East Tennessee State University Press. pp 55–75.

Halberg KA, Møbjerg N. 2012. First evidence for epithelialtransport in tardigrades: A comparative investigation of or-ganic anion transport. J Exp Biol 215:497–507.

Halberg KA, Persson D, Møbjerg N, Wanninger A, KristensenRM. 2009a. Myoanatomy of the Marine Tardigrade Halobiotuscrispae (Eutardigrada: Hypsibiidae). J Morph 270:996–1013.

Halberg KA, Persson D, Ramløv H, Westh P, Kristensen RM,Møbjerg N. 2009b. Cyclomorphosis in Tardigrada: Adaptationto environmental constraints. J Exp Biol 212:2803–2811.

Hejnol A, Schnabel R. 2005. The eutardigrade Thulinia stephaniaehas an indeterminate development and the potential to regulateearly blastomere ablations. Development 132:1349–1361.

Hejnol A, Schnabel R. 2006. What a couple of dimensions cando for you: Comparative developmental studies using 4D mi-croscopy—examples from tardigrade development. IntegrComp Biol 46:151–161.

Jager M, Murienne J, Clabaut C, Deutsch J, Le Guyader H,Manuel M. 2006. Homology of arthropod anterior appendagesrevealed by Hox gene expression in a sea spider. Nature441:506–508.

Jørgensen A, Faurby S, Hansen JG, Møbjerg N, KristensenRM. 2010. Molecular phylogeny of Arthrotardigrada (Tardi-grada). Mol Phylogenet Evol 54:1006–1015.

Kristensen RM. 1976. On the fine structure of Batillipes noerre-vangi Kristensen, 1976. 1: Tegument and molting cycle. ZoolAnz 197:129–150.

Kristensen RM. 1981. Sense organs of two marine arthrotardi-grades (Heterotardigrada, Tardigrada). Acta Zool 62:27–41.

Kristensen RM. 1982. The first record of cyclomorphosis in Tar-digrada based on a new genus and species from Arctic meio-benthos. Z Zool Syst Evol 20:249–270.

Kristensen RM. 2003. Comparative morphology: Do the ultra-structural investigations of Loricifera and Tardigrada supportthe clade Ecdysozoa? The new panorama of animal evolution.Proc 18th Int Congr Zoology, Athens, Greece. pp 467–477.

Kristensen RM, Higgins RP. 1984a. A new family of Arthrotar-digrada (Tardigrada: Heterotardigrada) from the AtlanticCoast of Florida, U.S.A. Trans Am Microsc Soc 103:295–311.

Kristensen RM, Higgins RP. 1984b. Revision of Styraconyx (Tar-digrada: Halechiniscidae) with descriptions of two new spe-cies from Disko Bay, West Greenland. Smithsonian ContribZool 391:1–32.

Kristensen RM, Higgins RP. 1991. Kinorhyncha. In: HarrisonFW and Ruppert EE, editors. Microscopic Anatomy of Inverte-brates, Volume 4: Aschelminthes. New York: Wiley-Liss, Inc.pp 377–404; Chapter 10.

Mallatt JM, Garey JR, Shultz JW. 2004. Ecdysozoan phylogenyand Bayesian inference: First use of nearly complete 28S and18S rRNA gene sequences to classify the arthropods and theirkin. Mol Phylogenet Evol 31:178–191.

Marcus E. 1929. Tardigrada, In: Bronn HG, editor, Klassen undOrdnungen des Tier-reichs. Akademische Verlagsgesellschaft,Leipzig. Vol. 5, Section 4, Part 3:1–609.

Mayer G, Hartzsch S. 2007. Immunolocalization of serotonin inOnychophora argues against segmental ganglia being an an-cestral feature of arthropods. BMC Evol Biol 7:118, 1–9.

Mayer G, Whitington PM, Sunnucks Pfluger HJ. 2010. A revi-sion of brain composition in Onychophora (velvet worms) sug-gests that the tritocerebrum evolved in arthropods. BMC EvolBiol 10:255, 1–9.

McInnes SJ. Tardigrada. In: Marguelis L, Chapman M, editors.Five Kingdoms: An Illustrated Guide to the Phyla of Life onEarth, 4th ed. (in press).

Muller MCM. 2006. Polychaete nervous systems: Ground pat-tern and variations-cLS microscopy and the importance ofnovel characteristics in phylogenetic analysis. Integr CompBiol 46:125–133.

Møbjerg N, Dahl C. 1996. Studies on the morphology and ultra-structure of the Malpighian tubules of Halobiotus crispaeKristensen, 1982 (Eutardigrada). Zool J Linn Soc 116:85–99.

Møbjerg N, Jørgensen A, Eibye-Jacobsen J, Halberg KA, Pers-son D, Kristensen RM. 2007. New records on cyclomorphosisin the marine eutardigrade Halobiotus crispae. J Limnol 66(Suppl. 1):132–140.

Møbjerg N, Halberg KA, Jørgensen A, Persson D, Bjørn M,Ramløv H, Kristensen RM. 2011. Survival in extreme envi-ronments—On the current knowledge of adaptations in tardi-grades. Acta Physiol 202:409–420.

Nielsen C. 2001. Animal evolution. Interrelationships of theLiving Phyla, 2nd ed. Oxford University Press, Oxford. pp226–231.

Nielsen C. 2011. Animal evolution. Interrelationships of the Liv-ing Phyla, 3rd ed. Oxford University Press, Oxford. pp 265–269.

Persson D, Halberg KA, Jørgensen A, Ricci C, Møbjerg N, Kris-tensen RM. 2011. Extreme stress tolerance in tardigrades:Surviving space conditions in low earth orbit. J Zool SystEvol Res 49:90–97.

Plate L. 1889. Beitrage zur Naturgeschichte der Tardigraden.Zool Jb Anat Ontog 3:487–550.

Pugh PJA, McInnes SJ. 1998. The origin of Arctic terrestrialand freshwater tardigrades. Polar Biol 19:177–182.

Ramazzotti G, Maucci. 1983. Il Phylum Tardigrada. Mem IstItal Idrobiol 41:1–1012.

Ramskold L, Hou X. 1991. New early Cambrian animal andonychophoran affinities of enigmatic metazoans. Nature 351:225–228.

Renaud-Mornant J. 1975. Deep-sea Tardigrada from the MeteorIndian Ocean Expedition. ‘Meteor’ Forschungsergebnisse. D21:54–61.

Renaud-Mornant J. 1982. Species diversity in marine Tardi-grada. In: Nelson DR, editor. Proceedings of the Third Inter-national Symposium on the Tardigrada, August 3–6, 1980,Johnson City, Tennessee. Johnson City: East Tennessee StateUniversity Press. pp 149–178.

Richter S, Loesel R, Purschke G, Schmidt-Rhaesa A, Sholtz G,Stach T, Vogt L, Wanninger A, Brenneis G, Doring C, FallerS, Fritsch M, Grobe P, Heuer CM, Kaul S, Møller OS, MullerCHG, Rieger V, Rothe BH, Stegner MEJ, Harzsch S. 2010. In-vertebrate neurophylogeny: Suggested terms and definitionsfor a neuroanatomical glossary. Front Zool 7:29, 1–49.

Rota-Stabelli O, Kayal E, Gleeson D, Daub J, Boore J, TelfordM, Pisani D, Blaxter M, Lavrov D. 2010. Ecdysozoan mitoge-nomics: Evidence for a common origin of the legged inverte-brates, the Panarthropoda. Genome Biol Evol 2:425–440.

Ruppert EE. 1982. Comparative ultrastructure of the Gastro-trich pharynx and the evolution of myoepithelial foreguts inAschelminthes. Zoomorphology 99:181–220.

Ruppert EE, Barnes RD. 1994. Invertebrate Zoology, 6th ed.Saunders College Publ. Harcourt Brace and Company, Or-lando, Florida. 1056 p.



Schmidt-Rhaesa A, Bartolomaeus T, Lemburg C, Ehlers U,Garey JR. 1998. The position of the Arthropoda in the phylo-genetic system. J Morph 238:263–285.

Scholtz G. 2002. The Articulata hypothesis—or what is a seg-ment? Org Divers Evol 2:197–215.

Scholtz G, Edgecombe GD. 2006. The evolution of arthropodheads: reconciling morphological, developmental and palaeon-tological evidence. Dev Genes Evol 216:395–415.

Storch V, Ruhberg H. 1993. Chapter 2. Onychophora. In: Harri-son FW, Rice ME, editors. Microscopic Anatomy of Inverte-brates, Volume 12: Onychophora, Chilopoda and Lesser Proto-stomata. New York: Wiley-Liss, Inc. pp 11–56.

Wiederhoft H, Greven H. 1996. The cerebral ganglia of Milne-sium tardigradum Doyere (Apochela, Tardigrada): Threedimensional reconstruction and notes on their ultrastructure.Zool J Linn Soc (Lond) 116:71–84.

Wright JC. 2001. Cryptobiosis 300 years on from van Leuwen-hoek: What have we learned about tardigrades? Zool Anz240:563–582.

Zantke J, Wolff C, Scholtz G. 2008. Three-dimensionalreconstruction of the central nervous system of M. hufe-landi (Eutardigrada, Parachela): implications for the phy-logenetic position of Tardigrada. Zoomorphology 127:21–36.



Documents

Neuroanatomy of