.
Monika C. M. Miiller Wolfgang Sterrer
9
Received: 4 December 2003 I Accepted: 17 March 2004 I Published online: 15 April 2004 O Springer-Verlag 2004
Abstract Musculature and nervous system of Gnathostomula peregrina (Gnathostomulida, Scleroperalia) were reconstructed from whole animals by immunohistochemistry and confocal laser scanning microscopy. The F-actin muscular subset, stained with FITC-labeled phal-loidin, consists of: (1) eleven pairs (four ventral, one ventrolateral, one dorsolateral, five dorsal) of longitudinal muscles; (2) two types of diagonal muscles (thin fibers throughout the body, and slightly thicker fibers of which seven pairs occur ventrally and two pairs dorsally); (3) evenly spaced thin circular fibers that gird the posterior half of the body, continuing less prominently into the anterior half; and (4) a complex pharyngeal and genital musculature. Dorsoventral muscles are absent. The organization of the FMRFamidergic nervous system shows: (1) a central nervous system with a frontal ganglion and one pair of longitudinal nerves ending in a terminal commissure, and one median ventral nerve; (2) eight to ten unipolar perikarya above, and up to ten bipolar perik a q a in front of the brain; (3) a total of five (one unpaired, two paired) longitudinal nerves of the peripheral nervous system with two to four accompanying perikarya; and (4) a buccal ganglion of the stomatogastric nervous system with six to eight perikarya above the pharyngeal bulbus. Our results reveal the musculature and nervous system of Gnathostornula to be more complex than hitherto reported. Keywords Phalloidin . F-actin . FMRFamide . Nervous system . Gnathostomulida . cLSM M. C. M. Miiller (€3) Spezielle Zoologic, Fachbereich 5, Universitat Osnabriick, 49069 O s n a b ~ c kGermany , e-mail:
[email protected] Tel.: 49-541-969285819692859 Fax: +49-541-9692587
W. Sterrer Bermuda Natural History Museum, Flatts FLBX, PO Box FI 145, Bermuda
Introduction Gnathostomulida, a small cosmopolitan taxon of microscopic acoelomate marine worms, are mainly distinguished by an exclusively monociliated epidermis and a bilaterally symmetric pharynx containing complex cuticular mouth parts (Sterrer 1996, 1998,2001; S0rensen and Sterrer 2002). Originally described as Plathelminthes (Ax 1956), Gnathostomulida are now considered to be related to Rotifera (Sterrer et al. 1985; Rieger and Tyler 1995) and, together with Acanthocephala and Micrognathozoa (Kristensen and Funch 2000), form the taxon Gnathifera within Protostomia-Spiralia (Ahlrichs 1995; Giribet 2002). While the morphology of gnathostornulid mouth parts (S0rensen and Sterrer 2002), epidemis (Rieger and Mainitz 1977; Lammert 1989), protonephridia (Lammert 1985), sensory organs (Lammert 1984), reproductive structures (Mainitz 1979, 1983; Knauss and Rieger 1979), and sperm (Graebner 1969; Sterrer 2001; Sterrer et al. 1985) has been documented, body musculature has only recently received attention (Tyler and Hooge 2001). The nervous system, apart from first reconstructions by Kristensen and Ngrrevang (1977), Sterrer et al. (1985), and Lammert (1986), remains largely unexplored. Here we describe previously unknown features in the body musculature, and present a reconstruction of the FMRFamidergic nervous system of Gnathostornula peregrina Kirsteuer, 1969.
Materials and methods Sediment samples were taken from the shallow subtidal of Somersct Long Bay, off Daniel's Hcad Beach Park, Bermuda. After extraction from the sediment, specimens o i C. pcregrino (syn. G. tuckeri Fmis, 1977; see Sterrer 1998) were relaxed for 10 min in MgCI2 solution (8%) and subsequently fixed on ice at a temperature of O°C overnight in 4% puaformaldehyde in 0.15 M phosphatebuffered saline (PBS, pH 7.4) containing 12% sucrose. Afcer several rinscs with PBS for at least 1 h, the specimens were preincubated for 1 h in PBS containing 0.1% Triton X-100, 0.25% bovine serum albumin (BSA), and 0.05% NaN3 (PBTB).
F-actin labeling (four specimens investigated)
In order to stain the muscle system, animals were incubated in phalloidin-fluorescein isothiocyanate (FITCj-labeled solution ( 5 pi 3.3 pM solution in 100 pi PBSj. Incubation was carried out for 40 min at room temperature in a wet-chamber in the dark. After several rinses in PBS, specimens were embedded and investigated as described below. Nervous system labeling (four specimens investigated) Thc primary antibodies, palycional rabbit anti-serotonin (Sigma, Heidelberg; dilution 1:100 in preincubation liquid without BSA) and polyclonal rabbit anti-FMRFamide (courtesy of C.J.P.G. Grimmelikhuijren, Copenhagen; dilution 1:2,000), were applied for 12 h at room temperature. Subsequently, the specimens were washed in several changes of PBS and incubated with secondary anubodies. These were directed against rabbit and conjugated with the fluorophores TRITC (Zymed, San Francisco), FITC, Cy3, or Alexa 488. All secondary antibodies were diluted 1:100 in preincubation solution without BSA. Finally the animals were rinsed in PBS and mounted between two coverslips in Citifluor (Piano, Wetzlar). Preparations were investigated with a confocal laser s c a n ~ n gmicroscope (cLSMj Zeiss LSM 410. One has to be aware that the antibody might not only bind to FMRFamide, but also to FMRFamide-like antigens. Specificity of primary antibody binding was tested by treating specimens as described above, but omitting the primary antibody. The reproducibilily of the results was checked by replicating the incubations. Series of "optical sections" were projected into maximum intensity pixel images. Figures I and 3 present depth-coded images in which the position of any structure within the stack can be estimated from the colored scale bars.
Results The body of Gnathostomulida can be divided into rostrum (preoral region), tmnk, and tail region (posterior to the male gonopore). While the rostrum is long, pointed, and thread-shaped in Filospermoidea, it is usually short, rounded, and head-shaped in Bursovaginoidea. In G. peregrina, the rostrum takes up about 7% (40-57 pm) of tile total body length (670 pm). The tail region is rounded or terminates in a more or less distinctive tail filament.
and end in the tail. Posteriorly some of these fibers bend ventrally at a right angle while the others continue dorsally into the tail (Fig. 1C). The innermost muscle pair (dml) bends medially approximately 100 pm behind the pharynx. Both fibers cross each other, decrease in thickness, and end before the bursa (Fig. 1A). Only the two outermost fibers, dmj, project into the rostrum; where they prolong ventrally [Figs. ID (change from caudally red to anteriorly green=dorsal to ventral), 2Bl. Dorsolatera1 fibers begin approximately 200 pm from the anterior and run along the lateral side of the body (Figs. IA, 2A, C). No longitudinal fibers were detectable in the lateral body wall (Fig. 1E). Four pairs of longitudinal muscles are visible on the ventral side (Figs. lB, 2D). Only the outermost pair of these fibers, vm4, arises in the head (Figs. IF, 2E). While these muscles represent one of the two thick bundles in the anterior half of the body, they diminish posteriorly and end at the level of the bursa (Fig. 1B). By contrast, the muscles vm3 start in the anterior third as thin fibers, increase in width posteriorly, then run alongside and end behind the proximal stylet sac. In the posterior part of the body vm3 represent the thickest bundles (Fig. 2F). The thin longitudinal muscles vm2 arise immediately behind the mouth opening, remain continuously slim, and end in front of the proximal stylet sac. The innermost muscle pair, vml, most prominent of all ventral muscles, originates approximately 50 pm behind the mouth opening and also ends at the stylet sac. The venhal longitudinal muscles of each side approach each other continuously from anterior to posterior, and in front of the stylet sac they form a single medioventral muscle band (Figs. lB, 2F). Ventrolateral longitudinal muscles (vlm) run from the head posteriorly (Fig. lB), splitting up shortly before the tail, into which they continue as a median and a lateral pair. At the level of the pharynx, the ventrolateral muscles bifurcate (Figs. 1B, 2G) and merge a few micrometers further anterior with the ventral muscles vm4. In the praeoral region (rostrum), ventral and ventrolateral muscle fibers project contralaterally to their attachment sites, crossing each other to form a rostral cross (Fig. 2H).
Musculature The trunk musculature of G. peregrina is dominated by strong longitudinal muscles surrounded by thinner diagonal and circular muscles. Dorsoventral muscles are absent. The longitudinal muscles are clearly cross-striated, a pattern not discernible in the other fibers. The strong musculature of the pharynx and the genital system partly superimposes on (and visually obliterates) more subtle structures. Longitudinal muscles Five pairs of longitudinal muscles can he distinguished dorsally in the midbody region (Figs. lA, 2A). The fibers dm2-5 are more or less equally wide and arise in the head
Diagonal muscles Two types of diagonal muscles can be distinguished in G. peregrina: numerous, thin, hardly visible fibers, and a few slightly thicker fibers. Seven pairs of distinct diagonal muscles (Fig. 1A diml-7, yellow numbers) occur on the ventral side. The fibers run from the ventrolateral longitudinal muscles of one side to a far more posterior position on the other side, each muscle forming a chiasma with the respective fibers of the other side in the midventral line (Figs. IB, 2D). The pairs dim6 and dim7 are more widely separated than the others. Instead of mnning contralaterally, the latter muscle pair joins the ventral longitudinal muscles and ends behind the stylet sac. Two pairs of these diagonal muscles were found on the dorsal side, crossing each other in the
Fig. lA-G Gnathostomulu peregrina, F-actin muscular subset, depth-coded images. A Dorsal view. Five pairs of dorsal longitudinal muscles (doml-5, white numbers) extend from the head posteriorly. In the periphery dorsolateral muscles (dlm) are visible. Two dorsal diagonal muscles appear anteriorly (yellow numbers). bu Bursa, pbu prebursa, ph pharynx, st stylet. B Ventral view. Four pairs of longitudinal muscles (vml--4) m n parallel from the head to the proximal stylet sac (psr); some prolong posteriorly. Jn the periphery ventrolateral muscles ( d m ) mn from the head into the tail (fa). Diagonal muscles (diml-7, yellow numbers) reach from one side posteriorly and contralaterally. mo Mouth opening. C Lateral
view of the posterior end. Arrow indicates dorsovenrral muscles. D Dorsal view of anterior end; only dm5 project into the rostrum. The fibers run from posterior and dorsal (red) anterior and ventral (green). inc Inclinator muscles, cms caudal muscle sac. E Lateral view of the anterior end. F The ventral longitudinal muscles stretch s h i g b t into the rostrum. The ventrolateral muscles elongate anteriorly and mcdially. bpr Basal plate rostralis, bpd basal plate dorsalis, mc mouth cavity, rc rostra1 cross. G Dorsolateral muscle arrangement with circular muscles (cm) and diagonal muscles (while arrowheuds)
middorsal line (Fig. lA). The thin diagonal fibers occur all along the body and surround it at different angles (Fig. 1G).
(Fig. 1G). Although this pattern is more obvious in the posterior half of the animal, it can also be found in the anterior body region (Fig. lA, B).
Outer circular muscles
Pharyngeal muscles
At higher magnification it becomes apparent that the circular muscles are spaced evenly, about 5-10 pm apart
Thin fibers connect the pharyngeal organ with the ventral and dorsal (Fig. IA) longitudinal muscles. Through fine
Fig. 2A-M Gnathostornula peregrina, F-actin muscular subset. A-F Cross-sections from image stacks. A-C Dorsal half. A Midbody; five dorsal (dml-5) and one dorsolateral (dlm) fibers are visible. B Rostrum; only dm5 is visible. ph Pharynx. C in front of the stylet sac, circular muscles (cm) surround the body. D-F Ventral half. D Midbody; crossing diagonal (dim), iour ventral ( v m l h l ) , and venholateral muscles ( d m ) are visible. E Rostrum, just behind the point where the venlrolateral muscles and vm4 join. mo Moulh opening. F In front of the stylet sac, the ventral muscles
form one band and in this region vm3 (3) is the chickest fiber. G Venhal and ventrolateral muscles join. H Ventral and venholateral muscles form a rostra1 cross (rc) in the rostmm. 1-M Pharynx muscul;lture. I Transverse fibers link the pharynx with venholateral muscles. bp Basal plate. J Ventral aspect with basal plate rostralis (bpr) and retractor (re). K Eight microns dorsal lo J. did Diductor. L Eight microns dorsal to K. rrp, lp, pp Anterior, lateral, and posterior pouch. M Dorsal view
transversal fibers it is also linked to the ventrolateral longitudinal muscles (Fig. 21). By scanning the pharyngeal bulb from ventral to dorsal one encounters the following structures. In the pharyngeal bulb the basal plate rostralis (bpr) and the basal plate dorsalis (bpd) form a semicircle around ihe anterior border of the mouth cavity (Figs. 1F. 25). The caudal muscle sac, with its pair of inclinator muscles (inc), can be observed in more dorsal sections. Fuaher dorsally, a pair of thick diductors (did) partly forms the anterior edges of the caudal muscle sac
(Fig. 2K). The caudal muscle sac itself, with its pair of inclinator muscles (inc), can be observed in more dorsal sections (Fig. 2L). The four non-fluorescing areas represent one anterior, one posterior, and two lateral fiberfree pouches. A dorsal aspect of the pharynx shows that V-shaped, cross-striated muscles make up the uppermost layer o i Lhe organ (Fig. 2M). No muscular grid surrounding the intestine could he found.
Reproductive sjstem Whereas the spherical praebursa and the elongated bursa are weakly stained (Fig. IB), the penial bulb fluoresces extremely brightly, owing to the densely packed muscles (Fig. IA). The utricular testes are surrounded by a mesh of fine fibers. Where they jointly open into the penial bulb, circular muscles form a distinct sphincter. The penis stylet shines even more brightly than the penial bulb. While it looks solid in a sagittal section, the cross-section demonstrates that it is tubular or U-shaped.
cells (5 pm in diameter) lies 20 ,um ahove and behind the brain (Figs. 3C red cells_E green cell, 4B). The other six to eight smaller cells lie in pairs in a transverse row directly ahove the brain commissures (Fig. 3D). Around ten nask-shaped bipolar perikarya (bpk) are located in front of the brain. Their dendrites reach the anterior margin of the head, and their axons projcct into the brain. IR-free. dark spherical spots within the soma indicate the location of the nuclei (Fig. 31)). Peripheral nervous system
Nervous system Only the FMRFamidergic subset of the nervous system could be successfully stained and will be described in the following. Neuronal structures in incubation directed against 5-HT (serotonin, not shown) could not he resolved because the gland-rich integument unspecifically bound the antibody, superimposing its bright fluorescence on the neural structures. In the following we distinguish between the central (CNS), peripheral (PNS), and stomatogastric (SNS) nervous systems. The CNS is characterized by its location and contains the supraesophageal ganglion, the circumesophageal connectives, and the ventrally located bauchmurk.
Five longitudinal nerves, situated laterally and dorsally, belong to the peripheral nervous system. The dorsomedian nerve (dmn) can only he traced for 50 pm. The IR of the neurites is very weak (Fig. 3F blue arrowheads), and the nerve is mainly visible thanks to the accompanying two perikarya (Figs. 3F red, H blue, blue encircled, 4B). The dorsolateral nerve pair (dln) is indicated by its eight perikarya (Fig. 3F, H yellow encircled). The dorsolaleral nerves reach twice the length of the dorsomedian nerve. The lateral nerve pair (In) is visible from the transition between head and trunk (Fig. 3H orange arrowheads) until 40 pm in front of the tail, where it terminates in a pair of big unipolar perikarya (Fig. 3G orange ellipses). In the anterior third of the body three perikarya are associated with each lateral nerve (Fig. 3H orange circles).
Central nervous system
Stornutogastric nervous system
Of eight longitudinal nerves (three CNS, five PNS) that showed positive FMRFamide immunoreactivity (IR), only the two main ventral nerves (vn) extend through the entire length of the body (Figs. 3A, 4A). These main nerves run approximately 25-30 pm apaa from and parallel to each other. In the hind end they fuse in what we consider, despite its acute angle, a terminal commissure. Whether the intense IR behind the fusion represents a nerve cell or only condensed fibers could not he clarified. No fibers penetrate the tail and no FMRFamidergic perikarya are allied with the main ventral nerves. Approximately 50 pm behind the brain some neurites branch off from each main ventral nerve (Fig. 3A arrowhead.^), join medially, and form the ventromedian nerve (vmn), with the same thickness and IR intensity as the main nerves. After 100 pm the ventromedian nerve ends blind. Two thick perikarya are visible, one immediately behind its V-shaped beginning and the second shortly before its end (Fig. 3A, H). Anteriorly the main nerves continue into the circumesophageal connectives (cc) that surround the mouth. At the level of the mouth, nerves branch laterally and medially off the connectives. The lateral branches arhorize whereas the median branches extend anteriorly and fuse to form a commissure. This commissure lies just behind the anterior commissure made up by the esophageal connectives (Figs. 3B, 4A). Approximately ten unipolar perikarya are positioned above the brain (Fig. 3C). One pair of large
A concentration of six to eight FMRFamide-positwe perikarya indicates the position of the buccal ganglion (Fig. 3B-D bg). A connection of this ganglion with the brain via buccal nerves could not he observed.
Discussion Musculature Our results confirm Tyler and Hooge's (2001) finding that the musculature of Gnathostornula armata Riedl, 1971 is dominated by thick longitudinal fibers; thin circular and thin as well as somewhat stronger diagonal fibers exist, while a dorsovenlral musculature is lacking. But there are several differences in number and arrangement: I. While Tyler and Hooge (2001) described a maximum of 14 longitudinal muscles in G. armata (8 dorsal, 2 lateral, 4 ventral; Fig. 5A). we found 22 in G. peregrim (10 dorsal, 2 dorsolateral, 2 ventrolateral, 8 ventral; Fig. 5B). Thus G. peregrinu possesses 2 more dorsal, 2 more dorsolateral and 4 more ventral longitudinal muscles than G. armata. Fusion of the dorsal innermost fibers (Fig. 5A gray fibers) into a median hand, ending in front of the bursa occurs in G. a m t a , hut not in G. peregrina.
2. A pair of lateral bundles stretching from head to tail are the strongest muscles in G. a n a t a , while in G. peregrina lateral muscles seem to be lacking altogether, and the ventral muscles (vml and vm4) appear strongest. In G. peregrina these same muscles, rather than running continuously from head to tail, either originate anteriorly and peter out posteriorly (vm4), or vice versa (vml). 3. Anterior to the pharynx there are two pairs (dorsal, lateral) of longitudinal muscle bundles in G. armata (Tyler and Hooge 2001), whereas there are three (dorsal, ventrolateral, and ventral) in G. peregrina. In both species these fibers create and anchor on a rostra1 cross. 4. For G. anmta the authors describe a pair of dorsolateral fihers that branches off the dorsal fibers in front of the bursa. In contrast, dorsolateral muscles appear in the anterior third of the trunk in G. peregrina and they do not originate from the dorsal muscles. 5. Whereas in C. a m t a dorsal and ventral longitudinal muscles attach to the copulatory organ, they reach over the organ and project into the tail in G. peregrina. 6. In G. annata, circular fihers are prominent in the posterior half of the hody hut totally absent in the anterior half. This difference is much less striking in G. peregrina, where (albeit thinner) circular fihers continue anteriorly to just behind the pharynx. 7. Two stronger pairs of diagonal muscles cross the ventral hody wall in G. armata, while there are seven such muscle pairs in G. peregrina. 8. Tyler and Hooge (2001) state 'The only c o ~ e c t i o nto the hodywall musculature appears to he by attachment
Fig. 3A-H
Gnathostornula peregrina, FMRFamidergic nervous system, depth-coded images. A Ventral view. The widely separated
nectivei C, D Dorsal view of the head. One pair of thickierik&ya is located dorsally and posterior of the brain (C, red), others lie in a row closer to and directly above the brain. Behind the transition of head and trunk, perikarya of the buccal ganglion (bg) are visible. Several bipolar perikarya (bpk) lie in front of the brain. E Lateral view of the head. osg Supraesophageal ganglion, ax axon. F Dorsal view, anterior end. Unipolar perikarya (pk) are located close to the brain, their axons (ax) project into the brain. A dorsomedian nerve (dmn, blue nrrowheadr) and two donolateral nerves (dln) are visible. The attended perikarya are encircled blue and yellow, respectively (colors correspond to H). Notice backpound staining of epidermal cell rows. G Posterior end. The lateral nerves (In) terminate with two large, unipolar perikarya (orange ellipses). H Lateral view, anterior end. From dorsal to ventral the following nerves are distinguishable: dorsomedian (dmn), dorsolateral (dln), lateral (In), main ventral (vn), and ventral median (vmn). The first three nerves are associated with two, four, and three perikarya (blue, yellow, and orange circles), respectively. Two thick perikarya belong to the ventral median nerve
of the sides of the pharynx to the passing dorsal and lateral longitudinal muscle pairs". For C. peregrina we demonstrated transverse fibers that link the pharynx with the ventrolateral longitudinal muscles and additional thin dorsal and ventral fibers extending from the organ posteriorly. Whether these differences between the two species are representative of muscle arrangement in the Gnathostomulida or whether they are, at least pmly, due to artifacts of preparation and analysis has to be answered by further investigations, which should include species of other Scerloperalia and of the Filospennoidea.
species similar in size to G. peregrina, Windoffer (1992) calculated that 5-7% of the brain perikarya contain FMRFamide. If this percentage is also valid for other iuvertebrates, the frontal ganglion of G. peregrina should contain 260-360 perikarya (18 FMRFamidergic cells assumed) and this number a p e s with the 250 cells Lammen (1986) assumed based on cross-sectioned fibers. Paired ventrolateral connectives are also the main nerves in the other gnathiferan taxa Rotifera, Acanthocephala, and Micrognathozoa (Lorenzen 1996; Kristenscn and Funch 2000). While a median nerve is not documented for these three gnathiferan taxa, it can be luuull in Fig. 5A, B Schematic cross-sections of midbody regions with ar- the ventral cords of Nematoda, Nematomorpha, and Prirangement of longitudinal muscles. A Rasrrngnathia macrostoma. The p a y muscles fuse funhcr posterior. B Gnuthosfornula pere- apulida (Nemathelmiuthes). In these taxa, however, the grim. dm, dlm Dorsal and dorsolateral muscles, lrn lateral muscles, ventromedian cord is the only main cord and, furthervrn, vlm ventral and ventrolateral muscles. (A after Kiistensen and more, is of the medullary type. The presence of single Nsrrevang 1977) accompanying perikarya (Lammert 1986; this study) does not justify referring to the longitudinal nerves of Differences between G. peregrina and G. armata in Gnathostomulida as medullary. Further investigations on body musculature do not require a re-interpretation of the other Gnathostomula species have to clarify whether the functional analysis by Tyler and Hooge (2001). Addi- median nerve is an autapomorphic character for the getional anterior circular muscles and diagonal and longi- nus, or even for the Bursovaginoidea. Whether the median tudinal muscles in G. peregrina may allow for greater nerve innervates the bursa could neither be excluded nor body flexibility. Similarly, the varying diameter of lon- proved; thus, for now, no explanation can be given for the gitudinal fibers along the body axis may permit differ- target of this nerve. The additional longitudinal nerve pairs in R. macrosential regional contraction. Anchoring of longitudinal muscles in a rostral cross has been described for Platy- tuma (two pairs; Kristensen and Norrevang 1977) and G. helminthes (Rieger et al. 1994) and various Polychaeta paradoxa (one pair; Lammert 1986) are regarded as be(Miiller 1999); in the case of G. peregrina it may permit a longing to the peripheral nervous system. Missing details sharper bending of the rostrum, and thus enhance ma- on the location of these nerves disables comparison between species. Ln G. peregrina we found two such pairs, neuverability in tight interstitial spaces. one lateral and one dorsolateral, and, moreover, an unpaired dorsomedian nerve. Within the Nemathelminthes Nervous system additional longitudinal nerves are only reported for Kinorhyncha and Loricifera (Lorenzen 1996), and are asIn their TEM analysis of Rastrognathiu macrostoma sumed for Micrognathozoa (Kristensen and Funch 2000). Kristensen and Ngrrevang, 1977, a distant relative of Application of immnnocytochemical staining, however, Gnathostomulu within Bursovaginoidea-Scleroperalia, might reveal peripheral nerves in other taxa as well. Kisteusen and Norrevang (1977) postulate a nervous system consisting of a brain (frontal ganglion), three pairs of longitudinal nerves, and one pair of buccal nerves Phylogenetic impact connecting to a buccal ganglion. Also on the basis of TEM series, Lammert (1986) reconstructed the nervous Kristensen and Ngrrevang (1977) speculated that the three system of Gnathostomula paradoxa Ax, 1956 as con- nerve pairs in the body wall, together with the buccal sisting of a frontal ganglion-which links to a buccal nerves, representing the transformed ventral pair, develganglion via a pair of buccal nerves-and at least two oped from an orthogonal system. In G. peregrina, eight pairs of longitudinal nerves of which the ventrolateral longitudinal nerves are already present in the body wall, pair, extending toward the tail, is interconnected by two but two of them are unpaired median fibers, which are not commissures, one ante* and the other posterior to the described for orthogonal systems. Futhermore, in an orpenis. A CNS oonsist~ngof frontal ganglion, circume- thogon, as originally described for Platyhelminthes by sophageal connectives, paired ventral cords, and two Reisingcr (1925), the longitudinal nerves are interconcommissures is also found in Filospermoidea (Lammert nected by perpendicular transverse commissures, which 1986) and is therefore, together with the prominent un- are missing in Gnathostomulida. Instead of orthogon this paired median nerve described here for the first time, pattern could be termed a "cordal nervous system", as assumed to be representative of the entire taxon. The described for Acoela (Raikova et al. 1998). dendrites of the bipolar FMRFamidergic perikarya loKristensen and Nclrrevang (1977, 1978) as well as cated in front of the brain might be identical with the Lammea (1986) described a pair of buccal nerves and "rostral nerves" described for both Bursovaginoidea and ganglia for the gnathostomulid stomatogastric nervous Filospermoidea by Lammert (1986). For two polychaete system. By contrast, Herlyn and Ehlers (1997) found
Lammsrt V (1984) The fine structure of spiral ciliary receptors in Gnathostomulida. Zoomorphology l04:36&364 Lammert V (1985) The h e structure of protonephridia in Gnathostomulida and their comparison with Bilateria. Zoomorphology 105308-316 Lmmcrt V (1986) Verglcichende Ultrastruktur-Untersuchungen an Gnathostomuliden und die phylogeneiische Bewertung ihrer Merkmale. PhD Thesis, Univeisital GRningen Lammcrt V (1989) Fine structure of the epidermis in Gnathostomulida. Zoomorphology 109: 131-144 Lorewen S (1996) Nemathelrninthes. In: Westheide W. Rieser R (eds) Spezielle Zoologic. Einzeller und Wirbellose Tiere. Fischer, SLungart, pp 682-736 Mainitz M (1979) The fine structure of gnathostomulid repmductive organs. I. New characters in the male copulatory organ of Scleroperalia. Zoomorphologie 92241-272 Mainia M (1983 Gnathostomulida. 1": Adiyodi KG, Adiyodi RG (eds) Rcproductive biology of invertebrates, 1. Oogenesis, oviposilion, and oosorption. Wiley, New York, pp 169-180 Miiller MC (1999) Das Newensystem der Polychaeten: Immunhistochemische Untersuchungen an ausgewiihlten Taxa. PhD Acknowledgements We wish to thank Prof. Dr. Wilfried WestThesis, Universitiit Osnabriick heide for taking the sediment samples in Bermuda and for valuable Miiller MC, Westheide W (2000) Structure of the nelvous system discussions about the manuscript. This is Contribution #51 from the of Myzosroma cirri&rum (Annelida) as revealed by imrnunoBermuda Biodiversity Project (BBP), Bermuda Aquarium Museum histochemistry and cLSM analyses. J Morphol 245:87-98 and Zoo. Miiller MCM, Westheide W (2002) Comparalive analysis of the nervous systems in presumptive progenetic dinophilid and dorvilleid polychaetes (Annelida) by immunohislochemisvy and cLSM. Acta Zool (Stockh) 83:33-48 References Raikova 01, Renter M, Kotikova EA, Gustfsson MKS (1998) A cornmissural brain1 The pattern of 5-HT immunoreactivity in Ahlrichs WH (1995) Ultrastruktur und Phylogenie von Seison Acoela (Plathelminthes). Zoomorphology 118:69-77 nebalioe (Grube 1859) nnd Seison annulatus (Claus 1876). Reisinger E (1925) Untersuchungen Nervensytem der ButhriHypothesen zu phylogenetischen Venvandt~chaftsverh2llnissen onlunrr s e m ~ e r Baun. i Z Momhol Okol Tiere 5:119-149 innerhalb der Bilateria. Cuvillier, Gottingen Ax P (1956) Die Gnathostomulida, eine ratsitaelhaftc Wurmgruppe aus dem Meeressand. Abh Akad Wiss u Lit Mainz MathNatunviss Kl 8:l-32 Evolutionsforsch -15:9-35 Giribel G (2002) Current advances in the phylogenetic recon- Rieger RM, Tyler S (1995) Sister-goup relationship of Gnathostornulida and Rotifera-Acanthocephala. Invertebrate Biol 114: neither such ganglia n o r nerves i n G. p a r a d o x a , a n d concluded that the Gnathostomulida lack such smcknres. T h e existence of a buccal ganglion in G. p e r e g n n a is documented b y staining eight t o t e n FMRFamidergic perikarya within this ganglion. Unexpectedly, the buccal nerves, which are "very large trunks" (Kristensen a n d Ngrrevang 1977). did not s h o w i m m u n e reactivity, although t h e antigen i s k n o w n to be widespread within invertebrate stomatogastric newour; systems (for example, Annelida: Miiller 1999: Miiller a n d Westheide 2000. 2002). presence o f a buccal ganglion in G. peregnna restores at least o n e autapomorphy f o r a monophyletic taxon Gnathifera and strengthens the basis of characters o n which "...a sister g r o u p relationship of the gnathostomulids a n d syndermates appears, f o r now, t o b e the best s u p p o r t e d (Jenner 2004 p 61).
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