Neural markers reveal a one-segmented head in tardigrades (water bears)

Abstract

Background: While recent neuroanatomical and gene expression studies have clarified the alignment of cephalic segments in arthropods and onychophorans, the identity of head segments in tardigrades remains controversial. In particular, it is unclear whether the tardigrade head and its enclosed brain comprises one, or several segments, or a non-segmental structure. To clarify this, we applied a variety of histochemical and immunocytochemical markers to specimens of the tardigrade Macrobiotus cf. harmsworthi and the onychophoran Euperipatoides rowelli.

Methodology/principal findings: Our immunolabelling against serotonin, FMRFamide and α-tubulin reveals that the tardigrade brain is a dorsal, bilaterally symmetric structure that resembles the brain of onychophorans and arthropods rather than a circumoesophageal ring typical of cycloneuralians (nematodes and allies). A suboesophageal ganglion is clearly lacking. Our data further reveal a hitherto unknown, unpaired stomatogastric ganglion in Macrobiotus cf. harmsworthi, which innervates the ectodermal oesophagus and the endodermal midgut and is associated with the second leg-bearing segment. In contrast, the oesophagus of the onychophoran E. rowelli possesses no immunoreactive neurons, whereas scattered bipolar, serotonin-like immunoreactive cell bodies are found in the midgut wall. Furthermore, our results show that the onychophoran pharynx is innervated by a medullary loop nerve accompanied by monopolar, serotonin-like immunoreactive cell bodies.

Conclusions/significance: A comparison of the nervous system innervating the foregut and midgut structures in tardigrades and onychophorans to that of arthropods indicates that the stomatogastric ganglion is a potential synapomorphy of Tardigrada and Arthropoda. Its association with the second leg-bearing segment in tardigrades suggests that the second trunk ganglion is a homologue of the arthropod tritocerebrum, whereas the first ganglion corresponds to the deutocerebrum. We therefore conclude that the tardigrade brain consists of a single segmental region corresponding to the arthropod protocerebrum and, accordingly, that the tardigrade head is a non-composite, one-segmented structure.

Figure 1. Relative size of a tardigrade compared to a single neuron of an insect.

Light micrographs; both images are to scale. Note that the entire anterior end of the tardigrade, including the brain and consisting of hundreds of cells , would fit into a single neuronal cell body of the insect. (A) Cobalt-filled dorsal unpaired median neuron (DUM neuron) from the thoracic ganglion of the locust Locusta migratoria. (B) Specimen of Macrobiotus cf. harmsworthi in ventral view. Abbreviation: so, neuronal soma. Scale bar: 100 µm (for A and B).

Figure 2. Organisation of the nervous system in the tardigrade Macrobiotus cf. harmsworthi.

See also Figures S1, S2 for the colour-blind. RFamide-like immunoreactivity (red) and DNA labelling (green). Maximum projections of confocal z-series. Anterior is up in all images except for B in which anterior is left. Note that no “suboesophageal” or “subpharyngeal ganglion” is evident in its presumptive position (asterisks in A–C). Arrowheads in A–C point to varicose swellings in the core of each trunk ganglion. Arrows in B and D indicate two anterior, extra-cerebral RFamide-like immunoreactive cell bodies. (A) Specimen in ventral view showing four trunk ganglia linked by somata-free connectives. (B) Specimen in lateral view revealing the dorsal position of the brain (dotted line). (C) Anterior end in ventral view. (D) Anterior end in dorsal view with details of central brain neuropil. Dotted line indicates the shape of the brain with its lobes. Abbreviations: br, brain; cn, connectives; ga1–ga4, trunk ganglia 1 to 4; il, inner brain lobe; le1–le4, walking legs 1 to 4; np, central brain neuropil; oc, outer connective; ol, outer brain lobe; ml, median brain lobe; so, neuronal somata. Scale bars: 25 µm (A), and 10 µm (B–D).

Figure 3. Neural structures in the anterior end of the tardigrade Macrobiotus cf. harmsworthi.

Combined anti-tyrosinated and anti-acetylated α-tubulin immunolabelling. Anterior is up. Arrowheads point to the nerve ring, which gives rise to numerous anterior neurites innervating the peribuccal/mouth lamellae. (A, B) Specimens in ventral and ventrolateral view, respectively. Maximum projections of confocal z-series. (C, D) Depth-coded projections of confocal z-series of anterior ends of an extended (in C) and a contracted specimen (in D). Note the complex fibre network within the central brain neuropil, which appears as a unitary structure with the nerve ring in the contracted specimen. Abbreviations: as, anterolateral sensory fields; bd, neurite bundles innervating the stylet musculature; cn, connective; co, commissure of the stomodeal complex (cf. Figure 5C); ga1, first trunk ganglion; ln, leg nerves; mp, macroplacoids; ne, neurites supplying the peribuccal/mouth lamellae; np, central brain neuropil; oc, outer connective; pn, peripheral nerve; ps, posterolateral sensory fields; un, unpaired posterior nerve/neurite. Scale bars: 20 µm (A–D).

Figure 4. Details of the brain in the tardigrade Macrobiotus cf. harmsworthi.

Specimens in dorsal view; anterior is up. Note the bilaterally symmetric arrangement of individually identifiable neuronal somata. (A) RFamide-like immunoreactivity; maximum projection of confocal z-series. Inset shows details of two extra-cerebral RFamide-like immunoreactive somata situated in front of the brain (asterisks). (B) Volume rendering of the same dataset as in A. The brain contours are illustrated in blue (autofluorescence), RFamide-like immunoreactive structures in yellow. Inset shows details of a ring-like structure surrounding the mouth. (C) Serotonin-like immunoreactivity; maximum projection of confocal z-series. Note the strong signal in the mouth cone. (D) Volume rendering of the same dataset as in C. Serotonin-like immunoreactive neural structures are illustrated in green, the mouth cone in light-blue, autofluorescent calcified stylet structures in magenta. Arrowheads in C and D point to two bilateral neurites, which join the dorsal portion of the ring nerve (see also Movie S2). Abbreviations: cn, connective; ex, cell bodies of extra-cerebral neurons; ga1, first trunk ganglion; mc, mouth cone; mp, macroplacoids; np, central brain neuropil; oc, outer connective; so, neuronal somata; st, stylet; un, unpaired posterior neurite/nerve. Scale bars: 10 µm (A–D).

Figure 5. Details of the stomodeal innervation in the tardigrade Macrobiotus cf. harmsworthi.

(A) Scanning electron micrograph showing the peribuccal lamellae surrounding the mouth opening. (B) Anterior end of a specimen labelled with phalloidin-rhodamine to reveal the stylet and pharyngeal musculature. Projection of a confocal z-series; anterior is up. (C) Stomodeal complex. Combined anti-tyrosinated and anti-acetylated α-tubulin immunolabelling. Projection of confocal z-series; anterior is up. For the sake of clarity, only elements of the stomodeal complex were selected from each optical section for the projection. Dotted lines demarcate the corresponding body region in the specimen labelled with phalloidin-rhodamine. Abbreviations: bd, neurite bundles innervating the stylet musculature; co, commissures of the stomodeal complex; ne, neurites supplying the peribuccal/mouth lamellae; nr, nerve ring; oe, oesophagus; ph, pharynx; pl, peribuccal/mouth lamellae; pr, projections linking the nerve ring and the neurite bundles innervating the stylet musculature; st, stylet. Scale bars: 3 µm (A), 25 µm (B), and 10 µm (C).

Figure 6. Characteristics of the stomatogastric ganglion supplying the oesophagus and midgut in the tardigrade Macrobiotus cf. harmsworthi.

Projections of confocal z-series. (A) Anterior half of a specimen in dorsal view. Anti-serotonin immunolabelling. Maximum projection of a confocal z-series. Note serotonin-like immunoreactivity in the stomatogastric ganglion. Arrowheads point to three posterior neurites. (B) Detail of the stomatogastric ganglion. Combined anti-tyrosinated and anti-acetylated α-tubulin immunolabelling. Neuronal cell bodies are marked by asterisks. (C) Detail of a lateral posterior neurite (arrowhead), which has an input into the second trunk ganglion (arrow). Anti-serotonin immunolabelling. Projection of a confocal z-series. Abbreviations: br, position of the brain; cn, connective; en, oesophageal neurite; ex, somata of extra-cerebral neurons innervating the mouth cone; ga1–ga2, position of the first and second trunk ganglia; lp, lateral neurite; mc, mouth cone; pp, posterior neurite; sg, stomatogastric ganglion; so, neuronal somata; un, unpaired posterior neurite/nerve. Scale bars: 10 µm (A–C).

Figure 7. Innervation pattern of pharynx, oesophagus and midgut in the onychophoran Euperipatoides rowelli.

See also Figure S9 for the colour-blind. Confocal micrographs. (A, B) Anti-serotonin immunolabelling of a dissected digestive tract. Anterior is left. (C–E) Triple-labelled vibratome cross-sections using an anti-acetylated α-tubulin antibody (green), phalloidin-rhodamine (red), and the DNA-selective dye Hoechst (blue). Dorsal is up. (A) Whole-mount preparation showing the pharyngeal loop nerve (lo). Inset, which is a projection of only a few focal planes, illustrates numerous serotonin-like immunoreactive somata associated with this nerve (arrowheads). (B) Detail of transition region between oesophagus and midgut in the same whole-mount preparation as in A. Note the scattered arrangement of neuronal cell bodies within the gut wall (arrowheads). Inset, which is a projection of only a few focal planes, shows a nerve fibre and details of serotonin-like immunoreactive somata of bipolar neurons within the gut wall (arrowheads). (A–E) Vibratome cross-sections of the pharynx (ph), oesophagus (oe) and midgut (mg) showing their innervation pattern. Arrows in C point to fibres supplying the pharyngeal musculature. Arrowheads in E indicate single neurons in the wall of the gut. Note the lack of nerves and neuronal cell bodies in the oesophagus wall in D. Abbreviations: bf, bundle of fibres in the pharyngeal wall; fi, serotonin-like immunoreactive fibre on the outer surface of the gut wall; lo, pharyngeal loop nerve; mg, midgut; nc, nerve cord; oe, oesophagus; ph, pharynx; sl, slime glands. Scale bars: 100 µm (A–E), and 25 µm (insets in A and B).

Figure 8. Innervation of the pharynx in embryos of the onychophoran Euperipatoides rowelli.

(A–C) Whole-mount preparations of almost fully developed embryos (stage 7) in dorsal (in A and B) and lateral views (in C). Maximum projections of confocal z-series; anti-acetylated α-tubulin immunolabelling. Note that there is no nerve tract connection between the pharynx and the midgut (arrows). Abbreviations: at, antenna; br, brain; ce, central brain neuropil; le, leg; lo, pharyngeal loop nerve; rc, ring commissure; sg, slime gland; sp, slime papilla. Scale bars: 300 µm (A), and 100 µm (B, C).

Figure 9. Diagrams summarising the innervation pattern of foregut and midgut structures in Tardigrada and Onychophora.

(A) Stomodeal complex innervating the mouth, the stylet and the pharynx (green) and the stomatogastric ganglion with its projections (magenta) in the tardigrade Macrobiotus cf. harmsworthi. (B) In the onychophoran Euperipatoides rowelli, the ectodermal pharynx is innervated by a loop-like, medullary pharyngeal nerve (blue), which is accompanied by neuronal somata. The endodermal midgut shows numerous neuronal cell bodies scattered in the gut wall (red). Abbreviations: at, antenna; br, brain; ga2, second trunk ganglion; jw, jaw; le1–le4, walking legs 1 to 4; mg, midgut; mo, mouth; nc, nerve cord; oe, oesophagus; ph, pharynx; sg, stomatogastric ganglion; sp, slime papilla.

Figure 10. Diagram showing the alignment of head segments and segmental regions of the nervous system in tardigrades and arthropods.

Circles filled with magenta indicate the position of the stomatogastric ganglia (often referred to as the “frontal”, “rostral” or “stomodeal ganglia” in some groups). Note that the protocerebrum, deutocerebrum and tritocerebrum do not form separate ganglia but are usually fused to a syncerebrum in adult arthropods. Note also the two alternative alignments for chelicerates, consistent with either the expression pattern of Hox genes , , (alternative 1) or with the anterior border and periodicity of expression of the pair-rule genes runt and paired , (alternative 2). According to the second alternative, the deutocerebral segment has been reduced in chelicerates (marked by white colour and light-grey lines). Small circles in the ocular segment indicate the position of eyes in tardigrades and median ocelli in arthropods. Hatched ovals illustrate the position of compound eyes in arthropods. Abbreviations: at, antenna; at1 and at2, first and second antennae; ch, chelicera; de, deutocerebrum; ga1–ga3, trunk ganglia 1 to 3; le, walking leg; md, mandible; mx, maxilla; pd, pedipalp; pr, protocerebrum; st, stylet; tr, tritocerebrum.