The organization and evolutionary implications of neuropils and their neurons in the brain of the onychophoran Euperipatoides rowelli

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Abstract

This account describes the organization of the brain of the adult Euperipatoides rowelli, a member of the Onychophora or “velvet worms.” The present account identifies three cerebral divisions, the first of which contains primary olfactory neuropils, visual neuropils, and brain regions that correspond anatomically to the mushroom bodies of annelids, chelicerates, myriapods, and insects. In common with the brains of many chelicerates, the onychophoran brain is supplied by many thousands of uniformly small basophilic perikarya. Other chelicerate-like features include mushroom body lobes that extend across the brain's midline, an unpaired arch-shaped midline neuropil, and visual pathways that supply midline neuropil and that of the mushroom bodies. These and other similarities with chelicerate brains are discussed in the context of arthropod evolution and with reference to recent molecular phylogenies.

Introduction

Reputedly an enigmatic group of animals, onychophorans were traditionally thought to occupy an intermediate phylogenetic position between arthropods and annelids (Snodgrass, 1938), a status also argued by other early students of invertebrate nervous systems (Fedorow, 1929, Hanström, 1935). Recent palaeontological observations have proposed that modern onychophorans, which are exclusively terrestrial, represent the vestiges of an ancient lineage of lobopod ecdysozoans whose greatest radiation occurred in the mid- and lower-mid Cambrian eras (Hou and Bergström, 1995), and which may be ancestral to modern arthropods (Budd, 2001).

Studies of segmentation gene expression, which have done much to establish homology across the arthropods and annelids, show that Hox genes that express in euarthropods (Ultrabithorax [Ubx] and abdominal-A [abd-A]) are present in onychophorans (Grenier et al., 1997) and annelids (Irvine and Martindale, 2000). Onychophorans express orthologous ftz (fushi tarazu) genes, a gene family that is restricted to a clade comprising the Euarthropoda (Grenier et al., 1997). Recently, phylogeneticists using gene sequences have placed onychophorans firmly within the ecdysozoans, some workers suggesting sister group relationships with the chelicerates (Ballard et al., 1992) although most situating the Onychophora as a sister group to the Euarthropoda (Regier et al., 2005).

These studies coupled with uncertainties regarding anatomical features underscore that resolving the phylogenetic position of the onychophorans may be central to an understanding of the origin and evolution of the arthropod body plan. If Onychophora indeed presages euarthropods with jointed limbs as proposed by Budd, 2001, Budd, 2002 it is reasonable to ask whether, despite their homonomous body plan, aspects of onychophoran morphology correspond to structures that are either shared across the Euarthropoda or are characteristic of a specific group within the euarthropods. Apart from at the head (referring here to the first three prestomodeal segments) and at the genital and anal segments, onychophorans lack segmental diversity and, again excepting the head, they lack modified appendages (Snodgrass, 1938). On the other hand, onychophorans possess a large brain, which according to both classic and recent studies posseses some neuropils comparable to those found in certain arthropods (Holmgren, 1916, Hanström, 1935, Schürmann, 1987, Schürmann, 1995, Eriksson et al., 2003, Strausfeld et al., in press). One objection that has been traditionally raised to dispute their affinity with arthropods has been that onychophorans differ from them in having an antennal commissure that lies dorsal to the optic nerves and olfactory glomeruli (Fedorow, 1929; discussed by Snodgrass, 1938). However, as will be considered later, the existence of such a commissure has never been revealed using methods that selectively stain axons. Furthermore, as will be described below, the optic nerve divides into two tributaries, one dorsal the other ventral to incoming antennal axons.

If brain organization in the Onychophora reveals affinities with euarthropods, then with which group? Does the onychophoran brain suggest affinities more to the brains of the Tetraconata (Richter, 2002) or to chilopods and diplopods, two groups that possess distinct heads but in other respects show less evidence of tagmosis? Or, as originally suggested by Holmgren (1916) and Hanström (1935) and recently by Strausfeld et al. (2006), might onychophoran brains be most similar to those of the chelicerates, a group that has retained many plesiomorphic characters within the Euarthropoda?

It has been long noted that the central nervous system of the Onychophora appears to be unusual in that segmental ganglia linked by paired ventral nerve cords consisting of bundled axons are absent in this group (Sedgwick, 1887). This situation is certainly unusual amongst euarthropods with the notable exception of embryonic and juvenile Limulus (see Mittmann and Scholtz, 2003). In onychophorans and young xiphosurans, the paired ventrolateral connectives do not form ganglia fused at the midline. Instead, they comprise synaptic neuropil along their lengths and are enlarged at each segment where the two connectives are heterolaterally connected by numerous axonal commissures that lie beneath the gut. A further peculiarity is that the onychophoran brain is surprisingly voluminous for an animal with such a simple body plan. The brain of an adult velvet worm of about 3 centimeters in length may possess many hundreds of thousands of neurons. Another oddity of the onychophoran nervous system is that the vast majority of its neuronal somata are small, intensely basophilic, and almost uniform in size except for occasional giant neuron somata (Schürmann, 1987), a feature also typical of many chelicerate brains (Table 1). However, large cell bodies associated with neuropils of the connectives are presumed to provide motor neurons as well as caudally directed wide-diameter axons peculiar to the ventral nervous system.

Observations by Hanström, 1935, Schürmann, 1987, Schürmann, 1995, and Eriksson et al. (2003) describe the onychophoran brain as composed of heterolateral neuropils that extend across the midline. Certain neuropils are clearly lateralized, however. Schürmann's 1987 account confirms descriptions by Holmgren (1916) and Hanström (1935) of pairs of stalked and lobed neuropils that originate from the exceedingly dense clusters of basophilic perikarya. It has been suggested that these centers are homologous to insect mushroom bodies because these also consist of lobes composed of parallel fibers that arise from small basophilic somata and because they receive connections from glomerular neuropils. In Onychophora, glomeruli have been ascribed chemosensory roles by analogy with glomerular neuropils in the antennal lobes of insects (Schürmann, 1987, Schürmann, 1995). Another component of the onychophoran brain that has been ascribed a specific functional attribute are the optic tracts, which originate from a distinctive layer of neuropil beneath the eye to reach the mushroom bodies and a prominent arch-shaped midline neuropil, as is also seen in some chelicerates. Innervation of the mouth and slime papillae suggests that their neural control resides in the segment from which their nerves originate (Eriksson et al., 2003).

In summary, descriptions of the onychophoran brain have until now identified only a few likenesses with the brains of euarthropods. A recent account of brain development in Euperipatoides kanangrensis suggests affinities with arthropod brain organization, particularly with regard to the circumesophageal nature of developing cephalic neuropil (Eriksson et al., 2003). Such organization is also typical of some annelids (Orrhage and Müller, 2005), insects, and crustaceans (Reichert and Boyan, 1997, Mittmann and Scholtz, 2003). Nor is the presence of mushroom bodies and glomeruli solely indicative of arthropods. Mushroom body-like neuropils are found in the brains of certain Platyhelminthes (Hadenfeldt, 1929, Strausfeld et al., 1995) as well as in polychaete annelids where they are associated with sensory terminals from the palpi and clusters of olfactory glomeruli (Hanström, 1927, Strausfeld et al., 1998). In considering possible affinities with either arthropod or annelid brains, Schürmann (1995) concluded that few similarities could be postulated between the onychophoran brain and the brains of arthropods, and he emphasized the difficulty of identifying discrete segmental elements that might correspond to the three prestomodeal neuromeres of the insect brain. Nevertheless, as shown here, discrete divisions of cerebral neuropils, and their relationships with peripheral nerves, suggest three neuromeres corresponding to the proto-, deuto- and tritocerebral neuromeres of euarthropods, including chelicerates.

The present account describes structures that suggest specific homologies between parts of the onychophoran brain and the tripartite brains of euarthropods. In particular, a layered arch-shaped midline neuropil, and projections from the eyes that reach this neuropil and the mushroom bodies, suggest that the onychophoran brain can be integrated into an euarthropod ground pattern. The innervation of the protocerebrum by the paired frontal appendages, referred to as antennae (Sedgwick, 1922), supports a hypothesized ancestral condition presumed for protoarthropods in which the first segment of the head is assumed to have been equipped with a pair of appendages that innervates the most anterior neuromere of the brain (Budd, 1996, Budd, 2002). In E. rowelli, the location of the eyes, optic nerves, optic centers, and glomeruli all suggest an ancestral condition in which visual and olfactory inputs together supply the protocerebrum.

Section snippets

Materials and methods

Specimens of Euperipatoides rowelli, between 1–3 cm in length, were collected from under the rotting bark of old felled eucalyptus trees in the Talaganda National Forest near Canberra, Australia. For reduced silver staining, animals were cooled and while comatose opened dorsally and then fixed using AAF (85% absolute ethanol, 10% formaldehyde, 5% glacial acetic acid). Brains were later dissected out under 70% ethanol, dehydrated through terpineol, rapidly embedded in Paraplast Plus, and

Histology

The neuropils of velvet worms stain reluctantly with reduced silver methods. Possibly, this is due to their neurons having extremely thin processes that offer few cytoskeletal components that can be coagulated to provide a reducing substrate. Unless the copper concentration is greatly reduced, favoring impregnation of the smallest axons, most Bodian-stained material appears amorphous pink or blue perforated by a syncytium of trachea-like vessels (Figs. 1A,D). Tissue is also resistant to many

Onychophoran neurons

Even without the advantages of Golgi impregnations, Schürmann (1987) correctly suggested that neurons in onychophorans are small and largely undifferentiated into clearly recognizable shapes. This lack of differentiation and the remarkable uniformity of neuronal perikarya in the brain distinguish this nervous system from another group of euarthropods with homonomous segmentation, the Chilopoda. In the centipede brain, neuron cell bodies vary considerably in size and in their staining

Acknowledgements

We thank Drs. Amy Maxmen, Gerhard Scholtz, Steffen Harzsch, and Georg Mayer for their many challenging comments and suggestions. This research was enabled by fellowships to NJS from the Paul Simon Guggenheim Foundation and the John D. and Catherine T. MacArthur Foundation, and a Visiting Research Fellowship from the Australian National University (ANU) enabled by Drs. Mandyam Srinivasan and Jochen Zeil. Preparative work for this study was done in 1995 and 2002 at the ANU's Research School of

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