A revision of brain composition in Onychophora (velvet worms) suggests that the tritocerebrum evolved in arthropods

Abstract

Background: The composition of the arthropod head is one of the most contentious issues in animal evolution. In particular, controversy surrounds the homology and innervation of segmental cephalic appendages by the brain. Onychophora (velvet worms) play a crucial role in understanding the evolution of the arthropod brain, because they are close relatives of arthropods and have apparently changed little since the Early Cambrian. However, the segmental origins of their brain neuropils and the number of cephalic appendages innervated by the brain--key issues in clarifying brain composition in the last common ancestor of Onychophora and Arthropoda--remain unclear.

Results: Using immunolabelling and neuronal tracing techniques in the developing and adult onychophoran brain, we found that the major brain neuropils arise from only the anterior-most body segment, and that two pairs of segmental appendages are innervated by the brain. The region of the central nervous system corresponding to the arthropod tritocerebrum is not differentiated as part of the onychophoran brain but instead belongs to the ventral nerve cords.

Conclusions: Our results contradict the assumptions of a tripartite (three-segmented) brain in Onychophora and instead confirm the hypothesis of bipartite (two-segmented) brain composition. They suggest that the last common ancestor of Onychophora and Arthropoda possessed a brain consisting of protocerebrum and deutocerebrum whereas the tritocerebrum evolved in arthropods.

Figure 1

Head composition and homology of cephalic appendages in Onychophora and Arthropoda. (A) Ventral view of onychophoran "head" showing three pairs of modified appendages: antennae (at), jaws (jw), and slime papillae (sp). Scanning electron micrograph. Scale bar: 500 μm. (B) Diagram of expression pattern of segment polarity gene engrailed in an onychophoran embryo in lateral view (based on fig. 1a from [25]). Scale bar: 200 μm. Note that there are only three anterior expression domains corresponding to posterior borders of antennal (as), jaw (js), and slime papilla segments (ss), in addition to eight trunk segments (numbered). (C) Alignment and serial homology of anterior appendages in Onychophora and the four major arthropod groups [after [2]]. Note that the onychophoran eyes (black filled circle) may be homologous to the median ocelli [62] rather than to the compound eyes of arthropods (checked ovals), although all these ocular structures belong to the same, anterior-most body segment. Abbreviations: as, antennal segment; at, antenna; at1, first antenna; at2, second antenna; ch, chelicera; jw, jaw; js, jaw segment; le, leg; md, mandible; mx, maxilla; pp, pedipalp; sp, slime papilla; ss, slime papilla segment.

Figure 2

Subdivision of arthropod brain and alternative possibilities for the position of neuronal cell bodies innervating the third pair of cephalic appendages in Onychophora. (A) Position of protocerebral (yellow), deutocerebral (red) and tritocerebral structures (green) in the brain of the fruitfly Drosophila melanogaster [modified from [32]]. (B) A position within the brain of neuronal cell bodies innervating the onychophoran slime papillae, as shown in this diagram, would support the existence of a region of the onychophoran brain equivalent to the arthropod tritocerebrum. (C) A position outside the brain of neuronal cell bodies innervating the onychophoran slime papillae, as shown in this diagram, would speak against the existence of a tritocerebrum in the onychophoran brain. Abbreviations: al, antennal lobe; an, antennal nerve; br, cerebral ganglion or brain; cc, central complex; jn, jaw nerve; lb, labral nerve; mb, mushroom body; nc, nerve cord; oc, ocellar nerve; ol, optic lobe; sn, slime papilla nerves.

Figure 3

Development of major neuropils in the onychophoran brain. Heads of embryos at progressively older developmental stages in dorsal view. Anti-acetylated α-tubulin immunolabelling. Confocal maximum projections (A, B) and depth-coded projections (C, D). Anterior is up. (A) Central neuropil (cn) arises from a single transverse commissure in the antennal segment in an early Euperipatoides rowelli embryo. Scale bar: 200 μm. (B-D) Further stages of brain development in embryos of Epiperipatus isthmicola. Scale bars: 100 μm. A second transverse neuropil (arrowhead in B) and a third neuropil (arrowheads in C and D) arise anterior to the original commissure. Abbreviations: an, antennal nerve; at, antenna; cn, developing central brain neuropil; jw, jaw anlage; np, future nerve cord neuropil; ph, pharynx; sp, slime papilla.

Figure 4

Position of neuronal cell bodies innervating segmental cephalic appendages in the onychophoran Euperipatoides rowelli. Differential staining by retrograde fills with dextran. Confocal projections. Scale bars: 50 μm. (A) Antennal nerve (an) filled with dextran-tetramethylrhodamine. Note the antero-median position of neuronal cell bodies (arrow) within the protocerebral brain region. Anterior is in the upper right corner. (B) Partial projection of the same stack as in A showing that some filled axons of the antennal nerve terminate in the antennal glomeruli (ag). (C) Detail of a jaw nerve (jn) filled with dextran-tetramethylrhodamine. (D) Detail of slime papilla nerves (sn) filled with dextran-fluorescein. Arrowheads indicate the position of neuronal cell bodies in C and D. Abbreviations: ag, antennal glomeruli; an, antennal nerve; ey, eye; jn, jaw nerve; nc, nerve cord; sn, slime papilla nerves.

Figure 5

Position of neuronal cell bodies innervating segmental cephalic appendages in Onychophora and posterior border of the onychophoran brain. (A) Overview of the differential staining of segmental cephalic nerves in Euperipatoides rowelli by retrograde fills with dextran (confocal projection). Jaw nerves (jn) and slime papilla nerves (sn) from both sides of the body were filled with dextran-tetramethylrhodamine (red) and dextran-fluorescein (green). Anterior is up. Scale bar: 200 μm. (B) Diagram summarising the location of neuronal cell bodies innervating the antennae (yellow), jaws (red), and slime papillae (green). Note that the cell bodies of neurons innervating the slime papillae lie outside the brain. (C) Anterior portion of the onychophoran nervous system (reconstruction based on confocal images of an immunolabelled embryo; see Additional file 1, Figure S1). The position of neurons innervating the segmental cephalic appendages (colour coding as in B) is mapped on the reconstructed nervous system. Blue dashed line indicates the posterior brain border behind the cerebral accumulation of neurons. Abbreviations: an, antennal nerve; br, cerebral ganglion or brain; cn, central neuropil; dc, deutocerebrum; ho, hypocerebral organ; jn, jaw nerve; ln, leg nerves; nc, nerve cord; pc, protocerebrum; sn, slime papilla nerves.

Figure 6

Implications of the current findings for the evolution of the arthropod brain. The brain of the last common ancestor of Onychophora and Arthropoda was composed of the protocerebrum and deutocerebrum whereas the tritocerebrum, as part of the brain, evolved in arthropods (chelicerates, myriapods, crustaceans, and hexapods). The phylogenetic position of Myriapoda is unresolved [42,63]. Double lines for Crustacea indicate that this group might not be monophyletic [64].