The visual system of harvestmen (Opiliones, Arachnida, Chelicerata) - a re-examination

Abstract

Background: The visual systems in chelicerates are poorly understood, even though they show strong variation in eye and visual neuropil architecture, thus may provide valuable insights for the understanding of chelicerate phylogeny and eye evolution. Comparable morphological characters are desperately sought for reconstructions of the phylogeny of Chelicerata, especially with respect to Arachnida. So far, reliable data exist only for Pycnogonida, Xiphosura, Scorpiones, and Araneae. The few earlier studies of the organisation of the visual system in harvestmen are contradictory concerning the number, morphology, and position of the visual neuropils.

Results: We undertook a descriptive and comparative analysis of the neuroanatomy of the visual system in several phalangid harvestmen species. Various traditional and modern methods were used that allow comparisons with previous results (cobalt fills, DiI/DiO labelling, osmium ethyl gallate procedure, and TEM). The R-cells (photoreceptor and arhabdomeric cells) in the eyes of Opiliones are linked to a first and a second visual neuropil. The first visual neuropil receives input from all R-cell axons, in the second only few R-cells terminate in the distal part. Hence, the second visual neuropil is subdivided in a part with direct R-cell input and a part without. The arcuate body is located in a subsequent position with direct contact to the second visual neuropil.

Conclusions: This re-examination comes to conclusions different from those of all previous studies. The visual system of phalangid Opiliones occupies an intermediate position between Pycnogonida, Xiphosura, and Scorpiones on the one side, and Araneae on the other side. The projection of the R-cells is similar to that in the former grouping, the general neuropil arrangement to that in the latter taxon. However, more research on the visual systems in other chelicerate orders is needed in order to draw inferences on phylogeny or eye evolution.

Keywords: Arachnida; Central projections; Chelicerata; Opiliones; Phylogeny; Visual system.

Fig. 1

Cobalt fills via both eyes of Leiobunum spec. (a-c) and Opilio canestrinii (d-g), transversal sections, dorsal is up. a right eye nerve and right first visual neuropil densely filled with cobalt, left nerve and neuropil less filled. Bar 100 μm. b three sections after A, in right hemisphere Cobalt-filled retinula axons terminate in first visual neuropil and in dorsal part of second visual neuropil, in second neuropil fewer fibres filled. Bar 100 μm. c detail of right hemisphere in B with border between both neuropils and Cobalt-filled retinula axons in dorsal part of second neuropil. Bar 50 μm. d Cobalt-filled retinula axons terminating via eye nerve in first and second visual neuropil. Bar 100 μm. e detail of right hemisphere in C with border between both neuropils and Cobalt-filled retinula axons in dorsal part of second neuropil. Bar 50 μm. f Cobalt-filled retinula axons with varicosities terminating in first and second visual neuropil. Bar 100 μm. g detail of left hemisphere in F with border between both neuropils and Cobalt-filled retinula axons with varicosities in dorsal part of second neuropil. Bar 50 μm. EN, eye nerve; M, median eye visual neuropil; NB, neuropil border

Fig. 2

Cobalt fills via both eyes of Opilio canestrinii (a-c, sagittal sections, dorsal is up) and Leiobunum spec. (d-g, frontal sections, anterior is up). a eye tubercle with eye and Cobalt-filled eye nerve, first and second visual neuropil. Bar 200 μm. b detail of first and second visual neuropil in A, second neuropil with fewer Cobalt-filled axons. Note eye nerve separating in several bundles after entering brain (arrow). Bar 50 μm. c one section after B, first and second visual neuropil with Cobalt-filled retinula axons, second neuropil with fewer Cobalt-filled axons. Bar 50 μm. d several Cobalt-filled eye nerve bundles projecting from eye to brain. Bar 50 μm. e five sections after D, eye nerve bundles fuse to one eye nerve. Bar 50 μm. f first visual neuropils packed with Cobalt-filled retinula axons. Bar 50 μm. g five sections after F, in right hemisphere first visual neuropil packed with Cobalt-filled retinula axons and second visual neuropil with few Cobalt-filled retinula axons. Bar 50 μm. EN, eye nerve; ENB, eye nerve bundles; EYE, eye; M, median eye visual neuropil; NB, neuropil border

Fig. 3

DiO (a, b, d, and e) and DiI (c) labelling via both eyes of Opilio canestrinii. (a-c, fluorescence microscope; d, e, CLSM; dorsal is up). a DiO labelled eye, eye nerve bundles, eye nerve, and visual neuropils; no distinction between first and second neuropil possible. Bar 200 μm. b DiO labelled eye nerve bundles, eye nerve, and visual neuropils; no distinction between first and second neuropil possible. Bar 200 μm. c DiI labelled eye nerve bundles, eye nerve, and visual neuropils; no distinction between first and second neuropil possible. Bar 200 μm. d DiO labelled eye nerve, and visual neuropils; no distinction between first and second neuropil possible. DAPI labelled cell bodies in green. Same specimen as in A. Bar 100 μm. e DiO labelled eye nerve, first, and second visual neuropil. Neuropil border between first and second neuropil visible (arrowheads), in second visual neuropil fewer DiO. Same specimen as in B. Bar 100 μm. EN, eye nerve; ENB, eye nerve bundles; EYE, eye; M, median eye visual neuropil

Fig. 4

General anatomy of protocerebrum and visual neuropils (Richardson (Platybunus pinetorum) and Wigglesworth stains (Leiobunum spec.), dorsal is up). a transversal section showing eye nerve, first and second visual neuropil with border in between (arrowhead). Bar 100 μm. b sagittal section showing eye nerve, first and second visual neuropil with border in between (arrowhead); note eye nerve separating in several bundles after entering brain (arrow). Bar 100 μm. c transversal section with eye nerve, first visual neuropil and on right hemisphere beginning of second visual neuropil; note second neuropil darker stained. Bar 100 μm. d seven sections after C; first and second visual neuropil with border in between (arrowhead). Bar 100 μm. e 16 sections after D; arcuate body in dorso-posterior position; note arcuate body darker stained. Bar 100 μm. f sagittal section with first visual neuropil in anterodorsal position and arcuate body in dorso-posterior position; note arcuate body darker stained. Bar 100 μm. g four sections after F; first and second visual neuropil in anterodorsal position with border in between (arrowhead) and arcuate body in dorso-posterior position in close vicinity to second visual neuropil; note second visual neuropil and arcuate body darker stained. Bar 100 μm. h three sections after G; second visual neuropil and arcuate body contact each other. Bar 100 μm. AB, arcuate body; EN, eye nerve; M, median eye visual neuropil

Fig. 5

3D serial reconstruction of eye and visual neuropils. Eye reconstructed on basis of semithin sections of Rilaena triangularis and visual neuropils on basis of Wigglesworth stains of Leiobunum spec.. a eye composed of lens (grey), vitreous body (dark blue), and retina (dark green); note several nerve bundles (orange) exit the eye and project to protocerebrum with retained relative positions representing subsets of retinula cells. b, c dorso-lateral and lateral view showing arrangement of neuropils; orange, eye nerve; light blue, first visual neuropil; purple, neuropil border; red, second visual neuropil; light green, arcuate body. AB, arcuate body; EN, eye nerve; ENB, eye nerve bundles; LE, lens; M, median eye visual neuropil; NB, neuropil border; OE, oesophagus; RE, retina; VB, vitreous body

Fig. 6

Transmission electron microscopy of Platybunus pinetorum of region where eye nerve enters brain and first visual neuropil (a, c, e, and g sagittal sections; b, d, f, transversal sections; dorsal is up). a eye nerve separates into several bundles after entering brain; with cell bodies between bundles (stitched image series). Bar 10 μm. b, c detail of eye nerve before entering the brain; note pattern of arrangement of retinula axons. Bars 10 μm and 5 μm, respectively. d, e detail of region where eye nerve separates into several nerve bundles with cell bodies in between; note no synapses in this region. Bars 10 μm and 5 μm, respectively. e, f detail of region where eye nerve bundles enter first visual neuropil. Bars 10 μm and 5 μm, respectively. EN, eye nerve; ENB, eye nerve bundles; M, median eye visual neuropil

Fig. 7

Transmission electron microscopy of first and second visual neuropil of Platybunus pinetorum (a, c, and f sagittal sections; b, d, e, g, and H transversal sections; dorsal is up). a, b showing arrangement of retinula axons (bright cells with high electron density) and dendrites of visual second order neurons (dark cells with low electron density) in first visual neuropil. Bars 5 μm and 10 μm, respectively. c detail of first visual neuropil with synapses (arrowheads) between retinula axons and visual second order neurons. Bar 2 μm. d, e transition area between first and second visual neuropil with neuropil border; note first and second neuropil with different anatomy and neuron gestalten; note several retinula axons traverse neuropil border (arrows). Bars 10 μm. f, g and h detail of transition area between first and second visual neuropil with various retinula axons traversing neuropil border (arrows). Bars 10 μm, 5 μm, and 5 μm, respectively. M, median eye visual neuropil; NB, neuropil border

Fig. 8

Comparison of the results of this study (a) with other taxa (b, c) and with previous studies (d–f). a Opiliones, this study, note region, that in previous studies (d–f) is described as M1, is indeed eye nerve and M1 lies deeper in the protocerebrum; b Scorpiones (Euscorpius italicus, E. hadzii; after Lehmann & Melzer [15]); c Araneae (Cupiennius salei; after Strausfeld et al. [22]); d Opiliones, after Saint Remy [25]; e Opiliones, after Hanström [29], note M2 subdivided into three layers (layer C, B, and C, see text) by Hanström; f Opiliones, after Breidbach & Wegerhoff [30], note M2 subdivided into two layers (internal and external, see text) by Breidbach & Wegerhoff. EN, eye nerve; L, lateral eye visual neuropil; LEN, lateral eye nerve; M, median eye visual neuropil; M/L2, region where M2 and L2 overlap; MEN, median eye nerve