G Model JCZ-25514; No. of Pages 9 ARTICLE IN PRESS Zoologischer Anzeiger xxx (2017) xxx–xxx Contents lists available at ScienceDirect Zoologischer Anzeiger jou rnal homepage: www.elsevier.com/locate/jcz A tiny visual system — retinula axons and visual neuropils of Neobisium carcinoides (Hermann, 1804) (Chelicerata, Arachnida, Pseudoscorpiones) a,b,∗ a,b,c Tobias Lehmann , Roland R. Melzer a Bavarian State Collection of Zoology – SNSB, Münchhausenstraße 21, 81247 Munich, Germany b Ludwig-Maximilians-Universität München, Department Biologie II, Großhaderner Straße 2, 82152 Planegg-Martinsried, Germany c LMU GeoBio-Center , Richard-Wagner-Straße 10, 80333 Munich, Germany a r t i c l e i n f o a b s t r a c t Article history: Only a few studies have examined the visual system of Pseudoscorpiones until now. To fill this knowledge Received 26 September 2017 gap we analysed the axonal trajectories and neuropil architecture of the visual system of the pseudoscor- Received in revised form pion Neobisium carcinoides (Hermann, 1804) with different neuroanatomical techniques. The R-cell axon 28 November 2017 terminals were identified with Cobalt fills and the morphology of the visual neuropils and the protocere- Accepted 28 November 2017 brum generally is described by means of the osmium-ethyl gallate procedure and TEM. N. carcinoides has Available online xxx two lateral eyes on each side of the prosoma. The R-cells of the eyes per hemisphere are linked to a first and a second visual neuropil, located in the dorsolateral protocerebrum. The first visual neuropil is sub- Keywords: Chelicerata divided into two hemineuropils, one for each lateral eye, while the second neuropil is not. Furthermore, − − Pseudoscorpiones the two-layered arcuate body is found isolated from the visual neuropils in the midline of the proto- Visual system cerebrum at a dorsoposterior position. These findings allow a detailed comparison of the pseudoscorpion Neuropils visual system with that of other previously studied taxa and shed new light on lateral eye evolution in Central projections Chelicerata. Phylogeny © 2017 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction was usually considered as superficial and without phylogenetic implications. However, Shultz (1990) proposed that Scorpiones are Pseudoscorpions, also known as false scorpions or book scor- closely related to Pseudoscorpiones and Solifugae and Sharma et al. pions, are very common but seldomly noticed soil animals. They (2014) favour a placement of Pseudoscorpiones within or sister to are typical representatives of the soil fauna and are found in leaf Arachnopulmonata (Scorpiones + Araneae + Amblypygi + Uropygi). litter, soil, moss, debris, or under bark. More than 3700 pseudoscor- Due to their hidden lifestyle preferentially in dark environ- pion species have been described so far (Harvey, 2013). The oldest ments, there is no doubt that their main sense organs are sensory known fossil pseudoscorpion dates back 380 million years to the setae, especially the trichobothria on the pedipalps (Weygoldt, Devonian period (Shear et al., 1989). 1969). Hence, the visual sense plays a minor role and is charac- The phylogenetic relationships of Pseudoscorpiones are unre- terized by structural and functional simplicity seen as reductions solved. A sister taxon relationship with Solifugae was frequently (Miether and Dunlop, 2016; Weygoldt, 1969). As the only repre- proposed (Shultz, 1990; Weygoldt, 1998; Weygoldt and Paulus, sentatives of the Chelicerata − with the exception of the eyeless 1979a; Weygoldt and Paulus, 1979b). Some molecular studies, Palpigradi and Ricinulei (however, lateral eyespots are present in however, showed a phylogenetic proximity with the parasitiform extant ricinuleids and some fossil ricinuleids retained two distinct branch of the diphyletic mites (Giribet et al., 2002; Regier et al., pairs of lateral eye lenses) (Miether and Dunlop, 2016) − pseu- 2010). The resemblance with scorpions − due to their common doscorpions lack median eyes, only lateral eyes are found dorsally and probably convergent possession of large chelate pedipalps − on the anterolateral corner of the carapace. There may be four, two, rudimentary or no lateral eyes at all (Roewer, 1940). These eyes are composed of a convex-concave dioptric apparatus com- posed of a lens and a hypodermis (Demoll, 1917; Roewer, 1940; ∗ Corresponding author at: Bavarian State Collection of Zoology – SNSB, Münch- Scheuring, 1913). Proximal to the preretinal membrane the ret- hausenstraße 21, 81247 Munich, Germany. inula cells are found, these were described as inverse or inverted, E-mail address: [email protected] (T. Lehmann). https://doi.org/10.1016/j.jcz.2017.11.014 0044-5231/© 2017 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/). Please cite this article in press as: Lehmann, T., Melzer, R.R., A tiny visual system — retinula axons and visual neuropils of Neobisium carcinoides (Hermann, 1804) (Chelicerata, Arachnida, Pseudoscorpiones). Zool. Anz. (2017), https://doi.org/10.1016/j.jcz.2017.11.014 G Model JCZ-25514; No. of Pages 9 ARTICLE IN PRESS 2 T. Lehmann, R.R. Melzer / Zoologischer Anzeiger xxx (2017) xxx–xxx so that the rhabdomeres are located proximally and the nuclei dis- 100% ethanol, 0.5 g gum arabic, and 0.02 g hydroquinone; pH value tally to the incoming light (Scheuring, 1913). However, Scheuring adjusted to between 2.6 and 3.1 using citric acid), and 20–30 min ◦ (1913) and Demoll (1917) described in pseudoscorpions an atypi- at 50 C in the dark in solution B (10 ml H2Odest, 3 ml 100% ethanol, cal situation for inverted eyes, as the axon leaves the retinula cell 0.5 g gum arabic, 0.02 g hydroquinone, 0.01 g AgNO3; pH value medially at the level of the nucleus. In genuine inverted eyes, the adjusted to between 2.6 and 3.1 using citric acid). Silver intensi- axon leaves the eyecup distally. Below the retinula cells, a tapetum fication was stopped in an acetic acid solution (50 ml 30% ethanol, is present in pseudoscorpions, which reflects the incoming light and 5 g glucose, pH value adjusted to between 2.6 and 3.1 using acetic increases the light sensitivity. It has been suggested that these eyes acid). After dehydration in a graded acetone series, the specimens can primarily detect light intensity and direction of the light source, were embedded in Glycidether 100, and sectioned with a rotary rather than generating a sharp image (Demoll, 1917; Roewer, 1940; microtome and stainless steel blade in the sagittal, frontal, and Weygoldt, 1969). transversal planes (14 ␮m). Finally, sections were silver intensified The development of pseudoscorpions and of their nervous in solution A and B for a second time. system was studied by Weygoldt (1964a), Weygoldt (1964b), Weygoldt (1965), Weygoldt (1968), and Weygoldt (1971). The 2.3. Wigglesworth technique (Osmium-ethyl-gallate procedure) visual neuropils of pseudoscorpions have been analysed in two studies in the past. Hanström (1928) reported one small visual Modified after Leise and Mulloney (1986), Mizunami et al. neuropil (“Sehmasse”) in the dorsolateral protocerebrum. Fur- (1997), and Wigglesworth (1957): Specimens were dissected and thermore, he described the arcuate body (“Zentralkörper”) in the ◦ fixed in 4% glutardialdehyde in 0.1 M cacodylate buffer at 4 C. After posterodorsal midline of the protocerebrum, but without any state- ◦ postfixation in 2% OsO4 in 0.1 M cacodylate buffer (3 h at 4 C) ment, whether or not the arcuate body is connected with the visual ◦ animals were stained for 20 h at 4 C in a saturated ethyl gallate neuropil. A somewhat more detailed description was provided by solution, dehydrated in a graded acetone series, embedded in Gly- Boissin and Cazal (1969). These authors also described one small cidether 100, and sectioned with a rotary microtome and stainless visual neuropil (“glomerules ocellaires”) in the same region as steel blade in the sagittal and transversal planes (7–9 ␮m). Hanström (1928), which is connected via a tract with the arcu- ate body (“corps central”). The arcuate body is characterised as arch-shaped and consisting of two layers, one small posteriodorsal 2.4. TEM layer and one larger anterioventral layer, which is connected to the visual neuropil. However, these putative visual neuropils had not After dissection, the specimens were fixed in 4% glutardialde- ◦ been identified using neuroanatomical markers or tracers, but with hyde in 0.1 M cacodylate buffer at 4 C. After postfixation in 2% OsO4 ◦ classical histology alone. in 0.1 M cacodylate buffer (3 h at 4 C), the specimens were dehy- Visual systems in other chelicerate taxa studied so far in detail drated in a graded acetone series and embedded in Glycidether 100. and with modern tracer techniques are: Xiphosura (Battelle, 2006; Ultra-thin sections of 70–100 nm thickness were made with a dia- Battelle et al., 2016; Calman et al., 1991; Chamberlain and Barlow, mond knife on an RMC-MTXL ultramicrotome. The sections were 1980), Araneae (Strausfeld and Barth, 1993; Strausfeld et al., 1993), stained with uranyl acetate and lead citrate and inspected in an FEI Pycnogonida (Lehmann et al., 2012; Lehmann et al., 2014), Scorpi- Morgagni transmission EM at 80 kV. ones (Lehmann and Melzer, 2013), and Opiliones (Lehmann et al., 2016a). 2.5. 3D-reconstruction In order to allow for comparisons with these modern studies, we analysed in the present work the number, form, connectivity The specimen (prepared as for Osmium ethyl gallate proce- and general morphology of the visual neuropils of pseudoscor- dure) was cut into a complete transversal series (8 ␮m). Slices pions, and located the target neuropils of the axon terminals of were mounted on glass slides, covered with cover slips, and pho- the retinula cells.