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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

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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. We used various neuroanatomical techniques

tographed under a conventional light microscope. Images were

(Cobalt fills, the Osmium ethyl gallate procedure, TEM, and AMIRA

contrast-enhanced in Adobe Photoshop, then aligned, segmented

3D-reconstruction) to examine the pseudoscorpion Neobisium car-

and rendered in Amira.

cinoides (Hermann, 1804). This species has two pairs of eyes, which

are − in comparison to most other pseudoscorpions − well devel-

oped (Scheuring, 1913). 3. Results

3.1. Cobalt fills

2. Material & methods

Cobalt fills via the anterior and posterior lateral eyes of Neobi-

2.1. Specimen collection

sium carcinoides revealed a visual neuropil complex as the target

region of the retinula axons (Fig. 1 ). The visual neuropil complex

Specimens of Neobisium carcinoides (Hermann, 1804) were col-

is located in the dorsolateral protocerebrum in each brain hemi-

lected in Munich (Angerlohe, München-Untermenzing) between

sphere. Dorsally and laterally, it is embedded in the cell body cortex

October and November 2016.

and ventrally in contact with the neuropil of the protocerebrum.

The dimensions of the visual neuropil complex are about 40 ␮m x

2.2. Cobalt fills 30 ␮m x 30 ␮m. The pathway of the eye nerve could not be traced

by means of the Cobalt fills. The R-cell terminals form synaptic

Modified after Altman and Tyrer (1980): CoCl2 crystals were varicosities all over their extension within the neuropil complex.

inserted into the eyes with a fine tungsten needle. After diffusion When cobalt was filled via the anterior lateral eye only, the lower

times between 2 and 4 h, cobalt was precipitated with a solution area of the visual neuropil complex is stained (Fig. 1A, B). Con-

of five drops of (NH4)2S in 10 ml H2Odest. After fixation of the versely, when filled via the posterior lateral eye only, the upper

cephalothorax in AAF (85 ml 100% ethanol, 10 ml 37% formalde- area of the visual neuropil complex is stained (Fig. 1C–F). Finally,

hyde, 5 ml glacial acetic acid), the specimen was silver intensified: when cobalt was filled via the anterior and posterior lateral eyes

◦

60 min at 50 C in the dark in solution A (10 ml H2Odest, 3 ml simultaneously, the entire neuropil complex is stained (Fig. 1G–J).

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

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Fig. 1. Cobalt fills via anterior, posterior, or both eyes of Neobisium carcinoides. Bars 25 ␮m. (A, B) Cobalt fills via the anterior lateral eye, two consecutive sagittal sections. First

and second lateral eye visual neuropil, located in dorsolateral protocerebrum, partly stained; viz. lower area of first visual neuropil stained only. (C, D) Cobalt fills via the poste-

rior lateral eye, two consecutive sagittal sections. First and second visual neuropil, located in dorsolateral protocerebrum, partly stained; viz. upper area of first visual neuropil

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

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However, a division into an upper and lower part is still visible one R-cell terminal is presynaptic to several second order neurons

(Fig. 1J). (Fig. 4D, E). However, synapses are also found frequently between

second order neurons (Fig. 4E).

3.2. Wigglesworth technique (osmium-ethyl-gallate procedure)

4. Discussion

By means of the Wigglesworth technique, the eye nerve within

the protocerebrum could be traced. It enters the protocerebrum 4.1. Comparison with previous studies

dorso-anteriorly in the middle of each hemisphere (Fig. 2B). The

visual neuropil complex, unequivocally identified with Cobalt fills, Hanström (1928) and Boissin and Cazal (1969) were the only

can also be recognised with osmium-ethyl-gallate staining, as dark- authors who studied the visual system of pseudoscorpions so far.

stained areas, as is typical for dense neuropils such as sensory They described one visual neuropil (Sehmasse or glomerules ocel-

neuropils (Fig. 2A, C, D). laires) in a dorsolateral position of the protocerebrum − at the

However, here a division into a first and second visual neu- corresponding position as in this study. However, a visual system

ropil becomes apparent. The neuropil complex is subdivided into a composed of only a single visual neuropil would be unexpected,

darker stained neuropil anteriorly, i.e. a first visual neuropil, and a since in all Chelicerata that have been studied in some detail there

brighter stained neuropil posteriorly, i.e. a second visual neuropil are always two neuropils, a first and a second one − in both

(Fig. 2A, C, D). In the first visual neuropil, as in the Cobalt fills, a the lateral and median eye visual systems (Calman et al., 1991;

subdivision into an upper and lower part, i.e. two hemineuropils, Chamberlain and Barlow, 1980; Lehmann et al., 2012; Lehmann

is also visible. In the second visual neuropil, this subdivision is not et al., 2016a; Lehmann and Melzer, 2013; Strausfeld and Barth,

evident (Fig. 2A, D). 1993; Strausfeld et al., 1993). The presence of only one neuropil

Furthermore, we identified the arcuate body, which is located in in pseudoscorpions, contrary to all other previously studied che-

the protocerebrum in a dorsoposterior position (Fig. 2E–H). It is of licerates, would therefore cast doubt on the relevance of such

arcuate shape and slightly bent ventrally. Transversal and sagittal studies for evolutionary considerations. However, this study comes

sections show that it is composed of two layers, a thinner dorsal to a different conclusion concerning the number and structure of

one and a thicker ventral one. In this study, we found no evidence the visual neuropils, i.e. that there are indeed the ‘expected’ two.

of a direct connection from the visual neuropil to the arcuate body. It will be our task below to show that this conclusion is not a

However, a tract that extends below the visual neuropils points in self-fulfilling prophecy, but a fact. On first impressions, the visual

the direction of the arcuate body. neuropil complex looks like one neuropil, especially when studying

conventional semithin sections. Even in the Cobalt fills, one can-

3.3. TEM not distinguish the tangential subdivision into a first and a second

neuropil, and only the radial subdivision into two hemineuropils is

Transmission electron microscopy confirmed the division into a visible. One reason for this is certainly the small size of the whole

first and a second visual neuropil (Fig. 3). The first visual neuropil neuropil complex with a diameter of only about 40 ␮m. However,

is characterised by a high number and density of large dark profiles the Wigglesworth technique and especially TEM revealed the full

with high electron density, i.e. retinula cell terminals, and a low differentiation of the neuropil complex. In Wigglesworth sections,

number and density of small bright profiles with low electron den- this area is subdivided into an anterior, dark and a posterior, bright

sity, i.e. dendrites of visual second order neurons. Furthermore, the stained region, representing the first and the second visual neu-

first visual neuropil is subdivided into two hemineuropils (Fig. 3A, ropil. In TEM sections, one can also distinguish between an anterior

C). Each hemineuropil is composed of 20–30 R-cell profiles (Fig. 3C). region predominantly with profiles with high electron density (i.e.

Between the large R-cell terminals is a network of the small den- R-cell terminals) and a posterior region predominantly with pro-

drites of second order neurons (Fig. 4A). In the first visual neuropil, files with low electron density (i.e. dendrites of visual second order

synapses are found regularly (Fig. 4B, C). Usually, the R-cell ter- neurons). Even the distribution of the synapses is different in the

minals are presynaptic to the second order neurons. However, two regions. In the anterior region, fewer synapses are found. R-

sometimes there are also synapses between R-cell terminals. cells are presynaptic to one or few visual second order neurons

Posterior to the first visual neuropil is directly adjacent the sec- and sometimes to other R-cells. In the posterior region, many more

ond visual neuropil. Genuine chiasmatic fibres between the first synapses are found. R-cells are also presynaptic to visual second

and second neuropil were not observed. The second visual neu- order neurons, but here one R-cell is connected to several visual

ropil is characterised by a high number and density of small bright second order neurons, a situation not found in the first visual neu-

profiles with low electron density, i.e. dendrites of visual second ropil. Additionally, synapses are found between visual second order

order neurons, and a low number and density of large dark pro- (or even higher order) neurons.

files with high electron density, i.e. retinula cell terminals. In the Hence, one can either consider these regions as one neuropil,

second visual neuropil, a subdivision into two hemineuropils is not as Hanström and Boissin et al. did, or as two neuropils. Due to

evident. We observed that only a fraction of the large profiles repre- the different morphology of the two regions, we concluded that

senting the retinula axons extend into the second visual neuropil. there are two visual neuropils − a first and a second visual neu-

Hence, it seems that there are short fibres with terminals in the ropil. TEM shows that both neuropils are characterised by different

first visual neuropil only and long fibres with terminals in both the cell types and synaptic connectivity. The first visual neuropil is

first and second visual neuropil. In the second visual neuropil, these mainly composed of R-cell terminals and the second visual neuropil

R-cell terminals are surrounded by several small second order neu- of dendrites of visual second order neurons. A reason why these

rons. Here synapses are found in great numbers (Fig. 4D, E). Usually, neuropils are not separated more obviously, may be the reduction

stained only. (E, F) Cobalt fills via the posterior lateral eye, two consecutive frontal sections. Upper area of first visual neuropil stained only; viz. unstained anterior eye

hemineuropil of first visual neuropil below stained posterior eye hemineuropil of first visual neuropil. (G, H) Cobalt fills via both lateral eyes, sagittal section. Entire first

and second lateral eye visual neuropils cobalt filled. (I) Overdyed cobalt fills via both lateral eyes, showing entire first and second visual neuropils stained (sagittal section).

(J) Cobalt fills via both lateral eyes, showing both, anterior/lower and posterior/upper eye hemineuropils of first visual neuropil stained (transversal section). L1 first lateral

eye visual neuropil, L1a first lateral eye visual neuropil − anterior eye hemineuropil, L1p first lateral eye visual neuropil − posterior eye hemineuropil, L2 second lateral eye

visual neuropil.

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

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Fig. 2. General anatomy of visual neuropils and protocerebrum of Neobisium carcinoides (Wigglesworth stains). Bars 25 ␮m. (A) Sagittal section of lateral eye visual neuropils.

First visual neuropil darker stained and subdivided into upper and lower hemineuropil, second visual neuropil brighter stained and no subdivision visible. (B) Eye nerve

(arrow) enters protocerebrum dorso-anteriorly in middle of each brain hemisphere. Asterisk indicates where visual neuropils appear two sections further posterior. (C,

D) Two consecutive transversal sections of first and second lateral eye visual neuropil (different specimen than in C and D). In first visual neuropil a subdivision visible

(arrowheads). (E) Transversal section of dorsoposterior protocerebrum, showing arcuate body composed of two layers. (F) Detail of two-layered arcuate body (transversal

section, different section than in E). (G, H) Sagittal sections of the arcuate body also showing two layers; G rather parasagittal plane, H rather midsagittal plane. AB arcuate

body, L1a first lateral eye visual neuropil − anterior eye hemineuropil, L1p first lateral eye visual neuropil − posterior eye hemineuropil, L2 second lateral eye visual neuropil.

of the visual system in N. carcinoides and in Pseudoscorpiones in for the anterior and one for the posterior eye. The two-layered

general (Weygoldt, 1969). One feature so far not described is the structure and the position of the arcuate body are in accordance

subdivision of the first visual neuropil in two hemineuropils, one with previous studies (Boissin and Cazal, 1969; Hanström, 1928;

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

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Fig. 3. Transmission electron microscopy of first and second lateral eye visual neuropil of Neobisium carcinoides. First visual neuropil characterised by high density of retinula

axons (dark profiles with high electron density), second visual neuropil characterised by high density of dendrites of visual second order neurons (bright profiles with low

electron density). Bars 5 ␮m. (A) Sagittal section of first and second lateral eye visual neuropil. In first visual neuropil division into anterior and posterior eye hemineuropil

clearly visible, in second visual neuropil no division recognisable. (B) Frontal section of first and second visual (hemi)neuropil of anterior lateral eye. (C) Transversal section of

first and second visual neuropil. In first visual neuropil division into anterior and posterior eye hemineuropil visible. 27 individual R-Cell profiles in anterior eye hemineuropil

labelled with red asterisk. L1a first lateral eye visual neuropil − anterior eye hemineuropil, L1p first lateral eye visual neuropil − posterior eye hemineuropil, L2 second lateral

eye visual neuropil.

Weygoldt, 1964a). However, a connection between the visual neu- be observed. Fig. 5 shows a summary of the visual system of the

ropils and the arcuate body could not be found. Furthermore, a lateral eyes in Neobisium carcinoides.

chiasma between the first and second visual neuropil could not

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

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Fig. 4. Detail of first (A-C) and second (D, E) lateral eye neuropil. Bars 1 ␮m. (A) First visual neuropil characterised by high density of retinula axons (dark profiles with high

electron density) surrounded by a net of dendrites of visual second order neurons (bright profiles with low electron density). (B, C) Synapses (arrowheads) between retinula

axons (presynaptic) and visual second order neurons (postsynaptic) in the first visual neuropil. (D) Synapses (arrowheads) between retinula axons (presynaptic) and visual

second order neurons (postsynaptic) in the second visual neuropil. Note: Often multiple second order neurons postsynaptic to one retinula axon. (E) Synapses between

retinula axons (presynaptic) and visual second order neurons (postsynaptic) (arrowheads) and between visual second order neurons (arrows) in the second visual neuropil.

4.2. Comparison with other arachnids Strausfeld and Barth, 1993). The neuropil arrangement, with two

successive visual neuropils in the lateral protocerebrum, is simi-

−

Chelicerates have two classes of eyes median and lateral eyes lar in these chelicerate clades (see above). Concerning the R-cell

(Paulus, 1979; Weygoldt and Paulus, 1979a). In Pseudoscorpiones, projections, two different configurations can be distinguished.

a taxon with only one of the two eye classes, the question must Xiphosura (lateral rudimentary eyes), Scorpiones, and Pseudoscor-

be asked, if these eyes are median or lateral eyes? The position piones on the one side have R-cell axon terminals in both the first

and structure of the eyes favour the classification as lateral eyes and the second visual neuropil, without crossing fibres in between

(Miether and Dunlop, 2016; Weygoldt and Paulus, 1979a). This (see below). Xiphosura (lateral compound eyes) and Araneae on

study comes to the same result. The position of the visual neu- the other side have R-cell axon terminals in the first visual neuropil

ropils is in the dorsolateral protocerebrum, median eye neuropils only. In Xiphosura the eccentric cells of the lateral compound eyes

are usually found in a more median position (see summary in project to first and second lateral eye visual eye neuropils (with

Lehmann et al. (2016b)). As in other chelicerates (Xiphosura, Scor- crossing fibres in between, see below) and also to second and first

piones, Araneae) the first and second visual neuropil of the lateral median eye visual neuropils.

eyes are successive and in close vicinity, while median eye visual The presence or absence of a chiasma between the first (lamina)

neuropils are sometimes further away from each other (Battelle, and the second (medulla) visual neuropil has been used in analyses

2006; Calman et al., 1991; Chamberlain and Barlow, 1980; Lehmann of phylogenetic relationships among arthropods (Harzsch, 2002;

et al., 2012; Lehmann and Melzer, 2013; Strausfeld and Barth, 1993; Harzsch et al., 2006; Strausfeld, 2005). In larval Xiphosura and

Strausfeld et al., 1993). Furthermore, the median eye visual system in all other studied chelicerate taxa, i.e. Pycnogonida, Scorpiones,

in chelicerates is closely linked with the arcuate body (Homberg, Opiliones and Pseudoscorpiones, no chiasma is present (Lehmann

2008; Loesel et al., 2002); in Neobisium, we found no evidence for et al., 2012; Lehmann et al., 2016a; Lehmann and Melzer, 2013).

such a link (Fig. 5A, B). So far, in chelicerates only in Araneae and adult Xiphosura a chi-

Other lateral eye visual systems in Chelicerata, which can asma opticum was described (Battelle, 2006; Strausfeld and Barth,

be compared to the findings here, were examined in Xipho- 1993). In Araneae, a chiasma between the second lateral eye visual

sura, Scorpiones, and Araneae only (Battelle, 2006; Calman et al., neuropil and the mushroom bodies − hence, in a different position

−

1991; Chamberlain and Barlow, 1980; Lehmann and Melzer, 2013; was reported by Strausfeld et al. (1993). In larval Xiphosura most

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

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JCZ-25514; No. of Pages 9 ARTICLE IN PRESS

8 T. Lehmann, R.R. Melzer / Zoologischer Anzeiger xxx (2017) xxx–xxx

Fig. 5. Comparison of lateral eye visual systems in (A-C) Pseudoscorpiones (Neobisium carcinoides), (D) Xiphosura (Limulus polyphemus), (E) Scorpiones (Euscorpius spp.,

Androctonus australis), and (F) Araneae (Cupiennius salei). Dorsal view, except (B) lateral view. (A, B) 3D serial reconstruction of the visual system of Neobisium carcinoides

based on Wigglesworth stain sections. (C) Retinula cells of lateral eyes terminate in first and second lateral eye visual neuropil. (D) left: terminals of the lateral rudimentary

eye have branches in first and second lateral eye visual neuropil; right: lateral compound eye photoreceptor cells terminate in first lateral eye visual neuropil only, eccentric

cells innervate first and second lateral visual eye neuropil, and continue to second and first median eye visual neuropil. After Calman et al. (1991), Chamberlain and Barlow

(1980), and Lehmann and Melzer (2013). (E) Retinula cells of lateral eyes terminate in first and second lateral visual eye neuropil. After Lehmann and Melzer (2013) (F)

Retinula cells of lateral eyes terminate in first lateral eye visual neuropil only. After Strausfeld and Barth (1993). EN eye nerve, LEN lateral eye nerve, L1 first lateral eye visual

neuropil, L2 second lateral eye visual neuropil, MEN median eye nerve, M1 first median eye visual neuropil, M2 second median eye visual neuropil, M/L2 region were M1 and

L1 overlap (in Scorpiones).

of the fibres that project from the first to the second visual neuropil lateral eyes were originally (semi-)compound, and that reduction

are axons of the rudimentary photoreceptors and these fibres run to only a handful of lenses is a homoplastic character state. Further-

parallel (Harzsch et al., 2006). In adult Xiphosura the axons of the more, Miether and Dunlop (2016) discussed, given the presence of

eccentric cells cross each other from the first to the second lateral rudimentary lateral eyes in Limulus (Fahrenbach, 1970) and in scor-

eye visual neuropil and form the adult chiasma. As in larvae, the pions (Spreitzer and Melzer, 2003), that these rudimentary eyes

axons from the lateral rudimentary eye in adults run parallel and were also present in the earliest arachnids, but have been largely

do not cross each other (Battelle, 2006; Harzsch et al., 2006). Hence, lost in the non-scorpion orders. However, since ultrastructural data

another similarity between the lateral rudimentary eyes of Xipho- on the eyes are lacking, we do not know for sure whether they are

sura and the lateral eyes of Pseudoscorpiones and Scorpiones is the present or absent in pseudoscorpions.

absence of a chiasma. Our results indicate that the lateral eye evolution is even more

complex. However, it is too early to solve this issue. At present,

there are two possibilities: the lateral eyes of Pseudoscorpiones

4.3. Evolutionary implications

could be offshoots of lateral compound eyes as Miether and Dunlop

(2016) stated or of lateral rudimentary eyes. Due to the similarities

Lately, Miether and Dunlop (2016) reviewed lateral eye evolu-

of the neuroanatomy and innervation pattern in lateral rudimen-

tion in arachnids. Accordingly, fossil data indicate that the arachnid

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

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T. Lehmann, R.R. Melzer / Zoologischer Anzeiger xxx (2017) xxx–xxx 9

−

tary eyes in Xiphosura and the lateral eyes in Pseudoscorpiones and Lehmann, T., Heß, M., Melzer, R.R., 2012. Wiring a periscope ocelli, retinula

axons, visual neuropils and the ancestrality of sea spiders. PLoS One 7.

Scorpiones, our data so far favour the latter hypothesis.

Lehmann, T., Heß, M., Wanner, G., Melzer, R.R., 2014. Dissecting a neuron network:

Hence, the lateral rudimentary eyes − instead of the lateral com-

FIB-SEM-based 3D-reconstruction of the visual neuropils in the sea spider

−

pound eyes become a focus of interest in the lateral eye evolution Achelia langi (Dohrn, 1881) (Pycnogonida). BMC Biol. 12, 59.

Lehmann, T., Lodde-Bensch, E., Melzer, R.R., Metz, M., 2016a. The visual system of

of arachnids. Little is known about their function, development,

harvestmen (Opiliones, Arachnida, Chelicerata) − a re-examination. Front.

and evolution. These eyes lack a dioptric apparatus, screening pig-

Zool. 13, 50.

ment and distinct rhabdom architecture, features that are found in Lehmann, T., Melzer, R.R., Hörnig, M.K., Michalik, P., Sombke, A., Harzsch, S., 2016b.

Arachnida (excluding Scorpiones). In: Schmidt-Rhaesa, A., Harzsch, S.,

pseudoscorpion eyes. One interpretation of the rudimentary eyes

Purschke, G. (Eds.), Structure and Evolution of Invertebrate Nervous Systems.

is that − similar to larval eyes in holometabolous insects − dur-

Oxford University Press, Oxford, pp. 453–477.

ing development this eye is the first part of the compound eye Leise, E.M., Mulloney, B., 1986. The osmium-Ethyl gallate procedure is superior to

and degenerates later (Melzer, 2009; Paulus, 1979; Weygoldt and silver impregnations for mapping neuronal pathways. Brain Res. 367, 265–272.

Loesel, R., Nässel, D.R., Strausfeld, N.J., 2002. Common design in a unique midline

Paulus, 1979a). This indicates that the lateral rudimentary eye is the

neuropil in the brains of arthropods. Arthropod Struct. Dev. 31, 77–91.

evolutionarily older part and the lateral compound eyes of Limulus

Melzer, R.R., 2009. Persisting stemma neuropils in Chaoborus crystallinus (Diptera:

(and probably of other fossil arachnid compound eyes) are already Chaoboridae): development and evolution of a bipartite visual system. J.

Morphol. 270, 1524–1530.

a derived condition (Weygoldt and Paulus, 1979a) and the lateral

Miether, S.T., Dunlop, J.A., 2016. Lateral eye evolution in the arachnids.

eyes in pseudoscorpions would be a different derived character

Arachnology 17, 103–119.

state compared to these rudimentary eyes. Mizunami, M., Iwasaki, M., Nishikawa, M., Okada, R., 1997. Modular structures in

the mushroom body of the cockroach. Neurosci. Lett. 229, 153–156.

Paulus, H.F., 1979. Eye structure and the monophyly of the Arthropoda. In: Gupta,

Acknowledgements

A.P. (Ed.), Arthropod Phylogeny. Van Nostrand Reinhold Company, New York,

pp. 299–383.

Regier, J.C., Shultz, J.W., Zwick, A., Hussey, A., Ball, B., Wetzer, R., Martin, J.W.,

This publication is part of the “Special Issue in honour of Peter

Cunningham, C.W., 2010. Arthropod relationships revealed by phylogenomic

Weygoldt”. Peter Weygoldt published various studies on mor-

analysis of nuclear protein-coding sequences. Nature 463, 1079–1083.

phology, ontogeny, phylogeny, taxonomy, and general biology of Roewer, C.F., 1940. Chelonethi oder Pseudoskorpione Leipzig.

Scheuring, L., 1913. Die Augen Der Arachnoideen. I. Zoologische Jahrbücher, 33.

Arachnida. His great impact is gratefully acknowledged here. Spe-

Abteilung für Anatomie und Ontogenie der Tiere, pp. 553–636.

cial thanks come from RRM for Weygoldt’s inspiring teaching

Sharma, P.P., Kaluziak, S.T., Pérez-Porro, A.R., González, V.L., Hormiga, G., Wheeler,

at the Zoological Institute in Freiburg. We thank the Deutsche W.C., Giribet, G., 2014. Phylogenomic interrogation of Arachnida reveals

systemic conflicts in phylogenetic signal. Mol. Biol. Evol. 31, 2963–2984.

Forschungsgemeinschaft for financial support (DFG LE3575/2-1)

Shear, W.A., Schawaller, W., Bonamo, P.M., 1989. Record of palaeozoic

and Dr. Jörg Spelda (ZSM) for help with species determination.

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Shultz, J.W., 1990. Evolutionary morphology and phylogeny of Arachnida.

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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