Serotonin-immunoreactivity in the ventral nerve cord of Pycnogonida – support for individually identifiable neurons as ancestral feature of the arthropod nervous system

Brenneis and Scholtz BMC Evolutionary Biology (2015) 15:136 DOI 10.1186/s12862-015-0422-1 RESEARCH ARTICLE Open Access Serotonin-immunoreactivity in the ventral nerve cord of Pycnogonida – support for individually identifiable neurons as ancestral feature of the arthropod nervous system Georg Brenneis* and Gerhard Scholtz Abstract Background: The arthropod ventral nerve cord features a comparably low number of serotonin-immunoreactive neurons, occurring in segmentally repeated arrays. In different crustaceans and hexapods, these neurons have been individually identified and even inter-specifically homologized, based on their soma positions and neurite morphologies. Stereotypic sets of serotonin-immunoreactive neurons are also present in myriapods, whereas in the investigated chelicerates segmental neuron clusters with higher and variable cell numbers have been reported. This led to the suggestion that individually identifiable serotonin-immunoreactive neurons are an apomorphic feature of the Mandibulata. To test the validity of this neurophylogenetic hypothesis, we studied serotonin-immunoreactivity in three species of Pycnogonida (sea spiders). This group of marine arthropods is nowadays most plausibly resolved as sister group to all other extant chelicerates, rendering its investigation crucial for a reliable reconstruction of arthropod nervous system evolution. Results: In all three investigated pycnogonids, the ventral walking leg ganglia contain different types of serotoninimmunoreactive neurons, the somata of which occurring mostly singly or in pairs within the ganglionic cortex. Several of these neurons are readily and consistently identifiable due to their stereotypic soma position and characteristic neurite morphology. They can be clearly homologized across different ganglia and different specimens as well as across the three species. Based on these homologous neurons, we reconstruct for their last common ancestor (presumably the pycnogonid stem species) a minimal repertoire of at least seven identified serotonin-immunoreactive neurons per hemiganglion. Beyond that, each studied species features specific pattern variations, which include also some neurons that were not reliably labeled in all specimens. Conclusions: Our results unequivocally demonstrate the presence of individually identifiable serotonin-immunoreactive neurons in the pycnogonid ventral nerve cord. Accordingly, the validity of this neuroanatomical feature as apomorphy of Mandibulata is questioned and we suggest it to be ancestral for arthropods instead. The pronounced disparities between the segmental pattern in pycnogonids and the one of studied euchelicerates call for denser sampling within the latter taxon. By contrast, overall similarities between the pycnogonid and myriapod patterns may be indicative of single cell homologies in these two taxa. This notion awaits further substantiation from future studies. Keywords: Sea spiders, Neuroanatomy, Neurophylogeny, Evolution, 5-hydroxytryptamine, Immunohistochemistry, Pycnogonum litorale, Meridionale, Cilunculus japonicus * Correspondence: Humboldt-Universität zu Berlin, Institut für Biologie/Vergleichende Zoologie, Philippstraße 13, 10115 Berlin, Germany © 2015 Brenneis and Scholtz. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Brenneis and Scholtz BMC Evolutionary Biology (2015) 15:136 Background Over the centuries, a vast number of neuroanatomical studies have yielded considerable details on arthropod nervous system architecture (e.g. [1–4]). Moreover, the last fifteen years have furthered deeper insights into the cellular basis of neurogenesis and its underlying genetic program in many arthropods other than insects and malacostracan crustaceans (e.g. [5–9]). This has allowed assessing similarities and differences between the four major arthropod groups (Chelicerata, Myriapoda, Hexapoda and paraphyletic crustaceans), ranging from the gross anatomical level all the way down to single identified cells. Notably, some of the findings have provided strong arguments in the ongoing debate on their phylogenetic relationships (e.g. [3, 10, 11]). In general, the arthropod central nervous system (CNS) has a basic segmental organization [2], being formed by neuromeres, i.e., segmental units of developing neural tissue [12]. These neuromeres give rise to the adult ganglia, whereby each ‘typical’ segmental ganglion is (among others) characterized by (1) a central neuropilar core surrounded by a cortex of the neural somata, (2) segmental nerves targeting an appendage pair (if present), and (3) commissural tracts between the neuropilar cores of the two body halves. By now, single stereotypic neurons have been individually identified in the ventral nerve cord (VNC) of several arthropod taxa [11, 13–19]. The identification of such stereotypic neurons is most commonly based on soma position and size, neurite morphology and target, use of specific neurotransmitters, developmental origin and gene expression, or sub-sets of these criteria (see [20]). Interestingly, comparisons across arthropods have revealed most similarities between identified neurons of hexapods and crustaceans, especially malacostracans (e.g. [11, 15, 21, 22]), which lends morphological support to the nowadays widely accepted Tetraconata hypothesis [23]. The different neuron types evaluated against a phylogenetic background include the serotonin-immunoreactive neurons of the VNC (e.g. [23–26]). These neurons have been considered promising candidates for ‘homology hunting’ [27], owing to their generally low number per segmental neuromere/ganglion, which facilitates comparisons at the single cell level. Today, serotonin-immunoreactivity has been investigated in the VNC of almost all major arthropod groups, including their close relatives Onychophora [28, 29] and Tardigrada [30, 31]. Notably, the number of serotonin-immunoreactive neurons has been found to be distinctly lower in hexapods and most studied crustaceans, as compared to myriapods and chelicerates. In the first two groups, up to four well-characterized neurons, often arranged in an anterior and a posterior pair, are present per ventral hemi-ganglion [24, 32–38]. In myriapods, on the other hand, between nine and twelve Page 2 of 21 stereotypically arranged segmental somata were detected, yet their neurite morphology remains largely unresolved [25]. Contrasting to this, the studied chelicerates show no indications for singly identifiable serotonin-immunoreactive neurons but instead neuron clusters with often variable and d (...truncated)