On the Serotonergic Nervous System of Two Planktonic Rotifers, Conochilus Coenobasis and C

ARTICLE IN PRESS Zoologischer Anzeiger 245 (2006) 53–62 www.elsevier.de/jcz On the serotonergic nervous system of two planktonic rotifers, Conochilus coenobasis and C. dossuarius (Monogononta, Flosculariacea, Conochilidae) Rick HochbergÃ Department of Biological Sciences, University of Massachusetts, One University Avenue, Lowell, MA 01854, USA Received 21 November 2005; received in revised form 4 April 2006; accepted 10 April 2006 Corresponding editor: M. Schmitt Abstract The serotonergic nervous systems of two non-colonial species of Conochilus were examined to obtain the first immunohistochemical insights into the neuroanatomy of species of Flosculariacea (Rotifera, Monogononta). Species of Conochilus, subgenus Conochiloides, were examined using serotonin (5-HT) immunohistochemistry, epifluorescence and confocal laser scanning microscopy, and 3D computer imaging software. In specimens of C. coenobasis and C. dossuarius, the serotonergic nervous system is defined by a dorsal cerebral ganglion, apically directed cerebral neurites, and paired nerve cords. The cerebral ganglion contains approximately four pairs of small 5-HT- immunoreactive perikarya; one pair innervates the posterior nerve cords and three pairs innervate the apical field. The most dorsal pair innervates a coronal nerve ring that encircles the apical field. Within the apical field is a second nerve ring that outlines the inner border of the coronal cilia. Together, both the inner and outer nerve rings may function to modulate ciliary activity of the corona. The other two pairs of perikarya innervate a region around the mouth. Specific differences in the distribution of serotonergic neurons between species of Conochilus and previously examined ploimate rotifers include the following: (a) a lack of immunoreactivity in the mastax; (b) a greater number of apically directed serotonergic neurites; and (c) a complete innervation of the corona in both species of Conochilus. These differences in nervous system immunohistochemistry are discussed in reference to the phylogeny of the Monogononta. r 2006 Elsevier GmbH. All rights reserved. Keywords: Rotifer; Nervous system; Immunohistochemistry; Serotonin 1. Introduction are solitary animals of the order Ploima, which contribute substantially to the trophic dynamics of Rotifers are some of the smallest free-living animals freshwater communities (Wallace and Snell 2001). in freshwater environments, and yet account for a Ploima is the largest and most diverse monophyletic large proportion of the taxonomic diversity of zoo- assemblage of monogonont rotifers, yet does not plankton in lentic and lotic systems (Nogrady et al. contain any colonial species. All colonial rotifers are 1993; Ricci and Balsamo 2000). Most planktonic rotifers restricted to the order Flosculariacea, a clade defined by the structure of their pharyngeal hardparts, the jaw-like ÃTel.: +1 978 934 2885; fax: +1 978 934 3044. malleoramate trophi (Nogrady et al. 1993). The clade E-mail address: [email protected]. has questionable relations to the Ploima, and to the 0044-5231/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jcz.2006.04.001 ARTICLE IN PRESS 54 R. Hochberg / Zoologischer Anzeiger 245 (2006) 53–62 order Collothecacea with which it is traditionally mind, recent attention has been directed at other organ aligned (Sørensen 2002). systems, namely the muscular system (Hochberg and Within the Flosculariacea are approximately 25 Litvaitis 2000; Kotikova et al. 2001, 2004; Sørensen species of rotifers that form colonies (Wallace 1987). et al. 2003; Santo et al. 2005; Sørensen 2005a, b) and Colony formation is generally described as autorecrui- nervous system (Kotikova 1995, 1997, 1998; Kotikova tive or allorecruitive, depending on whether partheno- et al. 2005), the latter of which generally shows a high genetic embryos develop alongside adults, often within a degree of conservation and may therefore provide clues gelatinous matrix, or settle on adults, arriving from to the origins and evolution of different lifestyles. More conspecifics outside of the colony (Nogrady et al. 1993). specifically, increasing attention has been directed at the Since colonies are essentially aggregations of solitary nervous systems of the most heterogeneous rotifers, the individuals, they lack any anatomical interconnections, ploimates (Kotikova, 1988, 1995, 1997, 1998; Kotikova and so do not share any metabolic demands or trophic et al., 2005). While no ploimates are colonial, their resources. However, the organic matrix that binds a diverse lifestyles and body morphologies provide colony together, whether it be made of a soft gelatinous an index against which to measure neuroanatomical medium or hardened secretions, may serve as protection variation – indeed, conservation within such diversity from predators, and large colony size may itself be a suggests that a Bauplan for the construction of the deterrent (Stemberger and Gilbert 1987; also reviewed in nervous system arose prior to the divergence in lifestyles. Wallace 1987). The number of individuals that make up Whether non-ploimate rotifers possess a fundamentally a colony is highly variable and sometimes species- different Bauplan or simply modifications to a common specific. Species of Lacinularia have been reported to pattern will require greater knowledge of species from comprise colonies of greater than 1000 individuals, while outside the Ploima. some species of Conochilus form colonies of only a few The purpose of the current analysis is to provide the individuals (Wallace 1987). Many of these colonies are first details on the organization of the nervous system in sessile and highly selective of their substratum, but other planktonic species of Conochilus. Specifically, solitary colonies form in the plankton, where they grow species of the subgenus Conochiloides are examined presumably by autorecruitment. There is apparently using anti-serotonin (5-HT) immunohistochemistry, little morphological difference between congeners that epifluorescence and confocal laser scanning microscopy, form sessile and planktonic colonies (Nogrady et al. and 3D imaging software. The distribution of seroto- 1993). nergic neurons is currently known for three species of The planktonic lifestyle is prevalent among species of Ploima (Kotikova et al. 2005) and provides a basis for Conochilidae, but coloniality is found in less than half comparing neural topologies and the innnervation of of the known species. The genus Conochilus contains specific organ systems. both colonial and solitary animals, the former desig- nated to the subgenus Conochilus and the latter to the subgenus Concohiloides. As noted in the cladistic 2. Material and methods analysis of Segers and Wallace (2000), the colonial lifestyle is hypothesized as the derived condition within the Flosculariacea, and probably arose several times 2.1. Collection and identification independently. While the embryos of solitary species Specimens of Conochilus were collected with a 64 mm may develop alongside their parents within a gelatinous plankton net from small lakes at the West Palm Beach matrix, as is present in all species of Conochilidae and Airport, Florida in February and March 2005. Rotifers Flosculariidae, adults generally lead a solitary lifestyle. were anaesthetized in 1% MgCl and photographed To date, our knowledge of the anatomical differences 2 alive as whole mounts on glass slides. Specimens were between solitary and colonial species, or planktonic and benthic species, is generally limited to differences in identified according to the taxonomic atlas of the Frank J. Meyers Rotifera Collection CD (The Academy of optically visible characteristics such as body shape and Natural Sciences). the organization of the corona. However, many such characters are often highly malleable and therefore ambiguous from a phylogenetic standpoint. Alterna- 2.2. Immunohistochemistry tively, pharyngeal hardparts have provided a solid foundation for making phylogenetic inferences because Twenty-three rotifers (Conochilus coenobasis Skorikov, of their conserved structure (Segers and Wallace 2000; 1914, N ¼ 15; C. dossuarius (Hudson, 1885), N ¼ 8) Sørensen 2002). Still, mastax structure alone provides wereanaesthetizedin1%MgCl2 andfixedin4% limited information on the evolution of lifestyle changes, paraformaldehye in 0.1 M PBS for 24 h at 4 1C. although it can presumably provide insights into habitat Specimens were next rinsed (3 Â ) over the course of 12 h partitioning through trophic specialization. With this in in 0.1 M PBS and then placed in IT Signal Enhancer ARTICLE IN PRESS R. Hochberg / Zoologischer Anzeiger 245 (2006) 53–62 55 (Ingenta) for 2 h to minimize non-specific staining. Twenty fluorescence was never detected in any embryos. specimens were transferred to anti-rabbit serotonin anti- Omission of the primary antibody did not produce any body (dilution 1:200, Sigma-Aldrich) in 1.5 ml centrifuge immunoreactivity, although secondary antibody stain- tubes at 4 1Conanorbitalshakerfor48h.Theantibody ing is present in small amounts on the coronal cilia and was diluted with PBT (0.1 M PBS plus 1% Triton X-100). the gel-like matrix that surrounds each individual rotifer Three specimens were used as controls and omitted from (not shown). Some autofluorescence is also present in the primary antibody. Specimens were then rinsed (3 Â )in the gut. The nervous systems of both species are similar 0.1 M PBS over 48 h and transferred to goat anti-rabbit and are therefore described

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