Topology of the nervous system of Notommata copeus (Rotifera: Monogononta) revealed with anti-FMRFamide, -SCPb, and -serotonin (5-HT) immunohistochemistry

The nervous system of the benthic freshwater rotifer, Notommata copeus, was examined using antibody probes, epifluorescence and confocal laser scanning microscopy, and digital imaging to highlight similarities with other monogonont rotifers. Immunoreactivity to anti-FMRFamide (Phe–Met–Arg–Phe–NH2), -SCPb (small cardioactive peptide b), and -serotonin (5-HT, 5-hydroxytryptamine) was present in the central, peripheral, and stomatogastric nervous system. Specifically, anti-FMRFamide and -SCPb staining was abundant in perikarya and neurites of the cerebral ganglion, ventrolateral nerve cords, and mastax. In addition, a single loop-like neurite was present in between the nerve cords at the posterior end of the body. Serotonergic neurites were also abundant, and highlighted several cerebral pathways that included connections to the nerve cords and possibly the mastax. Novel neural pathways were also present in the posterior trunk region, where serotonergic neurites innervated the foot and lateral body wall. The results presented herein also highlight the utility of 3D visualization software to gain further insights into the organization and architecture of the rotifer cerebral ganglion. Additional key words: rotifer, CLSM, cerebral ganglion Rotifera is a morphologically diverse taxon of aquatic, bilaterally symmetrical micrometazoans typical of lotic and lentic environments. Historically characterized as simple metazoans (Remane 1929–1933; Hyman 1951), rotifers have recently been shown to possess complex anatomies as revealed in the structure of their trophi (S^rensen 2002) and the organization of their muscular systems (Hochberg & Litvaitis 2000; Kotikova et al. 2001, 2004; S^rensen et al. 2003; Santo et al. 2005; S^rensen 2005a,b). Phylogenetic diversity of Rotifera is also more complex than previously thought, with the inclusion of the parasitic Acanthocephala based on molecular sequence and ultrastructural data (reviewed in Funch et al. 2005), and the recent discovery of Micrognathozoa, a group comprising microscopic species with pharyngeal hard parts similar to rotiferan trophi (Funch et al. 2005). Our knowledge of genomic diversity has also increased substantially, especially regarding the bdelloid rotifers (Arkhipova & Meselson 2000; Welch & Meselson 2000). Indeed, as methods for probing these micrometazoans increase in complexity, our knowledge of their anatomy and evolution is bound to improve and dramatically change our impressions about the apparent simplicity of their organization. Within the past two decades, advances in microscopy and histological probes have provided incredible opportunities to study a variety of organ systems that are difficult to visualize using traditional methodologies. In particular, the muscular and nervous systems, historically described using light optics and histological stains, are now being revealed with remarkable clarity using fluorescent probes, novel antibodies, and high-resolution fluorescence microscopy. For example, the use of fluorescent phallotoxin stains to label F-actin has provided new insights into the organization and function of the rotifer muscular system (Hochberg & Litvaitis 2000; Kotikova et al. 2001; S^rensen et al. 2003; Santo et al. 2005; S^rensen 2005a,b) and may eventually provide new characters for phylogenetic reconstruction (S^rensen 2005a). Similarly, histochemical studies of the rotifer nervous system have revealed unique neural topologies (Nogrady & Alai 1983; Raineri 1984; Keshmirian & Nogrady 1987, 1988; Kotikova 1995, 1997, 1998) and novelties in brain organization that may lead to a better understanding of rotifer phylogeny (Kotikova 1998). These early studies of nervous system organization relied almost exclusively on Invertebrate Biology 126(3): 247–256. r 2007, The Authors Journal compilation r 2007, The American Microscopical Society, Inc. DOI: 10.1111/j.1744-7410.2007.00094.x E-mail: [email protected] chemical reactions between fixatives and endogenous neurotransmitters to produce a histochemical fluorescence that was captured with standard epifluorescence microscopy. Nowadays, the availability of antibody probes for neurotransmitters and neural proteins, and the use of high-resolution optics with a computer interface, such as with confocal laser scanning microscopes, has the potential to dramatically increase our knowledge of the rotifer nervous system at both the structural and functional levels of organization (seeKotikova et al. 2005; Hochberg 2006). The aim of the present investigation is to gain new insights into the structure of the nervous system in a common freshwater rotifer, Notommata copeus EHRENBERG 1834. Fluorescence microscopy and antibodies to three neurotransmitters, FMRFamide (Phe–Met–Arg–Phe–NH2), SCPb (small cardioactive peptide b), and 5-hydroxytryptamine (5HT, serotonin), are combined with digital 3D imaging software to describe the topology of the nervous system. These patterns are then compared with the known distribution of neurons in other species of Monogononta in an attempt to discern their systematic value.