Received: 14 August 2018 Revised: 19 November 2018 Accepted: 21 November 2018 DOI: 10.1002/cne.24633 RESEARCH ARTICLE Neural system and receptor diversity in the ctenophore Beroe abyssicola Tigran P. Norekian1,2,3 | Leonid L. Moroz1,4 1Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, Florida Abstract 2Friday Harbor Laboratories, University of Although, neurosensory systems might have evolved independently in ctenophores, very little is Washington, Friday Harbor, Washington known about their organization and functions. Most ctenophores are pelagic and deep-water 3Institute of Higher Nervous Activity and species and cannot be bred in the laboratory. Thus, it is not surprising that neuroanatomical data Neurophysiology, Russian Academy of are available for only one genus within the group—Pleurobrachia. Here, using immunohistochem- Sciences, Moscow, Russia istry and scanning electron microscopy, we describe the organization of two distinct neural sub- 4Department of Neuroscience and McKnight Brain Institute, University of Florida, systems (subepithelial and mesogleal) and the structure of different receptor types in the comb Gainesville, Florida jelly Beroe abyssicola—the voracious predator from North Pacific. A complex subepithelial neural Correspondence network of Beroe, with five receptor types, covers the entire body surface and expands deep Dr. Leonid L. Moroz, The Whitney Laboratory, into the pharynx. Three types of mesogleal neurons are comparable to the cydippid Pleurobra- University of Florida, 9505 Ocean Shore chia. The predatory lifestyle of Beroe is supported by the extensive development of ciliated and Boulevard, St. Augustine, Florida 32080, USA. Email: [email protected] muscular structures including the presence of giant muscles and feeding macrocilia. The Funding information obtained cell-type atlas illustrates different examples of lineage-specific innovations within Human Frontier Science Program; National these enigmatic marine animals and reveals the remarkable complexity of sensory and effector Science Foundation, Grant/Award Numbers: systems in this clade of basal Metazoa. 1146575, 1557923, 1548121 and 1645219; National Institutes of Health, Grant/Award Number: R01GM097502; United States KEYWORDS National Aeronautics and Space basal metazoa, behaviors, cell atlas, cilia, ctenophora, development, evolution, feeding, Administration, Grant/Award Number: NASA- NNX13AJ31G macrocilia, mesoglea, Mnemiopsis, muscle system, nervous system, Pleurobrachia, sensory cells, stereocilia 1 | INTRODUCTION et al., 2012). But the field requires broader comparative data including the neuroanatomical mapping of ecologically and morphologically Ctenophores are one of the basal metazoan lineages comprising of diverse ctenophore species such as Beroe (Figure 1). about 200 marine carnivorous species (Hernandez-Nicaise, 1991; The placement of ctenophores within the animal tree of life is still Kozloff, 1990; Tamm, 1982). Most of them are pelagic and deep- highly debated (Dunn, 2017; Dunn, Leys, & Haddock, 2015; King & water organisms and cannot be bred in the laboratory. Thus, it is not Rokas, 2017; Simion et al., 2017; Telford, Moroz, & Halanych, 2016), surprising that neuroanatomical data are available for the only one with recent reconstructions supporting the hypothesis that Cteno- genus within the group—Pleurobrachia (Jager et al., 2011, 2013; Nore- phora is the sister clade to the rest of Metazoa (Arcila et al., 2017; Borowiec, Lee, Chiu, & Plachetzki, 2015; Halanych, Whelan, Kocot, kian & Moroz, 2016, 2019), which genome has been recently Kohn, & Moroz, 2016; Shen, Hittinger, & Rokas, 2017; Telford et al., sequenced (Moroz et al., 2014). This work, together with genome- 2016; Whelan et al., 2017; Whelan, Kocot, Moroz, & Halanych, 2015). scale analyses on cnidarians, sponges, and placozoans, provides Such a scenario means that the extant ctenophores are descendants molecular and phylogenetic evidence that neurons and muscles, as of the earliest animal lineage, which branched before the subsequent well as mesoderm, might evolve more than once (Jorgensen, 2014; divergence of nerveless sponges and placozoans as well as Eumetazoa Moroz, 2009, 2012, 2014; Moroz & Kohn, 2015, 2016; Steinmetz (a clade composed of cnidarians and bilaterians). The proposed rela- tionships among basal metazoans provide additional support for the Abbreviations: RRID:AB_325003, rat monoclonal anti-tubulin antibody (AbD Serotec Cat# MCA77G).; RRID:AB_2534074, goat anti-rat IgG antibodies; Alexa convergent evolution of neurons and synapses (Moroz, 2015; Moroz & Fluor 488 conjugated (Molecular Probes, Invitrogen, Cat# A11006). Kohn, 2016) and the hypothesis that neurons evolved from ancestral 1986 © 2019 Wiley Periodicals, Inc. wileyonlinelibrary.com/journal/cne J Comp Neurol. 2019;527:1986–2008. NOREKIAN AND MOROZ 1987 secretory cells as was summarized earlier (Moroz, 2009; specialized saber-shaped giant cilia (known as macrocilia) help to bite Moroz, 2014). off pieces of prey (Horridge, 1965a; Swanberg, 1974; Tamm, 1983). Molecular clock analyses suggest that the extant ctenophore Adaptations to predation also include specialized epithelial adhe- groups originated ~350 million years ago, followed by their rapid radi- sive mechanisms keeping the mouth reversibly closed without muscu- ation (Whelan et al., 2017). Consequently, numerous innovations lar or neuronal activity (Tamm & Tamm, 1991b). Synapses and within the clade are expected and frequently observed (Dunn et al., neurites were identified by electron microscopy at the end of these 2015; Kohn, Sanford, Yoshida, & Moroz, 2015; Moroz et al., 2014; specialized adhesive cells (Tamm & Tamm, 1991b). But even between Tamm, 2014; Whelan et al., 2017). For example, phylogenomic data two closely related Beroe species, there are substantial differences in recognize at least seven major ctenophore groups with Beroidae as the anatomy when the reversible epithelial adhesive ultrastructure the only lineage, which secondarily lost tentacles: possibly, because of was compared (Tamm & Tamm, 1991b). Additional adaptations are switching to different food sources. Both Beroe and Neis feed on large giant muscles (Bilbaut, Hernandez-Nicaise, Leech, & Meech, 1988), ctenophores (vs. small planktonic organisms as other comb jellies—see large mitochondria with very compact genome (Kohn et al., 2012) and Harbison, Madin, & Swanberg, 1978). bioenergetics, critical for active predation, as well as antimicrobial An active predatory lifestyle (Beroe always swim!) led to a sub- molecules, photoproteins (Burakova et al., 2016; Markova et al., 2012; stantial reorganization of the body plan in Beroidae. In addition to the Stepanyuk et al., 2013); and distinct developmental mechanisms loss of tentacles, there is an extensive development of the gut and com- (Carre, Rouviere, & Sardet, 1991; Carre & Sardet, 1984; Houliston, plex hunting behaviors. Upon contacts with the prey, Beroe quickly Carre, Johnston, & Sardet, 1993; Rouviere, Houliston, Carre, Chang, & reorients its mouth and engulfs the whole prey with gaping lips. The Sardet, 1994, 1998). FIGURE 1 The nontentaculate free swimming ctenophore Beroe abyssicola, Mortensen, 1927. Beroe has a highly muscular body with numerous diverticula of meridional canals (mc)—a part of the gastrovascular system. Beroe constantly swims with the help of eight ciliated comb plates, which show beautiful iridescence under illumination. Combs have one of the largest cilia in the animal kingdom. Beroe can actively search and catch its prey Bolinopsis infundibulum, swallowing them with a very wide mouth (m). Swimming and other behaviors can be controlled by the aboral organ (AO) with a gravity sensor. Beroe can rapidly change the shapes of the body; sizes of animals on the photo are 4–5 cm. See text for further details [Color figure can be viewed at wileyonlinelibrary.com] 1988 NOREKIAN AND MOROZ Despite great interest in Beroe, previous neuroanatomical studies between 1 and 4 cm, while full-grown animals could reach 10–12 cm were focused on distantly related cydippid ctenophores of the genus in length. Experiments were carried out at Friday Harbor Laboratories, Pleurobrachia; which has a passive feeding mechanism by capturing the University of Washington in the spring–summer seasons of small zooplankton with the net of tentacles and tentillae. It is esti- 2012–2018. The details of immunohistochemical and electron micros- mated that Pleurobrachia and Beroe lineages diverged about 250 mil- copy protocols have been described (Norekian & Moroz, 2016, 2019) lion years ago, around the time of the great Permian extinction event and we will provide only a summary. (Whelan et al., 2017). Here, we ask whether neural and receptive sys- tems of Beroe are comparable to Pleurobrachia. 2.2 | Scanning electron microscopy Previous transmission electron microscopy and vital staining with Adult, 0.5–4 cm, Beroe were fixed in 2.5% glutaraldehyde in 0.2 M reduced methylene blue indicated the presence of two nerve nets and phosphate-buffered saline (pH = 7.6) for 4 hr at room temperature characteristic synapses in Beroe (Hernandez-Nicaise, 1973a, 1973b, and washed for 2 hr in 2.5% sodium bicarbonate. Animals were then 1973c, 1991). Using immunohistochemistry and scanning electron dissected into a few smaller pieces. For secondary fixation, we used microscopy, we expand our earlier studies on Pleurobrachia bachei 2% osmium tetroxide in 1.25% sodium bicarbonate for 3 hr at room (Norekian