Contributions to Zoology 89 (2020) 324-352 CTOZ brill.com/ctoz From swimming towards sessility in two metamorphoses – the drastic changes in structure and function of the nervous system of the bay barnacle Amphibalanus improvisus (Crustacea, Thecostraca, Cirripedia) during development Paul Kalke Allgemeine & Spezielle Zoologie, Institut für Biowissenschaften, Universität Rostock, 18055 Rostock, Germany Current address: Georg-August-University Göttingen, Johann-Friedrich-Blumenbach Institute for Zoology & Anthropology, Animal Evolution and Biodiversity, Untere Karspuele 2, 37073 Göttingen, Germany Thomas Frase Allgemeine & Spezielle Zoologie, Institut für Biowissenschaften, Universität Rostock, 18055 Rostock, Germany [email protected] Stefan Richter Allgemeine & Spezielle Zoologie, Institut für Biowissenschaften, Universität Rostock, 18055 Rostock, Germany Abstract Knowledge about the development of the nervous system in cirripeds is limited, particularly with regard to the changes that take place during the two metamorphoses their larvae undergo. This study delivers the first detailed description of the development of the nervous system in a cirriped species, Amphibalanus improvisus by using immunohistochemical labeling against acetylated alpha-tubulin, and confocal laser scanning microscopy. The development of the nervous system in the naupliar stages corresponds largely to that in other crustaceans. As development progresses, the protocerebral sensory organs differentiate and the intersegmental nerves forming the complex peripheral nervous system appear, innervating the sensory structures of the cephalic shield. During metamorphosis into a cypris the lateral sides of the cephalic shield fold down into a bilateral carapace, which leads to a reorganization of the peripheral ner- vous system. The syncerebrum of the cypris exhibits the highest degree of complexity of all developmen- tal stages, innervating the frontal filaments, nauplius eye, compound eyes and the antennules. During settlement, when the second metamorphosis occur, the closely associated frontal filaments and © KALKE ET AL., 2020 | doi:10.1163/18759866-bja10003 This is an open access article distributed under the terms of the cc-by 4.0 License. Downloaded from Brill.com10/08/2021 07:29:42PM via free access <UN> From swimming towards sessility 325 compound eyes are shed together with the cuticle of the carapace and the antennules. In adults, the syn- cerebral structures are reduced while the ventral nerve cord and the peripheral nervous system increase in complexity. The peripheral nervous system plays an important role in processing sensory input and also in settlement. In summary, through the larval development we observed a structural and thus also functional increase of complexity in favor of the peripheral nervous system and the ventral nerve cord. Keywords Crustacea – Cirripedia – nervous system – development – metamorphosis – CLSM – immunohistology Introduction Fritsch et al. (2013) distinguished the filamen- tous external frontal filament from the under- The bay barnacle Amphibalanus improvisus lying frontal filament organ. In crustaceans, (Darwin, 1854) is the most common brackish- there are different names for this latter organ water cirriped (Lang, 1979), inhabiting low (e.g., Bellonci organ, x-organ; see Fritsch et al., and subtidal areas in estuaries all over the 2013) but the homology of these structures is world (Dineen & Hines, 1992). As in most generally accepted. Their function is still un- other Thoracica, the life cycle of A. improvisus der debate, but Walker (1974) considered the encompasses six naupliar stages followed by a frontal filaments to have either a mechano- highly specialized cypris larva and subsequent sensory or chemosensory function. The other metamorphosis into a sessile juvenile (Jones major sensory organs are the nauplius eye, & Crisp, 1954; Glenner & Høeg, 1993; Høeg & which becomes transformed and reorganized Møller, 2006; Høeg et al., 2009). The complex in the cypris stage into the ocelli of the adults development of cirripeds is accompanied by a (Takenaka et al., 1993), and the compound reduction, transformation and specialization eyes, which only start to develop in the late of sensory organs and the associated struc- nauplius, are fully developed in the cypris tures of the central nervous system (Doochin, larva and become completely reduced during 1951; Walley, 1969; Høeg, 1987; Høeg & Lützen, metamorphosis into the juvenile (Kauri, 1962; 1995; Høeg et al., 2012; Maruzzo et al., 2012; No- Walker & Lee, 1976). Other external sensory ever et al., 2016). There have been several stud- structures such as the lattice organs, sensory ies to date on the nervous system of cirriped setae, fronto-lateral horn pores and various nauplii (Walley, 1969; Semmler et al., 2008; features of the antennules (Rybakov et al., Ponomarenko, 2014) and cypris larvae (Gallus 2003; Glenner & Høeg, 1995; Walker & Lee, et al., 2005, 2009, 2012; Harrison & Sandeman, 1976; Walker, 1985; Blomsterberg et al., 2004; 1999). Among the sensory organs found in the Brickner & Høeg, 2010) allow A. improvisus cypris and nauplius larvae of barnacles are larvae to settle on a wide variety of substrates the pair of frontal filaments studied by Kauri (Lagersson et al., 2003). (1962, 1966), Walker (1974) and Obukhova et al. The sessility of adult Thecostraca is as- (2015, 2016). Frontal filaments are also found sociated with a drastic reduction of nervous in various crustacean groups including Bran- structures compared with their own vagile chiopoda (Fritsch & Richter, 2010), Remipedia larvae (Høeg et al., 2009). Although some (Fanenbruck et al., 2004), Copepoda (Elofsson, studies focused on the adult nervous sys- 1971) and several Ostracoda (Andersson, 1977). tem have shownDownloaded the changes from Brill.com10/08/2021 that take place 07:29:42PM via free access <UN> 326 KALKE ET AL. in the proto- and tritocerebrum and their from the harbor in Rostock. Larval specimens respective sensory organs and appendages were sampled using a plankton net of 100 μm (compound eyes and antenna) (Gwilliam & mesh. We kept all larval stages of A. improvisus Cole, 1979; Webster, 1998; Callaway & Stuart, in a small aquarium filled with water at room 1999), knowledge about the development of temperature taken from the Warnow River the nervous system in cirripeds is limited. It near the sampling point. All stages were fixed is still not clear how larval morphological in 4% paraformaldehyde (PFA, 16% stock so- features differentiate into the highly derived lution, Electron Microscopy Sciences) solu- morphology of adults, and our understand- tion in 0.1 M PBS (pH 7.4) at room temperature ing of the peripheral nervous system in cirri- before being preserved in 100% methanol. peds and crustaceans in general is particularly To determine species, adults from the quay inadequate. wall (where these barnacles appear in high In the present study, we aimed to describe densities) and specimens that had developed the development of the cirripedian nervous from larvae into adults in the aquarium were system in detail from early nauplius stages to identified using Luther (1987). In addition, the the young adult. By combining immunohis- mitochondrial cytochrome C oxidase subunit tochemical labeling with confocal laser scan- I (COI) of 20 larvae each in the nauplius and ning microscopy and 3D reconstruction, we cypris stages was sequenced using the “uni- are able to visualize the nervous system three- versal” DNA primers LCO 1490 and HCO 2198. dimensionally. We provide insights into the DNA extraction was performed using the in- development of the central nervous system nuPREP Forensic Kit (Analytik Jena). All ex- in nauplius larvae, and into the changes as- amined specimens proved to belong to the sociated with the metamorphoses into cypris species A. improvisus. larvae and later adults, whose nervous system is simultaneously reduced and modified. In Immunolabelling and mounting addition, we describe the remarkable pro- Immunohistochemical labelling was per- cesses of reduction and interconnection that formed as described by Fritsch & Richter take place in the peripheral nervous system (2010). Before antibody staining, larvae and throughout larval development. This study is adults were exposed to several short pulses in intended to complement earlier developmen- a bath ultrasonicator (Elmasonic One) to fa- tal studies on neuroanatomy in Malacostraca cilitate permeation. Further, specimens were (Harzsch, 2003, Vilpoux et al., 2006; Ungerer washed several times in 0.1 mol PBT (phosphat et al., 2011), Branchiopoda (Fritsch & Richter, buffered saline (PBS) with 0.3% Triton X-100, 2012; Fritsch et al., 2013; Frase & Richter, 2016), 1.5% dimethyl sulfoxid, 0.5% bovine serum Cephalocarida (Stegner & Richter, 2011, 2015), albumin) and pre-incubated in PBT contain- Remipedia (Fanenbruck et al., 2004; von Reu- ing normal goat serum (NGS). The primary mont et al., 2012) and Oligostraca (Brenneis & antibody monoclonal mouse anti- acetylated Richter, 2010) and thereby further our under- alpha-tubulin (clone 6 – 11 B-1, Sigma T6793, standing of crustacean evolution. dilution 1:100) in PBT+NGS was applied for two days. Subsequently, specimens were rinsed in PBT and incubated with a secondary Material and methods fluorochrome-conjugated antibody (goat anti- mouse Cy3, Jackson Immunoresearch 155-165- All larval stages of A. improvisus were sampled 003, dilution 1:200) in PBT+NGS for two days. between