Transition from marine to terrestrial ecologies: Changes in olfactory and tritocerebral neuropils in land-living isopods

Arthropod Structure & Development 40 (2011) 244e257

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Transition from marine to terrestrial ecologies: Changes in olfactory and tritocerebral neuropils in land-living isopods S. Harzsch a, b, *, V. Rieger a, b, J. Krieger a, F. Seefluth a, N.J. Strausfeld c, B.S. Hansson b a

Universität Greifswald, Fachbereich Biologie, Abteilung Cytologie und Evolutionsbiologie, J.-S.-Bach Strasse 11/12, D-17498 Greifswald, Germany Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, Beutenberg Campus, Hans-Knöll-Str. 8, D-07745 Jena, Germany c Department of Neuroscience and Center for Insect Science, University of Arizona, Tucson, AZ 85721, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 August 2010 Accepted 21 March 2011

In addition to the ancestors of insects, representatives of five lineages of crustaceans have colonized land. Whereas insects have evolved sensilla that are specialized to allow the detection of airborne odors and have evolved olfactory sensory neurons that recognize specific airborne ligands, there is so far little evidence for aerial olfaction in terrestrial crustaceans. Here we ask the question whether terrestrial Isopoda have evolved the neuronal substrate for the problem of detecting far-field airborne chemicals. We show that conquest of land of Isopoda has been accompanied by a radical diminution of their first antennae and a concomitant loss of their deutocerebral olfactory lobes and olfactory computational networks. In terrestrial isopods, but not their marine cousins, tritocerebral neuropils serving the second antenna have evolved radical modifications. These include a complete loss of the malacostracan pattern of somatotopic representation, the evolution in some species of amorphous lobes and in others lobes equipped with microglomeruli, and yet in others the evolution of partitioned neuropils that suggest modality-specific segregation of second antenna inputs. Evidence suggests that Isopoda have evolved, and are in the process of evolving, several novel solutions to chemical perception on land and in air. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Neurophylogeny Isopoda Brain Synapsin Olfactory system Antenna Evolution Amira 3D reconstruction Immunohistochemistry

1. Introduction Within Crustacea, representatives of five major lineages have succeeded in the transition from an aquatic to a terrestrial life style (reviews Bliss and Mantel, 1968; Powers and Bliss, 1983; Hartnoll, 1988; Greenaway, 1988, 1999). These include representatives of Isopoda (Edney, 1968; Wägele, 1989; Schmalfuss, 2003), Amphipoda (Hurley, 1968; Friend and Richardson, 1986; Morritt and Spicer, 1998), Anomura (Hartnoll, 1988; Greenaway, 2003), Brachyura (Hartnoll, 1988) and Astacoidea (Welch and Eversole, 2006). Although three of these lineages require aquatic larval stages, many species of Isopoda and Amphipoda do not, and hence are fully terrestrial. The degree to which Crustacea have partially or fully adapted to terrestrial life has been categorized into fives classes, T1 to T5 (Powers and Bliss, 1983; Hartnoll, 1988; Greenaway, 1999), T5 characterized as “a fully terrestrial species able to conduct all biological activities on land".

* Corresponding author. Universität Greifswald, Fachbereich Biologie, Abteilung Cytologie und Evolutionsbiologie, J.-S.-Bach Strasse 11/12, D-17498 Greifswald, Germany. Tel.: þ 49 3834 864124. E-mail address: [email protected] (S. Harzsch). 1467-8039/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2011.03.002

Amongst Crustacea, the most successful organisms in colonizing land are without any doubt members of the Oniscidea, a subgroup of the Isopoda (Powers and Bliss, 1983; Wägele, 1989; Kaestner, 1993; Schmalfuss, 2003; Schmidt, 2008). Oniscidea comprise around 3600 species (Schmalfuss, 2003), of which many have fully established the T5 grade of terrestrial life style according to Powers’ and Bliss’ grading of terrestrialness. The phylogeny of the Isopoda is a topic of ongoing research (e.g. Wägele, 1989; Wägele et al., 2003; Wetzer, 2002; Wirkner and Richter, 2003, 2007, 2008, 2010; Schmidt, 2008; Raupach et al., 2009; Richter et al., 2009; Wirkner, 2009), and it is still unclear if the Oniscidea invaded land several times or just once (Schmidt, 2008). Representatives that epitomize an ongoing transition from sea to land can be found amongst Ligiidae (e.g. Ligia oceanica, Ligia exotica, Ligia occidentalis). Members of this taxon lead an amphibious life style on rocky shores, guided by a well-developed visual system (Sinakevitch et al., 2003), and are frequently associated with washed-up sea wrack on which they feed. Depending on the species, these animals can move much faster on land than submerged (L. exotica and L. occidentalis, but not L. oceanica), although they walk in both habitats (Wägele, 1989). Fully terrestrial (T5) isopods are represented by, for example, Oniscidae (e.g. Oniscus asellus), Porcellionidae (e.g. Porcellio scaber), Armadillidiidae (e.g. Armadillidium

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Fig. 1. A: dorsal view of the marine hermit crab Pagurus bernhardus to show its cephalic sensory appendages, the first and second pair of antennae. B: higher magnification of the boxed area in A showing the aesthetascs on the tips of the first antennae. C, D: dorsal views of the head of the marine isopod Idotea baltica. E: Aesthetascs (arrows) on the distal segment of the first antennae of I. baltica. F: apical organ at the distal end of the second antennae in I. baltica. G, H: frontal views of the head of the desert isopod Hemilepistus reaumuri. I: higher magnification of the left first antenna shown in H. J: apical organ at the distal segment of the second antenna of H. reaumuri. Abbreviations: A1 antenna one, A2 antenna 2, AES aesthetascs, AO apical organ, CE compound eyes.

vulgare) and Trachelipodidae (e.g. the xerophilic desert isopod Hemilepistus reaumuri). The successful transition from marine to terrestrial life requires a number of physiological adaptations, all of which are important for survival out of water. These relate, for example, to gas exchange, salt

and water balance, nitrogenous excretion, thermoregulation, molting, and reproduction (Bliss and Mantel,1968; Powers and Bliss,1983; Burggren and McMahon, 1988; Greenaway, 1988, 1999, 2003). Aspects of the physiological ecology of Isopoda and those morphological adaptations related to land invasion are reviewed by Edney

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Fig. 2. Pagurus bernhardus (A, B) and Carcinus maenas (C, D), immunolocalization of RFamide-like neuropeptides (green) on vibratome sections of the brain counterstained with a probe for f-actin (red) and a nuclear marker (blue; A and B are optical sections with the Zeiss Apotome structured illumination device). A: P. bernhardus, right olfactory lobe (OL; anterior is toward the top) with olfactory glomeruli (OG) and antenna 2 neuropil (ANn). Inset: higher magnification of olfactory glomeruli to show their regionalization. B: P. bernhardus, transverse segmentation (arrows) of the antenna 2 neuropil. C: C. maenas, horizontal section showing an overview over the brain. D: C. maenas, higher magnification of the right olfactory lobe to show the radial arrangement of the cone-shaped glomeruli. Abbreviations: ANn antenna 2 neuropil, B base region, C cap, OG olfactory glomeruli, OL olfactory lobe, SC subcap, 9/11 cell cluster 9/11 that houses the somata of local olfactory interneurons.

(1968), Wägele (1989), Greenaway (1999), and Schmidt (2008). Isopoda can be somewhat arbitrarily arranged in an ascending order of terrestrial adaptation e Ligia - Oniscus - Porcellio - Armadillidium e Hemilepistus e to suggest the acquisition of evolutionary modifications required to meet the physiological challenge of the terrestrial habitat. Such adaptations include increasingly better protection against loss of water by transpiration, improving the capacity for osmoregulation and oxygen uptake, as well as acquiring a greater reproductive independence from water by highly modified brood care (Warburg, 1968; Powers and Bliss, 1983; Kaestner, 1993). With regard to the nervous system, the sensory organs of terrestrial species must be able to function in air rather than in water or be able to function in both if there is an aquatic phase of life (Hansson et al., 2011). For example, there is evidence that certain terrestrial and semiterrestrial crustaceans possess as good visual orientation abilities on land as in water. The visual system of fiddler crabs (Brachyura), for example, is remarkable for its tuning to master the visual world of inter-tidal mud flats (reviews e.g. Layne et al., 1997; Zeil and Hemmi, 2006). Furthermore, terrestrial anomurans (hermit crabs) have efficient aerial olfaction mediated by modified aesthetascs (Ghiradella et al., 1968; Greenaway, 2003; Stensmyr et al., 2005; Harzsch and Hansson, 2008; reviewed in: Hansson et al., 2011). Crustacea are equipped with two pairs of cephalic sensory appendages referred to as “antennae”. These are distinguished by the first pair, which is associated with the brain’s deutocerebrum.

The second “antennae” are associated with the tritocerebrum. A further distinction is that in Malacostraca the first antennal pair (termed the antennules or antennae 1) is equipped with olfactory sensilla (aesthetascs), whereas the second pair is not, although it may be equipped with bimodal chemo- and mechanosensilla (review Hallberg and Skog, 2010). The marine hermit crab Pagurus bernhardus (Fig. 1A) is shown here to represent the antennae of a decapod crustacean: the rather short pair of first antennae ("antennules") are laterally flanked by the much longer and more caudal second antennae. The tips of the first antennae bear arrays of aesthetascs that house the profusely branched dendrites of olfactory sensory neurons (Fig. 1B; see Ghiradella et al., 1968; also reviews by Hallberg et al., 1992; Hallberg and Hansson, 1999; Mellon, 2007; Hallberg and Skog, 2010; Schmidt and Mellon, 2010). The first antennae of marine representatives of the Isopoda such as Asellus aquaticus (Asellota) or Idotea baltica and Saduria entomon (Valvifera) are likewise equipped with these malacostracan chemosensory sensilla (aesthetascs probably are part of the malacostracan ground pattern; compare Hallberg and Skog, 2010), although fewer in numbers than in Decapoda (Guse, 1983; Heimann, 1984; Pynnönen, 1985). Fig. 1C and D show portraits of I. baltica to illustrate the distinct pair of first antennae, which are located medially relative to the pair of large mechanosensory second antennae, and whose tips are decorated with a few slender aesthetascs (arrows in Fig. 1E). However, within the terrestrial members of the Oniscoidea, the first

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Fig. 3. Idotea baltica, immunolocalization of RFamide-like neuropeptides (green) and synapsins (red; confocal laser-scan microscopy) on brain whole mounts. A: low power view of the brain, the dashed line indicates the midline. Z identifies an unidentified neuropil. B: higher magnification of the olfactory lobe showing spherical glomeruli (projection of five 0.95 mm optical sections). Inset: higher magnification of two olfactory glomeruli showing colocalization of both markers. C1-4: dorsal to ventral series of single optical sections (0.76 mm thickness), at 14.4, 19.8, 39.5 and 48.6 mm (stack covers 53 mm, 0 defined as dorsal). D: black-white inverted confocal image of RFamide-like immunorectivity. Arrows identify somata of labeled neurons. Double arrow identifies the entry point of the antennal nerve into the olfactory lobe. E: higher magnification of boxed area in D. F: segmentation of the antenna 2 neuropil as indicated by RFamide-like immunoreactive neurites (arrows; black-white inverted confocal image of RFamide-like immunoreactivity), projection of five optical sections of 0.95 mm thickness. Abbreviations: AnN antenna 2 neuropil, A1Nv antenna 1 nerve, CB central body, E esophageal foramen, LA lamina, LPC lateral protocerebrum, ME medulla, OG olfactory glomeruli, OL olfactory lobe, Z unidentified neuropil.

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antennae are greatly reduced in size and the second antennae are thought to function as major sensory organs (review Schmalfuss, 1998). This diminution of the antennules is shown in Fig. 1G and H featuring the xerophilic desert isopod H. reaumuri. In this species, the paired second antennae are its dominate cephalic sensory appendages, whereas the first antennae are minute and are located medially and rostrally above the base of the second antennae, as occurs in other terrestrial isopods (Hoese, 1989; Schmalfuss, 1998). Haug and Altner (1984) have shown that in the woodlouse P. scaber, the tips of the first antennae are equipped with ca.15-20 peg sensilla, which the authors suggest function as hygroreceptors. The first antennae of H. reaumuri have a similar morphological appearance (Fig. 1I). Nevertheless, Schmalfuss (1998) does not exclude that the first antennae of terrestrial isopods may also have a chemoreceptive function as they do in marine species. In marine isopods, all the segments of the second antennae bear numerous mechanoreceptive sensilla (Hoese, 1989). The terminal segment of the second antennae, bears a tuft of robust sensilla, as seen e.g. in I. baltica, (Fig. 1F), which Hoese called the “apical organ”. This organ is also present in terrestrial isopods where it has a characteristic apical cone-like shape and is interpreted as detecting both mechanical and chemical stimuli (Mead et al., 1976; Seelinger, 1977; Alexander, 1977; Hoese, 1989; review, Schmalfuss, 1998). In the desert isopod H. reaumuri, the apical organ (Fig. 1J) has been suggested to respond to both olfactory and gustatory stimuli (Seelinger, 1977, 1983). Although responses to distant olfactory stimuli have not yet been behaviorally demonstrated in this species (Seelinger, 1983), their apical organs are likely to play a key role in the perception of volatiles involved in social recognition, family cohesion and communication (review, Linsenmair, 2007). The general structure of the brains of representatives of the Isopoda is described by Gräber (1933), Walker (1935) and Hanström (1947). Classical morphological descriptions exist for the brains of two fully terrestrial members of the Oniscoidea, Armadillidium vulgare (Schmitz, 1989) and H. reaumuri (Kacem-Lachkar, 2000). Immunohistochemical studies on the isopod brain include the localization of neuropeptides in O. asellus (Nussbaum and Dircksen, 1995) and of serotonin in the same species and, in lesser detail, in three other isopods (Thompson et al., 1994). However, little information is available on the central olfactory system especially in marine species of isopods, such as Valvifera. With regards to Oniscidea, preliminary evidence suggests that their primary olfactory centers in the deutocerebrum, the olfactory lobes, are reduced in size as an adapatation to terrestrial life (Walker, 1935; Schmitz, 1989; Kacem-Lachkar, 2000). The present account examines this possible adaptation in more detail, using the brains of four isopod species including marine and terrestrial ones. In order to resolve the structure of the central olfactory pathway in marine versus terrestrial isopods we have used immunostaining against synaptic proteins and the neuropeptide RFamide. For comparison, we also include two representatives of the Decapoda.

Animals were anaesthetized for at least 1 h on ice before their brains were dissected under phosphate buffered saline (0.1M PBS, pH 7.4). Isolated brains were fixed overnight in 4% PFA in 0.1M PBS at 4  C. After fixation, tissues were washed for 4 h in several changes of PBS. The brains of P. bernhardus and C. maenas were subsequently sectioned (80 mm) with a HM 650 V vibratome (Microm) whereas the brains of Isopoda were processed as whole mounts. Overnight permeabilization in PBTx (0.3% Tx-100 in 0.1 M PBS, pH 7.4) at 4  C was followed by overnight incubation in primary antibodies at 4  C. The antisera used were: polyclonal rabbit anti-FMRFamide (1:1000; DiaSorin, Cat. No. 20091, Lot No. 923602); and monoclonal mouse anti-synapsin “SYNORF1" antibody (1:30 in PBS-TX; Klagges et al., 1996; antibody kindly provided by E. Buchner, Universität Würzburg, Germany). After incubation in the primary antisera, tissues were washed in several changes of PBS for 4 h at room temperature and incubated in secondary Alexa Fluor 488 or Alexa Fluor 546 IgGs (1:50, Invitrogen, Eugene, Oregon, USA) overnight at 4  C. All sections were routinely counterstained with the nuclear dye bisbenzimide (0.1%, Hoechst H 33258) for 15 min at room temperature. Some sections were processed with a high-affinity probe for actin by adding Phallotoxin conjugated to Alexa Fluor 546 (Molecular Probes; concentration 200 units/ml) to the secondary antibody in a dilution 1:50. Finally, tissues were washed for at least 2 h in several changes of PBS and mounted in GelMount (Sigma). The localization of synapsin and actin as general markers of neuropils was chosen to provide a good overview over the general brain architecture and as robust methods that are well suited to study crustacean neuroanatomy (e.g. Harzsch and Hansson, 2008). Specifically, synapsin immunohistochemistry will reliably label synaptic neuropil, which is a characteristic for primary olfactory centers and therefore is a good probe to detect the absence or presence of certain neuropils. Immunohistochemistry against RFamidelike peptides labels subsets of neurons and allows the visualization of their neurites. These markers have been applied in a wide range of crustaceans thereby allowing interspecific comparisons. Specimens were viewed with a Zeiss AxioImager equipped with the Zeiss Apotome structured illumination device for optical sectioning ("grid projection"; http://www.zeiss.de/apotome). Digital images were processed with the Zeiss AxioVision software package. In addition, specimens were analyzed with the laser scanning microscope Zeiss LSM 510 Meta. Double-labeled specimens were generally analyzed in the multi-track mode in which the two lasers operate sequentially, and narrow band-pass filters were used to assure a clean separation of the labels and to avoid any crosstalk between the channels. All images were processed in Adobe Photoshop using global picture enhancement features (brightness/contrast).

2. Materials and methods

2.3. Specificity of the antisera

2.1. Animals

The tetrapeptide FMRFamide and FMRFamide-related peptides (FaRPs) are widely distributed among invertebrates and vertebrates and form a large neuropeptide family with more than fifty members, all of which share the RFamide motif (reviews e.g.: Price and Greenberg, 1989; Greenberg and Price, 1992; Nässel, 1993; Homberg, 1994; Dockray, 2004; Nässel and Homberg, 2006; Zajac and Mollereau, 2006). In malacostracan Crustacea, at least twelve FaRPs have been identified and sequenced from crabs, shrimps, lobsters and crayfish (Mercier et al., 2003; Huybrechts et al., 2003). These peptides range from seven to twelve amino acids in length and most of them share the carboxy terminal sequence LRFamide.

The marine hermit crab P. bernhardus (Decapoda, Reptantia, Anomura), the marine “green crab” Carcinus maenas (Decapoda, Reptantia, Brachyura) and the marine isopod I. baltica (Peracarida, Isopoda, Valvifera) were obtained from the Biologische Anstalt Helgoland, Germany (http://www.awi.de/de/institut/allgemeine_ dienste/biologischer_materialversand/). The terrestrial isopods P. scaber and A. vulgare (Peracarida, Isopoda, Oniscidea) were collected in and around the greenhouses of the Max Planck Institute for Chemical Ecology in Jena, Germany. The desert isopod

H. reaumuri (Peracarida, Isopoda, Oniscidea) was kindly collected by Dr. M. Knaden (Jena) in a saltpan close to Menzel Chaker, Tunisia. 2.2. Immunohistochemistry

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The antiserum we used was generated in rabbit against synthetic FMRFamide (Phe-Met-Arg-Phe-NH2) conjugated to bovine thyroglobulin (DiaSorin, Cat. No. 20091, Lot No. 923602). According to the manufacturer, staining with this antiserum is completely eliminated by pretreatment of the diluted antibody with 100 g/ml of FMRFamide. We repeated this experiment and preincubated the antiserum with 100 g/ml FMRFamide (Sigma; 16 h, 4  C) and this preincubation abolished all staining. Because the crustacean FaRPs known so far all share the carboxy terminal sequence LRFamide we conclude that the DiaSorin antiserum that we used most likely labels any peptide terminating with the sequence RFamide. Therefore, we will refer to the labeled structures in our specimens as "RFamide-like immunoreactive neurons" throughout the paper. The monoclonal mouse anti-Drosophila synapsin "SYNORF1" antibody (provided by E. Buchner, Universität Würzburg, Germany) was raised against a Drosophila GST-synapsin fusion protein and recognizes at least four synapsin isoforms (ca. 70, 74, 80, and 143 kDa) in western blots of Drosophila head homogenates (Klagges et al., 1996). In an analysis of crayfish homogenates, this antibody stains a single band at ca. 75 kDa (Sullivan and Beltz, 2004). In a western blot analysis comparing brain tissue of Drosophila melanogaster and Coenobita clypeatus, the antibody provided identical results for both species staining one strong band around 80e90 kDa and a second weaker band slightly above 148 kDa (Harzsch and Hansson, 2008) suggesting that the epitope which SYNORF 1 recognizes is strongly conserved between the fruit fly and the hermit crab. Similar to D. melanogaster, the antibody consistently labels brain structures in representatives of all major subgroups of the malacostracan crustaceans (Beltz et al., 2003; Harzsch et al., 1997, 1998, 1999; Harzsch and Hansson, 2008) in a pattern that is consistent with the assumption that this antibody does in fact label synaptic neuropil in Crustacea. The antibody also labels neuromuscular synapses both in Drosophila and in Crustacea (Harzsch et al., 1997) and synaptic neuropil in ancestral clades of protostomes, Chaetognatha (Harzsch and Mueller, 2007) and platyhelminths (Cebria, 2008), suggesting that the recognized epitope is conserved over wide evolutionary distances. In additional control experiments for possible nonspecific binding of the secondary antiserum, we omitted the primary antiserum, replaced it with blocking solution, and followed the labeling protocol as above. In these control experiments, staining was absent.

2.4. 3D-reconstruction The three-dimensional reconstructions of the olfactory lobe were based on a synapsin-labeled specimen that was scanned with a confocal microscope and were prepared by using Bitplane’s AutoAligner and Imaris 3D reconstruction software operated on a FS Celsius work station. In each optical section, contours of the neuropils were demarcated and a three-dimensional model, in which individual neuropils could be identified, was generated.

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2.6. Nomenclature In insects, olfactory glomeruli denote spherical neuropil units in their antennal lobes where the afferents of olfactory receptor neurons from their antennae interact with the dendrites of projection neurons and local olfactory interneurons. Although in malacostracan crustaceans superficially similar neuronal elements interact in their olfactory subunits (Schachtner et al., 2005), it remains unclear how far structural, physiological and functional similarities of the insect and crustacean olfactory neuropil units exist (discussed in Strausfeld 2012). Therefore, we use the word “olfactory glomeruli” for these neuropil units in malacostracan crustaceans throughout this paper in the sense and tradition as crustacean neurobiologists have used it for almost hundred years (e.g. Sandeman et al., 1992, 1993, Schmidt and Mellon 2010) but at the same time explicitly state that our usage of this term is NOT meant to imply a homology of crustacean and insect olfactory glomeruli. Other than this aspect, the neuroanatomical nomenclature in this contribution is in accordance with the recently proposed standard for invertebrate neuroanatomy (Richter et al., 2010). 3. Results 3.1. P. bernhardus and C. maenas These species were chosen as a representatives of two major taxa within Decapoda, the Anomura and the Brachyura. As in other Decapoda whose olfactory systems are described (Sandeman et al., 1992, 1993; reviews Mellon and Alones, 1993; Sandeman and Mellon, 2002; Schachtner et al., 2005; Schmidt and Mellon, 2010), P. bernhardus and C. maenas have bilaterally paired, spherical olfactory lobes (OL) that receive the axons of olfactory sensory neurons, and which are characterized by radially arranged neuropil units, the olfactory glomeruli (OG; Fig. 2A, C, D). This configuration of the olfactory system may be part of the decapods ground pattern. In P. bernhardus, olfactory glomeruli have elongated finger-like shapes similar to the glomeruli in the related terrestrial hermit crab, C. clypeatus (Harzsch and Hansson, 2008). The synaptic neuropil of glomeruli is subdivided into a cap, subcap and core region (inset Fig. 2A), a feature typical of many other decapod crustaceans (Schmidt and Ache, 1997; Schachtner et al., 2005; Schmidt and Mellon, 2010). In C. maenas, the glomeruli are not as elongated but more or less shaped like a cone (Fig. 2C, D). The bilaterally paired antenna 2 neuropils (AnN) of P. bernhardus are the recipients of mechanosensory inputs from the relevant antennae. They are well separated from the olfactory lobes and are part of the more caudal tritocerebral neuromere (Fig. 2A). These neuropils in P. bernhardus have a characteristic architecture comprising transverse divisions lined up along their medio-lateral axes. The arrows in Fig. 2B identify more than nine of these compartments which are lined up side by side. This feature is shared with other aquatic decapod crustaceans (Tautz and Müller-Tautz, 1983). 3.2. I. baltica

2.5. Other procedures Brains of the littoral isopod L. occidentalis were fixed in AAF (in 85 ml 98% ethanol, 5 ml glacial acetic acid, 10 ml formalin), dehydrated, cleared in terpineol, and embedded in Paraplast Plus (Sherwood Medical, St. Louis, MO) and serial sectioned at 10 mm. After dewaxing, sections were incubated in silver proteinate and stained according to Bodian’s (1936) original reduced silver procedure to reveal neuropils, cell bodies, primary neuritis, axons and dendrites.

The general appearance of the brain of I. baltica as revealed by double labeling against synapsins and RFamide-like neuropeptides is shown in Fig. 3. The overall layout is distinct from that of P. bernhardus in that its deuto-, and tritocerebral neuropils are not well separated but instead are confluent (Fig. 3A). Distinctive protocerebral neuropils include the optic lobes’ lamina and medulla (compare Sinakevitch et al., 2003 for the optic neuropils of L. occidentalis). However, a lobula and lobula plate, which are present in the littoral isopod L. occidentalis (Sinakevitch et al.,

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Fig. 4. Idotea baltica, immunolocalization of synapsins (confocal laser-scan microscopy) on brain whole mounts. A1-3: three confocal optical sections from the stack on which the reconstruction in C is based. The A and B lobes are identified. Medial is to the left, anterior to the top. B1-3: three confocal optical sections from the scan of a different specimen than that in A. Same orientation as in A. C1-3: Three-dimensional reconstruction from a stack of 109 confocal optical sections of the specimen shown in A1-3. The glomeruli of the A lobe are shown in blue, and the B lobe in orange. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

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Fig. 5. Armadillidium vulgare (A, B) and Porcellio scaber (CeE), immunolocalization of RFamide-like neuropeptides (green), synapsins (red) on whole mounts of the brain counterstained with a nuclear marker (blue; A1, C: Zeiss Apotome structured illumination device, A2, 3, B, D, E: confocal laser-scan microscopy). The double arrow identifies the rudimentary deutocerebrum. A, B: Armadillidium vulgare; A1: low power view showing all three labels. A2: same as A1 but showing a black-white inverted image of the synapsin immunoreactivity only. A3: same as A1 but showing a black-white inverted image of RFamide-like immunoreactivity only. The single arrow labels a conspicuous cluster of immunolabeled neuronal somata. B: higher magnification of the antenna 2 neuropil. CeE: Porcellio scaber; C: low power view showing all three labels (asterisk labels an artificial rupture of the tissue). D: RFamide-like immunoreactivity in the antenna 2 neuropil, black-white inverted image (projection of 20 optical sections of 0.86 mm thickness). Arrows identify immunolabelled somata. E: same as D, but showing synapsin immunoreactivity. Abbreviations: AnN antenna 2 neuropil, E esophageal foramen, LA lamina, LPC lateral protocerebrum, ME medulla, MD mandibular neuromere.

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2003), if present at all, are embedded in the median protocerebrum (compare Gräber, 1933). There is no clear boundary that distinguishes the lateral protocerebrum from its medial portions. However, the central body (CB) is revealed as a transverse unpaired neuropil stretching across the midline and provides a landmark in the medial part of the protocerebrum (compare Strausfeld, 1998; Loesel et al., 2002 for L. occidentalis). Bilaterally paired olfactory lobes (OL) are present in the deutocerebrum of I. baltica. These are composed of several dozen roughly spherical or irregularly shaped olfactory glomeruli (OG) of various sizes, which are strongly immunoreactive against both synapsins and RFamide-like peptides (Fig. 3B and inset, C1-4). A z-stack of optical sections through single olfactory lobes confirms the roughly spherically shape of the glomeruli (Fig. 4A, B) but a 3D- reconstruction shows that, with synapsin immunohistochemistry, many glomeruli seem to touch and be confluent so that it is difficult to separate single glomeruli (Fig. 4C1-3). The reconstruction also demonstrates that the glomeruli are arranged in two groups: an anterior A lobe, and a posterior B lobe (Fig. 4C). Fig. 4A1-3 and B1-3 show single optical sections from confocal scans of the two lobes at a higher magnification (synapsin immunohistochemistry). Whereas dorsally the A (blue) and B (orange) lobes are clearly separate, they converge ventrally. The glomeruli ventrally are arranged radially around a joint fibrous core, and dorsally around the two separate A and B cores. Although the two specimens shown here (Fig. 4A, B) display the same arrangement of A and B lobes, it was not possible to resolve uniquely identifiable glomeruli in the two specimens as can be done for insect antennal lobe glomeruli (see, Rospars and Chambille, 1980). The antenna 1 nerve (A1Nv), which is visible because it is associated with fine, weakly RFamide-like immunoreactive profiles, enters the olfactory lobes from a lateral/anterior direction (Fig. 3D double arrows; 3E). The tritocerebrum is dominated by the large, bilaterally paired antenna 2 neuropil (AnN; Fig. 3D), located either side of the esophageal foramen. As in P. bernhardus, this neuropil is transversely divided into at least eight segment-like domains that are revealed by RFamide-like immunolocalization (arrows Fig. 3F).

3.3. A. vulgare and P. scaber These two species are discussed together here because of the similarities of their brains, the general layout of which also resembles I. baltica. The optic neuropils of the lamina and medulla can be clearly identified as distinct in both A. vulgare and P. scaber (Fig. 5). The lobula and lobula plate could not be differentiated from the lateral protocerebrum, however. In both species, a conspicuous cluster of neuronal somata with RFamide-like immunoreactivity is located dorsally in the protocerebrum close to its midline (dashed circle in Fig. 5A1, A3, C). These somata give rise to a dense network of RFamide-like immunoreactive neurites in the protocerebrum (Fig. 5A3) including the central body (Fig. 5C). The major difference between these terrestrial species and the marine I. baltica is that in the former distinct olfactory lobes are missing. An inconspicuous lateral protrusion is the only indication of a rudimentary deutocerebrum (double arrow in Fig. 5) that is confluent anteriorly with the protocerebrum and posteriorly with the tritocerebrum without showing any distinct border. As in the marine species I. baltica, antenna 2 neuropils are large, flanking the esophageal foramen. Anti-synapsin immunohistochemistry does not reveal any partitions within antenna 2 neuropils (Fig. 5A2, E), which are densely innervated by RFamide-like immunoreactive neurons (Fig. 5A3, single arrows in Fig. 5D) whose neurites form an irregular network. Caudally, the mandibular neuromere (MD) adjoins the brain (Fig. 5). A conspicuous bilaterally paired group of three RFamide-

like immunoreactive somata is associated with the mandibular neuromere (arrows Fig. 5A3, C). 3.4. H. reaumuri The overall layout of the brain in this xerophilic desert species is similar to that in the other two terrestrial isopods described above. Most notably, a defined olfactory lobe is also absent in this species (Fig. 6A). One major difference concerns the antenna 2 neuropil. In H. reaumuri, immunolocalisation of synapsin reveals a texture of the neuropil that seems to be locally condensed into small spherical glomeruli, sometimes referred to as “microglomeruli” (Fig. 6B and inset), although RFamide-like immunoreactive structures do not exhibit any such pattern (Fig. 6B). Optical sectioning reveals that dorsally this neuropil is divided in an anterior and a posterior synaptic zone (Fig. 6C1), whereas more ventrally these two zones are fused (Fig. 6C2). 4. Discussion 4.1. Some terrestrial members of Isopoda have lost a deutocerebral system for processing airborne olfactory cues In decapod crustaceans, chemosensory inputs to the brain from olfactory sensory neurons on the paired first antennae are processed in conspicuous deutocerebral neuropil centers. These are the bilaterally arranged olfactory lobes consisting of cone-like areas of dense synaptic neuropil, called “glomeruli”, that form the thick synaptic layer of the lobe with their apices pointing inwards (see: Sandeman and Luff, 1973; Blaustein et al., 1988; Mellon and Alones, 1993; Sandeman et al., 1992; Schmidt and Ache, 1992, 1996a; reviews: Schachtner et al., 2005; Schmidt and Mellon, 2010). Studies on representatives of the basal malacostracan taxon Nebalia (Leptostraca) suggest spherical glomeruli to be part of the malacostracan ground pattern (Kenning, Harzsch; unpublished results; Strausfeld, 2012) whereas in Decapoda the glomeruli take on the shape of a barrel or cone (Schachtner et al., 2005; Schmidt and Mellon, 2010). Mechanosensory and non-olfactory chemosensory input from the first antennae of decapod crustaceans is processed in quite different neuropils; namely, the lateral antenna 1 neuropil and the median antenna 1 neuropil (Schmidt and Ache, 1992; Schmidt and Ache, 1996b; Harzsch and Hansson, 2008). As has been well documented from crayfish, clawed and clawless lobsters, and hermit crabs, in the olfactory lobe each glomerulus is stratified to provide an outer cap, a subcap, and a base (Sandeman and Luff, 1973; Schmidt and Ache, 1997; Sandeman and Sandeman, 1994; Langworthy et al., 1997; Wachowiak et al., 1997; Harzsch and Hansson, 2008). The brains of P. bernhardus and C. maenas conform to this general architecture of the decapod olfactory system. Pynnönen (1985) reported for the marine isopod S. entomon that each of the first antennae is equipped with up to 60 aesthetascs. This author also found that S. entomon orients toward distant food sources. Using ablation experiments, it was shown that aesthetascs were required to mediate movement toward chemical cues. In the closely related marine species I. baltica, Guse (1983) found 16-18 aesthetascs per antenna 2, each of which contained 60-80 olfactory sensory neurons. Hence, in this species, between 1000 and 1400 olfactory sensory neurons provide an input into each of the olfactory lobes. Although this number is fairly low in comparison to most decapod Crustacea (Mellon and Alones, 1993; Beltz et al., 2003; Hallberg and Skog, 2010), it nevertheless provides an explanation that distinct olfactory lobes are present in this species. Our present data show that olfactory lobes are absent from all three terrestrial isopod species studied. Together with information

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on additional species covered in the classical studies by Gräber (1933) and Walker (1935) it seems reasonable to conclude that all terrestrial representatives of the Oniscoidea have probably lost their olfactory lobes and that sensory perception by the remnant antennules supports modalities other than odor perception. For example, Haug and Altner (1984) proposed the peg sensilla on the first antennae of terrestrial isopods to function as hygroreceptors. Even though their axons presumably target remnant deutocerebral areas, together these findings suggest that the deutocerebrum of terrestrial isopods has almost entirely lost any functional role compared to the deutocerebrum in their marine cousins. Apparently, in colonizing terrestrial habitats, Isopoda did not retain deutocerebral olfactory neuropils. What evolutionary constraints permitted this loss is a matter of conjecture. However, terrestrial isopods are generalist and opportunist scavengers that rely on contact chemoreception. This mode of life is similar to that of Diplopoda, whose single pair of antennae also possesses what have been described as terminal cone organs (Chung and Moon, 2006). In contrast to the loss of antennular olfaction in Isopoda, there is clear evidence that terrestrial species of Anomura (hermit crabs) have retained their antennules and have successfully met the physiological challenges of olfactory perception in air (review, Hansson et al., 2011). Behavioral, neuroethological and neuroanatomical studies provide solid evidence that associated with their conquest of land, terrestrial hermit crabs preserved their marine olfactory system but evolved specific modifications that allow aerial olfaction. These include foreshortening of the aesthetascs to provide mechanical stability and the provision of a permeable cuticle just on one surface of recumbent aesthetascs to minimize dessication (Ghiradella et al., 1968; also, Stensmyr et al., 2005; Harzsch and Hansson, 2008). However, strictly speaking, even though the somata and initial dendritic segments have evolved to lie within the antennule proper, such modifications are not truly convergent with insect olfactory sensilla, which are equipped with pores, and in which multiple dendritic branches occur in only few crown taxa. In addition, Coenobitidae use their first antennae to touch and sample the ground (Greenaway, 2003), which suggests the additional presence of contact chemoreceptors on the antennules. Nevertheless, the enormous olfactory lobes in C. clypeatus, as an adult a fully terrestrial tropical hermit crab, demonstrate that olfaction is the major sensory modality processed by the brain (Harzsch and Hansson, 2008). 4.2. A new role for the tritocerebral sensory pathway in terrestrial isopods?

Fig. 6. Hemilepistus reaumuri, immunolocalization of RFamide-like neuropeptides (green) and synapsins (red) on whole mounts of the brain (confocal laser-scan microscopy). A: low power view. The double arrow identifies the rudimentary deutocerebrum. B: higher magnification of the antenna 2 neuropil. Inset: higher magnification of the boxed area in B. C1, 2: black-white inverted images of single optical sections (0.44 mm thickness) showing synapsin immunoreactivity (C1 is dorsal to C2). Abbreviations: AnN antenna 2 neuropil, CB central body, LPC lateral protocerebrum.

The antenna 2 neuropils of Decapoda, which belong to the tritocerebrum, receive their afferent input from the second antennae and contain the motor neurons that control their movements (Tautz and Müller-Tautz, 1983; Sandeman et al., 1992). Antenna 2 neuropils of decapods with long antennae exhibit a repeated geometrical structure in which the neuropil is transversely segmented (Tautz and Müller-Tautz, 1983). DC Sandeman and coworkers suggested that there could be a spatiotopic (i.e. somatotopic) mapping of the antennal mechanoreceptors along the length of the antennal neuropil. Behavioral studies on attack behavior that can be released by lightly touching the antennae of blinded crayfish suggest that the animals know where on the antenna they have been stimulated because they direct the chelae quite precisely to that point of contact (Zeil et al., 1985; Sandeman and Varju, 1988). Such segmentation is also present in the antenna 2 neuropils of the marine isopod I. baltica, suggesting that these animals may also be able to locate spatial cues perceived by the second antenna. In the present terrestrial isopod species, antenna 2 neuropils are not structured in such a transverse pattern indicating that in the

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Fig. 7. Bodian stained antennal 2 lobes of the tritocerebrum. The tritocerebral lobe of Ligia occidentalis (A), supplied by the antennal 2 nerve (A2Nv) shows three glomerulus-like domains (arrowed). The overall organization of the lobe in L. occidentalis radically departs from that of the homologous lobe shared by Stomatopoda (Lysiosquillina, B) and also from that of Decapoda, where antennal 2 afferents supply a striate and columnar lobe.

context of a terrestrial ecology this neuropil may serve different analytical functions. The data presented here, together with previous analyses (reviewed by Schmalfuss, 1998), indicate that in terrestrial isopods the deutocerebrum plays no role for aerial olfaction and that the tritocerebrum has taken over the function of processing both mechanosensory and other input modalities. The question, then, is this: do terrestrial isopods detect airborne ligands; and, if they do, are such cues perceived by their second antennae? In other words, might terrestrial isopods have evolved olfactory sensors de novo or have they shifted the expression of olfactory sensors to their second antennae? Thus far, evidence that the second antennae may have acquired aerial olfaction comes from observations of one littoral species, L. occidentalis, and a single terrestrial species, the desert dwelling isopod H. reaumuri. In L. occidentalis, the architecture of the large antenna 2 neuropils is distinct from that of the corresponding neuropils of decapods and marine isopods. The latter have lobes composed of layers and columns, clear evidence of somatotopic organization of sensory terminals and relay neurons (Figs. 2, 3 and Fig. 7B). In contrast, the homologous neuropil in L. occidentalis looks entirely different: it is partitioned into discrete neuropil units, some of which are glomerular (Fig. 7A): an organization that suggests that sensory neuron terminals from the second antenna sort out not according to their peripheral spacing but according to their modal identity, just as occurs in segmental ganglia of the arthropod body

(see Murphey et al., 1989). Amongst the many partitions of neuropil in the tritocerebrum of L. occidentalis are 3e5 discrete glomeruli. Whether these are indicative of olfactory processing is not yet known. However, it is of interest that antennation movements by the second antennae of L. occidentalis clearly sample more the air than the substrate and look remarkably similar in action to antennation by the antennae of cockroaches. One isopod species that is adapted to a fully terrestrial life is the desert isopod H. reaumuri. This species has evolved a highly developed system of kin recognition (for review, see Linsenmair, 2007). In addition to being monogamous, both parents engage in intensive care of their offspring, which they raise in burrows. In a series of studies, Linsenmair (1972, 1984, 1985, 1987) showed that the parents are able to distinguish their own young from those belonging to other families and that the young isopods also exhibit parental recognition. This discrimination depends on a set of polar, mainly non-volatile cuticle compounds produced in epidermal glands, which are blended as family-specific signatures (Linsenmair, 2007). The animals touch each other with the tip of their second antennae, which, as mentioned above, in terrestrial isopod species is differentiated into a highly specialized chemosensory organ, the apical cone (Seelinger, 1977, 1983; review Schmalfuss, 1998). Behavioral studies have not yet shown that the desert isopods respond to distant odor cues. However, two observations suggest that some receptors have this capability.

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First, Seeliger’s 1983 recordings of action potentials from axons originating from H. reaumuri’s apical cone, which he drew from a panel of 37 olfactory and gustatory stimuli, showed that in principle receptors could detect airborne ligands. Stimuli included volatiles such as benzaldehyde and geraniol, several amines, acids, sugars and salts. Although most sensory neurons tested by Seelinger (1983) were activated by contact chemoreception, and neither benzaldehyde nor geraniol were effective airborne stimuli, some receptors nevertheless responded to volatiles. For example, sensory cells of the second antennae that responded to butyric acid also responded to volatile short-chain fatty acids and aldehydes. Sensory cells responding to amines were sensitive to volatilized short-chain monoamines and diamines. However, the responses to volatiles were not as pronounced as physiological responses elicited by water-soluble substances, a general class of compounds that are the relevant sensory cues of an ancestral aquatic setting. Second, isopods are reported to be able to locate each other and food sources from a distance, an ability that suggests that either they can recognize chemical tracks or they can detect odors. The latter ability is suggested from studies of the terrestrial isopod, P. scaber, which lacks olfactory lobes but detects airborne metabolites generated by leaf litter bacteria (Schmitz, 1989; Zimmer et al., 1996). Behavioral observations showing spatial memory by A. vulgare of nutrient rich versus nutrient poor location also imply a role for far-field chemoreception (Tuck and Hassall, 2005). Olfactometer studies by Kuenen and Nooteboom (1963) using O. asellus, P. scaber and A. vulgare demonstrated that odors given off by aggregates of conspecifics selectively trigger motor actions by an individual of that species, whereas odors from a different species aggregation does not. These observations, along with Seeliger’s study, admit the possibility that the second antennae may serve distance olfaction. This needs to be further tested using carefully controlled experiments. Returning to H. reaumuri, it is notable that its tritocerebral neuropil, which processes inputs from the apical organ, is composed of units that are reminiscent of small glomeruli (Fig. 6B,C), sometimes referred to as “microglomeruli,” that typify certain insect antennal lobes, such as those of locusts (Anton et al., 2002). This finding raises the interesting possibility that distance olfaction may have newly evolved e or is in the process of evolving e in certain terrestrial and littoral isopods in association with the second antennae and its target neuropils in the tritocerebrum. 4.3. Divergent evolution of olfactory systems in terrestrial crustaceans? In conclusion, it is a given fact that different kinds of ecologies exert their own suites of selective pressures, including those on sensory systems that detect chemicals. Sensilla and sensory neurons used for contact chemoreception may not differ much between terrestrial and marine animals, but does this hold when it comes to the perception of chemicals that are distributed in the medium? It might seem that adaptations to an ecology, in which volatiles play a crucial role in behavior on land, would exert different demands on sensory neurons compared to a marine ecology, in which chemicals are dissolved in seawater. However, there are many conceptual problems with such a view. As shown for land hermit crabs of the genera Birgus and Coenobita, rather few modifications of a sensillary system that evolved in the sea are required for aerial olfaction. However, it is important to remember that land hermit crabs are actually amphibious: they depend on the sea to reproduce, and as juveniles, they lead a marine life, emerging on land later as preadults. It is likely that their aesthetascs play a major role at the marine stages of their life cycle. Isopods, however, appear to have evolved differently during

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their colonization of land. They are indeed fully terrestrial species that have entirely abandoned marine reproduction: their entire life cycle is terrestrial. These obvious differences imply that the ligands, to which the aesthetascs of land hermit crabs are tuned when used in the marine context, are completely irrelevant to terrestrial Isopoda. Evidence suggests that when wholly terrestrial, these crustaceans detect airborne volatiles and that this ability has evolved in response to exclusively non-aqueous ecologies that are not experienced by other "terrestrial" crustaceans, with the possible exception of some species of amphipods (see Hansson et al, 2011). We suggest that such a complete transition to terrestrial life has selected against retention of aesthetascs and has resulted in the evolution of modified chemoreceptor neurons on the second antennae that now assume a role in airborne ligand detection. Whether the acquisition of an antennal-tritocerebral olfactory system by terrestrial isopods may represent a convergent alternative to an insect-like antennal lobe is still an open question. Broader surveys of antennal sensilla and tritocerebral lobes across terrestrial isopod species should provide answers to such questions. Acknowledgments This study was funded by the Max Planck Society and by the grant I/84 176 from the Volkswagenstiftung. We wish to thank M. Knaden (Jena) for collecting live specimens of H. reaumuri in Tunisia and A. Sombke (Greifswald) for assistance with 3D reconstruction. E. Buchner (Biozentrum Universität Würzburg) is gratefully acknowledged for providing the anti-synapsin antibody. References Anton, S., Ignell, R., Hansson, B.S., 2002. Developmental changes in the structure and function of the central olfactory system in gregarious and solitary desert locusts. Microscopy Research and Technique 56, 281e291. Alexander, C.G., 1977. Antennal sense organs of the isopod Ligia oceanica (Linn.). Marine Behavior & Physiology 5, 61e77. Beltz, B.S., Kordas, K., Lee, M.M., Long, J.B., Benton, J.L., Sandeman, D.C., 2003. Ecological, evolutionary, and functional correlates of sensilla number and glomerular density in the olfactory system of decapod crustaceans. Journal of Comparative Neurology 455, 260e269. Blaustein, D.N., Derby, C.D., Simmons, R.B., Beall, A.C., 1988. Structure of the brain and medulla terminals of the spiny lobster Panulirus argus and the crayfish Procambarus clarkii with an emphasis on olfactory centers. Journal of Crustacean Biology 8, 493e519. Bliss, D.E., Mantel, L.H., 1968. Adaptations of crustaceans to land: a summary and analysis of new findings. American Zoologist 8, 673e685. Bodian, D., 1936. A new method for staining nerve fibers and nerve endings in mounted paraffin sections. Anatomical Record 69, 153e162. Burggren, W.W., McMahon, B.R., 1988. Biology of the Land Crabs. Cambridge University Press. Cebria, F., 2008. Organization of the nervous system in the model planarian Schmidtea mediterranea: an immunocytochemical study. Neuroscience Research 61, 375e384. Chung, K.-H., Moon, M.-J., 2006. Fine structure of the antennal sensilla of the millipede Orthomorphella pekuensis (Polydesmida: Paradoxosomatidae). Entomological Research 36, 172e178. Dockray, G.J., 2004. The expanding family of -RFamide peptides and their effects on feeding behaviour. Experimental Physiology 89, 229e235. Edney, E.B., 1968. Transition from water to land in isopod crustaceans. American Zoologist 8, 309e326. Friend, J.A., Richardson, A.M.M., 1986. Biology of terrestrial amphipods. Annual Reviews of Entomology 31, 25e48. Ghiradella, H., Case, J., Cronshaw, J., 1968. Fine structure of the aesthetasc hairs of Coenobita compressus Edwards. Journal of Morphology 124, 361e385. Gräber, H., 1933. Über das Gehirn der Amphiopoden und Isopoden. Zeitschrift für Morphologie und Ökologie der Tiere 26, 334e371. Greenaway, P., 1988. Ion and water balance. In: Burggren, W.W., McMahon, B.R. (Eds.), Biology of the Land Crabs. Cambridge University Press, pp. 211e248. Greenaway, P., 1999. Physiological diversity and the colonization of land. In: Schram, F.R., von Vaupel Klein, J.C. (Eds.), Proceedings of the Fourth International Crustacean Congress, Amsterdam. Brill Academic Publishers, Leiden, pp. 823e842. Greenaway, P., 2003. Terrestrial adaptations in Anomura (Crustacea: Decapoda). Memoirs of Museum Victoria 60, 13e26.

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