Neuropeptide complexity in the crustacean central olfactory pathway: immunolocalization of A-type allatostatins and RFamide-like peptides in the brain of a terrestrial hermit crab

The general layout of the brain in C. clypeatus based on synapsin labelling was analysed in detail by Harzsch and Hansson[18] and therefore will be only briefly reviewed here (Figure1). In many respects it closely conforms to that of other decapod malacostracan crustaceans[8, 9, 47‐49]. It is composed of a protocerebrum that is divided into a lateral part housed in the eyestalk and associated with the compound eyes and a median part that houses the central complex. The deutocerebrum receives afferents from the first pair of antennae, the mechanosensory input of which is processed in the lateral and median antennae 1 neuropils. The chemosensory input from the same appendages is processed in the olfactory lobes, which are closely associated with the accessory lobe. The tritocerebrum is associated with the second pair of antennae that provide input to the antenna 2 neuropil. The immunolocalization of A-type allatostatins (ASTs; green in Figure1) combined with a general marker for synaptic neuropil (red in Figure1) and nuclear counter stain (blue in Figure1) reveals a wide distribution of this peptide throughout the median brain of Coenobita clypeatus, especially in the central complex of the protocerebrum and the olfactory lobes, but also the eyestalk neuropils (see below). A dorsal to ventral section series featuring A-type allatostatin immunoreactivity (ASTir; green) and the nuclear (blue) counter stain (Figure2) provides evidence for intense labelling for the neuropeptide in the synaptic compartments of the olfactory lobe, the olfactory glomeruli. Most ASTir somata that innervate the glomeruli are located in cell cluster (9). The ASTir somata that innervate the protocerebrum are located in cell cluster (6). The nomeclature of cell clusters with numbers in brackets is according to that suggested by Sandeman and co-workers[47, 48].

The protocerebrum is filled with a loose network of ASTir fibers that shows the typical subdivision of the protocerebral neuropil into an anterior and a posterior component, the anterior (AMPN) and posterior medial protocerebral neuropils (PMPN; Figure3A-D). This subdivision is most obvious at the level of the central body (see below), whereas more ventrally and more dorsally such a distinction is not possible. The source of this labeling includes fibres associated with the protocerebral tract (PT; Figure3G) and ca. 20 ASTir somata in cluster (6) that extend their axons into the protocerebral neuropils and the central body (Figures1,2H,3B-F). The central body (CB) is an unpaired neuropil extending across the midline, embedded between the anterior and posterior medial protocerebral neuropils and displaying strong ASTir across its entire width (Figure3). A thick ASTir commissural fiber bundle accompanies the central body posteriorly (Figure1,3E, H). Behind these commissural fibers, the cerebral artery (CA) pierces the brain in a dorso-ventral direction (Figures1,3D, E). The animal’s protocerebral bridge (PB) also displays strong ASTir and is associated with distinct immunolabelled fibres in the protocerebral tract (Figure3B, C). Slightly dorsal to the central body lies the chiasm of the olfactory globular tract (x in Figures2D, E,3B). This tract does not display any ASTir but is visible as a negative imprint (Figure3B) in the otherwise labeled protocerebral neuropils. This tract originates in the olfactory lobes and carries the axons of olfactory projection neurons (cluster 10) to the lateral protocerebrum (see Harzsch and Hansson for details[18]). In the protocerebral tract that links the anterior median protocerebral neuropil to the lateral protocerebrum, numerous ASTir fibers are present (Figure3B-G). Double labeling reveals the presence of many neurons with RFamide-like immunorectivity (RFir) in cell cluster (6) but not any co-labelling with ASTir in neuronal somata. However, both ASTir and RFir are closely associated in the protocerebral neuropils and most likely some co-labelling is present (Figure3G).

The bipartite olfactory lobes (OL; see Figures2,4A, D) dominate the brain of C. clypeatus[18]. They show intense ASTir and the corresponding somata are localized in cell clusters (9) and (11) (Figure2G, H; and arrowheads in Figure4B), which are known to house local olfactory interneurons[8, 9]. We could distinguish two morphological types of ASTir interneurons. Type one is located in cluster (9), has a soma diameter of around 10 μm and amounts to at least one hundred per side (Figures2H,4C,5A,B,6C). The axons of these interneurons penetrate into the olfactory lobe via the medial foramen (mF) in a massive bundle (Figures1,2D-F,5A,B). The olfactory lobes are composed of numerous column-like units, the “olfactory glomeruli”. These are elongate, cylindrical structures, where the distal part is slightly larger than the proximal part. They are arranged in a radial array around the periphery of the bipartite olfactory lobe (Figures2,4D-F, H). Despite their columnar shape we will refer to these neuropil elements as glomeruli since this term is well introduced in the literature[8, 9]. The afore mentioned bundle of ASTir axons splits up to target the olfactory glomeruli and gives rise to strong immunoreactivity in these. Fine neurites with strong ASTir are present throughout the entire volume of the elongate glomeruli (Figure4A-H).

The type two of ASTir neurons, located in cluster (11) has a soma diameter of around 20 μm. The axons do not penetrate into the core of the OL as type 1 axons, but spread around the outside of the olfactory lobe in various directions (asterisks in Figure5C,D; and schematic drawing Figure7) to target glomeruli from the outside. Cell cluster (10), which houses the somata of the olfactory projection neurons that give rise to the olfactory globular tract[18], is devoid of any ASTir but can be visualized with the nuclear stain (Figure2A,4D).

At a higher magnification, and using confocal laser-scan microscopy instead of conventional fluorescence microscopy, it becomes clear from the ASTir that the glomeruli are regionalized in two dimensions. First, looking at (optical) cross sections (Figure4G) the label is clearly concentrated in a small rim around the periphery of the glomeruli, while the core shows a medium labelling density. Second, an analysis of the longitudinal (optical) sections of the glomeruli reveals a regionalization in cap, subcap and base regions (Figure4H), as it is also known in other decapod crustaceans[9]. The cap region is devoid of ASTir and shows the synapsin label only (Figure4H1; and inset), whereas the subcap is strongly immunoreactive for A-type allatostatins. Compared to the subcap, the label is weaker in the base region and shows the lowest intensity in the proximal end of the base (Figure4H2). Horizontal sections through the olfactory lobes (Figure5) show that once the axon bundle that emerges from the cluster (9) ASTir neurons has passed the medial foramen, it splits up into several smaller bundles (Figure2D-F,5A, B) that cross the coarse non-synaptic neuropil in the centre of the lobes to target the glomeruli. From our preparations it does not appear as if these ASTir axon bundles target the base of the glomeruli, but rather that the axon bundles penetrate the glomerular array in a few places to then spread out tangentially to invade the subcap regions (Figure5A, B). This would account for the intense labeling in the subcap region as seen in longitudinal sections of the glomeruli (Figure4H2). This tangential spreading is also visible in Figure4G where ASTir material is visible in between the glomeruli.

Harzsch and Hansson[18]. already described immunoreactivity against RFamide-related peptides (RFir) in C. clypeatus. With double labeling experiments we wanted to explore the spatial relationship of RFir with ASTir in the olfactory system and found a close association of these two neuropeptide systems (Figure6A). Large RFir somata are located in immediate vicinity to the type two ASTir somata (Figure6B) in cluster (11). Furthermore, around one hundred RFir somata are located in cluster (9) close to the ASTir type 1 neurons. Interestingly, there is a clear spatial separation with the ASTir neurons arranged medially within cluster (9) and RFir neurons surrounding these (Figure7). This spatial separation persists within the glomeruli. Whereas in longitudinal (optical) sections it appears that ASTir and RFir are co-expressed in much of the subcap and base (Figure6E, F), transverse (optical sections) show that ASTir is concentrated in the outer ring of the subcap (see above and Figure4G), whereas RFir material is concentrated more medially in the core (Figure6D). Furthermore, in the longitudinal (optical) sections it becomes clear that that RFir material is also localized in a narrow region at the interface of cap and core but that this interface region is devoid of ASTir (Figure6E, F).

The deutocerebrum also comprises the median antenna 1 neuropil (MAN) that extends across the brain posterior to the protocerebrum behind the cerebral artery. The MAN is on both sides flanked by the arms of the olfactory globular tract. Together with the lateral antenna 1 neuropils (LAN; see Figure1) it is devoted to processing mechanosensory input from the first pair of antennae. The MAN and LAN display a moderate level of ASTir that originates from five to ten posteriorly located somata (arrowheads in 3A, B). We did not detect any ASTir in the accessory lobe (AcN; see Figure1), which is a conspicuous neuropil of the C. clypeatus brain and is composed of about 50–80 small, spherical glomeruli (see Harzsch and Hansson for details[18]).

The main structure of the tritocerebrum in the C. clypeatus brain is the antenna 2 neuropil (AnN) which receives mechanosensory afferents from the second pair of antennae and contains the motoneurons that control the movements of the second antennae[47, 50]. This neuropil is filled with a network of weakly AST-immunolabelled fibres (Figure2D).

The medial protocerebrum is connected to the eyestalk neuropils via the protocerebral tract (PT; Figure1). The eyestalks house a substantial part of the brain including the lateral protocerebrum, which is composed of the medulla terminalis (MT) and the hemiellipsoid body (HE), two neuropils that in addition to receiving visual input are also targeted by the axons of olfactory projection neurons. Furthermore, the four optic neuropils lamina (La), medulla (Me), lobula (Lo) and a small proximal lobula neuropil (LoP) are enclosed in the eyestalks (Figure8,; and compare Harzsch and Hansson[18]). Because we could not determine the dorso-ventral orientation of dissected neuropils we found it impossible to generate sections that are unambiguously oriented e.g. in the horizontal plane.

An analysis of the lateral protocerebrum showed strong immunolabeling of rather coarse neurites throughout the entire medulla terminalis (Figure8D). Because of our difficulties to obtain a reliable orientation of the sections, we could not recognize any conspicuous substructures within the medulla terminalis. The hemiellipsoid body is a large spherical neuropil (ca. 300 μm in diameter; compare[18, 44]), that is associated with a compact, laterally situated cluster of densely packed neurons, the lateral protocerebral interneurons (LPI; Figure8D). The hemiellipsoid body can be easily subdivided into the peripheral cap neuropil (Cap), and the core neuropils I-III (Co1-Co3; compare[44]) separated by an unlabelled first and second intermediate layer (IL1 and IL2, respectively) (Figure8E) that are made up of neurite bundles from the lateral protocerebral interneurons (“globuli cells”; see[44]). Cap, core I and core II neuropils all display ASTir that appear homogenously distributed in low power views, where single fibres were not recognizable (data not shown). The source of this innervation with neurites that contain A-type allatostatins could not be determined. The cluster of lateral protocerebral interneurons that is associated with the hemiellipsoid body did not contain any ASTir somata. The core III neuropil was devoid of any ASTir. Higher magnifications of the hemiellipsoid body with confocal laser-scan technique provided a clearer picture of the immunoreactive fibers in Cap, Co1 and Co2 (Figure8F-G). Neurites with ASTir in Co1 are aligned in parallel tangential layers (Figure8G) reminiscent of the neuropil lamellae that had previously been observed by synapsin labeling[18] and Golgi impregnations[44]. However, a different type ASTir material seems to be arranged such that it crosses the tangential fibres at a right angle (Figure8F1, F2) suggesting a grid-like architecture of Co2 as it has been recently described based on other markers[44].

In C. clypeatus, like in other decapod crustaceans, the visual system consists of the compound eyes and three columnar optic neuropils, lamina (La), medulla (Me) and lobula (Lo) as well as the recently discovered lobula plate. Comparing synapsin labeling, which allows for the visualization of the neuropil organization, with the distribution of ASTir reveals that the peptide is widely distributed in lamina, medulla and lobula (unclear for the lobula plate). The most distal of the three optic neuropils, the lamina, appears as a thin layer of ASTir profiles in a geometrical layout that reflects the arrangement of optic cartridges (Figure8A-C). The origin of these peptidergic innervations could not be determined. Higher magnifications reveal that ASTir material in the medulla is arranged in three parallel layers, labeled by single, double, and triple arrowheads in Figure8A. One layer is located at the distal margin of that neuropil (single arrowhead), the second in the centre (double arrowhead) and the third at the proximal margin of the neuropil (triple arrowhead). A tangential section through the medulla is shown in Figure8B, where the arrowheads identify the distal layer of ASTir neurites. Thick immunolabelled fibres that reach the medulla from a proximal direction seem to be the source of this innervation (Figures.8B, C). Furthermore, ASTir somata located close to the medulla seem to contribute to its peptidergic innervations (arrown in Figure8B, C). The lobula is also immunoreactive for A-type allatostatins. The signal is mostly concentrated in a layer that is located in the centre of the lobula (Figure8,9A).

General immunolocalization studies have provided evidence that, in principle, A-type allatostatins are present in the olfactory lobes of various malacostracan crustaceans, such as the crayfish Procambarus clarkii[17] and Orconectes limosus[23], the prawn Penaeus monodon[51], the shore crab Carcinus maenas and the marine hermit crab Pagurus bernhardus[40], and the giant robber crab Birgus latro[39], a close relative of C. clypeatus. Here, we provide the first higher resolution analysis of this peptide`s distribution in the crustacean olfactory system. They are also present in antennal lobes of various insect species, including honeybee Apis melifera[46], the cockroach Diploptera punctata[20], the tobacco hornworm Manduca sexta[52], and the wingless hexapod Lepismachilis y-signata (Archaeognatha,[53]). In C. clypeatus and the crayfish[17, 23] the ASTir fibres that target the olfactory glomeruli in the OL from the proximal side emerge from local olfactory interneurons in cell cluster (9). The olfactory interneurons in this cell cluster are involved in modulation of olfactory processing and, as is known mostly from studies on crayfish, clawed lobsters and spiny lobsters, synthesize a large variety of neurotransmitters (Figures9,10) including serotonin, histamine and GABA as well as many different neuropeptides such as RFamide related peptides, substance P, small cardiactive peptide b, orcokinins, SIFamide, and tachykinin-related peptides[10‐18], review[19]. For C. clypeatus, so far RFamide related peptides, allatostatins and serotonin have been shown for cluster (9) neurons ([18]; and present study). The diversity of neurotransmitter phenotypes of cluster (9) neurons is mirrored in the characteristic regionalization of malacostracan olfactory glomeruli. In longitudinal sections, cap, subcap, and base regions can be distinguished (Figure10; and[13, 14]). In cross sections of the subcap region, a concentric arrangement of three compartments is present, the central rod, core, and outer ring regions. These compartments are defined by the projection patterns of afferent olfactory sensory neurons, local olfactory interneurons, and olfactory projection neurons. For example, the central rod of the subcap is characterized by substance P expression whereas the core displays RFamide-like immunoreactivity (Figure10). However, the fine morphology of local interneuron branchings and their synaptic connectivity as well as the whole neuronal network in the olfactory lobe of C. clypeatus are poorly understood and require further studies involving backfill and intracellular techniques. The present study is the first study that allowed a localization of allatostatin-like immunoreactivity from cluster (9) neurons to the outer rim region of the glomeruli (Figure10). The second type of ASTir neurons with their large somata in cluster (11) course over the surface of the olfactory lobe and most likely penetrate into the glomeruli from the periphery. The olfactory lobes of the moth Heliothis virescens are also targeted by two different types of ASTir neurons one with large the other with smaller somata[54]. In the spiny lobster, Wachowiak and co-workers[55] described the physiology and anatomy of a certain type of local olfactory interneuron, the “rim neurons” (type II; see Figure10), with large somata and neurites that travel around the periphery of the olfactory lobe to innervate the cap region of the glomeruli. The anatomy of these neurons conforms to that of the type of ASTir neurons with their large somata in cluster (11) that we describe in the present report.

The distribution of serotonin and RFamide-like peptides in the glomeruli seems to be similar to the pattern in crayfish and spiny lobsters. Double labeling of allatostatin-like peptides with FMRFamide-like antibodies shows no co-expression of these two neuropeptides in the local olfactory interneurons, rather on the contrary: subsets of FMRFamide-like- and ASTir somata of local interneurons form distinct separate clusters that match their localized expression in the core versus outer rim region of the glomerular subcap (Figure10). The available information as summarized in Figure10 suggest that crustacean olfactory glomeruli are targeted by the axons of the olfactory receptor neurons from the outside, possibly in a multiglomerular pattern[8, 9] (dashed line in Figure10A). In addition, the neurites of the large ASTir neurons and the neurites of GABAergic neurons travel around the outside of the olfactory lobes to enter the cap of the glomeruli from the outside. This stream of incoming receptor axons and neurites of GABAergic and ASTir local interneurons from the outside meets the neurites of local interneurons with substance P, serotonin and histamine that come from the inside of the olfactory lobes to dive into the base of the glomeruli. A third class of local interneurons has neurites that target the subcap region and express RFamide-like peptides, ASTir or show immunoreactivity for small cardioactive peptide B (Figure10). Projection neurons that target the secondary olfactory centers in the protocerebrum (the hemiellipsod bodies; see below) are the major output channel of the system. In summary, we can expect that the crustacean olfactory glomeruli house complex local circuits the connectivity within which as yet is completely unclear. Because the glomeruli of C. clypeatus and its close relative B. latro belong to the longest known for decapod crustaceans[39], and hence display a distinct pattern of regionalization, their olfactory systems form an ideal model to explore the functional relevance of glomerular compartments and diversity of local olfactory interneurons for olfactory processing in crustaceans.

Although the insect studies mentioned above address different questions on the role of ASTs in the brain and thus do not always provide a detailed description of ASTir in the central olfactory pathway, there are nevertheless obvious similarities with our results in C. clypeatus. In insects, ASTir somata are found in a cluster housing local olfactory/antennal lobe interneurons, whose axons in honeybees invade the core of their spherical glomeruli without an evident overlap with afferent projections[46]. In general, regionalization of the mostly spherical insect olfactory glomeruli is much less pronounced than that in crustaceans, suggesting that the latter may have more elaborate local computing capacities within their glomeruli. What is more, the number of glomeruli in malacostracans also seems to be considerably higher than that in insects, amounting to well over thousand in several malacostracan species[56, 57]. Based on the pattern of ASTir and our general knowledge on the architecture of all other involved neuronal players, the cap of bee glomeruli may be equivalent to the cap in decapods crustacean glomeruli, and the bee core may be equivalent to the crustacean base region. In bee glomeruli, Kreissl et al.[46] reported a distinct border region between cap and core, where ASTir profiles are concentrated. Based on our analysis of ASTir in the C. clypeatus glomeruli, we suggest that the outer ring of the subcap region of malacostracan glomeruli corresponds to this border region in the bee.

Neither in Malacostraca nor in insects, ASTir has been observed in the somata of projection neurons, which means that ASTs are not responsible for transferring olfactory information to higher brain centers (mushroom bodies in case of insects, hemiellipsoid body and terminal medulla in crustaceans), but rather are involved in a modulation of the olfactory signal at the level of the deutocerebrum. Though ASTs are documented to exert an inhibitory myomodulatory function[34, 36], their precise role in olfaction is so far unclear and AST-like peptide receptors in crustaceans is yet to be characterized. In the honeybee, a subset of local interneurons coexpress ASTs with GABA, which could suggest that ASTs also act as inhibitory neuromodulators and help to sharpen the olfactory signal[46].

In malacostracan crustaceans, the axons of olfactory projection neurons relay olfactory information from first-order processing areas, the olfactory lobes, to second-order olfactory neuropils, the hemiellipsoid bodies in the protocerebrum (Figure9;[8, 9, 49]). We already noted previously that both first- and second-order olfactory neuropils in C. clypeatus are substantially enlarged in comparison to marine decapods crustaceans[18], an observation that was later verified in Coenobita’s larger relative, the giant robber crab Birgus latro[39]. In C. clypeatus, the estimated number of projection neurons is 20,000 per side[44], and ca. 160,000 in Birgus latro[39]. In the hemiellipsod bodies, the projection neuron input interacts with intrinsic interneurons in a rectilinear array[44], ca. 125,000 neurons per side in C. clypeatus and ca. 250,000 in B. latro[39]. Both species devote an enormous amount of neuronal tissue to processing chemosensory input. In a recent comparative study[44] we showed that the neuronal networks in the C. clypeatus hemiellipsoid bodies share more similarities with that in insect mushroom bodies, also second-order olfactory neuropils, than we had previously expected. Comparisons of the morphology, ultrastructure, and immunoreactivity of the hemiellipsoid body of C. clypeatus and the mushroom body of the cockroach P. americana revealed both a layered motif provided by rectilinear arrangements of extrinsic and intrinsic neurons as well as a microglomerular organization. These findings suggest that the central olfactory pathways of malacostracan crustaceans (Figure9) and insects share an ancestral computational circuit: in the olfactory glomeruli in the deutocerebrum, afferents from olfactory sensory neurons from antenna one interact with various local olfactory interneurons and dendrites of olfactory projection neurons. The axons of the latter project to protocerebral areas where they interact with neurites of intrinsic cells (called Kenyon cells in insects) in a rectilinear array. A convergent multiplication of this basic circuit during evolution of the divergent insect and malacostracan lineages to increase computational power has generated different morphological phenotypes of the olfactory centers that have masked the deep homology of their basic neuronal networks.

Adult specimens of Coenobita clypeatus (Herbst, 1791; Anomura, Coenobitidae) were obtained from the “Zoologischer Großhandel Peter Hoch” (August Jeanmaire Str. 12, 79183 Waldkirch, Germany;http://​www.​hoch-rep.​com/​). The animals (ca. 5–8 cm total length) were anaesthetized for at least one hour on ice and then their brains were dissected in phosphate buffered saline (0.1M PBS, pH 7.4). Killing of the animals was carried out in accordance with the national ethical guidelines (“genehmigungsfreien Versuchsvorhabens nach § 8a Abs. 1 und 2 des Tierschutzgesetzes Deutschland vom 18. Mai 2006 BGBl. I S. 1206”) including notification and consent of the responsible administrative authorities of the University of Greifswald.

The isolated brains and eyestalks were fixed overnight in 4% PFA in 0.1M PBS, pH 7.4 at 4°C. After fixation the tissues were washed for 4 hours in several changes of PBS and subsequently processed as whole mount after carefully removing the neurolemma using forceps or sectioned (80 μm) with a HM 650 V Vibrating Blade Microtome (Microm). Overnight permeabilization in PBTx (0.3% Tx-100 in 0.1 M PBS, pH 7.4) at 4°C of the specimens was followed by an incubation in 1% NGS in PBS TX for at least 2 hours and an incubation in the primary antibodies overnight at 4°C (sections) or for 2 days at 4°C (whole mounts). We used the following reagents for the immonohistolabelling labeling experiments: polyclonal rabbit or monoclonal mouse anti-type A allatostatin (dilution 1:1000[45]), in some experiments combined with monoclonal mouse anti-synapsin “SYNORF1“ antibody (dilution 1:20[58] or rabbit anti-FMRFamide antiserum (Immunostar Inc., dilution 1:100). Subsequently, all tissues were washed in several changes of PBS for 2 hours at room temperature and incubated in a mix of the secondary anti-rabbit Alexa Fluor 488 antibodies (Invitrogen, Molecular Probes), and secondary anti-mouse Cy3 antibodies (Jackson ImmunoResearch Laboratories Inc.) and the nuclear dye bisbenzimide as a histochemical counterstain (0.05%, Hoechst H 33258) for another 4 hours. Finally, the tissues were washed for at least 2 hours in several changes of PBS at room temperature and mounted in Mowiol (sections) or dehydrated in an ethanol series (50, 70, 90, 2 x 100%, 10 min each), and mounted in methylsalicilate (whole mounts). The 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).

The tetrapeptide FMRFamide and FMRFamide-related peptides (FaRPs) are prevalent among invertebrates and vertebrates and form a large neuropeptide family with more than 50 members all of which share the RFamide motif[1, 59‐61]. In malacostracan Crustacea, at least twelve FaRPs have been identified and sequenced from crabs, shrimps, lobsters, and crayfish[26, 61], which range from seven to twelve amino acids in length and most of them share the carboxy-terminal sequence Leu-Arg-Phe-amide. The utilized antiserum was generated in rabbit against synthetic FMRFamide (Phe-Met-Arg-Phe-amide) conjugated to bovine thyroglobulin (DiaSorin; Cat. No. 20091; Lot No. 923602). According to the manufacturer, immunhistochemistry with this antiserum is completely eliminated by pretreatment of the diluted antibody with 100 μg/ml of FMRFamide. We repeated this experiment and preincubated the antiserum with 100 μg/ml FMRFamide (Sigma; 16 h, 4°C) resulting in a complete abolishment of all staining. In another approach we omitted the primary antibody which also 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 immunoreactivity” (RFir) throughout the paper.

The monoclonal mouse anti-Drosophila synapsin “SYNORF1” antibody (kindly 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[58]. In Western blot analysis of crayfish homogenates, this antibody stains a single band at ca. 75 kDa[62]. We have conducted a western blot analysis comparing brain tissue of Drosophila and Coenobita clypeatus[18] in which the antibody provided identical results for both species staining one strong band around 80–90 kDa and a second weaker band slightly above 148 kDa. This previous analysis strongly suggested that the epitope which SYNORF 1 recognizes is strongly conserved between the fruit fly and the hermit crab. Similar to Drosophila, the antibody consistently labels brain structures in representatives of all major subgroups of the malacostracan crustaceans[39, 40, 56, 63‐67] 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[64]. These close parallels in the labeling pattern of SYNORF1 between Drosophila and various Crustacea strengthens the claim that it also recognizes crustacean synapsin homologs. According to the preceded terminology, we will refer to the labelled structures as “synapsin-like immunoreactivity (SYNir)” throughout this manuscript.

The neuroanatomical nomenclature, used in this manuscript is based on that proposed by Sandeman and co-workers[47, 48] with some modifications adopted from Harzsch and Hansson[18]. Cell clusters are numbered in brackets following the syntax proposed by Sandeman and co-workers[47, 48]. All other aspects of the basic neuroanatomical nomenclature are according to the glossary by Richter et al.[68].