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Arthropod Structure & Development 32 (2003) 175–188 www.elsevier.com/locate/asd

The peripheral and central antennular pathway of the Caribbean stomatopod crustacean Neogonodactylus oerstedii Charles D. Derby*, Jennifer K. Fortier, Paul J.H. Harrison, Holly S. Cate1 Department of Biology and Center for Behavioral Neuroscience, Georgia State University, P.O. Box 4010, Atlanta, GA 30302-4010, USA Received 22 March 2003; accepted 10 June 2003

Abstract Although stomatopod crustaceans use their chemical senses in many facets of behavior, little is known about their chemosensory neural pathways, especially in comparison to the better-studied decapod crustaceans. We examined the stomatopod Neogonodactylus oerstedii to determine organizational aspects of peripheral and central neural pathway of antennules, which is a major chemosensory organ. We describe the three flagella of the triramous antennule as the medial, dorsolateral, and ventrolateral flagella. The primary branch point is between the medial flagellum and lateral flagella, and the secondary branch point is at the junction of the dorsolateral and ventrolateral flagella. The antennule bears at least three types of setae, based on their external morphology. Simple setae are present only on the medial flagellum and ventrolateral flagellum, organized as a tuft of 10 – 15 setae on each flagellar annulus. Aesthetasc setae and asymmetric setae occur only on the distal annuli of the dorsolateral flagellum, with each annulus bearing a row of three aesthetascs and one asymmetric seta. DiI fills of the antennular nerve near the junction of the flagella show that sensory neurons in the antennular flagella project to two neuropils in the ipsilateral midbrain—the olfactory lobe (OL) and lateral antennular neuropil (LAN). The OL is glomerular and has rich serotonergic innervation, a characteristic of the OL in decapods. The LAN is bi-lobed and stratified as it is in decapods. However, the LAN of stomatopods differs from that of decapods in being relatively large and containing extensive serotonergic innervation. The median antennular neuropil of stomatopods has sparse serotonergic innervation, and it is more diffusely organized compared to decapods. No accessory lobes were found in N. oerstedii. Thus, the stomatopod antennular flagella have the same two, highly organized parallel pathways common to decapods—the OL pathway and the LAN pathway. q 2003 Elsevier Ltd. All rights reserved. Keywords: Antennule; Olfaction; Chemoreception; Mechanoreception; Mantis shrimp; Crustacea; Olfactory lobe; Lateral antennular neuropil

1. Introduction Stomatopod crustaceans, commonly called mantis shrimp, have interested biologists due to their fascinating and complex behavior (Ferrero, 1989). Their visually mediated behaviors, including prey capture, recognition of conspecifics, color vision, and depth perception, have been especially well studied (Schaller, 1953; Cronin et al., 1994; Schiff, 1997) and shown to be linked to a highly developed visual system (Marshall and Land, 1993; Schiff, 1997; Chiao et al., 2000; Cronin and Marshall, 2001). Their chemosensory systems, including the antennular system, * Corresponding author. Tel.: þ 1-404-651-3058; fax: þ1-404-651-2509. E-mail address: [email protected] (C.D. Derby), http://www.gsu.edu/ ~biocdd/. 1 Present address: The Walter and Eliza Hall Institute of Medical Research, The Royal Melbourne Hospital, Vic. 3050, Australia. 1467-8039/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1467-8039(03)00048-3

also mediate important behaviors, including locating food (Schaller, 1953; Caldwell et al., 1989), avoiding predators (Caldwell and Lamp, 1981), recognizing and mediating social interactions between other species, conspecifics, and even individuals with whom they have had social experiences (Caldwell, 1979, 1982, 1985, 1987). Nonetheless, their antennular chemosensory system is poorly understood, especially compared to their better-studied relatives, the decapod crustaceans. For example, studies of the antennule (e.g. Gru¨nert and Ache, 1988; Carr et al., 1990; Ache and Zhainazarov, 1995; Gomez et al., 1999; Derby, 2000; Cate and Derby, 2001; Harrison et al., 2001) and antennular brain centers (e.g. Mellon and Munger, 1990; Mellon and Alones, 1993; Sandeman et al., 1992, 1993; Schmidt and Ache, 1996a,b; Wachowiak et al., 1996; Beltz, 1999; Harzsch et al., 1999; McClintock et al., 2000; Benton and Beltz, 2001; Sullivan and Beltz, 2001) of decapod crustaceans

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have revealed much about molecular, cellular, and systems solutions to perireception, transduction, coding, and control of behavior. Comparable studies of stomatopod crustaceans are rare. For example, little is known about the antennular regions of stomatopod brains since the work of Bellonci (1878, 1883) and Hanstro¨m (1931) (reviewed in Bullock and Horridge, 1965). Although there have been brief descriptions of the morphology of the antennule of stomatopods based on light microscopy (e.g. Manning and Provenzano, 1963; Provenzano and Manning, 1978; Bauer, 1989), not until recent studies of Gonodactylus mutatus by Mead and colleagues (Mead et al., 1999; Mead and Koehl, 2000; Stacey et al., 2002; Mead and Weatherby, 2002; Mead, 2002) based on scanning and transmission electron microscopy have the antennules been examined in much detail. Mead and colleagues provide a description of the antennular setae of G. mutatus, especially the aesthetascs, with a focus on chemosensory sampling by the aesthetascs. A comparison of antennular systems of stomatopods and decapods would be particularly interesting because, while both have complex behaviors, they are from phylogenetically different groups. Though the phylogeny of Crustacea is controversial, recent analyses suggest that both decapods and stomatopods are eumalacostracan crustaceans (Watling et al., 2000). However, the decapods, which include shrimp, crabs, and lobsters, are a member of the Carida, a more recent eumalacostracan group than the Hoplocarida, to which the stomatopods belong (Watling et al., 2000). Comparisons of the antennular system within the groups of decapods are available (Sandeman, 1982; Hallberg et al., 1992, 1997; Sandeman et al., 1992, 1993; Cate and Derby, 2002b), but there are no such comparisons between the stomatopods and decapods. Consequently, we examined the organization of the antennular system of the Caribbean stomatopod, Neogonodactylus oerstedii, in comparison to that of decapods. We are particularly interested in the relationship between the peripheral and central pathways, including the following issues. First, how is the stomatopod antennule organized? In decapods, the antennule is typically biramous, having two flagella, typically called the lateral and medial flagella (Gru¨nert and Ache, 1988; Hallberg et al., 1992; Cate and Derby, 2001; Richter and Scholtz, 2001). While most decapod crustaceans have two antennular flagella, some do have three (e.g., the prawn Macrobrachium rosenbergii and the mysid shrimp Neomysis integer: Hallberg et al., 1992). The lateral flagellum is distinctive in having at its distal end a tuft of specialized setae that include aesthetasc sensilla. The aesthetasc is ubiquitous among decapods and unique in that it is innervated densely and only by chemoreceptor neurons (Gru¨nert and Ache, 1988; Hallberg et al., 1992, 1997; Cate and Derby, 2001). Outside this aesthetasc tuft region, the lateral flagellum is morphologically similar to the medial flagellum, as both bear numerous types of setae, collectively called non-aesthetasc setae (Cate and Derby, 2001). Many of these non-aesthetasc setae are bimodal,

being innervated by both chemoreceptor neurons and mechanoreceptor neurons (Cate and Derby, 2001, 2002a, b). The stomatopod, on the other hand, has three flagella, a character that it shares with representatives of its fossil relatives (Richter and Scholtz, 2001). Therefore, the first goal of our study was to describe the organization of the antennule of stomatopods. Specifically, we wanted to identify the homologues of the lateral and medial flagella of decapods, and to describe the structure and distribution of aesthetascs and non-aesthetasc setae on these flagella. Our second major question is, what is the organization of the regions of the stomatopod brain that receive antennular input? In decapods, there are three neuropils that receive antennular input: olfactory lobes (OLs), lateral antennular neuropils (LANs), and median antennular neuropil (MAN). The paired OLs receive input predominately or exclusively from aesthetasc chemoreceptor neurons (Mellon and Munger, 1990; Sandeman et al., 1992; Mellon and Alones, 1993; Schmidt and Ache, 1992, 1996b). OLs have a glomerular organization, in which aesthetasc chemoreceptor neurons synapse with interneurons in hundreds to thousands of neuropil regions called glomeruli (Sandeman et al., 1992; Mellon and Alones, 1993; Schmidt and Ache, 1992, 1996b). On the other hand, the paired LANs receive input from nonaesthetasc chemoreceptor neurons and mechanoreceptor neurons on the antennular lateral and medial flagella, as well as antennular motor neurons (Maynard, 1966; Schmidt et al., 1992; Schmidt and Ache, 1996a). The LANs of decapods typically have a horseshoe or bi-lobed shape, its lobes have a stratified neuropil, and each lobe is thought to receive ipsilateral input from only receptors from one of the two antennular flagella (Hanstro¨m, 1931; Schmidt et al., 1992; Sandeman et al., 1993; Schmidt and Ache, 1996a). The unpaired MAN receives input from sensillar receptor neurons in the statocysts and on the proximal segments of antennules, as well as output in the form of antennular motor neurons (Sandeman and Okajima, 1972; Schmidt et al., 1992; Sandeman et al., 1993; Schmidt and Ache, 1996a; Cate and Roye, 1997). The functional roles of OL, LAN, and MAN have been inferred from electrophysiological and anatomical studies (e.g. Maynard, 1966; Schmidt and Ache, 1993, 1996a,b; Sandeman et al., 1993) and explored by recent behavioral studies (Steullet et al., 2001, 2002; Derby et al., 2001; Horner and Derby, 2002; Wroblewska et al., 2002). Our knowledge on the CNS of stomatopods is limited to the early work of Bellonci (1878, 1883) and Hanstro¨m (1931) on Squilla mantis, which was based on general histological techniques. Their work clearly identified glomerularly organized OLs and horseshoe-shaped LANs. Hanstro¨m (1931) did not identify in stomatopods a clearly defined MAN that could be differentiated from surrounding neuropil, although he did describe an unstructured, diffuse neuropil that received antennular input and thus might be related to the decapod MAN. Bellonci (1878, 1883) and Hanstro¨m (1931) did not identify accessory lobes (ALs),

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and indeed the ALs may be a derived feature of the decapods that are absent in all other crustaceans (Hanstro¨m, 1931; Sandeman et al., 1992, 1993). Consequently, the second major goal of our study was to examine the organization of the antennular regions of the brain of stomatopods using more modern neuroanatomical techniques. Specifically, we wanted to investigate the glomerular organization of the stomatopod OL, determine if stomatopods have ALs and MAN, and examine whether the stomatopod LAN is bipartite, as in the decapods which typically have a biramous antennule, or tripartite, to accommodate the stomatopod’s triramous antennule.

2. Materials and methods 2.1. Animals N. oerstedii (Hansen, 1895) in the family Gonodactylidea (Ahyong and Harling, 2000; Barber and Erdmann, 2000) was used in our study. Males and females of 30 –40 mm length were collected in the Florida Keys and shipped to Georgia State University. They were held in aquaria containing recirculating, filtered, and aerated Instant Oceane (Aquarium Systems, Mentor, OH) at 23 –28 8C, and fed shrimp and squid. 2.2. Scanning electron microscopy of antennules The external morphology of the antennules and their setae was examined using scanning electron microscopy. Antennules were removed from the animal, fixed in 70% ethanol, dehydrated through a graded series of ethanol, immersed in absolute acetone, and then infiltrated with dimethoxypropane (Electron Microscopy Sciences, Fort Washington, PA). The dehydrated tissue was mounted onto a stub, vacuum dried, sputter-coated with gold/palladium (Desk II sputter coater, Denton Vacuum Inc., Moorestown, NJ), and examined using a Leica S420 scanning electron microscope (Cambridge, UK). Digital images were captured, imported, placed, and labeled using Adobe Photoshop and Illustrator software (Adobe Systems, Inc., Mountain View, CA). 2.3. Vibratome sections of brains Brains were dissected from the animal and fixed overnight in 4% paraformaldehyde in 0.2 M phosphate buffered saline (PBS). Tissue was then twice rinsed briefly in 0.2 M PBS and washed for 1 h in PBS. Brains were then embedded in 14% gelatin (Sigma) in PBS. The gelatinembedded brains were fixed in 4% paraformaldehyde in 0.2 M PBS overnight. They were then rinsed twice in PBS and washed for 1 h in PBS. Brains were mounted on stubs and sectioned at 50 mm using a Vibratome Series 1000 (St. Louis, MO). Each section was placed into an individual well

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of a 24-well cell culture dish filled with PBS, and subsequently either stained with toluidine blue or processed for serotonin immunocytochemistry. For toluidine blue staining, the PBS was aspirated from the wells and replaced with fresh toluidine blue. Sections were stained for 30 min. Toluidine blue was gently aspirated from each well; sections were rinsed twice in PBS, once in 70% ethanol for 5 min to differentiate the staining, and then rinsed in PBS. Sections were aligned on microscope slides and mounted in Cytoseal (Stephens Scientific, Kalamazoo, MI). 2.4. Paraffin sections stained with toluidine blue For paraffin sections, brains were dissected in crustacean saline (in mM: 458 NaCl, 13.4 KCl, 13.6 CaCl2, 9.8 MgCl2, 14.1 Na2SO4, 3 HEPES, 1.9 glucose, 1.2 NaOH, pH adjusted to 7.4), and fixed overnight in 4% paraformaldehyde at room temperature. The brains were rinsed (3 £ 30 min) in 0.2 M PBS, dehydrated through a graded alcohol series: 30%, 50, 70, 90, 100, 100 dry, 100 dry (1 h each step), cleared in xylene (3 £ 30 min), and infiltrated with paraffin at 58 8C in an oven using the following 1-h steps: 50:50, 25:75, 12.5:87.5, 0:100, 0:100 xylene:wax. Each brain was aligned in a mold and allowed to set at room temperature. Molds were then placed in ice water for approximately 10 min, removed from the mold, trimmed, and stored for use. Blocks were mounted on metal stubs and sectioned at 6 mm on a Reichert– Jung histomicrotome. Ribbons were floated in warm water bath, aligned on Superfrost slides (VWR, West Chester, PA), and air-dried overnight. Wax was removed from sectioned tissue by rinsing in xylene (3 £ 10 min), and the tissue was rehydrated to 0.2 M PBS through a series of alcohol: 100% dry, 100, 90, 70, 50, 30, PBS, PBS (10 min each step). Slides were immersed in toluidine blue for 30 min at 58 8C, and then rinsed in PBS until no toluidine blue was seen leaching from the tissue and dehydrated through the above alcohol series (5 min each step). This shorter time was used to limit the time for toluidine blue to be removed by alcohol. The tissue was then cleared in xylene and mounted in Cytoseal. 2.5. Immunocytochemistry An anti-serotonin antibody was used to identify neuropils, describe their organization, and label individual neurons. Serotonin immunocytochemistry has been used in many crustaceans to label preferentially the OLs, ALs, and olfactory globular tract neuropils (OGTNs) (Sandeman and Sandeman, 1994; Sandeman et al., 1995; Schmidt and Ache, 1997; Benton and Beltz, 2001). For anti-serotonin immunocytochemistry, brains were dissected and fixed overnight in 4% paraformaldehyde (in 0.2 M PBS, pH 7.4), rinsed in PBS, then embedded in gelatin and sectioned at 50 mm on a vibratome. The sections were rinsed in PBTA (0.2 M phosphate buffer containing 0.3% Triton X-100 and

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0.1% sodium azide), incubated for 1 h in PBS containing 0.3% Triton X-100 and 5% normal goat serum, and then incubated overnight in rabbit serotonin antisera (ImmunoStar, Hudson, WI) at a 1:200 dilution in PBTA. The sections were then rinsed in PBTA, incubated overnight in Texas red conjugated goat anti-rabbit antiserum (1:50 in PBTA), and rinsed again in PBTA. The sections were viewed and photographed on a Zeiss confocal microscope, using LSM 510 software.

in petroleum jelly was applied to the cut end of the nerve. The preparation was returned to 4% paraformaldehyde in PBS and incubated in a 37 8C oven for 2– 3 weeks. After this time, the brains were rinsed with PBS, counterstained by incubating in Neurotrace Green (Molecular Probes) for 20 min, rinsed in PBS, and then viewed and photographed in wholemount on a Zeiss confocal microscope, using LSM 510 software.

2.6. DiI filling of antennular nerve

3. Results

The brain and attached antennules were dissected from animals, pinned out in petri dishes coated with Sylgard (Dow Corning Corp.), and fixed overnight in 4% paraformaldehyde in 0.2 M PBS. After rinsing the tissue with PBS, the antennular nerve was exposed at the junction between antennular segments III and IV (see Fig. 1). The nerve was cut and dabbed dry with tissue paper. A small crystal of DiI (Molecular Probes, Inc., Eugene, Oregon) thoroughly mixed

3.1. External morphology of the antennule A characteristic of the stomatopod antennule is that the fourth antennular segment is composed of three flagella (Fig. 1). This is in contrast to the biramous (two flagella) segment of most decapods. Fig. 1(C) shows a schematic of the stomatopod’s triramous antennule. This figure is based on light (Fig. 1) and scanning electron micrographs (Fig. 2).

Fig. 1. Organization of the stomatopod head and antennule. (A) Dorsal view of the head, showing the eyes, antennules (first antennae), and antennae (second antennae). (B) Higher-power image of the triramous antennules of the boxed-area in (A). A branch in the basal annulus of the lateral flagellum gives rise to the ventrolateral and dorsolateral flagella. (C) Diagram of the triramous antennule. This is a left antennule drawn from the medial perspective (looking at the inner margin). The medial flagellum branches from the lateral flagellum in the horizontal plane. The ventrolateral and dorsolateral flagella branch in the vertical plane. The two lateral flagella share a common basal annulus. Aesthetascs extend forward (distal) from the inner margin of the dorsolateral flagellum. A tuft of simple setae extends downward from each annulus on the medial and the ventrolateral flagellum. The medial flagellum is similar to the ventrolateral flagellum in both its length (number of annuli) and its complement of setae. Scale bars: A, 2 mm; B, 0.5 mm.

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Fig. 2. Scanning electron micrographs showing setal types on the antennular flagella. (A) Aesthetasc sensilla (a) form a tuft of ca. three sensilla per annulus on the dorsolateral flagellum (dl); also shown is the ventrolateral flagellum (vl) and the medial flagellum (m). (B) Higher-power view of aesthetascs (a) and asymmetric setae (as). (C) Tuft of simple setae (indicated by arrow) on the distal edge of each annulus of the medial flagellum (m). (D) Single tuft of simple setae on the medial flagellum. Scale bars: A, 100 mm; B, 50 mm; C, 100 mm; D, 10 mm.

In decapods, a single branch point at the base of the exopodite results in two flagella that branch along the horizontal plane. These are referred to as the medial (or inner) and lateral (or outer) flagella (Gleeson, 1982; Gru¨nert and Ache, 1988; Cate and Derby, 2001). A similar branch point occurs on the stomatopod exopodite (Fig. 1(C): primary branch distal to antennular segment III), and branches are referred to here as medial and lateral flagella. A second branch point occurs on the lateral flagellum immediately distal to the primary branch point (Fig. 1(C): secondary branch distal to antennular segment IV) and thus results in a triramous exopodite. This secondary branch occurs in the vertical plane to produce a dorsolateral flagellum and a ventrolateral flagellum. This organization is

consistent with descriptions by Manning and Provenzano (1963) and Provenzano and Manning (1978). These authors reported that the antennule bifurcates early in development to become biramous, and later in development the proximal segment of the outer ramus (referred to here as lateral flagellum) bifurcates to form the triramous antennule. 3.2. Antennular setae The setal composition on the flagella of the stomatopod’s triramous antennule is far less complex than that of many decapods, such as the spiny lobster for which 10 antennular setal types have been described (Cate and Derby, 2001). At least three setal types are distinguished based on external

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morphology and location, all of which are ‘simple’ in form in that they lack setules (Watling, 1989). The first type is aesthetascs, which is a setal type ubiquitous across many groups of crustaceans (Gru¨nert and Ache, 1988; Hallberg et al., 1992, 1997; Cate and Derby, 2001), including stomatopods (Mead et al., 1999; Mead and Koehl, 2000; Mead and Weatherby, 2002; Mead, 2002). The aesthetascs of N. oerstedii have a typical aesthetasc external structure and organization (Figs. 1 and 2). Each aesthetasc has a bulbous base and more slender shaft (Fig. 2(A) and (B)). They form a single row of about three setae on each annulus in the distal portion of the flagellum and are absent from the proximal annuli (Figs. 1(C), 2(A) and (B)). They insert at the distal edge of annulus and point toward the distal end of the flagellum (Figs. 1(C), 2(A) and (B)). There are approximately 40 aesthetascs on each dorsolateral flagellum of N. oerstedii of 30 – 40 mm length. The aesthetascs occur exclusively on the dorsolateral flagellum (Figs. 1 and 2). The second setal type is the asymmetric seta, which has been described in several decapod crustaceans, including Achelata (spiny lobsters), Homarida (marine clawed lobsters), Brachyura (crabs) (Gleeson, 1982; Gleeson et al., 1993; Cate and Derby, 2001), and stomatopods (Mead et al., 1999; Mead and Weatherby, 2002). Asymmetric setae occur exclusively on the dorsolateral flagellum in N. oerstedii (Fig. 2(B)). A single asymmetric seta is associated with each row of aesthetascs (Fig. 2(A) and (B)). As in other crustaceans, the asymmetric seta is oriented oblique to the row of aesthetascs. The third setal type is a simple seta, which are organized into tufts of 10– 15 setae (Fig. 2). These tufts are located only on the medial and ventrolateral flagella, and are present as a single tuft on the distal end of each annulus of the medial and ventrolateral flagella (Figs. 1(C), 2(C) and (D)). In summary, the stomatopod has at least three setal types based on external morphology. The dorsolateral flagellum has only aesthetasc and asymmetric setae. The medial and ventrolateral flagella are devoid of aesthetasc and asymmetric setae, but instead have a tuft of simple setae on each flagellar annulus. 3.3. Antennular innervation of the CNS Antennular innervation of the CNS was examined in two ways. First, brain morphology was examined in paraffin sections, specifically to describe major neuropils, soma

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clusters, and tracts. We used toluidine blue stained tissue and serotonin immunocytochemistry to study this. Second, DiI filling of the antennular nerve in whole-mount brains was used to examine antennular input. 3.3.1. Brain morphology The midbrain, or deutocerebrum, was the main focus of our study, since it is known to be the site of antennular innervation in a variety of crustaceans (Sandeman et al., 1992). Deutocerebral neuropils of decapods include the paired OLs, paired ALs, paired LANs, unpaired MAN, and paired OGTNs (Sandeman et al., 1992; Mellon and Alones, 1993; Schmidt and Ache, 1992, 1996a,b; Schmidt et al., 1992; and Section 1). Our results using toluidine blue stained sections of brains of N. oerstedii (Fig. 3) reveal many neuropils, tracts, and somata clusters of the deutocerebrum similar to those of decapods. These include the OL, LAN, MAN, the olfactoryglobular tract (OGT) (which connects the OL and ALs to the next neuronal level in the eyestalk: e.g. Sullivan and Beltz, 2001), and the lateral somata cluster ( ¼ cluster 10, which are somata for output interneurons of the OLs: Sandeman et al., 1992; Wachowiak et al., 1996). The medial somata cluster ( ¼ cluster 9, which are somata for intrinsic interneurons of the OLs: Sandeman et al., 1992; Wachowiak et al., 1996), AL, and OGTN were not identified. The OL of N. oerstedii is glomerular in structure, as are their decapod counterparts. OL glomeruli are the sites of synapses between aesthetasc olfactory receptor neurons and interneurons in crustaceans (Sandeman et al., 1992). The glomeruli of N. oerstedii are evident in toluidine-blue stained sections (Fig. 3(B), (C) and (F)), DiI-filled antennular nerves (Fig. 4(C)), and brains labeled with an anti-serotonin-antibody (Fig. 5(A)). These glomeruli are spherical in shape, each has a diameter of ca. 50– 80 mm (volume of ca. 110,000 mm3), and there are ca. 60 – 80 glomeruli per OL. The LAN of N. oerstedii (Figs. 4(A), (B) and 5(C)) is bilobed or horseshoe shaped, as it is in decapods (Sandeman et al., 1992). There is no indication of a tripartite organization. Notably, the LAN is proportionally much larger in stomatopods than decapods. In N. oerstedii, the LAN is oriented such that the arms of the horseshoe point away from the midline of the brain. The LAN of N. oerstedii, like that of decapods (Schmidt and Ache, 1993, 1996a; Schmidt et al., 1992), has a stratified appearance. This is in the form of fibers running in the

Fig. 3. Stomatopod brain. (A)–(D) Longitudinal views. (E) and (F) Transverse views. (A) and (E) Schematic diagrams of brain, showing select neuropils and somata clusters (shaded dark). (B)–(F) 6-mm paraffin sections stained with toluidine blue. (C) Olfactory lobe, showing its spherical glomeruli ( p ), input (antennular nerve), and output (olfactory-globular tract). (D) High-magnification view of median antennular neuropil. (E) and (F) Olfactory deutocerebrum, showing olfactory lobe with spherical glomeruli ( p ), lateral antennular neuropil, lateral cluster somata and neurites to the olfactory lobe. The antennal neuropil (in the tritocerebrum) receives input from the second antenna. AMPN, anterior medial protocerebral neuropil; AnN, antennal neuropil; CB, central body; LAN, lateral antennular neuropil; LC, lateral cluster ( ¼ somata cluster 10); MAN, median antennular neuropil; OGT, olfactory-globular tract; OL, olfactory lobe; PMPN, posterior medial protocerebral neuropil; PB, protocerebral bridge. Scale bars: A, B, C, E: 200 mm; D, F: 100 mm.

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middle of each of the lobes, with neural processes perpendicular to them, and is apparent from DiI (Fig. 4(B)) and serotonin labeling (Fig. 5(B)). N. oerstedii appears to have a MAN (Fig. 3(B) and (D)). It is located in the same approximate position as in decapods –medial and slightly anterior to the LANs. However, the boundary of the MAN is not distinct, as was described for S. mantis by Hanstro¨m (1931). Although not the focus of our study, neuropils in the protocerebrum and tritocerebrum of N. oerstedii were also identified (Fig. 3). In the protocerebrum, these include the anterior medial protocerebral neuropil (AMPN), posterior medial protocerebral neuropil (PMPN), protocerebral bridge (PB), and central body (CB). In the tritocerebrum, they include the antennal neuropils (AnNs). 3.3.2. DiI fills DiI filling of the antennular nerve at the junction of antennular segments III and IV (i.e. just proximal to the flagella) labeled only the ipsilateral OL and LAN (Fig. 4). The entire neuropil of each was labeled. No other neuropils, including the MAN or the contralateral OL or LAN, were labeled. 3.3.3. Serotonin immunocytochemistry Labeling the brain with an anti-serotonin antibody allowed us to identify deutocerebral neuropils and somata clusters and to define the distribution of serotonin-containing neurons in these neuropils and clusters. Extensive serotonin labeling was especially evident in the OLs (Fig. 5(A)) and LANs (Fig. 5(B)). Serotonin labeling highlights the glomerular organization of the OLs, since the glomeruli but not the antennular nerve layer are labeled (Fig. 5(A)). Serotonin also highlights the stratified organization of the LANs (Fig. 5(B)). The MAN had very little serotonin labeling. Dorsal giant neurons (DGNs) are distinctive serotonin immunoreactive cells in the medial cluster of OL interneurons in many decapod species (Sandeman and Sandeman, 1987; Sandeman et al., 1988, 1995; Schmidt and Ache, 1997). These neurons were not identified by serotonin immunoreactivity in the brain of N. oerstedii. However, a cluster of serotonin-immunoreactive neurons located near the LAN was identified (Fig. 5(C) and (D)). These are smaller than expected for DGNs, are located near the LANs, and appear to innervate the LANs instead of the OLs (Fig. 5(D)).

Fig. 4. Whole-mount, longitudinal views of confocal images of the stomatopod brain after DiI filling of the antennular flagella, showing labeling of the ipsilateral olfactory lobe (OL) and lateral antennular neuropil (LAN). DiI-filled projections are labeled yellow/red; background tissue is labeled green by fluorescein (in (A)) and DAPI (in (B) and (C)). Box in (A) is shown in higher magnification in (B). AnN, antennal neuropil; LC, lateral cluster; OC, oesophageal connective; OL, olfactory lobe; p , glomeruli. Scale bars: A, 500 mm. B, 200 mm; C, 100 mm.

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Fig. 5. Serotonin immunoreactivity in the stomatopod brain. Shown are confocal images from 50-mm sections in the longitudinal plane. (A) Olfactory lobe (OL). (B) Lateral antennular neuropil (LAN). (C) LAN, antennal neuropil (AnN), and associated somata. (D) Different confocal slice of boxed area in (C). Arrow indicates soma. Scale bars: A –C, 100 mm; D, 50 mm.

Serotonin-immunoreactive labeling was also present in the protocerebrum and tritocerebrum. Ascending and descending serotonin-immunoreactive fiber tracts extend throughout the protocerebrum. In the tritocerebrum, serotonin-immunoreactive varicosities were present in the AnN and in the somata cluster immediately between the AnNs and the LANs (Fig. 5(C) and (D)).

4. Discussion Our study on the peripheral and central antennular pathways of N. oerstedi was inspired by that fact that relatively little is known about the stomatopod antennular neural pathway, even though stomatopods use their chemical senses, including their antennules, in many facets of their behavior. Thus, an aim of our study was to provide a comparative perspective to the antennular system of eumalacostracan crustaceans. In this study, we have explored two questions using N. oerstedii. First, how is

the antennule organized? Second, how is the stomatopod brain organized to receive sensory input from this triramous antennule? 4.1. Peripheral antennular pathway Our results show that the triramous antennule of N. oerstedii has three flagella that bear distinct setae—the medial, dorsolateral, and ventrolateral flagella (Fig. 1). Based on external morphology, there are at least three types of setae on these flagella. One type—the simple seta—is present only on the medial and ventrolateral flagella, and occurs as a tuft of 10 –15 setae on each flagellar annulus. The two other types—aesthetasc and asymmetric setae—are present only on the dorsolateral flagellum and occur on the distal annuli as a row of three aesthetascs and one asymmetric seta (Fig. 2). Of course, there may be diversity in the innervation of the setae belonging to a single morphological type, such as in the number of sensory neurons and/or response properties of the neurons. If so,

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there might be more than three types of setae. Examination of this possibility will require transmission electron microscopic and electrophysiological studies of these setae. The setal organization of the dorsolateral flagellum suggests that it is homologous to the lateral flagellum (or outer flagellum) of decapod crustaceans (Gleeson, 1982; Gleeson et al., 1993). The development of these flagella has been described for N. oerstedii ( ¼ Gonodactylus oerstedii) (Manning and Provenzano, 1963; Provenzano and Manning, 1978), although distinguishing the identity of the three flagella from their drawings and labels is difficult. For example, Provenzano and Manning (1978) describe an ‘inner flagellum’, ‘outer (ventral) flagellum’, and ‘middle flagellum’, with the middle flagellum budding from the outer flagellum. This would suggest that their inner, middle, and outer flagella are equivalent to our medial, ventrolateral, and dorsolateral flagella, respectively. Yet they also state that aesthetascs are present on the inner flagellum, though their figures indicate this to be their outer flagellum (their Figs. 4(c) and 6(b)). Mead and colleagues (Mead et al., 1999; Mead and Koehl, 2000; Mead and Weatherby, 2002) describe on G. mutatus a medial flagellum, a lateral flagellum, and a ‘filament’ that extends from the base of the lateral flagellum. This filament bears aesthetasc and asymmetric setae in a similar arrangement as in N. oerstedii, and thus is then equivalent to our ‘dorsolateral flagellum’, and their ‘lateral flagellum’ is equivalent to our ‘ventrolateral flagellum’. Thus, we believe that the medial flagellum and the dorsolateral flagellum of stomatopods are homologous with the medial flagellum and the lateral flagellum, respectively, of decapods with biramous antennules. The branching that forms the dorsolateral and ventrolateral flagella in stomatopods may correspond to the splitting of the lateral flagellum into one flagellum possessing aesthetascs and into another branch without aesthetascs, as has been described in the larval development of the triramous M. rosenbergii (Ling, 1969; Hallberg et al., 1992). The antennule, which is likely to mediate many complex behaviors (Schaller, 1953; Caldwell, 1979, 1982, 1985, 1987; Caldwell and Lamp, 1981; Caldwell et al., 1989), is obviously well developed, yet the number of setal types on the antennules is low relative to some decapod crustaceans. For example, in N. oerstedii, we identify only three types of setae based on external morphology, all belonging to the simple category (i.e. without any branching) (Watling, 1989). In contrast, some decapods have many more morphological types of antennular setae (Derby, 1989; Watling, 1989). For example, the Caribbean spiny lobster Panulirus argus has at least 10 setal types on its antennular flagella (Cate and Derby, 2001). The significance of these differences in sensillar diversity is not fully understood. Obviously, the structure of a sensillum can influence the physiological response properties of its sensory neurons; however, the determinants of afferent responsiveness are numerous and often complex such that they often cannot be predicted from changes in a single feature (Kanou et al.,

1988; Ka¨mper, 1992; Devarakonda et al., 1996). Consequently, until we know more about the innervation of sensillar types, the response properties and central connections of these sensory neurons, and the behaviors mediated by them, the reasons for the differences in the number of setal types in different groups of eumalacostracans will remain unknown. 4.2. Central antennular system Several questions are addressed in our CNS study. Do stomatopods have the deutocerebral neuropils typical of caridean crustaceans—OLs, LANs, and MAN—or other present in more advanced carideans—ALs? Is the structure of these neuropils organized in a similar fashion to that of decapods? Does the stomatopod LAN have a tripartite organization, reflecting the sensory input from its triramous antennule, or does it have a bipartite organization as do decapods, which typically have biramous antennule? We found that most of the major neuropils of the deutocerebrum of most decapods—the OLs, LANs, and MAN—are also present in stomatopods (Fig. 3). This is not surprising given the relatively conservative nature of evolutionary changes to nervous systems despite significant changes in external morphology (Sandeman et al., 1992, 1993; Beltz et al., 2002). We did not find ALs in N. oerstedii. Indeed, ALs are not ubiquitous in the crustaceans, even in the decapods. They are present in Achelata (spiny lobsters), Homarida (marine clawed lobsters), Astacida (freshwater crayfish), and Brachyura (crabs) (though reduced in the Brachyura), but not present in Dendrobranchiata (shrimps), and Anomura (Sandeman et al., 1992, 1993; Scholtz and Richter, 1995; Wachowiak et al., 1996). 4.2.1. Olfactory lobe The organization of the OL of N. oerstedii is similar in many respects to that of decapod crustaceans. The stomatopod OL receives sensory input from the ipsilateral antennular flagellum, as demonstrated by our DiI fills (Fig. 4). Although we did not identify which antennular sensory neurons project to the OLs, we assume that they are predominantly aesthetasc chemosensory neurons, as this has been experimentally demonstrated in other crustaceans (e.g. Mellon and Munger, 1990; Mellon and Alones, 1993; Schmidt and Ache, 1992, 1996b). The synaptic neuropil of the OL of N. oerstedii is organized into glomeruli, as it is in other crustaceans. These glomeruli appear to have approximately the same volume, but are more spherical and less numerous, than those of decapod crustaceans. For example, compared to 17 decapod crustaceans examined by Beltz et al. (2002), the volume of each glomerulus of N. oerstedii (ca. 110,000 mm3) is near the median value of the decapod species but the number of glomeruli (ca. 60 –80) is lower than in all of the decapods. Additionally, the number of aesthetascs per antennule (ca. 40) is at the low end of the range for the 17 decapods

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examined by Beltz et al. (2002). Consequently, N. oerstedii has an aesthetasc:glomerulus convergence ratio of about 0.5, which is very near the median for 17 decapod crustaceans (Beltz et al., 2002). We estimate a chemosensory neuron:glomerulus convergence ratio of 10:1, based on an assumption of 12 – 20 chemosensory neurons per aesthetasc—a value derived from a study of aesthetascs from a different stomatopod species, G. mutatus (Mead and Weatherby, 2002). Are the dorsal giant neurons (DGNs) and medial cluster (cluster 9/11) somata present in stomatopods and are they serotonergic? The OLs of many decapod crustaceans (clawed lobsters: Langworthy et al., 1997; spiny lobsters: Schmidt and Ache, 1997; crayfish: Sandeman and Sandeman, 1987; Sandeman et al., 1988) as well as the homologous neuropils of insects (the antennal lobes: Sun et al., 1993; Bicker, 1999) contain significant amounts of serotonin. Several types of decapod crustacean OL interneurons have been identified as containing serotonin. The DGNs, which branch in the OL and AL, are the most prominent serotonin-containing neurons in the decapods (Johansson, 1991; Beltz, 1999; Antonsen and Paul, 2001), but interneurons of the OLs are also typically serotonergic. In N. oerstedii, the OL also has significant serotonergic labeling (Fig. 5(A)), and the labeling is concentrated in the glomeruli. However, we could not identify DGNs or medial cluster cells. This is fitting with previous phylogenetic analyses of crustaceans in which only one somata cluster has been identified in the earlier crustacean lineages, including the stomatopods (Hanstro¨m, 1931; Sandeman and Scholtz, 1995; Richter and Scholtz, 2001). 4.2.2. Lateral antennular neuropil The LAN of stomatopods has most of the same features as those in decapods, although there are interesting differences. Sensory neurons from the antennular flagella of stomatopods project to the ipsilateral LAN (Fig. 4), as is the case in decapods (Schmidt et al., 1992; Schmidt and Ache, 1996a). The sensory input to the LAN in decapods is thought to be from non-aesthetasc chemosensory neurons and mechanosensory neurons (Schmidt et al., 1992; Schmidt and Ache, 1996a). A key finding is that the LAN of N. oerstedii occupies a relatively large proportion of the deutocerebrum. This may be related to the importance of antennular mechanosensory input to prey striking and capturing behavior by stomatopods (Schaller, 1953) and to the concomitant well-developed proprioceptive system in the antennules (Sandeman, 1964). Although the stomatopod LAN receives input from a triramous antennule, it is still bi-lobed (Figs. 3– 5). The biramous decapods also have a bi-lobed LAN, and it is thought that each lobe receives input from only one flagellum (Schmidt et al., 1992; Schmidt and Ache, 1996a). It is likely that in stomatopods, the medial flagellum is represented in one of the LAN’s lobes, and the dorsolateral and ventrolateral flagella are represented in

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the other lobe. How the two branches of the lateral flagellum are represented within the LAN—e.g. are their projections overlapping or discrete—remains to be determined, as our attempts to fill individual antennular flagella with DiI were unsuccessful. The stratified or laminated structure of the LAN of N. oerstedii is also present in LANs of other stomatopod species (Hanstro¨m, 1931). This stratified structure is well described in spiny lobsters (Schmidt et al., 1992; Schmidt and Ache, 1996a) and may represent a topotopic map from antennular receptors to the CNS. In decapods, there is evidence for a pathway from non-aesthetasc sensilla to the stratified LAN that is sufficient to mediate odor-activated orientation, odor learning, and odor discrimination (Derby et al., 2001; Steullet et al., 2001, 2002; Horner and Derby, 2002). The serotonergic labeling in the LANs of N. oerstedii is extensive (Fig. 5(B)). This was not expected, since most crustacean species examined to date have relatively little serotonin labeling in the LAN (Sandeman et al., 1988; Langworthy et al., 1997; Schmidt and Ache, 1997; Antonsen and Paul, 2001). The serotonergic branching in the LAN of N. oerstedii is stratified, reflecting the general organization of the LAN. 4.2.3. Median antennular neuropil The stomatopod MAN is small and indistinct in comparison to decapods. Our data on DiI filling of the antennular nerve support the view that the MAN of stomatopods does not receive input from the antennular flagella (Fig. 4). Evidence in decapods suggests that the MAN receives input from the proximal segments of the antennules, especially the statocysts (Sandeman and Okajima, 1972; Sandeman et al., 1992; Schmidt and Ache, 1996a; Schmidt et al., 1992; Cate and Roye, 1997). However, since we did not attempt to fill with DiI the antennular proximal segments, we do not know if the stomatopod MAN also receives such input. The small size of the MAN may be because stomatopods are not reported to have statocysts, at least not on their antennules. The MAN of N. oerstedii is similar to that of most crustaceans in that the extent of serotonin-containing neurons is low and generally much less than in the protocerebral neuropils, OLs, and ALs (Langworthy et al., 1997; Schmidt and Ache, 1997; Sandeman et al., 1988). In summary, the stomatopod antennule is different from the caridean antennule in that it has three flagella, due to the fact that its lateral flagellum is bifurcated. Nonetheless, the stomatopod antennule has setal types common to the carideans, including aesthetasc, asymmetric, and simple setae. The stomatopod brain has many of the same neuropils as the carideans, including deutocerebral neuropils—the OLs, LANs, and MAN. Although these neuropils are present in stomatopods, they have some differences from those of the decapods: the LANs are relatively large and have extensive serotonergic innervation, while the MAN is

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relatively small and diffuse. Thus, it appears that stomatopods have the same two, highly organized, parallel antennular pathways as do the decapod crustaceans—the OL pathway and the LAN pathway.

Acknowledgements We thank Keith Burkmann for supplying stomatopods, Vivian Ngo for technical assistance, and Dr Manfred Schmidt for critical comments on a draft of the manuscript. This work was supported by the National Science Foundation under grant IBN-0077474, National Institute on Deafness and Other Communication Disorders grant DC00312, the Georgia Research Alliance, and the Georgia State University Research Program Enhancement Fund.

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