Insect mushroom bodies, particularly those of Drosophila, are the most accessible models for elucidating molecular and computational algorithms underlying learning and memory within genetically and connectomically defined circuits (e.g. Aso et al., 2014a; Senapati et al., 2019; Jacob and Waddell, 2020; Modi et al., 2020). An advantage of Drosophila is that it exemplifies a ground pattern organization that is largely consistent across hexapod lineages (Li and Strausfeld, 1997; Ito et al., 1998; Ito et al., 2014; Sinakevitch et al., 2001; Groh and Rössler, 2011; Montgomery and Ott, 2015).

Because of the importance of mushroom bodies in understanding the relevance of synaptic organization in sentience and cognition, recognizing evolutionary divergence of these centers would be expected to yield experimentally testable predictions about evolved modifications of circuitry in relation to ecological demands imposed on the species. However, insect mushroom bodies show a remarkably conserved organization, which makes them relatively unsuited for neuroevolutionary studies. There are some minor exceptions, to be sure: in honey bees and ants, calycal domains receive modality-specific afferents; in beetles, dietary generalists have more elaborate calyces than specialists; in aquatic beetles, a modality switch has replaced olfactory input with a visual input into the calyces (Gronenberg, 2001; Farris and Schulmeister, 2011; Lin and Strausfeld, 2012). Less attention has been given to neural arrangements comprising the mushroom body lobes (columns), although some studies have addressed distinctions in basal groups such as silverfish (Zygentoma), dragonflies and mayflies (Farris, 2005; Strausfeld et al., 2009).

The evolutionary stability of the insect mushroom body contrasts with the recent demonstration that mushroom bodies of malacostracan crustaceans have undergone substantial and often dramatic modification of the mandibulate (ancestral) mushroom body ground pattern (Stegner and Richter, 2011; Wolff and Strausfeld, 2015; Wolff et al., 2017; Strausfeld et al., 2020). Whereas Stomatopoda (mantis shrimps), the sister group of Eumalacostraca, possesses mushroom bodies that correspond to those of insects, equipped with calyces from which arise prominent columns (Wolff et al., 2017), the trend over geological time has been a reduction of those columnar components such that the ancestral ground pattern organization of parallel fibers and orthogonal Hebbian networks has morphed to provide planar arrangements within domed centers lacking columns (Wolff and Strausfeld, 2015; Sayre and Strausfeld, 2019; Strausfeld et al., 2020). These neuronal adaptations, nevertheless, share the property with insect mushroom bodies of being immunopositive to an antibody raised against the catalytic subunit of protein kinase A, encoded by the Drosophila gene DC0, that is required for effective learning and memory (Kalderon and Rubin, 1988; Skoulakis et al., 1993). Antibodies raised against DC0 are reliable identifiers of neuropils mediating learning and memory in arthropods and other phyla (Strausfeld et al., 2009; Wolff et al., 2012; Wolff and Strausfeld, 2015).

Whereas the evolutionary shift from columnar mushroom bodies to noncolumnar homologues is demonstrated across most decapod crustaceans, one lineage has until now defied unambiguous identification of a columnar or even a noncolumnar center. This is Brachyura, known in the vernacular as true crabs, here represented by Hemigrapsus nudus. Brachyura is a comparatively young lineage, recognized from fossils dating from the mid-Jurassic (Schweitzer and Feldmann, 2010; Guinot, 2019). Phylogenomics places the origin of Brachyura also as mid-to-late Jurassic (Wolfe et al., 2019). Brachyura is the most species-rich decapod clade, comprising 6793 currently known species (Ng et al., 2008). It is also a hugely successful lineage; extant species occupy benthic, littoral, estuarine, brackish, fresh water, terrestrial and even arboreal habitats. Many of these ecologies are defined by complex topographies (Hartnell, 1988; Lee, 2015).

Historically, identifying a mushroom body homologue in the crab’s brain has been problematic. Claims for homologous centers range from paired neuropils in the brain’s second segment, the deutocerebrum, later attributed to the olfactory system (Bethe, 1897), to an insistence that the crab’s reniform body is a mushroom body (Maza et al., 2016; Maza et al., 2021). Demonstrated 138 years ago in stomatopod crustaceans, the reniform body is a morphologically distinct center that coexists in the brain’s lateral protocerebrum adjacent to its columnar mushroom bodies (Bellonci, 1882; Thoen et al., 2020).

Until the present, observations of the varunid shore crab Hemigrapsus nudus have identified large anti-DC0-reactive domains occupying almost the entire rostral volume of its lateral protocerebrum but no evidence for mushroom bodies (Thoen et al., 2020). The affinity of these domains to anti-DC0 suggested, however, a cognitive center far more expansive than found in any other arthropod of comparable size, with the possible exception of the domed mushroom body of the land hermit crab Coenobita clypeatus (Wolff et al., 2012). Here we demonstrate that in the shore crab there are indeed paired mushroom bodies. But these have been ‘hiding in plain sight,’ having undergone an entirely unexpected neurological transformation that is opposite to the evolutionary trend toward a domed noncolumnar morphology shown in other lineages.

Here, we provide evidence at the level of neuronal arrangements showing that in the shore crab paired mushroom bodies have undergone an evolved transformation that is possibly unique to Arthropoda. They are inverted: their calyces reside deep within the lateral protocerebrum, a location allowing outward expansion of the mushroom body columns, which reach expanded cortex-like folds beneath the brain’s surface. The entire disposition of the shore crab’s mushroom bodies is opposite to that of any other crustacean, or insect, in which the calyces are situated under the rostral surface of the lateral protocerebrum with their columns extending downwards into deeper neuropil (Strausfeld et al., 2009; Wolff et al., 2017; Sayre and Strausfeld, 2019).

Traits defining neuronal organization demonstrate that the mushroom bodies of Stomatopoda phenotypically correspond to those defining the mushroom bodies of Drosophila (Wolff et al., 2017). The following description uses complementary methods that resolve those traits in Hemigrapsus nudus. Reduced silver staining demonstrates spatial arrangements of calycal and columnar neuropils and their detailed neuroarchitecture. Osmium-ethyl gallate treatment of intact brains resolves neuronal densities; and serial sections of these preparations provide the three-dimensional reconstructions shown throughout this account (see Materials and methods). Golgi mass-impregnations enable crucial insights into the exceptionally elaborate organization of mushroom body intrinsic neurons, identified as the phenotypic homologues of insect Kenyon cells. Immunohistology has been used to resolve DC0-immunopositive components of the mushroom body calyces and columns, and antibodies raised against GAD (glutamic acid decarboxylase) reveal putative levels of local inhibition. Antibodies raised against tyrosine hydroxylase (TH) and 5-hydroxytryptophan (5HT) demonstrate arborizations consistent with those of output and input neurons intersecting different levels of the Drosophila and stomatopod mushroom body columns.

We show that despite the varunid mushroom body’s unparalleled intricacy and its unique transformation of the ancestral ground pattern, its defining traits nevertheless demonstrate phenotypic homology with the mushroom bodies of insects and those of other crustaceans. We close with a Discussion proposing that the evolution of such intricacy, and the expansion of the lateral protocerebrum to accommodate mushroom body enlargement, is likely to have contributed novel circuits and, ultimately, to cognitive flexibility permitting varunid Brachyura to exploit complex ecological topographies at the interface of marine and terrestrial biotopes.

The Results are organized into eight sections, the first describing the overall location and structure of the varunid mushroom body. This is followed by the identification of its neural components and their arrangements as elaborate networks comprising the paired calyces. Descriptions of the internal organization of the calyces’ rostral extensions, and their two prominent columns, culminates with an explanation of the cortex-like organization of overlying gyri to which the columns project. The final observations address the relationship of the mushroom body to the reniform body, a center common to malacostracan crustaceans. To provide a three-dimensional understanding of this highly intricate system, descriptions refer to locations and arrangements within a reconstructed lateral protocerebrum and its major components (see Materials and methods).

The varunid lateral protocerebrum (Figures 1–3)

In crabs, as is typical of stomatopods and most decapod crustaceans, the most anterior part of its brain, the protocerebrum, is flanked each side by a lateral outgrowth that expands into an enlargement of the eyestalk immediately proximal to the retina (Figure 1A). This volume of brain comprises the lateral protocerebrum (Figures 1A and 2A). The lateral protocerebrum is connected to the midbrain proper by the eyestalk nerve composed of axons relaying information to and from the midbrain.

Figure 1

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Figure 2

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Brains treated with osmium-ethyl gallate or with reduced silver (Figures 2A and 3) resolve the varunid lateral protocerebrum as a hump-shaped volume divided into a rostral and caudal part. The rostral lateral protocerebrum (RLPR), once referred to as the terminal medulla, a now defunct term (Strausfeld, 2020), is dominated by two centers: the mushroom bodies and the smaller reniform body. There are a small number of other satellite neuropils near the origin of the eyestalk nerve. The mushroom body is immediately recognizable by its intensely osmiophilic calyces from which arise columns that extend outwards, branching into a cortex-like neuropil composed of gyri (Figure 2A). These occupy the outermost volume of the RLPR. The reniform body comprises an independent system of interconnected neuropils situated laterally constrained to a volume of the RLPR that extends from its dorsal to its ventral surface (Thoen et al., 2020). In longitudinal sections of the RLPR parallel to its rostro-caudal plane, the reniform body’s prominent axon bundle, the pedestal, is seen in cross-section (RB in Figures 2 and 3).

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The crab’s RLPR corresponds to that volume of the insect protocerebrum containing the mushroom body and its immediately associated neuropils, such as the lateral horn (Figure 1B,C). As in stomatopods and decapods, the crab’s RLPR receives terminals of olfactory projection neurons originating from both the ipsi- and contralateral olfactory (antennular) lobes located in the brain’s second segment, the deutocerebrum (Figure 1). The axons of more than a thousand projection neurons are organized into several discrete fascicles bundled together to provide the olfactory globular tract (OGT). The left and right OGTs extend superficially across the midbrain to meet just dorsal to the central body. There, each tract bifurcates to provide inputs to each lateral protocerebrum from both the ipsi- and contralateral olfactory lobes (Figure 1). The eyestalk nerve also carries other efferent axons to the lateral protocerebrum: these include relay neurons from other sensory neuropils, relays from the opposite lateral protocerebrum and optic lobes, and interneurons relaying from various midbrain neuropils.

The caudal volume of the lateral protocerebrum (CLPR) is mainly associated with outputs from the optic lobe neuropils (Figure 2A). Most of these originate from the lobula, each bundle of outgoing axons representing a population of one of the numerous morphological types of columnar neurons. Bundles segregate to a system of interconnected neuropils called optic glomeruli, as occurs in insects (Okamura and Strausfeld, 2007). Further relays from the glomeruli send information centrally to the midbrain via the eyestalk nerve. The nerve also carries to the midbrain the axons of efferent neurons from the lateral protocerebrum’s rostral neuropil, from the mushroom bodies and their associated gyri and from CLPR neuropils associated with the optic glomeruli.

The calyces and their origin from globuli cells (Figures 2–4)

If centers are claimed as phenotypic homologues of insect mushroom bodies, then those centers are expected to have the following categories of neurons irrespective of their divergence from the ancestral ground pattern as represented in the allotriocarid clade Cephalocarida (Stegner and Richter, 2011). These categories are: (1) intrinsic neurons, the most populous of which are Kenyon cells defining the mushroom body’s volume, shape, and subdivisions; (2) extrinsic neurons providing inputs from sensory centers (Kanzaki et al., 1989; Lin and Strausfeld, 2012; Li et al., 2020a), as well as protocerebral regions encoding high-level multisensory information (Li and Strausfeld, 1999); and (3) output neurons that encode information computed by levels of the mushroom body lobes (Li and Strausfeld, 1999; Turner et al., 2008). The latter are referred to here as MBONs, adopting the term from studies on Drosophila (Aso et al., 2014a; Aso et al., 2014b).

Intrinsic neurons supplying the Hemigrapsus nudus calyces correspond to insect Kenyon cells. As do insect Kenyon cells, these originate from the brain’s smallest and most crowded population of neuronal cell bodies, the globuli cells (Figures 2C, 3A,D and 4). In other pancrustaceans, globuli cell perikarya have been shown as dense clusters situated at the rostral surface of the lateral protocerebrum, usually near its proximal margin or immediately above the mushroom body calyces, as in Drosophila or Stomatopoda, or, in malacostracans with eyestalks, close to the lateral protocerebrum’s attachment to the eyestalk nerve. In H. nudus, however, globuli cell perikarya are found in a completely different and surprising locality: a densely populated ‘hidden’ volume tucked between the proximal margin of the lobula and the most distal border of the lateral protocerebrum (Figures 2C, 3A and 4A–C). As in other pancrustaceans, these perikarya are the smallest perikarya of the brain, in the varunid ranging from 5 to 7 μm in diameter (compared in Figure 3A). In crabs used for these studies (carapace widths of 4–6 cm), we estimate that this perikaryal cluster accounts for at least 22,000 mushroom body intrinsic neurons shared by the two calyces (see Materials and methods). Cytological distinctions amongst cells in the cluster, denoted by their appearance after osmium-ethyl gallate staining (Wigglesworth, 1957) or reduced silver (Figure 4D–G), suggest that new globuli cells are being continuously generated from ganglion mother cells situated at the cluster’s periphery (Figure 4D–G). Continuous generation of interneurons supplying the antennular lobes, and in the lateral protocerebrum, has been previously reported for the shore crab Carcinus maenas (Schmidt, 1997).

Figure 4

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Cell body fibers (neurites) extending from the globuli cell cluster are tightly packed into two prominent bundles, called globuli cell tracts, one projecting obliquely ventrally the other obliquely dorsally to supply each of the two adjacent calyces (Figure 3—figure supplement 1). Neurites comprising these tracts fan out to supply each calyx with its ensembles of mostly orthogonally arranged dendritic networks (Figure 3B; Figure 5—figure supplement 1). The networks, which are resolved by silver stains, comprise overlapping populations of intrinsic neuron processes extending amongst a mosaic of many thousands of discrete microglomeruli (Figures 3B and 5A). These are assumed to be sites of synaptic convergence (Figure 5—figure supplement 1), corresponding to microglomerular organization in the calyces of insect, stomatopod and caridean mushroom bodies (Yasuyama et al., 2002; Sjöholm et al., 2006; Groh and Rössler, 2011; Wolff et al., 2017; Sayre and Strausfeld, 2019). As described in the following section, components of microglomeruli include the terminals of afferent relays originating from the olfactory lobes, terminals from neuropils associated with the visual system, including the reniform body, and the endings of centrifugal neurons from the midbrain.

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Serial sections, cut parallel to the rostro-caudal plane of the lateral protocerebrum and stained with reduced silver, suggest the calyces are planar neuropils (Figure 3B). But this is deceptive, as shown by sections demonstrating how the deep calycal cytoarchitecture extends rostrally to merge with the base of its column (Figure 3C). The total extent and volume of calycal neuropil is surprisingly large as can be seen in brains treated with osmium-ethyl gallate, a technique that selectively colors cell membranes black (Wigglesworth, 1957). The method reveals volumes of neuropil in shades of black to light gray, the darker the neuropil, the more densely packed its neuronal processes (Figures 2A and 3D,E; Figure 6—figure supplement 1B). The darkness of the calyces reflects the dense packing of the many thousands of intrinsic neurons comprising them. Volumes that are paler are composed of neuronal processes that are stouter and contribute less stainable membrane, as in the overlying gyri.

Neuronal organization of the calyces (Figures 5–8)

Computer-generated reconstructions and rotations of osmium-ethyl gallate stained sections (see Materials and methods and Figure 3—figure supplement 1) demonstrate that the calycal volumes are topographically elaborate. Each calyx is stratified at deeper levels of the lateral protocerebrum's rostral volume, but, where calycal neuropils extend forwards, their upper borders are defined by irregular arrangements of small peaks and troughs. Two prominent columns, one from each calyx, extend outwards and branch into the overlying gyri (Figure 3E). Reduced silver material and Golgi impregnations resolve their elaborate internal organization, which is described in Section 5, below.

Intrinsic neuron processes and terminals of efferent interneurons are the main constituents of the varunid calyces (Figure 5B, Figure 5—figure supplement 1; Figure 6B–G, Figure 6—figure supplement 1). They receive inputs from neurons primarily from the deutocerebrum (Figure 5—figure supplement 1B), but also from the optic lobes and from its associated neuropils, such as the reniform body (Figure 6—figure supplement 2), as also shown for Stomatopoda (Thoen et al., 2020). Golgi impregnations and osmium-ethyl gallate staining demonstrate that calyces, across their horizontal and vertical extent, are composed of discrete domains of morphologically distinct networks comparable to the discrete modality-specific zones of Kenyon cell dendrites that define the insect calyces (Ehmer and Gronenberg, 2002; Strausfeld, 2002).

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Most calycal neuropil is characterized by its dense arrangement of small almond-shaped subunits (Figure 5A), here referred to as microglomeruli following the term given to comparable elements described from insect mushroom body calyces (Yasuyama et al., 2002; Sjöholm et al., 2006; Groh and Rössler, 2011). However, some peripheral volumes of the calyces comprise more distributed arrangements (Figure 5A,B). These appear to receive terminals from axons of relay neurons emerging from fascicles in the eyestalk nerve that are not part of projections from the olfactory lobes (Figure 3B).

Discrete fields comprising intrinsic cell processes are recognized by their distinctive and particular morphologies (Figure 6B–G, Figure 6—figure supplement 1). Each morphological type is defined by the patterning of its initial branches from its globuli cell neurite and the subsequent reticular trajectories made by their tributaries down to the details of their final specializations. The latter indicate points of functional connection and are morphologically comparable to the claws, clasps, and dendritic spines that decorate the dendritic branches of Kenyon cells in Drosophila and other insects (Ito et al., 1998; Strausfeld and Li, 1999; Strausfeld, 2002) and the dendrites of homologous intrinsic neurons in the mushroom bodies of marine hermit crabs (Strausfeld and Sayre, 2020). As in those taxa, in the varunid calyx these specializations invariably relate to the distribution and patterning of its microglomeruli (Figure 5).

Microglomeruli revealed by reduced silver beautifully match the size and mosaic arrangement of microglomeruli resolved by their intense immunoreactivity to antibodies raised against DC0 (Figure 5). The bundled branches of intrinsic neurons resolved by antibodies against α-tubulin (Figure 5D,E) further resolve microglomeruli as participants of an elaborate organization of orthogonal networks that correspond to distinct morphological types of intrinsic neurons revealed as single cells by Golgi-impregnation (Figure 6B,C, Figure 6—figure supplement 1). Each microglomerulus signifies the site of an incoming terminal’s presynaptic bouton or varicosity (Figure 5—figure supplement 1). Combined anti-synapsin immunoreactivity and F-actin labeling (Figure 5F–G) further demonstrates the profusion of postsynaptic sites belonging to intrinsic neurons that converge at each microglomerulus. The densely packed presynaptic specializations further confirm the status of a microglomerulus as the site of massive transfer of information from presynaptic terminals onto many postsynaptic channels.

As remarked, the shapes and arrangements of intrinsic neurons and their synaptic specializations contribute to orthogonal networks in the calyces and define calycal domains. However, the domains are not arranged as neatly adjacent territories as in the insect calyx (Gronenberg, 2001). Instead, the varunid calyx can reveal domains that extend vertically or horizontally (Figure 5—figure supplement 1), or comprise unexpectedly elaborate arrangements that are understood only after they are matched to an appropriately oriented osmium-ethyl gallate section (Figure 6—figure supplement 1A,B). One of the most challenging of these arrangements consists of a population of intrinsic neurons defining a cylindrical domain that splays out as it extends toward the proximal margin of the lateral protocerebrum (Figure 6A). The entire ensemble comprises densely branched narrow-field arborizations that surround the initial branches of a subset of globuli cell neurites (Figure 6A,C,D). The core leads to a terminal expansion that is confluent with the base of one of the two large columns that extends to the overlying gyri. Serial reduced-silver sections reveal this as originating only from calyx 2, indicating that calyx 2 differs from calyx 1 with regard to at least one population of intrinsic neurons. As in many insect lineages where there are paired calyces, it is such subtle distinctions that demonstrate disparate organization across paired calyces (Farris and Strausfeld, 2003). Reduced-silver stains showing axon tracts reveal inputs to calyx 2 originating from a tract that recruits its axons from the reniform body and the medulla. Collaterals from this tract spread into a deep level of calyx 2 neuropil where they spread across microglomeruli bordering one side of the incoming neurites from the globuli cell tract (Figure 6—figure supplement 2). The correspondence of this arrangement with insect calyces is considered in the Discussion.

There is one critical exception to the generalization that calycal subdivisions are denoted by different types of intrinsic neurons. The exception is that there is a system of neurons that arborizes uniformly throughout both calyces. These arborizations belong to GAD-immunoreactive local processes originating from a dorsal cluster of large cell bodies, each of which provides an anaxonal field that arborizes throughout both calyces, and into each of its domains. (Figure 7A,B). These densely packed processes are exceptional in that they do not obey the orthogonal leitmotif of the calyx, as revealed by anti-α-tubulin (Figure 7C,D), nor do they reflect the mosaic arrangement of microglomeruli (Figure 7C–E). Instead, anti-GAD-immunoreactive branchlets populate all the calycal domains and do so in great numbers, the totality suggesting that local inhibitory elements are distributed amongst all intrinsic cell processes irrespective of whether they are in immediate proximity to or distant from a microglomerulus (Figure 7C–E).

Figure 7

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Whereas GAD immunohistology does not distinguish local calycal domains, these are indicated by the inhomogeneity of fields of 5HT- and TH-immunoreactivity across the calyces (Figure 8). The neurons providing these fields also appear to be specific to the calyces and not part of a wider system that extends to other regions of the lateral protocerebrum. Those more distributed systems include densely branched arborizations that populate the optic lobe’s lobula and 5HT-immunoreactive neurons that arborize in certain neuropils of the caudal lateral protocerebrum as well as the gyri. These are likewise distinct from the 5HT- and TH-immunoreactive fields that intersect the mushroom body columns, which are described next.

Figure 8

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Transition from the calyces to the columns (Figures 9 and 10)

Thus far, we have demonstrated the composition of the calyces as approximately orthogonal networks provided by intrinsic neurons and their arrangements with microglomeruli.

Stomatopod and shrimp mushroom bodies demonstrate a clear transition of neuronal organization between the calyx and its columnar extensions (Wolff et al., 2017; Sayre and Strausfeld, 2019). In Hemigrapsus nudus there is no abrupt transition (Figure 3). This ambiguity is seconded in brains treated with osmium-ethyl gallate, which show lateral protocerebral neuropil supplied by globuli cell neurites gradually fading from black to gray, signifying decreasing densities of neuronal membrane, finally giving way to the uniformly pale aspect of the overlying gyri (Figures 2A and 3D,E). 3D reconstructions of the dark volumes show a layered arrangement at deeper layers of the calyces changing to a more corrugated appearance, particularly at its outer surface (Figure 3—figure supplement 1). Silver impregnations show that the base and some of the initial length of a column are clearly reticulated (Figure 9C). However, these are not microglomeruli. Double Golgi impregnation shows that these networks receive contributions from two sources: the extensions of axon-like processes from intrinsic neuron fields in the calyx; and intrinsic arborizations, many extremely complicated, that originate from thin neurites, presumed to belong to globuli cells (Figure 9—figure supplement 1). An additional system of networks extends outwards to flank each calyx’s column (Figure 9B). These finger-like extensions originate from a tangle of processes situated at a deeper level of the calyces (Figure 9B) that does not reflect any relationships with microglomeruli. The likely origin of this system (Figure 9B) is not from globuli cells but from a small cluster of cell bodies, some DC0-immunoreactive, situated caudally close to the inner margin of the lobula (Figure 2C). We interpret this tangle of processes (Figure 9B) as belonging to interstitial local interneurons providing elements to the columns at a level limited to their emergence from the calyces. The topology of these arrangements, including the small column-specific intrinsic arborizations (Figure 9—figure supplement 1E–K) is reminiscent of interstitial neurons interposed between the calyces and pedunculus of the honeybee (Strausfeld, 2002).

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The origins of the columns and smaller extensions from the calyces are further demonstrated by selected levels of silver-stained brains. Serial sections resolve the uneven rostral border of the calyces and the two columns reaching outwards toward the overlying gyri (Figure 9—figure supplement 2). The series also demonstrates the agreement between how silver and osmium-ethyl gallate resolve the origins of the columns from the two calyces. These aspects emphasize that the seemingly irregular morphology of the rostral part of the calyces is not irregular by happenstance but is a defining feature of the lateral protocerebrum.

Reduced silver preparations demonstrate that a column is subdivided into parallel components as in insect mushroom bodies, where each subdivision has a distinctive arrangement of intrinsic neurons (Sjöholm et al., 2005; Strausfeld, 2002; Tanaka et al., 2008) and is defined by distinctive patterns of gene expression (Yang et al., 1995). What is unexpected is the degree of elaboration observed in the varunid mushroom body. Golgi impregnations demonstrate that, compared with other pancrustaceans, mushroom bodies in a crab are stunningly complex with regard to both their calyces and columns. The varunid’s columns consist of parallel intrinsic cell fibers that are almost obscured by numerous other arrangements, the variety of which is compatible with the variety of their morphological fields in the calyces. A silver-stained column originating from calyx 2 (Figure 9C) and one revealed by mass Golgi impregnation (Figure 9D) both suggest longitudinal subdivisions of the columns. Golgi impregnations resolve discrete and complicated systems of processes, some providing bridgelike connections across them (Figure 9E), others having irregular branches, lateral prolongations, and bundled parallel fibers (Figure 9E,F). In addition, larger dendrite-like elements arising from stout processes within the columns suggest that those processes may provide outputs to the overlying gyri (Figure 9D,F), a feature to which we return in Section 7.

Golgi impregnation has its own peculiar limitations: it is stochastic, revealing probably less than 1% of the total population of neurons, even after double impregnation. Nevertheless, mass impregnation further suggests that the columns from the two calyces are not identical. Although networks of intrinsic cells in both columns receive terminals from branches of the olfactory globular tract, there are clear differences between the columns from calyx 1 and calyx 2. Column 2 is broader and more difficult to discern in reduced silver, in part because its intrinsic processes are extremely thin, many less than a micron in diameter (see section Transitions to gyri below). At first sight, the organization of processes comprising column 2 appears to be chaotic. However, closer inspection reveals subsets of different arrangements of its dendrites and collaterals disposed across its width and at different levels along it (Figure 9—figure supplement 1A–C). Reciprocal channels to the column from the gyri are suggested by the presence of descending axons flanking the column, providing varicose collaterals into it (Figure 9—figure supplement 1A). These varieties of columnar arrangements are further considered in the Discussion.

Fields of 5HT- and TH-immunoreactive dendrites extend across columns. Here we show fields relating to column 1 from calyx 1 that provide clear resolution of their arrangements from the calycal origin to the interface with gyri (Figure 10). Fields are arranged at specific levels along the column, and they intersect different domains across the column, as occurs across the parallel subdivisions of the columns (lobes) of mushroom bodies in Drosophila and other insects (Nässel and Elekes, 1992; Liu et al., 2012; Aso et al., 2014a; Hamanaka et al., 2016; Tedjakumala et al., 2017). The first of these fields occurs at the origin of the column from the calyx (Figure 10A,B) above where the neat orthogonal arrangements seen at deeper levels give way to the first appearance of looser arrangements, including parallel fibers (Figure 10C,F). Anti-5HT- and anti-TH-immunoreactive fields are clearly segregated (Figure 10A and D) at or near the column origin but begin to overlap at more rostral levels closer to the gyri, at locations where osmium-ethyl gallate preparations show the columns branching, with tributaries extending to overlying gyri. At those levels, immunoreactive processes appear to spread laterally into the gyri themselves (Figure 10G,H).

Figure 10

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Organization of gyriform neuropils (Figures 11 and 12)

Five distinct gyri comprise the outer layer of the rostral lateral protocerebrum (Figure 11A). But only four gyri are associated with the two calyces. A fifth gyrus, termed here the proximal gyrus, is situated near the entry of the eyestalk nerve. Its neuronal organization distinguishes it as entirely separate from the mushroom body (Figure 11A,F).

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Mass impregnations demonstrate at this outer level of the lateral protocerebrum large numbers of efferent neurons and their dendritic processes, many of which originate from clusters of loosely arranged patches of perikarya that extend over the outer surface of the lateral protocerebrum (Figure 11—figure supplement 1). Although stochastically impregnated, the abundance and variety of these neurons suggests the gyri contain dense arrangements of efferent (output) and local interneurons (Figure 11—figure supplement 1D–I). Golgi impregnations reveal swathes of axons extending across the gyri, suggesting lateral interactions amongst them. Oblique palisades of dendritic trees, which have branching patterns peculiarly reminiscent of vertebrate pyramidal cells (Figure 11B), send axons to fascicles that extend to the midbrain via the eyestalk nerve.

Like deeper levels of the lateral protocerebrum, gyri comprising this superficial level of the lateral protocerebrum are supplied by terminals of afferent neurons, the axons of which radiate out from the eyestalk nerve. Many of these terminals originate from discrete fascicles that comprise the olfactory globular tract, denoting not only their identity as axons of projection neurons from the olfactory lobes but suggesting that different classes of projection neurons comprise different fascicles (Figure 11D,E). The terminals of projection neurons are denoted by their swollen boutons, the different morphologies of which are distinctive. For example, terminals spreading out over the surface of gyrus 1, belonging to calyx 1, have rough ball-shaped profiles that are distinct from larger smooth boutons from extremely slender axons supplying the proximal gyrus (pGy in Figure 11A,B,E). That gyrus’s large allatostatin-immunoreactive processes and the paucity, even absence, of GAD-immunoreactive networks suggests its complete independence from the mushroom body’s organization (Figure 11F). Nor, as shown by osmium-ethyl gallate, is this gyrus associated with any of the calyces’ outward extensions.

In addition to axons extending from the eyestalk nerve providing afferents to superficial levels in the gyri, all levels in the rostral lateral protocerebrum receive an abundant supply of terminals, including many penetrating to deep levels of the calyces (Figure 11G–K). Again, it is worth bearing in mind when viewing the images in Figure 9G,H that Golgi impregnations likely reveal not more than a few percent of the constituent neurons providing these already dense arrangements of incoming fiber tracts.

Transitions to gyri

We have shown that four gyri overlie two large columns, each extending from one of the two calyces, as do occasional smaller finger-like extensions. Osmium-ethyl gallate demonstrates that the columns bifurcate close to the gyri (Figures 2A and 3E), a feature also resolved in serial reduced-silver-stained sections (Figure 9—figure supplement 1). However, despite the dark staining by osmium that suggests a well-defined transition between columns and gyri, neither silver staining nor Golgi impregnations provide a sharply defined inner margin of a gyrus or an obvious termination of the column. Here we consider neural arrangements that denote the ambiguity of the interface between the mushroom body’s columns and their corresponding gyri.

Tangential sections across the gyri demonstrate novel and as yet puzzling aspects of their composition (Figure 12). One is the presence of dendritic trees, as in Figure 12B, here represented by a neuron, the axon of which joins with others extending to the lateral protocerebral root of the eyestalk nerve. Its dendrites overlap elaborate networks of finely branching processes that terminate in small bead-like swellings suggestive of presynaptic specializations. This network (Figure 12B) derives from extremely thin processes that ascend into the gyrus from outward extensions of the calyx (lower bracket in Figure 12B). Further examples (Figure 12C) show that the dendrites of a gyrus's efferent neurons overlap other arrangements of putative ascending presynaptic fields. In this example, the fields do not appear to be part of an intervening synaptic network, but instead their disposition with the efferent dendritic trees suggests that they likely terminate there (Figure 12C). In addition, highly restricted systems of mixed spiny and beaded arborizations suggest local anaxonal neurons that may provide synaptic interfaces between such terminal systems arising from the columns and the dendrites of gyral efferent neurons (Figure 12D,E). Evidence that arborizations predominantly equipped with presynaptic specializations likely originate from the calyces is indicated by densely packed elements situated distally in the columns. These are equipped with short digiform specializations arranged as small basket-like receptacles matching afferent terminals of unknown origin (Figure 12G,H).

Figure 12 with 1 supplement see all

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The rare Golgi impregnation that resolves one of the columns as a recognizable ensemble of heterogeneous elements within the column’s boundaries also demonstrates that its terminus is 'open', meaning contiguous with the gyral neuropil into which some of its fibers project (Figure 12—figure supplement 1A,B). Also fortuitous are preparations that demonstrate the terminus of a column coinciding with bundled endings from the olfactory globular tract. Such a preparation (Figure 12—figure supplement 1B) also shows that the terminus of the column is open to gyral neuropil around it and that processes extend from the column into the gyrus. Combined labeling with anti-synapsin and actin at the terminus of the column (Figure 12—figure supplement 1C) further indicates that sizes and densities of sites indicating synaptic convergence differ systematically across it. The flanking gyral neuropils also show substantial variation in synaptic densities. Inputs to the gyri do not, however, derive solely from calycal columns and afferents from the olfactory globular tract. Reduced silver preparations show that tracts from caudal neuropil associated with the optic lobe extend rostrally to the superficial levels of a gyrus (Figure 12—figure supplement 1D), yet another indication of the complexity of organization and its multimodal nature at all levels of the mushroom body and its gyri. The possible significance of this is considered below in the Discussion.

The reniform body (Figure 13)

We have shown that the lateral protocerebrum is divided into a rostral and caudal part (RLPR and CLPR), the former containing the paired mushroom bodies. The mushroom bodies are not, however, the sole components of the RLPR. The gyrus nearest the RLPR’s junction with the eyestalk nerve, termed the proximal gyrus (pGy), is distinct and probably to some degree functionally separate from the gyri associated with the mushroom bodies (Figure 11F). In addition, another prominent system of circumscribed neuropils is situated near the lateral border of the RLPR, arising from a dorsal group of relatively small perikarya almost adjacent to the optic lobes. These neuropils comprise the reniform body, a center identified in stomatopods by Bellonci, 1882, who distinguished it from the very prominent mushroom bodies in that taxon. The same center has been described in detail in the stomatopods Neogonodactylus oerstedii, Gonodactylus smithii, and G. chiragra, and the carideans Lebbeus groenlandicus and Alpheus bellulus. In all these species, the reniform body has the same morphology: a densely populated bundle of axons, referred to as the pedestal, extends from the dorsal surface of the RLPR to its ventral surface, providing four discrete neuropils called the initial (dorsal), lateral, and the ventrally situated proximal and distal zones (Wolff et al., 2017; Thoen et al., 2020; Sayre and Strausfeld, 2019; Strausfeld et al., 2020). In all the cited taxa, the reniform body is situated between the columnar mushroom body and the lobula.

Precisely the same placement is observed in the varunid lateral protocerebrum (Figure 13). The reniform body occupies a location lateral to the ensembles of neurons that constitute the mushroom body gyri (Figures 2 and 13). Reconstructed from serial osmium-ethyl gallate sections, the reniform body’s pedestal lies immediately above, that is, rostral to, the two tracts of neurites from the globuli cell cluster (the globuli cell tract) that provide intrinsic neurons to the mushroom body calyces (Figure 13C). In Hemigrapsus nudus, the reniform body pedestal extends from a cluster of cell bodies at the dorsal surface of the lateral protocerebrum and extends almost to the lateral protocerebrum’s ventral surface, bifurcating into two main tributaries about half way along its length (Figure 13F). These give rise to the lateral, distal and proximal zones, each zone composed of substantial volumes of arborizations (Figure 13D). The reniform body is thus entirely distinct from the mushroom bodies and is situated distally with respect to their centrally disposed calyces (Figure 13E). The reniform body’s thick pedestal, albeit columnar in form, is distinct from a mushroom body column in that its constituent axons are naked, lacking spines or other specializations that define a mushroom body. The reniform body’s four zones of branching collaterals contribute to structures reminiscent of microglomeruli. However, they are of larger girth, and synapsin-actin labeling shows them to have significantly fewer converging presynaptic elements than a calycal microglomerulus (Figure 13—figure supplement 1D,E). Despite these clear differences from the mushroom body, the reniform body is strongly immunoreactive to anti-DC0, showing that it too is a learning and memory center, as proposed by Maza et al., 2016.

Figure 13 with 2 supplements see all

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Source data are preserved on vibratome immunostained sections maintained at 4° C in investigator's laboratory (NJS). Likewise, original data of selectively impregnated neurons (Golgi material) and silver stains are curated and maintained in the NJS laboratory.

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Author details

1. Nicholas Strausfeld

   Department of Neuroscience, University of Arizona, Tucson, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   flybrain@neurobio.arizona.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1115-1774

2. Marcel E Sayre

   1. Lund Vision Group, Department of Biology, Lund University, Lund, Sweden
   2. Department of Biological Sciences, Macquarie University, Sydney, New South Wales, Australia

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2667-4228

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