Response to Comment on "The lower Cambrian lobopodian Cardiodictyon resolves the origin of euarthropod brains".

Budd et al. challenge the identity of neural traces reported for the Cambrian lobopodian Cardiodictyon catenulum. Their argumentation is unsupported, as are objections with reference to living Onychophora that misinterpret established genomic, genetic, developmental, and neuroanatomical evidence. Instead, phylogenetic data corroborate the finding that the ancestral panarthropod head and brain is unsegmented, as in C. catenulum.

The lower Cambrian lobopodian Cardiodictyon resolves the origin of euarthropod brains.

For more than a century, the origin and evolution of the arthropod head and brain have eluded a unifying rationale reconciling divergent morphologies and phylogenetic relationships. Here, clarification is provided by the fossilized nervous system of the lower Cambrian lobopodian Cardiodictyon catenulum, which reveals an unsegmented head and brain comprising three cephalic domains, distinct from the metameric ventral nervous system serving its appendicular trunk. Each domain aligns with one of three components of the foregut and with a pair of head appendages. Morphological correspondences with stem group arthropods and alignments of homologous gene expression patterns with those of extant panarthropods demonstrate that cephalic domains of C. catenulum predate the evolution of the euarthropod head yet correspond to neuromeres defining brains of living chelicerates and mandibulates.

A robust method for particulate detection of a genetic tag for 3D electron microscopy.

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.

Shore crabs reveal novel evolutionary attributes of the mushroom body.

Neural organization of mushroom bodies is largely consistent across insects, whereas the ancestral ground pattern diverges broadly across crustacean lineages resulting in successive loss of columns and the acquisition of domed centers retaining ancestral Hebbian-like networks and aminergic connections. We demonstrate here a major departure from this evolutionary trend in Brachyura, the most recent malacostracan lineage. In the shore crab Hemigrapsus nudus, instead of occupying the rostral surface of the lateral protocerebrum, mushroom body calyces are buried deep within it with their columns extending outwards to an expansive system of gyri on the brain's surface. The organization amongst mushroom body neurons reaches extreme elaboration throughout its constituent neuropils. The calyces, columns, and especially the gyri show DC0 immunoreactivity, an indicator of extensive circuits involved in learning and memory.

Mushroom bodies in Reptantia reflect a major transition in crustacean brain evolution.

Brain centers possessing a suite of neuroanatomical characters that define mushroom bodies of dicondylic insects have been identified in mantis shrimps, which are basal malacostracan crustaceans. Recent studies of the caridean shrimp Lebbeus groenlandicus further demonstrate the existence of mushroom bodies in Malacostraca. Nevertheless, received opinion promulgates the hypothesis that domed centers called hemiellipsoid bodies typifying reptantian crustaceans, such as lobsters and crayfish, represent the malacostracan cerebral ground pattern. Here, we provide evidence from the marine hermit crab Pagurus hirsutiusculus that refutes this view. P. hirsutiusculus, which is a member of the infraorder Anomura, reveals a chimeric morphology that incorporates features of a domed hemiellipsoid body and a columnar mushroom body. These attributes indicate that a mushroom body morphology is the ancestral ground pattern, from which the domed hemiellipsoid body derives and that the "standard" reptantian hemiellipsoid bodies that typify Astacidea and Achelata are extreme examples of divergence from this ground pattern. This interpretation is underpinned by comparing the lateral protocerebrum of Pagurus with that of the crayfish Procambarus clarkii and Orconectes immunis, members of the reptantian infraorder Astacidea.

The reniform body: An integrative lateral protocerebral neuropil complex of Eumalacostraca identified in Stomatopoda and Brachyura.

Mantis shrimps (Stomatopoda) possess in common with other crustaceans, and with Hexapoda, specific neuroanatomical attributes of the protocerebrum, the most anterior part of the arthropod brain. These attributes include assemblages of interconnected centers called the central body complex and in the lateral protocerebra, situated in the eyestalks, paired mushroom bodies. The phenotypic homologues of these centers across Panarthropoda support the view that ancestral integrative circuits crucial to action selection and memory have persisted since the early Cambrian or late Ediacaran. However, the discovery of another prominent integrative neuropil in the stomatopod lateral protocerebrum raises the question whether it is unique to Stomatopoda or at least most developed in this lineage, which may have originated in the upper Ordovician or early Devonian. Here, we describe the neuroanatomical structure of this center, called the reniform body. Using confocal microscopy and classical silver staining, we demonstrate that the reniform body receives inputs from multiple sources, including the optic lobe's lobula. Although the mushroom body also receives projections from the lobula, it is entirely distinct from the reniform body, albeit connected to it by discrete tracts. We discuss the implications of their coexistence in Stomatopoda, the occurrence of the reniform body in another eumalacostracan lineage and what this may mean for our understanding of brain functionality in Pancrustacea.

Mushroom bodies in crustaceans: Insect-like organization in the caridid shrimp Lebbeus groenlandicus.

Paired centers in the forebrain of insects, called the mushroom bodies, have become the most investigated brain region of any invertebrate due to novel genetic strategies that relate unique morphological attributes of these centers to their functional roles in learning and memory. Mushroom bodies possessing all the morphological attributes of those in dicondylic insects have been identified in mantis shrimps, basal hoplocarid crustaceans that are sister to Eumalacostraca, the most species-rich group of Crustacea. However, unless other examples of mushroom bodies can be identified in Eumalacostraca, the possibility is that mushroom body-like centers may have undergone convergent evolution in Hoplocarida and are unique to this crustacean lineage. Here, we provide evidence that speaks against convergent evolution, describing in detail the paired mushroom bodies in the lateral protocerebrum of a decapod crustacean, Lebbeus groenlandicus, a species belonging to the infraorder Caridea, an ancient lineage of Eumalacostraca.

Multiple spectral channels in branchiopods. I. Vision in dim light and neural correlates.

Animals that have true color vision possess several spectral classes of photoreceptors. Pancrustaceans (Hexapoda+Crustacea) that integrate spectral information about their reconstructed visual world do so from photoreceptor terminals supplying their second optic neuropils, with subsequent participation of the third (lobula) and deeper centers (optic foci). Here, we describe experiments and correlative neural arrangements underlying convergent visual pathways in two species of branchiopod crustaceans that have to cope with a broad range of spectral ambience and illuminance in ephemeral pools, yet possess just two optic neuropils, the lamina and the optic tectum. Electroretinographic recordings and multimodel inference based on modeled spectral absorptance were used to identify the most likely number of spectral photoreceptor classes in their compound eyes. Recordings from the retina provide support for four color channels. Neuroanatomical observations resolve arrangements in their laminas that suggest signal summation at low light intensities, incorporating chromatic channels. Neuroanatomical observations demonstrate that spatial summation in the lamina of the two species are mediated by quite different mechanisms, both of which allow signals from several ommatidia to be pooled at single lamina monopolar cells. We propose that such summation provides sufficient signal for vision at intensities equivalent to those experienced by insects in terrestrial habitats under dim starlight. Our findings suggest that despite the absence of optic lobe neuropils necessary for spectral discrimination utilized by true color vision, four spectral photoreceptor classes have been maintained in Branchiopoda for vision at very low light intensities at variable ambient wavelengths that typify conditions in ephemeral freshwater habitats.

Representation of the stomatopod's retinal midband in the optic lobes: Putative neural substrates for integrating chromatic, achromatic and polarization information.

Stomatopods have an elaborate visual system served by a retina that is unique to this class of pancrustaceans. Its upper and lower eye hemispheres encode luminance and linear polarization while an equatorial band of photoreceptors termed the midband detects color, circularly polarized light and linear polarization in the ultraviolet. In common with many malacostracan crustaceans, stomatopods have stalked eyes, but they can move these independently within three degrees of rotational freedom. Both eyes separately use saccadic and scanning movements but they can also move in a coordinated fashion to track selected targets or maintain a forward eyestalk posture during swimming. Visual information is initially processed in the first two optic neuropils, the lamina and the medulla, where the eye's midband is represented by enlarged regions within each neuropil that contain populations of neurons, the axons of which are segregated from the neuropil regions subtending the hemispheres. Neuronal channels representing the midband extend from the medulla to the lobula where populations of putative inhibitory glutamic acid decarboxylase-positive neurons and tyrosine hydroxylase-positive neurons intrinsic to the lobula have specific associations with the midband. Here we investigate the organization of the midband representation in the medulla and the lobula in the context of their overall architecture. We discuss the implications of observed arrangements, in which midband inputs to the lobula send out collaterals that extend across the retinotopic mosaic pertaining to the hemispheres. This organization suggests an integrative design that diverges from the eumalacostracan ground pattern and, for the stomatopod, enables color and polarization information to be integrated with luminance information that presumably encodes shape and motion.

An insect-like mushroom body in a crustacean brain.

Mushroom bodies are the iconic learning and memory centers of insects. No previously described crustacean possesses a mushroom body as defined by strict morphological criteria although crustacean centers called hemiellipsoid bodies, which serve functions in sensory integration, have been viewed as evolutionarily convergent with mushroom bodies. Here, using key identifiers to characterize neural arrangements, we demonstrate insect-like mushroom bodies in stomatopod crustaceans (mantis shrimps). More than any other crustacean taxon, mantis shrimps display sophisticated behaviors relating to predation, spatial memory, and visual recognition comparable to those of insects. However, neuroanatomy-based cladistics suggesting close phylogenetic proximity of insects and stomatopod crustaceans conflicts with genomic evidence showing hexapods closely related to simple crustaceans called remipedes. We discuss whether corresponding anatomical phenotypes described here reflect the cerebral morphology of a common ancestor of Pancrustacea or an extraordinary example of convergent evolution.