Steffen Harzsch

A Systematic Nomenclature for the Insect Brain

Despite the importance of the insect nervous system for functional and developmental neuroscience, descriptions of insect brains have suffered from a lack of uniform nomenclature. Ambiguous definitions of brain regions and fiber bundles have contributed to the variation of names used to describe the same structure. The lack of clearly determined neuropil boundaries has made it difficult to document precise locations of neuronal projections for connectomics study. To address such issues, a consortium of neurobiologists studying arthropod brains, the Insect Brain Name Working Group, has established the present hierarchical nomenclature system, using the brain of Drosophila melanogaster as the reference framework, while taking the brains of other taxa into careful consideration for maximum consistency and expandability. The following summarizes the consortiums nomenclature system and highlights examples of existing ambiguities and remedies for them. This nomenclature is intended to serve as a standard of reference for the study of the brain of Drosophila and other insects.

From variable to constant cell numbers: cellular characteristics of the arthropod nervous system argue against a sister-group relationship of Chelicerata and “Myriapoda” but favour the Mandibulata concept

In the new debate on arthropod phylogeny, structure and development of the nervous system provide important arguments. The architecture of the brain of Hexapoda, Crustacea and Chelicerata in recent years has been thoroughly compared against an evolutionary background. However, comparative aspects of the nervous systems in these taxa at the cellular level have been examined in only a few studies. This review sets out to summarize these aspects and to analyse the existing data with respect to the concept of individually identifiable neurons. In particular, mechanisms of neurogenesis, the morphology of serotonergic interneurons, the number of motoneurons, and cellular features and development of the lateral eyes are discussed. We conclude that in comparison to the Mandibulata, in Chelicerata the numbers of neurons in the different classes examined are much higher and in many cases are not fixed but variable. The cell numbers in Mandibulata are lower and the majority of neurons are individually identifiable. The characters explored in this review are mapped onto an existing phylogram, as derived from brain architecture in which the Hexapoda are an in-group of the Crustacea, and there is not any conflict of the current data with such a phylogenetic position of the Hexapoda. Nevertheless, these characters argue against a sister-group relationship of “Myriapoda” and Chelicerata as has been recently suggested in several molecular studies, but instead provide strong evidence in favour of the Mandibulata concept.

A new look at embryonic development of the visual system in decapod crustaceans: neuropil formation, neurogenesis, and apoptotic cell death.

In recent years, comparing the structure and development of the central nervous system in crustaceans has provided new insights into the phylogenetic relationships of arthropods. Furthermore, the structural evolution of the compound eyes and optic ganglia of adult arthropods has been discussed, but it was not possible to compare the ontogeny of arthropod visual systems, owing to the lack of data on species other than insects. In the present report, we studied the development of the crustacean visual system by examining neurogenesis, neuropil formation, and apoptotic cell death in embryos of the American lobster, Homarus americanus, the spider crab, Hyas araneus, and the caridean shrimp, Palaemonetes argentinus, and compare these processes with those found in insects. Our results on the patterns of stem cell proliferation provide evidence that in decapod crustaceans and hemimetabolous insects, there exist considerable similarities in the mechanisms by which accretion of the compound eyes and growth of the optic lobes is achieved, suggesting an evolutionary conservation of these mechanisms.

The phylogenetic significance of crustacean optic neuropils and chiasmata: A re-examination†

Recent molecular data challenge the traditional hypotheses of arthropod phylogeny founded on morphologic characters. In this discussion, the structure of the visual systems in Pterygota (Hexapoda) and Decapoda (Malacostraca, Crustacea) is an important argument. Although many components of their visual systems depict structural homology, differences exist between Pterygota/Decapoda on the one side and Branchiopoda (Entomostraca) on the other in that the latter do not have a third optic neuropil or optic chiasmata. Therefore, the goals of the current study were to explore whether the third optic neuropils in Pterygota and Decapoda are homologous, to examine the formation of the first two optic neuropils and the chiasmata in Crustacea, and to compare these processes with Pterygota. For this purpose, five species of entomostracan and malacostracan crustaceans were analyzed by examination of serial sections, fluorescence labeling with phallotoxins, and anti‐histamine immunohistochemistry. We found that the chiasmata of Decapoda and Pterygota are characterized by striking similarities regarding both the level of individually identifiable classes of neurons and ontogenetic mechanisms, which are clearly different from those in Branchiopoda. Furthermore, the third optic neuropil of Decapoda and Pterygota, the lobula, shares an ontogenetic protocerebral origin and an innervation by corresponding sets of histamine‐immunoreactive neurons, suggesting homology of the lobula in these two groups. In conclusion, the characteristics of the visual system are in conflict with the traditional classification of Arthropoda. Instead, they support a sister‐group relationship of Hexapoda and Malacostraca, as suggested by some of the molecular studies. J. Comp. Neurol. 453: 10–21, 2002.

Brain architecture in the terrestrial hermit crab Coenobita clypeatus (Anomura, Coenobitidae), a crustacean with a good aerial sense of smell

BackgroundDuring the evolutionary radiation of Crustacea, several lineages in this taxon convergently succeeded in meeting the physiological challenges connected to establishing a fully terrestrial life style. These physiological adaptations include the need for sensory organs of terrestrial species to function in air rather than in water. Previous behavioral and neuroethological studies have provided solid evidence that the land hermit crabs (Coenobitidae, Anomura) are a group of crustaceans that have evolved a good sense of aerial olfaction during the conquest of land. We wanted to study the central olfactory processing areas in the brains of these organisms and to that end analyzed the brain of Coenobita clypeatus (Herbst, 1791; Anomura, Coenobitidae), a fully terrestrial tropical hermit crab, by immunohistochemistry against synaptic proteins, serotonin, FMRFamide-related peptides, and glutamine synthetase.ResultsThe primary olfactory centers in this species dominate the brain and are composed of many elongate olfactory glomeruli. The secondary olfactory centers that receive an input from olfactory projection neurons are almost equally large as the olfactory lobes and are organized into parallel neuropil lamellae. The architecture of the optic neuropils and those areas associated with antenna two suggest that C. clypeatus has visual and mechanosensory skills that are comparable to those of marine Crustacea.ConclusionIn parallel to previous behavioral findings of a good sense of aerial olfaction in C. clypeatus, our results indicate that in fact their central olfactory pathway is most prominent, indicating that olfaction is a major sensory modality that these brains process. Interestingly, the secondary olfactory neuropils of insects, the mushroom bodies, also display a layered structure (vertical and medial lobes), superficially similar to the lamellae in the secondary olfactory centers of C. clypeatus. More detailed analyses with additional markers will be necessary to explore the question if these similarities have evolved convergently with the establishment of superb aerial olfactory abilities or if this design goes back to a shared principle in the common ancestor of Crustacea and Hexapoda.

Early embryonic development of the central nervous system in the Australian crayfish and the Marbled crayfish (Marmorkrebs)

This study sets out to provide a systematic analysis of the development of the primordial central nervous system (CNS) in embryos of two decapod crustaceans, the Australian crayfish Cherax destructor (Malacostraca, Decapoda, Astacida) and the parthenogenetic Marbled crayfish (Marmorkrebs, Malacostraca, Decapoda, Astacida) by histochemical labelling with phalloidin, a general marker for actin. One goal of our study was to examine the neurogenesis in these two organisms with a higher temporal resolution than previous studies did. The second goal was to explore if there are any developmental differences between the parthenogenetic Marmorkrebs and the sexually reproducing Australian crayfish. We found that in the embryos of both species the sequence of neurogenetic events and the architecture of the embryonic CNS are identical. The naupliar neuromeres proto-, deuto-, tritocerebrum, and the mandibular neuromeres emerge simultaneously. After this “naupliar brain” has formed, there is a certain time lag before the maxilla one primordium develops and before the more caudal neuromeres follow sequentially in the characteristic anterior–posterior gradient. Because the malacostracan egg-nauplius represents a re-capitulation of a conserved ancestral information, which is expressed during development, we speculate that the naupliar brain also conserves an ancestral piece of information on how the brain architecture of an early crustacean or even arthropod ancestor may have looked like. Furthermore, we compare the architecture of the embryonic crayfish CNS to that of the brain and thoracic neuromeres in insects and discuss the similarities and differences that we found against an evolutionary background.

Serotonin-immunoreactive neurons in the ventral nerve cord of Crustacea: a character to study aspects of arthropod phylogeny

The number of serotonin-expressing neurons in the nervous system of Euarthropoda is small and their neurites have a characteristic branching pattern. They can be identified individually, which provides a character well suited for phylogenetic analyses. In order to gain data that may be useful in the ongoing discussion on insect-crustacean relationships, we documented the pattern of serotonin immunoreactive neurons in the ventral nerve cord of four crustacean species: the phyllocarid malacostracan Nebalia bipes Fabricius, 1780 (Phyllocarida, Leptostraca) and the entomostracans Artemia salina Linnaeus, 1758 (Branchiopoda, Anostraca, Sarsostraca), Triops cancriformis Bosc, 1801 (Branchiopoda, Phyllopoda, Calmanostraca, Notostraca), and Leptestheria dahalacensis Rüppell, 1837 (Branchiopoda, Phyllopoda, Diplostraca, Conchostraca, Spinicaudata). In the entomostracan taxa investigated, the pattern of serotonergic cells in the thoracic hemiganglia comprises an anterior and a posterior bilateral pair of neurons with ipsi- and/or contralateral neurites. Comparing these data to existing information on serotonin-immunoreactivity in the ventral nerve cord of other malacostracan and entomostracan groups enabled us to determine several features of these thoracic neurons being part of the ground pattern of these taxa. Our data demonstrate that studying individually identifiable neurons in Arthropoda can be used to analyse the phylogeny of this taxon.

An immunohistochemical study of structure and development of the nervous system in the brine shrimp Artemia salina Linnaeus, 1758 (Branchiopoda, Anostraca) with remarks on the evolution of the arthropod brain.

Brain morphology is an important character in the discussion of arthropod relationships. While a large body of literature is available on the brains of Hexapoda and Malacostraca, the structure of the brain has been rarely studied in representatives of the Entomostraca. This account examines the morphology and development of the nervous system in the brine shrimp Artemia salina Linnaeus, 1758 (Crustacea, Branchiopoda, Anostraca) by classical histology and immunohistochemistry against synaptic proteins (synapsins), and the neurotransmitters serotonin and histamine. The results indicate that the shape of the developing larval brain in A. salina (a circumstomodeal ring of neuropil) closely resembles that in malacostracan embryos. Furthermore, the organization of the central complex as well as the tritocerebral innervation pattern of the labrum is homologous in this species and in Malacostraca. Nevertheless, differences exist in the layout of the deutocerebrum, specifically in the absence of olfactory glomeruli in A. salina while the glomerular organization of the olfactory lobe is a character in the ground pattern of Malacostraca. These findings are compared to the brain structure in other Euarthropoda and possible phylogenetic implications are discussed.

Neurogenesis in the crustacean ventral nerve cord: homology of neuronal stem cells in Malacostraca and Branchiopoda?

SUMMARY In Insecta and malacostracan Crustacea, neurons in the ventral ganglia are generated by the unequal division of neuronal stem cells, the neuroblasts (Nbs), which are arranged in a stereotyped, grid‐like pattern. In malacostracans, however, Nbs originate from ectoteloblasts by an invariant lineage, whereas Nbs in insects differentiate without a defined lineage by cell‐to‐cell interactions within the neuroectoderm. As the ventral ganglia in entomostracan crustaceans were thought to be generated by a general inward proliferation of ectodermal cells, the question arose as to whether neuroblasts in Euarthropoda represent a homologous type of stem cell. In the current project, neurogenesis in metanauplii of the entomostracan crustaceans Triops cancriformis Fabricius, 1780 (Branchiopoda, Phyllopoda) and Artemia salina Linné, 1758 (Branchiopoda, Anostraca) was examined by in vivo incorporation of the mitosis marker bromodeoxyuridine (BrdU) and compared to stem cell proliferation in embryos of the malacostracan Palaemonetes argentinus Nobili, 1901 (Eucarida, Decapoda). The developmental expression of synaptic proteins (synapsins) was studied immunohistochemically. Results indicate that in the ventral neurogenic zone of Branchiopoda, neuronal stem cells with cellular characteristics of malacostracan neuroblasts are present. However, a pattern similar to the lineage‐dependent, grid‐like arrangement of the malacostracan neuroblasts was not found. Therefore, the homology of entomostracan and malacostracan neuronal stem cells remains uncertain. It is now well established that during arthropod development, identical and most likely homologous structures can emerge, although the initiating steps or the mode of generation of these structures are different. Recent evidence suggests that adult Entomostraca and Malacostraca share corresponding sets of neurons so that the present report provides an example that those homologous neurons may be generated via divergent developmental pathways. In this perspective, it remains difficult at this point to discuss the question of common patterns of stem cell proliferation with regard to the phylogeny and evolution of Atelocerata and Crustacea.

Potential and limitations of X-Ray micro-computed tomography in arthropod neuroanatomy: A methodological and comparative survey

Classical histology or immunohistochemistry combined with fluorescence or confocal laser scanning microscopy are common techniques in arthropod neuroanatomy, and these methods often require time‐consuming and difficult dissections and sample preparations. Moreover, these methods are prone to artifacts due to compression and distortion of tissues, which often result in information loss and especially affect the spatial relationships of the examined parts of the nervous system in their natural anatomical context. Noninvasive approaches such as X‐ray micro‐computed tomography (micro‐CT) can overcome such limitations and have been shown to be a valuable tool for understanding and visualizing internal anatomy and structural complexity. Nevertheless, knowledge about the potential of this method for analyzing the anatomy and organization of nervous systems, especially of taxa with smaller body size (e.g., many arthropods), is limited. This study set out to analyze the brains of selected arthropods with micro‐CT, and to compare these results with available histological and immunohistochemical data. Specifically, we explored the influence of different sample preparation procedures. Our study shows that micro‐CT is highly suitable for analyzing arthropod neuroarchitecture in situ and allows specific neuropils to be distinguished within the brain to extract quantitative data such as neuropil volumes. Moreover, data acquisition is considerably faster compared with many classical histological techniques. Thus, we conclude that micro‐CT is highly suitable for targeting neuroanatomy, as it reduces the risk of artifacts and is faster than classical techniques. J. Comp. Neurol. 523:1281–1295, 2015.