Nervous system development in the fairy shrimp Branchinella sp. (Crustacea: Branchiopoda: Anostraca): Insights into the development and evolution of the branchiopod brain and its sensory organs

Using immunohistochemical labeling against acetylated a‐tubulin and serotonin in combination with confocal laser scanning microscopy and 3D‐reconstruction, we investigated the temporary freshwater pond inhabitant Branchinella sp. (Crustacea: Branchiopoda: Anostraca) for the first time to provide detailed data on the development of the anostracan nervous system. Protocerebral sense organs such as the nauplius eye and frontal filament organs are present as early as the hatching stage L0. In the postnaupliar region, two terminal pioneer neurons grow from posterior to anterior to connect the mandibular neuromeres. The first protocerebral neuropil to emerge is not part of the central complex but represents the median neuropil, and begins to develop from L0+ onwards. In stage L3, the first evidence of developing compound eyes is visible. This is followed by the formation of the visual neuropils and the neuropils of the central complex in the protocerebrum. From the deutocerebral lobes, the projecting neuron tract proceeds to both sides of the lateral protocerebrum, forming a chiasma just behind the central body. In the postnaupliar region, the peripheral nervous system, commissures and connectives develop along an anterior–posterior gradient after the fasciculation of the terminal pioneer neurons with the mandibular neuromere. The peripheral nervous system in the thoracic segments consists of two longitudinal neurite bundles on each side which connect the intersegmental nerves, together with the ventral nervous system forming an orthogon‐like network. Here, we discuss, among other things, the evidence of a fourth nauplius eye nerve and decussating projecting neuron tract found in Branchinella sp., and provide arguments to support our view that the crustacean frontal filament (organ) and onychophoran primary antenna are homologous. J. Morphol. 277:1423–1446, 2016. © 2016 Wiley Periodicals, Inc.     

Immunocytochemical studies on the naupliar nervous system of Balanus improvisus (Crustacea, Cirripedia, Thecostraca)

The nervous system of nauplii of the crustacean taxon Cirripedia was analysed in the species Balanus improvisus Darwin, 1854 using for the first time immunocytochemical staining against serotonin, RFamide and α-tubulin in combination with confocal laser scanning microscopy. This approach revealed a circumoesophageal neuropil ring with nerves extending to the first and second antennae and to the mandibles, all features typical for Crustacea. In addition, RFamidergic structures are present in the region of the thoraco-abdomen. A pair of posterior nerves and a pair of lateral nerves run in anterior-posterior direction and are connected by a thoracic nerve ring and a more posteriorly situated commissure. A median nerve is situated along the ventral side of the thoraco-abdomen. The innervation of frontolateral horns and the frontal filaments are α-tubulin-positive. Several pairs of large neurons in the protocerebrum, along the circumoesophageal connectives and in the mandibular ganglion stain only for serotonin. Due to the almost complete absence of comparable data on the neuroanatomy of early (naupliar) stages in other Crustacea, we include immunocytochemical data on the larvae of the branchiopod, Artemia franciscana Kellogg, 1906 in our analysis. We describe several characteristic neurons in the brains of the nauplius larvae of both species which are also found in decapod larvae and in adult brains of other crustaceans. Furthermore, our data reveal that the naupliar brain of cirripedes is more complex than the adult brain. It is concluded that this ontogenetic brain reduction is related to the sessile life style of adult Cirripedia.     

Filling the gap between identified neuroblasts and neurons in crustaceans adds new support for Tetraconata

The complex spatio-temporal patterns of development and anatomy of nervous systems play a key role in our understanding of arthropod evolution. However, the degree of resolution of neural processes is not always detailed enough to claim homology between arthropod groups. One example is neural precursors and their progeny in crustaceans and insects. Pioneer neurons of crustaceans and insects show some similarities that indicate homology. In contrast, the differentiation of insect and crustacean neuroblasts (NBs) shows profound differences and their homology is controversial. For Drosophila and grasshoppers, the complete lineage of several NBs up to formation of pioneer neurons is known. Apart from data on median NBs no comparable results exist for Crustacea. Accordingly, it is not clear where the crustacean pioneer neurons come from and whether there are NBs lateral to the midline homologous to those of insects. To fill this gap, individual NBs in the ventral neuroectoderm of the crustacean Orchestia cavimana were labelled in vivo with a fluorescent dye. A partial neuroblast map was established and for the first time lineages from individual NBs to identified pioneer neurons were established in a crustacean. Our data strongly suggest homology of NBs and their lineages, providing further evidence for a close insect-crustacean relationship.     

Embryonic development of the histaminergic system in the ventral nerve cord of the Marbled Crayfish (Marmorkrebs)

The embryonic development of neurotransmitter systems in crustaceans so far is poorly understood. Therefore, in the current study we monitored the ontogeny of histamine-immunoreactive neurons in the ventral nerve cord of the Marbled Crayfish, an emerging crustacean model system for developmental studies. The first histaminergic neurons arise around 60% of embryonic development, well after the primordial axonal scaffold of the ventral nerve cord has been established. This suggests that histaminergic neurons do not serve as pioneer neurons but that their axons follow well established axonal tracts. The developmental sequence of the different types of histaminergic neurons is charted in this study. The analysis of the histaminergic structures is also extended into adult specimens, showing a persistence of embryonic histaminergic neurons into adulthood. Our data are compared to the pattern of histaminergic neurons in other crustaceans and discussed with regard to our knowledge on other aspects of neurogenesis in Crustacea. Furthermore, the possible role of histaminergic neurons as characters in evolutionary considerations is evaluated.     

The formation of the nervous system during larval development in Triops cancriformis (Bosc) (crustacea,Branchiopoda): An immunohistochemical survey

We provide data of the development of thenervous system during the first five larval stages of Triops cancriformis. We use immunohistochemical labeling (against acetylated α‐tubulin, serotonin, histamine, and FMRFamide), confocal laser scanning microscopy analysis, and 3D‐reconstruction. The development of the nervous system corresponds with the general anamorphic development in T. cancriformis. In larval stage I (L I), all brain parts (proto‐, deuto‐, and tritocerebrum), the circumoral connectives, and the mandibular neuromere are already present. Also, the frontal filaments and the developing nauplius eye are already present. However, until stage L III, the nauplius eye only consists of three cups. Throughout larval development, the protocerebral network differentiates into distinct subdivisions. In the postnaupliar region, additional neuromeres and their commissures emerge in an anteroposterior gradient. The larval nervous system in L V consists of a differentiated protocerebrum including a central body, a nauplius eye comprising four cups, a circumoral nerve ring, mandibular‐ and postnaupliar neuromeres up to the seventh thoracic segment, each featuring an anterior and a posterior commissure, and two parallel connectives. The presence of a protocerebral bridge is questionable. The distribution of neurotransmitters in L I is restricted to the naupliar nervous system. Over the course of the five stages of development, neurotransmitter distribution also follows an anteroposterior gradient. Each neuromere is equipped with two ganglia innervating the locomotional appendages and possesses a specific neurotransmitter distribution pattern. We suggest a correlation between neurotransmitter expression and locomotion. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Architecture of the nervous system in mystacocarida (Arthropoda,crustacea)—An immunohistochemical study and 3D reconstruction

Mystacocarida is a species‐poor group of minute crustaceans with unclear phylogenetic affinities. Previous studies have highlighted the putative “primitiveness” of several mystacocarid features, including the architecture of the nervous system. Recent studies on arthropod neuroarchitecture have provided a wealth of characters valuable for phylogenetic reconstructions. To permit and facilitate comparison with these data, we used immunohistochemical labeling (against acetylated α‐tubulin, serotonin and FMRFamide) on the mystacocarid Derocheilocaris remanei, analyzing it with confocal laser‐scanning microscopy and 3D reconstruction. The mystacocarid brain is fairly elongated, exhibiting a complicated stereotypic arrangement of neurite bundles. However, none of the applied markers provided evidence of structured neuropils such as a central body or olfactory glomeruli. A completely fused subesophageal ganglion is not present, all segmental soma clusters of the respective neuromeres still being delimitable. The distinct mandibular commissure comprises neurite bundles from more anterior regions, leading us to propose that it may have fused with an ancestral posterior tritocerebral commissure. The postcephalic ventral nervous system displays a typical ladder‐like structure with separated ganglia which bears some resemblance to larval stages in other crustaceans. Ganglia and commissures are also present in the first three limbless “abdominal” segments, which casts doubt on the notion of a clear‐cut distinction between thorax and abdomen. An unpaired longitudinal median neurite bundle is present and discussed as a potential tetraconate autapomorphy. Additionally, a paired latero‐longitudinal neurite bundle extends along the trunk. It is connected to the intersegmental nerves and most likely fulfils neurohemal functions. We report the complete absence of serotonin‐ir neurons in the ventral nervous system, which is a unique condition in arthropods and herein interpreted as a derived character. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Immunohistochemical mapping of histamine, dopamine, and serotonin in the central nervous system of the copepod Calanus finmarchicus (Crustacea; Maxillopoda; Copepoda)

Calanoid copepods constitute an important group of marine planktonic crustaceans that often dominate the metazoan biomass of the world’s oceans. In proportion to their ecological importance, little is known about their nervous systems. We have used immunohistochemical techniques in a common North Atlantic calanoid to localize re-identifiable neurons that putatively contain the biogenic amines histamine, dopamine, and serotonin. We have found low numbers of such cells and cell groups (approximately 37 histamine pairs, 22 dopamine pairs, and 12 serotonin pairs) compared with those in previously described crustaceans. These cells are concentrated in the anterior part of the central nervous system, the majority for each amine being located in the three neuromeres that constitute the brain (protocerebrum, deutocerebrum, and tritocerebrum). Extensive histamine labeling occurs in several small compact protocerebral neuropils, three pairs of larger, more posterior, paired, dense neuropils, and one paired diffuse tritocerebral neuropil. The most concentrated neuropil showing dopamine labeling lies in the putative deutocerebrum, associated with heavily labeled commissural connections between the two sides of the brain. The most prominent serotonin neuropil is present in the anterior medial part of the brain. Tracts of immunoreactive fibers of all three amines are prominent in the cephalic region of the nervous system, but some projections into the most posterior thoracic regions have also been noted.     

Neuromuscular development in Novocrania anomala: evidence for the presence of serotonin and a spiralian-like apical organ in lecithotrophic brachiopod larvae

SUMMARY The phylogenetic position of Brachiopoda remains unsettled, and only few recent data on brachiopod organogenesis are currently available. In order to contribute data to questions concerning brachiopod ontogeny and evolution we investigated nervous and muscle system development in the craniiform (inarticulate) brachiopod Novocrania anomala . Larvae of this species are lecithotrophic and have a bilobed body with three pairs of dorsal setal bundles that emerge from the posterior lobe. Fully developed larvae exhibit a network of setae pouch muscles as well as medioventral longitudinal and transversal muscles. After settlement, the anterior and posterior adductor muscles and delicate mantle retractor muscles begin to form. Comparison of the larval muscular system of Novocrania anomala with that of rhynchonelliform (articulate) brachiopod larvae shows that the former has a much simpler muscular organization. The first signal of serotonin-like immunoreactivity appears in fully developed Novocrania anomala larvae, which have an apical organ that consists of four flask-shaped cells and two ventral neurites. These ventral neurites do not stain positively for the axonal marker α-tubulin in the larval stages. In the juveniles, the nervous system stained by α-tubulin is characterized by two ventral neurite bundles with three commissures. Our data are the first direct proof for the presence of an immunoreactive neurotransmitter in lecithotrophic brachiopod larvae and demonstrate the existence of flask-shaped serotonergic cells in the brachiopod larval apical organ, thus significantly increasing the probability that this cell type was part of the bauplan of the larvae of the last common lophotrochozoan ancestor.     

Development of the nervous system in the "head" of <Emphasis Type="Italic">Limulus polyphemus</Emphasis> (Chelicerata: Xiphosura): morphological evidence for a correspondence between the segments of the chelicerae and of the (first) antennae of Mandibulata

We investigated brain development in the horseshoe crab Limulus polyphemus and several other arthropods via immunocytochemical methods, i.e. antibody stainings against acetylated alpha-tubulin and synapsin. According to the traditional view, the first appendage-bearing segment in chelicerates (the chelicerae) is not homologous to the first appendage-bearing segment of mandibulates (first antenna, deutocerebrum) but to the segment of the second antenna (tritocerebrum) or the intercalary segment in hexapods and myriapods. Accordingly, the segment of the deutocerebrum in chelicerates would be completely reduced. The main arguments for this view are: (1) the postoral origin of the cheliceral ganglion, (2) a poststomodaeal commissure, and (3) a connection of the cheliceral ganglion to the stomatogastric system. Our data show that these arguments are not convincing. During the development of horseshoe crabs there is no evidence for a former additional segment in front of the chelicerae. Instead, comparison of the brain structure (neuropil ring) between chelicerates, crustaceans and insects shows remarkable similarities. Furthermore, the cheliceral commissure in horseshoe crabs runs mainly praestomodaeal, which would be unique for a tritocerebral commissure. An unbiased view of the developing nervous system in the "head" of chelicerates, crustaceans and insects leads to a homologisation of the cheliceral segment and that of the (first) antenna (= deutocerebrum) of mandibulates that is also congruous to the interpretation of the Hox gene expression patterns. Thus, our data provide morphological evidence for the existence of a chelicerate deutocerebrum.     

Phylogenetic implications of the Crustacean nauplius

The plesiomorphic mode of crustacean development is widely accepted to be via a larva called the nauplius. Extant taxa like the Cephalocarida, Branchiopoda, Ostracoda, Mystacocarida, Copepoda, Cirripedia, Ascothoracida, Facetotecta, Euphausiacea and Penaeidea hatch from an egg as a free-living nauplius. Other crustaceans show an embryonic phase of development suggestive of a naupliar organization. Several features of the nauplius larva have been proposed as diagnostic characters for the Crustacea: a median (nauplius) eye; at least three pairs of head appendages (antennules, antennae, mandibles); a posteriorly directed fold (the labrum) extending over the mouth and a cephalic (nauplius) shield. The relationship between trilobite protaspis with at least four appendages and the crustacean nauplius remains unclear, but reports of a copepod orthonauplius with four appendages are rejected. Swimming is suggested to represent the underived mode of locomotion for the crustacean nauplius, and that naupliar swimming directly results in naupliar feeding which also is underived.     

Commissure formation in the embryonic insect brain

The primary axon scaffold of the insect brain is established early in embryogenesis and comprises a preoral protocerebral commissure, a postoral tritocerebral commissure and longitudinal fiber pathways linking the two. In both grasshopper and fly its form is approximately orthogonal and is centered around the stomodeum. We show how pioneer fibers from the protocerebrum and tritocerebrum cross the brain midline directly via their respective commissures. The deutocerebrum, however, lacks its own commissure and we describe how deutocerebral pioneers circumnavigate the gut to cross the midline either via the protocerebral commissure or the tritocerebral commissure. In contrast to all other commissures of the central nervous system, the protocerebral commissure persists, albeit in reduced form, in the commissureless mutation in the fly. Besides the com gene, a further, as yet unidentified, mechanism must regulate this commissure. The formation of the tritocerebral commissure involves labial, a member of the Hox gene group. Genetic rescue experiments in labial mutants reveal that the formation of this commissure can be rescued by all other Hox genes except Abdominal-B. However, only in the labial and Deformed null mutants are the commissures associated with the respective expression domains (tritocerebral, mandibular, respectively) absent. This suggests that the molecular mechanisms regulating postoral brain commissure formation are distinct from those in the neuromeres of the ventral nerve cord.     

Localization patterns of dopamine active transporter synthesizing cells during development of brine shrimp

There have been many studies on dopamine active transporter (DAT) in humans and laboratory animals; however, there is a lack of information on DAT in brine shrimp. In this study, we demonstrated the neuronal and nonneuronal characteristics of DAT‐synthesizing (DAT⁺ cells) during development of brine shrimp. In neuronal cells, the DAT⁺ neurons in the central body and lobes of a protocerebrum (PC) controlled the deutocerebrum. The sensory cells of nauplius eyes projected their decussated axons to the PC, and the DAT⁺ cells at the posterior region were associated with migration and control of the 10 posterior neurons during the early nauplius stage. In nonneuronal cells, the five types of glands, that is, the salt, antennal, mandible, and accessory glands and posterior gland1 and gland2 synthesized DAT protein. In addition, the gut and rectum dilator muscles and renal cells expressed DAT protein. Thus, DAT protein acts in the development of several types of cells during development of brine shrimp.     

The nauplius eye complex in 'conchostracans'(Crustacea, Branchiopoda: Laevicaudata, Spinicaudata, Cyclestherida) and its phylogenetic implications

The nauplius eye in Cyclestherida, Laevicaudata and Spinicaudata (previously collectively termed Conchostraca) consists of four cups of inverse sensory cells separated by a pigment layer and a tapetum layer. There are two lateral and two medial cups, a ventral medial cup and a posterior medial cup. The pigment and tapetum layers contain two different kinds of pigment granules, the inner pigment layer relatively large, dark (and electron dense) granules, and the outer tapetum layer light, reflective pigment granules. The presence of four cups and two different kinds of pigment granules are interpreted as autapomorphies of Phyllopoda. The position and shape of the nauplius eye in Spinicaudata is very distinct and herein interpreted as an autapomorphy of this taxon.Additional frontal eyes might be present dorsally or ventrally in varying proximity to the nauplius eye, but they have separate nerves from their sensory cells to the nauplius eye centre in the protocerebrum. Rhabdomeric structures are present in all these frontal eyes, evidencing their light sensitivity. In Lynceus biformis and L. tatei (Laevicaudata), two pairs of frontal eyes were found. In Cyclestheria hislopi (Cyclestherida), an unpaired ventral frontal eye is present. We did not find additional frontal eyes in Limnadopsis parvispinus and Caenestheriella sp. (Spinicaudata).     

The pineal gland of the mole-rat (Spalax ehrenbergi,Nehring)

Summary A comparative investigation of the distribution of monoaminergic neurons in non-malacostracan crustaceans was performed with the histochemical fluorescence method of Falck-Hillarp.Two fluorophores were found: the more widespread of the two emits a green fluorescence; and the more sparsely distributed emits a yellow to brown-yellow fluorescence.Specific green fluorescent areas were shown to exist in the protocerebrum. The central body and the optic ganglia of the compound eye (where present) are always fluorescent. Moreover, the centre of the nauplius eye may have a green fluorophore, as in ostracods, and a neuropile area, here called the frontal area. These neuropile centres are known from ordinary histological studies of the nervous system. In addition, there are specific monoaminergic centres, such as the so-called dorsal area of phyllopods and anostracans as well as the copepod specific areas. Specific monoaminergic areas appear in the deutocerebrum and the suboesophageal ganglion where they are particularly well developed.Presumed sensory neurons in the cavity receptor organ of Artemia salina are shown to be monoaminergic. Monoaminergic sensory neurons have not been described previously in Arthropods.Presumed motor innervation of hind-gut and trunk muscles is also found, and it is concluded that in crustaceans neurons of every type (sensory, internuncial, motor) may be monoaminergic.We have enjoyed unrestricted laboratory facilities at the Department of Histology, Faculty of Medicine, and with great pleasure express our sincere thanks to Prof. Bengt Falck. — Grants from the Swedish Natural Science Research Council (2760-007), the Swedish Medical Research Council (04X-712), the Royal Swedish Academy of Science (Hierta-Retzius), the Royal Physiographic Society of Lund, and the University of Lund supported the work.     

The Organ of Bellonci in Ostracodes: An Ultrastructural Study of the Rod-shaped,or Frontal,Organ

Ultrastructural observations of the rod-shaped organ in Cypridina norvegica and Paraconchoecia elegans indicate homology with the organ of Bellonci of other crustaceans. In C. norvegica the organ is situated close to the ventral cup of the nauplius eye. Distally in the organ, several ciliary ramifications of the sensory neurons protrude into internal cavities formed by bordering cells. Six dendrites, with cell bodies within and in front of the brain, form the proximally bifurcated nerve, which enters the protocerebrum in the region of the medullae terminales. In this species the organ represents the deep receptor of the organ of Bellonci complex. In P. elegans the external part of the organ is situated between the proximal parts of the antennulae. Four dendrites in two groups emerge from the protocerebrum. Distally, they form branching cilia that are in close contact which the cuticle of the organ, thus forming a receptor similar to the superficial receptor of the organ of Bellonci complex of other crustaceans. It is suggested that the terms frontal organ and rod-shaped organ be abandoned in favour of the term organ of Bellonci.     

The Nauplius Larva of Crustaceans: Functional Diversity and the Phylotypic Stage

SYNOPSIS. The crustacean nauplius larva is a development stagecharacterized by the presence of three pairs of head appendages.All crustaceans pass through the naupliar stage whether embryonicallyor as freeliving larvae. The nauplius is thought to be the phylotypicstage and represent a fundamental developmental constraint incrustaceans. However, free-living nauplii are primitive andI present evidence that this form is functionally plastic, e.g.,locomotory modes are diverse even in closely related species.I argue that this functional plasticity allowed the persistenceof nauplii in the early evolution of crustaceans and, as a consequence,naupliar development became a deep-seated feature of crustaceans.Thus, we see nauplii as phylotypic. This suggests that, in spiteof the presence of phylotypic stages in various phyla, phylotypyitself may not represent a similar, underlying developmentalconstraint in every case.     

Pioneer neurons: A basis or limiting factor of lophotrochozoa nervous system diversity?

It has been demonstrated by us and other authors that first nervous cells in developing larvae from various trochozoan groups differentiate at the periphery. These pioneer neurons are distinguished by the set of characters. They are located outside the forming central ganglia; outgrowing fibers of central neurons use their processes as a “scaffolding” transmitter expression in these neurons is transient. On the one hand, pioneer neurons mark the “frame” of the adult nervous system and thus play a limiting role. On the other hand, pioneering navigation provides possible mechanisms for evolutional plasticity of the nervous system in adults. In addition, pioneer neurons can underlie functional adaptation of trochophore animals, which minimizes fitness decrease during the transition from the larval to the adult form during metamorphosis.     

The crustacean eye: dark/light adaptation, polarization sensitivity, flicker fusion frequency, and photoreceptor damage

Compound eyes, nauplius eyes, frontal organs, intracerebral ocelli, and caudal photoreceptors are the main light and darkness detectors in crustaceans, but they need not be present all at once in an individual and in some crustaceans no photoreceptors whatsoever are known. Compound eye designs reflect on their functions and have evolved to allow the eye to operate optimally under a variety of environmental conditions. Dark-light-adaptational changes manifest themselves in pigment granule translocations, cell movements, and optical adjustments which fine-tune an eye's performance to rapid and unpredictable fluctuations in ambient light intensities as well as to the slower and predictable light level changes associated with day and night oscillations. Recycling of photoreceptive membrane and light-induced membrane collapse are superficially similar events that involve the transduction cascade, intracellular calcium, and membrane fatty acid composition, but which differ in aetiology and longterm consequence. Responses to intermittant illumination and linearly polarized light evoke in the eye of many crustaceans characteristic responses that appear to be attuned to each species' special needs. How the visual responses are processed more centrally and to what extent a crustacean makes behavioural use of e-vector discrimination and flickering lights are questions, however, that still have not been satisfactorily answered for the vast majority of all crustacean species. The degree of light-induced photoreceptor damage depends on a large number of variables, but once manifest, it tends to be progressive and irreversible. Concomittant temperature stress aggravates the situation and there is evidence that free radicals and lipid hydroperoxides are involved.     

Ciliary and nervous structures in juvenile females of the annelid Dinophilus gyrociliatus (O. Schmidt, 1848) (Annelida: Polychaeta)

Ciliary and nerve structures were described in juvenile female Dinophilus gyrociliatus (O. Schmidt, 1848) after immunochemical staining with tubulin, serotonin, and FMRFamide antibodies. Anti-tubulin antibodies revealed the following external structures: two head and seven body ciliary bands, a ventral ciliary band, and head ciliary fields. Gut cilia and five pairs of protonephridia were detected inside the body. The nervous system consists of an oval headed neuropile with anterior and posterior nerves extending from it, seven longitudinal nerve cords, commissures, and circular nerves. Anti-serotonin antibodies revealed the head neuropile, neurons at the base of the ventral ciliary band, an oesophageal ring, and seven longitudinal ventral cords. Anti-FMRFamide antibodies revealed approximately ten neurons in the cerebral ganglion, five longitudinal cords, and the oesophageal and caudal-nerve rings. The presented data suggest the simplification of the nervous system structure in D. gyrociliatus, which probably reflects pedomorphosis.     

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.