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.     

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.     

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.     

Hox genes in sea spiders (Pycnogonida) and the homology of arthropod head segments

The pycnogonids (or sea spiders) are an enigmatic group of arthropods, classified in recent phylogenies as a sister-group of either euchelicerates (horseshoe crabs and arachnids), or all other extant arthropods. Because of their bizarre morpho-anatomy, homologies with other arthropod taxa have been difficult to assess. We review the main morphology-based hypotheses of correspondence between anterior segments of pycnogonids, arachnids and mandibulates. In an attempt to provide new relevant data to these controversial issues, we performed a PCR survey of Hox genes in two pycnogonid species, Endeis spinosa and Nymphon gracile, from which we could recover nine and six Hox genes, respectively. Phylogenetic analyses allowed to identify their orthology relationships. The Deformed gene from E. spinosa and the abdominal-A gene from N. gracile exhibit unusual sequence divergence in their homeodomains, which, in the latter case, may be correlated with the extreme reduction of the posterior region in pycnogonids. Expression patterns of two Hox genes (labial and Deformed) in the E. spinosa protonymphon larva are discussed. The anterior boundaries of their expression domains favour homology between sea spider chelifores, euchelicerates chelicerae and mandibulate (first) antennae, in contradistinction with previously proposed alternative schemes such as the protocerebral identity of sea spider chelifores or the absence of a deutocerebrum in chelicerates. In addition, while anatomical and embryological evidences suggest the possibility that the ovigers of sea spiders could be a duplicated pair of pedipalps, the Hox data support them as modified anterior walking legs, consistent with the classical views.Supplementary material is available for this article at and is accessible for authorized users.Guest editors Jean Deutsch and Gerhard Scholtz     

On predatory tendencies in the feeding ecology of the fairy shrimp Streptocephalus proboscideus (Frauenfeld, 1873) (Crustacea: Anostraca)

The feeding habits of the filter-feeding fairy shrimp Streptocephalus proboscideus are documented experimentally by offering them ciliates, Volvox, rotifers, nematodes and small crustaceans as prey. Escape capabilities (e.g. swimming speed) rather than size or shape were found to determine these animals' vulnerability to predation by the fairy shrimp. Ingestion rates for Volvox increased hyperbolically with size and, at the high temperatures in which they live, fairy shrimps may daily remove the equivalent of their body weight from the environment.     

Muscle precursor cells in the developing limbs of two isopods (Crustacea, Peracarida): an immunohistochemical study using a novel monoclonal antibody against myosin heavy chain

In the hot debate on arthropod relationships, Crustaceans and the morphology of their appendages play a pivotal role. To gain new insights into how arthropod appendages evolved, developmental biologists recently have begun to examine the expression and function of Drosophila appendage genes in Crustaceans. However, cellular aspects of Crustacean limb development such as myogenesis are poorly understood in Crustaceans so that the interpretative context in which to analyse gene functions is still fragmentary. The goal of the present project was to analyse muscle development in Crustacean appendages, and to that end, monoclonal antibodies against arthropod muscle proteins were generated. One of these antibodies recognises certain isoforms of myosin heavy chain and strongly binds to muscle precursor cells in malacostracan Crustacea. We used this antibody to study myogenesis in two isopods, Porcellio scaber and Idotea balthica (Crustacea, Malacostraca, Peracarida), by immunohistochemistry. In these animals, muscles in the limbs originate from single muscle precursor cells, which subsequently grow to form multinucleated muscle precursors. The pattern of primordial muscles in the thoracic limbs was mapped, and results compared to muscle development in other Crustaceans and in insects. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Architecture of the nervous system in two Dactylopodola species (Gastrotricha, Macrodasyida)

Immunohistochemical stainings have become standard tools to describe the nervous system, but usually only singular or few markers are used and consequently show only subsets of neurons within the nervous system. We investigated two species of Dactylopodola (Gastrotricha, Macrodasyida) with a broad set and combination of markers, to represent the nervous system in a more holistic approach. We suggest that markers for both neurotubuli (tubulin) and neurotransmitters (e.g. serotonin, FMRF-amides, histamine) should be used. Combinations with markers for the musculature (phalloidin) and nuclei (propidiumiodide or other markers) help to reveal spatial patterns and when used with TEM can provide a more precise picture of the spatial relationships of particular nerves. Species of Dactylopodola have a brain consisting of a solid dorsal commissure and a fine ventral commissure. Cell somata of brain cells are arranged lateral to the dorsal commissure and form a dumbbell-like brain. Additionally, projections into the head region, head sensory organs, one pair of lateroventral nerve cords with three commissures and stomatogastric nerves are described. Obviously, some longitudinal transmitter-specific fibres run in parallel to the main longitudinal nerve and represent additional longitudinal fibres. In comparison with the nervous system architecture of other gastrotrich species and that of different bilaterian animals it is speculated that the gastrotrich nervous system retains several ancestral features, such as being commissural and not a compact brain.     

On the serotonergic nervous system of two planktonic rotifers, Conochilus coenobasis and C. dossuarius (Monogononta, Flosculariacea, Conochilidae)

The serotonergic nervous systems of two non-colonial species of Conochilus were examined to obtain the first immunohistochemical insights into the neuroanatomy of species of Flosculariacea (Rotifera, Monogononta). Species of Conochilus, subgenus Conochiloides, were examined using serotonin (5-HT) immunohistochemistry, epifluorescence and confocal laser scanning microscopy, and 3D computer imaging software. In specimens of C. coenobasis and C. dossuarius, the serotonergic nervous system is defined by a dorsal cerebral ganglion, apically directed cerebral neurites, and paired nerve cords. The cerebral ganglion contains approximately four pairs of small 5-HT-immunoreactive perikarya; one pair innervates the posterior nerve cords and three pairs innervate the apical field. The most dorsal pair innervates a coronal nerve ring that encircles the apical field. Within the apical field is a second nerve ring that outlines the inner border of the coronal cilia. Together, both the inner and outer nerve rings may function to modulate ciliary activity of the corona. The other two pairs of perikarya innervate a region around the mouth. Specific differences in the distribution of serotonergic neurons between species of Conochilus and previously examined ploimate rotifers include the following: (a) a lack of immunoreactivity in the mastax; (b) a greater number of apically directed serotonergic neurites; and (c) a complete innervation of the corona in both species of Conochilus. These differences in nervous system immunohistochemistry are discussed in reference to the phylogeny of the Monogononta.     

First description of the serotonergic nervous system in a bdelloid rotifer: Macrotrachela quadricornifera Milne 1886 (Philodinidae)

Class Bdelloidea of phylum Rotifera comprises aquatic microinvertebrates that are known for both obligate parthenogenesis and for resisting desiccation through a dormant reversible state. In the frame of an investigation about the role of the nervous system in controlling life cycle, reproduction and dormancy, we describe the serotonergic system of a bdelloid, Macrotrachela quadricornifera, using serotonin immunohistochemistry and confocal laser scanning microscopy. Serotonin immunoreactivity is present in the cerebral ganglion, lateral nerve cords and peripheral neurites. The cerebral ganglion consists of perikarya that send neurites cephalically to the rostrum and corona. A pair of neurites exits the cerebral ganglion as lateral nerve cords, and proceeds caudally to the pedal ganglion where additional neurites enter the foot. Based on the location of serotonergic immunoreactivity, we hypothesize that the neurotransmitter is involved in both motor activity (e.g., ciliary beating, inchworm-like locomotion) and sensory activity. A comparison between the serotonergic nervous systems of M. quadricornifera and species of Monogononta reveals differences in the numbers and patterns of cerebral perikarya, peripheral perikarya, and periperhal neurites. These differences may have functional significance for understanding adaptations to specific environments and/or systematic significance for reconstructing the rotiferan ground pattern.     

Fenvalerate induced changes in evoked potentials from the ventral nerve cord (VNC) of cockroach, Periplaneta americana

Changes in evoked potentials from the VNC of P. americana were recorded under in vitro and topical application of sublethal doses of fenvalerate. In this study significant changes in physical characteristics of action potentials like threshold voltage, duration, latency and amplitude were noticed. In in vitro studies the effects were found to be dose dependent, while in topical exposure maximum effect was noticed at 3h, followed by recovery during subsequent periods of exposure. Moreover, changes were more pronounced in in vitro than topical exposure.     

When an epithelium ceases to exist – an ultrastructural study on the fate of the embryonic coelom in Epiperipatus biolleyi (Onychophora, Peripatidae)

It is an accepted fact that fusion between the coelomic cavities and the primary body cavity occurs during development in the Arthropoda. However, such a fusion is much disputed in the Onychophora. In order to clarify this subject, the fate of embryonic coelomic cavities has been studied in an onychophoran. Ultrastructural investigations in this paper provide evidence that embryonic coelomic cavities fuse with spaces of the primary body cavity in Epiperipatus biolleyi. During embryogenesis, the somatic and splanchnic portions of the mesoderm separate and the former coelomic linings are transformed into mesenchymatic tissue. The resulting body cavity therefore represents a mixture of primary and secondary (coelomic) body cavities, i.e. the ‘mixocoel’. The nephridial anlage is already present, when the ‘mixocoel’ is formed, although there is no trace of a sacculus yet. The lumen of the nephridial anlage, thus, communicates with the newly formed ‘mixocoel’. Accordingly, the lumen of the nephridial sacculus cannot be regarded as a kind of ‘persisting coelomic cavity’ in E. biolleyi. Our findings support the hypothesis that the ‘mixocoel’ was already present in the common stem species of the Onychophora and Euarthropoda.     

Development of embryonic membranes in the silverfish Lepisma saccharina linnaeus (insecta: Zygentoma, Lepismatidae)

Development and fate of embryonic membranes in the silverfish Lepisma saccharina was examined throughout embryogenesis. The amnioserosal folds first arise as serosal folds that are completed by the later addition of the amnion from the embryo's margins as in archaeognaths. The close link between production of the amnion and formation of the folds should not be assigned to Dicondylia but to Pterygota as an autapomorphy. During fold formation, folding of embryonic membranes beneath the embryo is less extensive and the ventral cupping of the embryo plays a larger role comparable to that occurring in archaeognath embryos. In L. saccharina, the embryonic membrane pore (the amniopore) varies in its manner of closure, either by complete fusion of serosal folds or by formation of a serosal cuticular plug between them as in archaeognaths. Although, in many aspects of its embryogenesis, L. saccharina retains the primitiveness of archaeognaths, its amnioserosal folds persist and are well integrated into its embryogenesis as the amnioserosal fold-amniotic cavity system is established and as occurs in many pterygote embryos; this may be thus regarded as an autapomorphy of Dicondylia.     

Structure of identified neurons innervating the lateral cardiac nerve cords in the migratory locust,Locusta migratoria migratorioides (Reiche and Fairmaire) (Orthoptera,Acrididae)

Our knowledge about the morphology of neurons innervating the lateral cardiac nerve cords (LCNCs) in migratory locusts, Locusta migratoria migratorioides (R. and F.) (Orthoptera, Acrididae) has increased considerably during recent years, mainly owing to immunocytochemical studies using antisera directed against members of insect neuropeptide families. In principle, there are three morphological types of neurons located within the CNS, which innervate the LCNCs in locusts: abdominal ganglia contain (i) bilaterally projecting, possibly unpaired neurons (BPNs) and (ii) paired, unilaterally projecting neurons. In addition, (iii) the LCNCs receive innervation from a pair of neurons, which is located within the suboesophageal ganglion. The axons of all three types of neurons project into the LCNCs via the segmental heart nerves, the most distal extensions of the dorsal segemental nerves of abdominal ganglia. When estimating the number of axons contained in one segmental heart nerve and formed by all central neurons so far identified, this number exceeds the number of axon profiles previously seen using the electron microscope. This indicates that most, or perhaps all central neurons projecting into the LCNCs, have been identified in these insects.     

Localization of [14C]DDT in the ventral nerve cord of the cockroach,Periplaneta americana (L.)

[¹⁴C]DDT was used as a probe to determine the subcellular localization of DDT in the ventral nerve cord (VNC) of the cockroach, Periplaneta americana (L.). Male cockroaches were injected intra-abdominally with [¹⁴C]DDT and their VNCs removed at 1 h post-injection. The VNCs were then subjected to homogenization and differential centrifugation to isolate plasma membrane, mitochondrial, and microsomal fractions. Results indicate that the plasma membrane fraction contained the greatest amount of [¹⁴C]DDT, with the mitochondrial and microsomal fractions containing significantly less. Calculations and a comparison with I₅₀ values for oligomycin-sensitive (OS)Mg-ATPase from the literature support the prediction that an insufficient amount of DDT reaches the ventral nerve cord mitochondria of a cockroach to effect an I₅₀ level of inhibition of the (OS)Mg-ATPase.     

Pathfinding of peripheral neurons in the central nervous system of an embryonic grasshopper (Chorthippus biguttulus)

The peripheral leg nerves of grasshoppers are initially formed by a set of pioneer neurons and guidepost cells. These cells are used as guiding structures for later-arising axons of sensory neurons. The development of the central projections of the pioneer cells, the guidepost cells and some sensory cells is shown with Lucifer Yellow injection or with DiI application. The axons of the pioneer cells Ti1 enter the central nervous system at 38% of embryonic development. They turn anteriorly close to the midline and ascend with no major branching to the brain. The axons of the guidepost cells Fe1 and Tr1 follow the same path but do not ascend to the brain. Sensory axons of the subgenual organ and the femoral organ probably do not follow the central path pioneered by the former neurons. They end ipsilaterally in the respective thoracic neuromere, as is found in the adult.     

Organisation of the lamprey (Lampetra fluviatilis) embryonic brain: insights from LIM-homeodomain, Pax and hedgehog genes

To investigate the embryonic development of the central nervous system of the lamprey Lampetra fluviatilis, we have isolated and analysed the expression patterns of members of the LIM-homeodomain, Pax, Hedgehog and Nkx2.1 families. Using degenerate RT-PCR, single representatives of Lhx1/Lhx5, Lhx2/Lhx9, Pax3/Pax7 and Hedgehog families could be isolated in L. fluviatilis. Expression analysis revealed that the lamprey forebrain presents a clear neuromeric pattern. We describe the existence of 4 embryonic diencephalic prosomeres whose boundaries can be identified by the combined and relative expressions of LfPax37, LfLhx15 and LfLhx29. This suggests that the embryonic lamprey and gnathostome forebrain are patterned in a highly similar manner. Moreover, analysis of the LfHh gene, which is expressed in the hypothalamus, zona limitans intrathalamica and floor plate, reveals the possible molecular origin of this neuromeric brain pattern. By contrast, LfHh and LfNkx2.1 expressions suggest major differences in patterning mechanisms of the ventral telencephalon when compared to gnathostomes. In summary, our findings highlight a neuromeric organisation of the embryonic agnathan forebrain and point to the possible origin of this organisation, which is thus a truly vertebrate character. They also suggest that Hh/Shh midline signalling might act as a driving force for forebrain evolution.