A developmental study of serotonin-immunoreactive neurons in the embryonic brain of the Marbled Crayfish and the Migratory Locust: Evidence for a homologous protocerebral group of neurons

It is well established that the brains of adult malacostracan crustaceans and winged insects display distinct homologies down to the level of single neuropils such as the central complex and the optic neuropils. We wanted to know if developing insect and crustacean brains also share similarities and therefore have explored how neurotransmitter systems arise during arthropod embryogenesis. Previously, Sintoni et al. (2007) had already reported a homology of an individually identified cluster of neurons in the embryonic crayfish and insect brain, the secondary head spot cells that express the Engrailed protein. In the present study, we have documented the ontogeny of the serotonergic system in embryonic brains of the Marbled Crayfish in comparison to Migratory Locust embryos using immunohistochemical methods combined with confocal laser-scan microscopy. In both species, we found a cluster of early emerging serotonin-immunoreactive neurons in the protocerebrum with neurites that cross to the contralateral brain hemisphere in a characteristic commissure suggesting a homology of this cell cluster. Our study is a first step towards a phylogenetic analysis of neurotransmitter system development and shows that, as for the ventral nerve cord, traits related to neurogenesis in the brain can provide valuable hints for resolving the much debated question of arthropod phylogeny.     

Unravelling the evolution of neural stem cells in arthropods: Notch signalling in neural stem cell development in the crustacean Daphnia magna

The genetic regulatory networks controlling major developmental processes seem to be conserved in bilaterians regardless of an independent or a common origin of the structures. This has been explained by the employment of a genetic toolkit that was repeatedly used during bilaterian evolution to build the various forms and body plans. However, it is not clear how genetic networks were incorporated into the formation of novel structures and how homologous genes can regulate the disparate morphological processes. Here we address this question by analysing the role of Notch signalling, which is part of the bilaterian toolkit, in neural stem cell evolution in arthropods. Within arthropods neural stem cells have evolved in the last common ancestor of insects and crustaceans (Tetraconata). We analyse here for the first time the role of Notch signalling in a crustacean, the branchiopod Daphnia magna, and show that it is required in neural stem cells for regulating the time of neural precursor production and for binary cell fate decisions in the ventral neuroectoderm. The function of Notch signalling has diverged in the ventral neuroectoderm of insects and crustaceans accompanied by changes in the morphogenetic processes. In the crustacean, Notch controlled mechanisms of neuroblast regulation have evolved that are surprisingly similar to vertebrates and thus present a remarkable case of parallel evolution. These new data on a representative of crustaceans complete the arthropod data set on Notch signalling in the nervous system and allow for reconstructing how the Notch signalling pathway has been co-opted from pre-existing structures to the development of the evolving neural stem cells in the Tetraconata ancestor.     

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.     

A good eye for arthropod evolution

Insect and crustacean lineages diverged over 500 Myr ago, and there are continuing uncertaintles about whether they evolved from a common arthropod ancestor or, alternatively, they evolved independently from annelid worms. Despite the diversity of their limbs and lifestyles, the nervous systems of insects and crustaeeans share many common features both in development and in function. Cellular and molecular embryology techniques reveal good evidence for homologies in the developing segmental ganglia. In the visual system, this seemingly common programme of insect and crustacean CNS development culminates in common adult neural function. Comparisons of the cellular anatomy and physiology of animals as diverse as flies and crayfishes indicate that the neural circuits in the lamina of their optic lobe have been inherited largely unchanged from a common ancestor with good compound eyes.     

HOM/Hox genes of Artemia: implications for the origin of insect and crustacean body plans

BACKGROUND: Insects and crustaceans are generally assumed to derive from a segmented common ancestor that had a distinct head but uniform, undifferentiated trunk segments. The subdivision of the body into functionally distinct regions (e.g. thorax and abdomen) is thought to have evolved independently in these two lineages. In insects, the differences between segments in the trunk are controlled by the Antennapedia-like genes of the homeotic gene clusters. Study of these genes in crustaceans should provide a basis for comparing body plans and assessing their evolutionary origin. RESULTS: Using a polymerase chain reaction (PCR) / inverse PCR strategy, we have isolated six genes of the HOM/Hox family from the crustacean Artemia franciscana. Five of these are clearly identifiable as specific homologues of the insect homeotic genes Dfd, Scr, Antp, Ubx and abdA. The sixth appears to have no close counterpart in insects. CONCLUSION: All the homeotic genes that specify middle body regions in insects originated before the divergence of the insect and crustacean lineages, probably not later than the Cambrian (about 500 million years ago). A commonly derived groundplan may underlie segment diversity in these two groups.     

Hox genes and the phylogeny of the arthropods

The arthropods are the most speciose, and among the most morphologically diverse, of the animal phyla. Their evolution has been the subject of intense research for well over a century, yet the relationships among the four extant arthropod subphyla - chelicerates, crustaceans, hexapods, and myriapods - are still not fully resolved. Morphological taxonomies have often placed hexapods and myriapods together (the Atelocerata) [1, 2], but recent molecular studies have generally supported a hexapod/crustacean clade [2-9]. A cluster of regulatory genes, the Hox genes, control segment identity in arthropods, and comparisons of the sequences and functions of Hox genes can reveal evolutionary relationships [10]. We used Hox gene sequences from a range of arthropod taxa, including new data from a basal hexapod and a myriapod, to estimate a phylogeny of the arthropods. Our data support the hypothesis that insects and crustaceans form a single clade within the arthropods to the exclusion of myriapods. They also suggest that myriapods are more closely allied to the chelicerates than to this insect/crustacean clade.     

Axogenesis in the stomatopod crustacean Gonodactylaceus falcatus (Malacostraca)

Abstract. The formation of the central nervous system of the stomatopod crustacean Gonodactylaceus falcatus is described by means of antibody stainings against synapsin and α-tubulin. It is shown that the longitudinal fiber tracts of the ventral nervous system are formed by two centers of origin comprising a number of pioneer neurons, one at the posterior part of the forming brain, the other in the area of the telson anlage at the posteriormost region of the embryo. In addition to the lateral anlagen of the connectives, a median longitudinal nerve is formed beginning in the mandibular segment neuromere. In contrast to those of other segments, the mandibular ganglia are connected by a single commissure. The brain forms a circumoral ring. There is evidence that the deutocerebrum possesses praestomodeal and poststomodeal commissural fibers. The anlage of the nauplius eye reveals a specific pattern of pigment and sensory cells with the two pigment cells expressing synapsin. Clear differences between the expression patterns of synapsin and α-tubulin recommend the combination of a variety of antibodies to gain a complete picture of embryonic neuroanatomy. Our results show overall similarities to other malacostracan and non-malacostracan crustaceans. The comparisons with other crustaceans and arthropods indicate homology of crustacean nauplius eyes, a circumoral deutocerebrum, and a more widespread occurrence of posterior pioneer neurons forming the axon scaffold of the ventral central nervous system than previously thought.     

A structural and functional comparison of nematode and crustacean PDH-like sequences

The elucidation of the whole genome of the nematode Caenorhabditis elegans allowed for the identification of ortholog genes belonging to the pigment dispersing hormone/factor (PDH/PDF) peptide family. Members of this peptide family are known from crustaceans, insects and nematodes and seem to exist exclusively in ecdysozoans where they play a role in different processes, ranging from the dispersion of integumental and eye (retinal) pigments in decapod crustaceans to circadian rhythms in insects and locomotion in C. elegans. Two pdf genes (pdf-1 and pdf-2) encoding three different peptides: PDF-1a, PDF-1b and PDF-2 have been identified in C. elegans. These three C. elegans PDH-like peptides are similar but not identical in primary structure to PDHs from decapod crustaceans. We investigate whether this divergence has an influence on the pigment dispersing function of the peptides in a decapod crustacean, namely the shrimp Palaemon pacificus. We show that C. elegans PDF-1a and b peptides display cross-functional activity by dispersing pigments in the epithelium of P. pacificus at physiological doses. Moreover, by means of a comparative amino acid sequence analysis of nematode and crustacean PDH-like peptides, we can pinpoint several potentially important residues for eliciting pigment dispersing activity in decapod crustaceans. Although there is no sequence information on a receptor for PDH in decapod crustaceans, we postulate that there is general conservation of the PDH/PDF signaling system based on structural similarities of precursor proteins and receptors (including those from a branchiopod crustacean and from C. elegans).     

The Engrailed-expressing secondary head spots in the embryonic crayfish brain: examples for a group of homologous neurons in Crustacea and Hexapoda?

Hexapoda have been traditionally seen as the closest relatives of the Myriapoda (Tracheata hypothesis) but molecular studies have challenged this hypothesis and rather have suggested a close relationship of hexapods and crustaceans (Tetraconata hypothesis). In this new debate, data on the structure and development of the arthropod nervous system contribute important new data ("neurophylogeny"). Neurophylogenetic studies have already provided several examples for individually identifiably neurons in the ventral nerve cord that are homologous between insects and crustaceans. In the present report, we have analysed the emergence of Engrailed-expressing cells in the embryonic brain of a parthenogenetic crayfish, the marbled crayfish (Marmorkrebs), and have compared our findings to the pattern previously reported from insects. Our data suggest that a group of six Engrailed-expressing neurons in the optic anlagen, the so-called secondary head spot cells can be homologised between crayfish and the grasshopper. In the grasshopper, these cells are supposed to be involved in establishing the primary axon scaffold of the brain. Our data provide the first example for a cluster of brain neurons that can be homologised between insects and crustaceans and show that even at the level of certain cell groups, brain structures are evolutionary conserved in these two groups.     

Segmentation,neurogenesis and formation of early axonal pathways in the centipede,Ethmostigmus rubripes (Brandt)

Summary We have examined the embryo of the centipedeEthmostigmus rubripes to determine the degree of evolutionary conservatism in the developmental processes of segmentation, neurogenesis and axon formation between the insects and the myriapods. A conspicuous feature of centipede embryogenesis is the early separation of the left and right sides of the ganglionic primordia by extra-embryonic ectoderm. An antibody to the protein encoded by theDrosophila segmentation geneengrailed binds to cells in the posterior margin of the limb buds in the centipede embryo, in common with insect and crustacean embryos. However, whereas in insects and crustaceans this protein is also expressed in a subset of cells in the neuroectoderm, the anti-engrailed antibody did not bind to cells in the ganglionic primordia of the centipede embryo. Use of the BrdU labelling technique to mark mitotically active cells revealed that neuroblasts, the ubiquitous neuron stem cell type in insects, are not present in the centipede. The earliest central axon pathways in the centipede embryo do not arise from segmentally repeated neurons, as is the case in insects, but rather by the posteriorly directed growth of axons originating from neurons located in the brain. Axonogenesis by segmental neurons begins later in development; the pattern of neurons involved is not obviously homologous to the conservative set of central pioneering neurons found in insects. Our observations point to considerable differences between the insects and the myriapods in mechanisms for neurogenesis and the formation of central axon pathways, suggesting that these developmental processes have not been strongly conserved during arthropod evolution.     

Juvenile hormone as a physiological regulator mediating phenotypic plasticity in pancrustaceans

Phenotypic plasticity and polyphenism, in which phenotypes can be changed depending on environmental conditions, are common in insects. Several studies focusing on physiological, developmental, and molecular processes underlying the plastic responses have revealed that similar endocrine mechanisms using juvenile hormone (JH) are used to coordinate the flexible developmental processes. This review discusses accumulated knowledge on the caste polyphenism in social insects (especially termites), the wing and the reproductive polyphenisms in aphids, and the nutritional polyphenism and sexual dimorphism in stag beetles. For the comparison with non-insect arthropods, extensive studies on the inducible defense (and reproductive polyphenism) in daphnids (crustacean) are also addressed. In all the cases, JH (and methyl farnesoate in daphnids) plays a central role in mediating environmental stimuli with morphogenetic processes. Since the synthetic pathways for juvenoids, i.e., the mevalonate pathway and downstream pathways to sesquiterpenoids, are conserved across pancrustacean lineages (crustaceans and hexapods including insects), the evolution of developmental regulation by juvenoids that control molting (ecdysis) and metamorphosis is suggested to have occurred in the ancestral arthropods. The discontinuous postembryonic development (i.e., molting) and the regulatory physiological factors (juvenoids) would have enabled plastic developmental systems observed in many arthropod lineages.     

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.     

Arthropod evolution: great brains, beautiful bodies

While arthropod phylogeny remains controversial, comparative studies of the genetic control of segmentation and of the nervous system have begun to throw light on how mandibulate arthropods (myriapods, crustaceans and insects) reached their current level of morphological and behavioural complexity. Insects and crustaceans show remarkable similarities in the construction of their brains, suggesting that their common ancestor had typically arthropod behaviour, while developmental genetic studies are consistent with this ancestor having had distinct head, trunk and tail regions. This conclusion contrasts with the influential view, drawn from comparative embryology and functional anatomy, that insects and crustaceans evolved independently from a simple worm-like organism, perhaps resembling an annelid.     

Brain organization and the origin of insects: an assessment

Within the Arthropoda, morphologies of neurons, the organization of neurons within neuropils and the occurrence of neuropils can be highly conserved and provide robust characters for phylogenetic analyses. The present paper reviews some features of insect and crustacean brains that speak against an entomostracan origin of the insects, contrary to received opinion. Neural organization in brain centres, comprising olfactory pathways, optic lobes and a central neuropil that is thought to play a cardinal role in multi-joint movement, support affinities between insects and malacostracan crustaceans.     

Genome size in arthropods; different roles of phylogeny,habitat and life history in insects and crustaceans

Despite the major role of genome size for physiology, ecology, and evolution, there is still mixed evidence with regard to proximate and ultimate drivers. The main causes of large genome size are proliferation of noncoding elements and/or duplication events. The relative role and interplay between these proximate causes and the evolutionary patterns shaped by phylogeny, life history traits or environment are largely unknown for the arthropods. Genome size shows a tremendous variability in this group, and it has a major impact on a range of fitness‐related parameters such as growth, metabolism, life history traits, and for many species also body size. In this study, we compared genome size in two major arthropod groups, insects and crustaceans, and related this to phylogenetic patterns and parameters affecting ambient temperature (latitude, depth, or altitude), insect developmental mode, as well as crustacean body size and habitat, for species where data were available. For the insects, the genome size is clearly phylogeny‐dependent, reflecting primarily their life history and mode of development, while for crustaceans there was a weaker association between genome size and phylogeny, suggesting life cycle strategies and habitat as more important determinants. Maximum observed latitude and depth, and their combined effect, showed positive, and possibly phylogenetic independent, correlations with genome size for crustaceans. This study illustrate the striking difference in genome sizes both between and within these two major groups of arthropods, and that while living in the cold with low developmental rates may promote large genomes in marine crustaceans, there is a multitude of proximate and ultimate drivers of genome size.     

On the phylogenetic position of insects in the Pancrustacea clade

The current views on the phylogeny of arthropods are at odds with the traditional system, which recognizes four independent arthropod classes: Chelicerata, Crustacea, Myriapoda, and Insecta. There is compelling evidence that insects comprise a monophyletic lineage with Crustacea within a larger clade named Pancrustacea, or Tetraconata. However, which crustacean group is the closest living relative of insects is still an open question. In recent phylogenetic trees constructed on the basis of large gene sequence data insects are placed together with primitive crustaceans, the Branchiopoda. This topology is often suspected to be a result of the long branch attraction artifact. We analyzed concatenated data on 77 ribosomal proteins, elongation factor 1A (EF1A), initiation factor 5A (eIF5A), and several other nuclear and mitochondrial proteins. Analyses of nuclear genes confirm the monophyly of Hexapoda, the clade uniting entognath and ectognath insects. The hypothesis of the monophyly of Hexapoda and Branchiopoda is supported in the majority of analyses. The Maxillopoda, another clade of Entomostraca, occupies a sister position to the Hexapoda + Branchiopoda group. Higher crustaceans, the Malacostraca, in most analyses appear a more basal lineage within the Pancrustacea. We report molecular synapomorphies in low homoplastic regions, which support the clade Hexapoda + Branchiopoda + Maxillopoda and the monophyletic Malacostraca including Phyllocarida. Thus, the common origin of Hexapoda and Branchiopoda and their position within Entomostraca are suggested to represent bona fide phylogenetic relationships rather than computational artifacts.     

How far does cell lineage influence cell fate specification in crustacean embryos?

In crustaceans, invariant cell lineages have been shown to occur (i) in early cleavages of several taxa and (ii) in the course of formation and differentiation of the post-naupliar germ bands in malacostracans. Work on early cleavages is still in its infancy. In contrast, the generation and proliferation of mesoteloblasts and ectoteloblasts and the subsequent proliferation and differentiation of bandlet cells have been studied in members of several subgroups of Malacostraca. Similarities and differences have been determined in order to interpret the interdependencies of the steps in the differentiation process. Some of these steps are highly conserved, as in the case of the generation of four pairs of mesoteloblasts, others are prone to phylogenetic change, as in the case of the primary ring of 19 ectoteloblasts which has been altered at least twice in evolution. A stereotyped cleavage pattern in the germ band has been shown to be independent of the origin of the precursor cells. The question whether neuroblasts in crustaceans and insects are homologous or are the result of convergent evolution is still open. However, the homology of early differentiating neurons in crustaceans and insects seems to be well established. In addition, similarities in the expression patterns of the engrailed gene are likely to be homologous and point to a close relationship between these two groups.