Carsten Wolff

Three-dimensional reconstruction of the central nervous system of Macrobiotus hufelandi (Eutardigrada, Parachela): implications for the phylogenetic position of Tardigrada

The morphology of the central nervous system of the tardigrade species Macrobiotus hufelandi was analysed with anti α-tubulin immunostaining in combination with confocal-laser-scanning-microscopy and computer aided three-dimensional reconstruction. The brain anatomy is unexpectedly complex with distinct tracts and highly intermingled nerve fibres. In contrast to older descriptions, we could not detect a suboesophageal ganglion. Furthermore, we found no evidence for a tripartite/three-segmented brain organisation. The median part of the brain is directly connected to the first pair of trunk ganglia via circumoesophageal connectives. Surprisingly, the four paired ventral ganglia do not show segmental commissures typical for the ladder-like nervous system of arthropods. Our findings constrain the phylogenetic position of Tardigrada. The much simpler organisation of the central nervous system of Tardigrada compared to that of Onychophora and Euarthropoda and some similarities to the nervous system of Cycloneuralia support a phylogenetic position of Tardigrada outside an Onychophora/Euarthropoda clade. This means that Tardigrada might be either the sister group to all other Arthropoda or they are more closely related to Cycloneuralia.

Embryonic development and staging of the cobweb spider Parasteatoda tepidariorum C. L. Koch, 1841 (syn.: Achaearanea tepidariorum; Araneomorphae; Theridiidae)

The cobweb spider Parasteatoda tepidariorum (C. L. Koch, 1841; syn.: Achaearanea tepidariorum) has become an important study organism in developmental biology and evolution as well as in genetics. Besides Cupiennius salei, it has become a chelicerate model organism for evo-devo studies in recent years. However, a staging system taking into account the entire development, and detailed enough to apply to modern studies, is still required. Here we describe the embryonic development of P. tepidariorum and provide a staging system which allows easy recognition of the distinct stages using simple laboratory tools. Differences between P. tepidariorum and other chelicerates, primarily C. salei, are discussed. Furthermore, cocoon production and the first postembryonic moulting procedure are described. Schematic drawings of all stages are provided to ease stage recognition.

The house spider genome reveals an ancient whole-genome duplication during arachnid evolution.

BackgroundThe duplication of genes can occur through various mechanisms and is thought to make a major contribution to the evolutionary diversification of organisms. There is increasing evidence for a large-scale duplication of genes in some chelicerate lineages including two rounds of whole genome duplication (WGD) in horseshoe crabs. To investigate this further, we sequenced and analyzed the genome of the common house spider Parasteatoda tepidariorum.ResultsWe found pervasive duplication of both coding and non-coding genes in this spider, including two clusters of Hox genes. Analysis of synteny conservation across the P. tepidariorum genome suggests that there has been an ancient WGD in spiders. Comparison with the genomes of other chelicerates, including that of the newly sequenced bark scorpion Centruroides sculpturatus, suggests that this event occurred in the common ancestor of spiders and scorpions, and is probably independent of the WGDs in horseshoe crabs. Furthermore, characterization of the sequence and expression of the Hox paralogs in P. tepidariorum suggests that many have been subject to neo-functionalization and/or sub-functionalization since their duplication.ConclusionsOur results reveal that spiders and scorpions are likely the descendants of a polyploid ancestor that lived more than 450 MYA. Given the extensive morphological diversity and ecological adaptations found among these animals, rivaling those of vertebrates, our study of the ancient WGD event in Arachnopulmonata provides a new comparative platform to explore common and divergent evolutionary outcomes of polyploidization events across eukaryotes.

The clonal composition of biramous and uniramous arthropod limbs

We present the first comparative cell lineage analysis of uniramous and biramous limbs of an arthropod, the crustacean Orchestia cavimana. Via single cell labelling of the cells that are involved in limb development, we are able to present the first complete clonal composition of an arthropod limb. We show that the two main branches of crustacean limbs, exopod and endopod, are formed by a secondary subdivision of the growth zone of the main limb axis. Additional limb outgrowths such as exites result from the establishment of new axes. In contrast to general belief, uniramous limbs in Orchestia are not formed by the loss of the exopod but by suppression of the split into exopod and endopod. Our results offer a developmental approach to discriminate between the different kinds of branches of arthropod appendages. This leads to the conclusion that a ‘true’ biramous limb comprising an endopod and an exopod might have occurred much later in euarthropod evolution than has previously been thought, probably either in the lineage of the Mandibulata or that of the Tetraconata.

External morphology of limb development in the amphipod Orchestia cavimana (Crustacea, Malacostraca, Peracarida)

The external morphology of limb development in Orchestia cavimana is examined by scanning electron microscopy and fluorescence staining from the appearance of the first limb buds until hatching. As other amphipods, O. cavimana undergoes direct development and the degree of segmental differentiation shows a more or less continual decrease in anteroposterior direction. Limbs form ventrally as small buds, which elongate and divide into podomeres early in development. This early subdivision largely corresponds with the limb segmentation of the hatchling. When the post-naupliar limbs start to develop, the germ band begins to split into two halves along the midline, so that the trunk limbs transiently occupy a very dorsolateral position. After the germ band has closed again, the differentiation into the characteristic amphipodan tagmata (cephalothorax, pereon, pleon) takes place and the limb podomeres lose their round-shape. The late embryo is covered by a so-called intermediate cuticle, which is formed after an embryonic moult and shed after hatching. The early development of O. cavimana reveals the Anlage of a vestigial seventh pleonic segment that is assumed to belong to the ground pattern of malacostracans, but is retained as a free, limbless segment only in adult Leptostraca. A transient subdivision of the proximal segment of the pleopods suggests the occurrence of a coxa and a basis in these limbs. The mandible attains its upright, adult position via a characteristic bending process that is strikingly similar to that in Archaeognatha (Insecta).

The embryonic development of the malacostracan crustacean Porcellio scaber (Isopoda, Oniscidea).

To examine the evolution of development and put it into a phylogenetic context, it is important to have, in addition to a model organism like Drosophila, more insights into the huge diversity of arthropod morphologies. In recent years, the malacostracan crustacean Porcellio scaber Latreille, 1804 has become a popular animal for studies in evolutionary and developmental biology, but a detailed and complete description of its embryonic development is still lacking. Therefore, the embryonic development of the woodlouse P. scaber is described in a series of discrete stages easily identified by examination of living animals and the widely used technique of nuclei staining on fixed specimens. It starts with the first cleavage of the zygote and ends with a hatched manca that eventually leaves the mother’s brood pouch. Classical methods like normal light microscopy, scanning electron microscopy and fluorescence microscopy are used, in addition to confocal LCM and computer-aided 3D reconstruction in order to visualise important processes during ontogeny. The purpose of these studies is to offer an easy way to define the different degrees of development for future comparative analyses of embryonic development amongst crustaceans in particular, as well as between different arthropod groups. In addition, several aspects of Porcellio embryonic development, such as the mouth formation, limb differentiations and modifications or the formation of the digestive tract, make this species particularly interesting for future studies in evolutionary and developmental biology.

Cell lineage analysis of the mandibular segment of the amphipod Orchestia cavimana reveals that the crustacean paragnaths are sternal outgrowths and not limbs

The question of arthropod head segmentation has become one of the central issues in Evolutionary Developmental Biology. The number of theories pertaining to head segments progressively enlarges, old concepts have been revitalized, and nearly every conceivable composition of the arthropod head has at some point received discussion. One contentious issue involves a characteristic mouthpart in crustaceans – the lower lips or the so-called paragnaths. The paragnaths build the posterior border of the mouth region antagonistic to the upper lip – the labrum. We show here the development of the appendage-like structures in the mandibular region of the amphipod crustacean Orchestia cavimana at a high level of cellular resolution. The embryos are examined during development of the mouthparts using in vivo labeling. An invariant cell division pattern of the mandibular segment was detected by 4D-microscopy and a preliminary model for pattern of the first cleavages in the mandibular region created. With this indispensable precondition single ectodermal cells of the grid-like pattern were labeled with DiI – a lipophilic fluorescent dye – to trace cell lineages and determine the clonal composition of the developing mouthparts, especially the mandibular segment. From our data it is evident that the paragnaths are sternal outgrowths of the mandible segment. The assumption of the limb nature of paragnaths and the presence of an additional head segment between the mandibular and the second antennal segments are clearly refuted by our data. Our results show the power of cell lineage and clonal analyses for inferences on the nature, origin and thus homology of morphological structures. With this kind of investigation morphological and gene expression data can be complemented.We discuss notable similarities of paragnath anlagen to those of the hypopharynx complex in myriapods and hexapods. The fact that both structures grow out as two lateral buds in the same region of the mandibular sternite during development, and their important role in the formation of the feeding apparatus as a highly specialized chewing chamber in adults of crustaceans, myriapods, and hexapods argue for the paragnaths/hypopharynx anlagen being an additional potential apomorphy of Mandibulata.

Development and staging of the water flea Daphnia magna (Straus, 1820; Cladocera, Daphniidae) based on morphological landmarks.

BackgroundCrustaceans of the genus Daphnia are one of the oldest model organisms in ecotoxicology, ecology and evolutionary biology. The publication of the Daphnia pulex genome has facilitated the development of genetic tools to answer long-standing questions in these research fields (Science 331: 555-561, 2011). A particular focus is laid on understanding the genetic basis of the striking ability of daphnids to change their phenotype in response to environmental stressors. Furthermore, Daphnia have recently been developed into crustacean model organisms for EvoDevo research, contributing to the ongoing attempt to resolve arthropod phylogeny. These problems require the comparative analyses of gene expression and functional data, which in turn require a standardized developmental staging system for Daphnia.ResultsHere we provide a detailed staging system of the embryonic development of Daphnia magna based on morphological landmarks. The staging system does not rely on developmental hours and is therefore suitable for functional and ecological experiments, which often cause developmental delays in affected embryos and thus shifts in time reference points. We provide a detailed description of each stage and include schematic drawings of all stages showing relevant morphological landmarks in order to facilitate the application of this staging scheme.ConclusionWe present here a staging system for Daphnia magna, which is based on morphological landmarks. The staging system can be adopted for other daphnids with minor variations since the sequence of development is highly conserved during early stages and only minor heterochronic shifts occur in late embryonic stages.

Arthropod Embryology: Cleavage and Germ Band Development

The overwhelming diversity of arthropod morphology and lifestyles finds it correspondence in a comparatively impressive variety of developmental trajectories. These ontogenetic differences concern all embryonic stages, steps, and levels from gene expression, cleavage and gastrulation, germ band formation and growth, to segmentation and morphogenesis (Weygoldt 1960a, 1963; Anderson 1973; Scholtz 1997; Akam 2000; Hughes and Kaufman 2002a). Likewise, postembryonic development reveals all sorts of growth patterns, direct and indirect development and within the latter a great variety of larval types with a wide spectrum of lifestyles comparable to those of the adult forms (see Chap. 5). However, it has to be stressed that variation in development is not necessarily directly correlated or even causally linked to adult diversity. Similar adult body organization and shapes can result from very different ontogenies, whereas similar ontogenies can result in highly diverse adults (Scholtz 2005).

Axogenesis in the central and peripheral nervous system of the amphipod crustacean Orchestia cavimana

We describe the formation of the major axon pathways in the embryonic central and peripheral nervous systems of the amphipod crustacean Orchestia cavimana Heller, 1865 by means of antibody staining against acetylated alpha-tubulin. The data add to a long list of previous studies of various other aspects of development in Orchestia and provide a basis for future studies of neurogenesis on a deeper cellular and molecular level. Orchestia exhibits a tripartite dorsal brain, which is a characteristic feature of euarthropods. Its anlagen are the first detectable structures in the developing nervous system and can be traced back to distinct neuronal cell clusters in the early embryo. The development of the ventral nervous system proceeds with an anteroposterior gradient of development. In each trunk segment, the longitudinal connectives and the anterior commissure form first, followed by the intersegmental nerve, the posterior commissure and segmental nerves, respectively. A single commissure of a vestigial seventh pleonal segment is found. In the peripheral nervous system we observe a spatial and temporal pattern of leg innervation, which is strikingly similar in both limb types, the uniramous pereopods and the biramous pleopods. A proximal leg nerve splitting distally into two separated nerves probably reflects a general feature of crustaceans.