Appendage patterning in the primitively wingless hexapods Thermobia domestica (Zygentoma: Lepismatidae) and Folsomia candida (Collembola: Isotomidae)

Arthropod appendages are among the most diverse animal organs and have been adapted to a variety of functions. Due to this diversity, it can be difficult to recognize homologous parts in different appendage types and different species. Gene expression patterns of appendage development genes have been used to overcome this problem and to identify homologous limb portions across different species and their appendages. However, regarding the largest arthropod group, the hexapods, most of these studies focused on members of the winged insects (Pterygota), but primitively wingless groups like the springtails (Collembola) or silverfish and allies (Zygentoma) are underrepresented. We have studied the expression of a set of appendage patterning genes in the firebrat Thermobia domestica and the white springtail Folsomia candida. The expressions of Distal-less (Dll) and dachshund (dac) are generally similar to the patterns reported for pterygote insects. Modifications of gene regulation, for example, the lack of Dll expression in the palp of F. candida mouthparts, however, point to changes in gene function that can make the use of single genes and specific expression domains problematic for homology inference. Such hypotheses should therefore not rely on a small number of genes and should ideally also include information about gene function. The expression patterns of homothorax (hth) and extradenticle (exd) in both species are similar to the patterns of crustaceans and pterygote insects, but differ from those in chelicerates and myriapods. The proximal specificity of hth thus appears to trace from a common hexapod ancestor and also provides a link to the regulation of this gene in crustaceans.     

A forgotten homology supporting the monophyly of Tracheata: The subcoxa of insects and myriapods re-visited

The current discussion about the relationships of higher arthropod taxa has led to a conflict between the traditional Tracheata (=Atelocerata) concept (hexapods united with myriapods), the Tetraconata concept (hexapods united with crustaceans, excluding myriapods), and the Paradoxopoda or Myriochelata concept (myriapods united with cheliceratans), with major contradictions between morphological and molecular data. We have analyzed a character set which apparently has completely vanished from the recent debate, namely the equipment of the trunk pleura of myriapods and insects with a characteristic set of concentric sclerites around the leg base and accompanying muscles. Based on the work of Heymons (1899) these sclerites were thought to be remains of the first appendage article, then denominated “subcoxa”. We have re-visited this old idea and show the arrangement of the much discussed pleural structures by SEM for the first time. Obviously a characteristic pattern of concentric pleural plates around the leg-base is present in all major myriapod taxa, including for the first time evidence for their presence in Progoneata. Because of their equal structure and orientation, the pleural sclerites present in entognathous and ectognathous insects may be derived from the same ground pattern. We conclude that the pleurites of Hexapoda and Myriapoda seem to be homologous structures, and there is evidence that the “subcoxa” of Tracheata is homologous with the coxa of crustaceans. Since no other arthropods show these sclerites, the transformation of the crustacean coxa to the pleural region in myriapods and insects is probably a synapomorphy congruent with the traditional Tracheata-hypothesis. Further investigations of recently published molecular work using the phylogenetic network software SplitsTree V.4 indicate that information content of several data sets is not convincing.     

Larval leg integrity is maintained by Distal-less and is required for proper timing of metamorphosis in the flour beetle, Tribolium castaneum

The dramatic transformation from a larva to an adult must be accompanied by a coordinated activity of genes and hormones that enable an orchestrated transformation from larval to pupal/adult tissues. The maintenance of larval appendages and their subsequent transformation to appendages in holometabolous insects remains elusive at the developmental genetic level. Here the role of a key appendage patterning gene Distal-less (Dll) was examined in mid- to late-larval stages of the flour beetle, Tribolium castaneum. During late larval development, Dll was expressed in appendages in a similar manner as previously reported for the tobacco hornworm, Manduca sexta. Removal of this late Dll expression resulted in disruption of adult appendage patterning. Intriguingly, earlier removal resulted in dramatic loss of structural integrity and identity of larval appendages. A large amount of variability in appendage morphology was observed following Dll dsRNA injection, unlike larvae injected with dachshund dsRNA. These Dll dsRNA-injected larvae underwent numerous supernumerary molts, which could be terminated with injection of either JH methyltransferase or Methoprene-tolerant dsRNA. Apparently, the partial dedifferentiation of the appendages in these larvae acts to maintain high JH and, hence, prevents metamorphosis.     

Of mites and millipedes: Recent progress in resolving the base of the arthropod tree

Deep‐level arthropod phylogeny has been in a state of upheaval ever since the emergence of molecular tree reconstruction approaches. While a consensus has settled in that hexapods are more closely related to crustaceans than to myriapods, the phylogenetic position of the latter has remained a matter of debate. Mitochondrial, nuclear, and genome‐scale studies have proposed rejecting the long‐standing superclade Mandibulata, which unites myriapods with insects and crustaceans, in favor of a clade that unites myriapods with chelicerates and has become known as Paradoxapoda or Myriochelata. Here we discuss the progress, problems, and prospects of arriving at the final arthropod tree.     

Neurogenesis in myriapods and chelicerates and its importance for understanding arthropod relationships

Several alternative hypotheses on the relationships betweenthe major arthropod groups are still being discussed. We reexaminehere the chelicerate/myriapod relationship by comparing previouslypublished morphological data on neurogenesis in the euarthropodgroups and presenting data on an additional myriapod (Strigamiamaritima). Although there are differences in the formation ofneural precursors, most euarthropod species analyzed generateabout 30 single neural precursors (insects/crustaceans) or precursorgroups (chelicerates/myriapods) per hemisegment that are arrangedin a regular pattern. The genetic network involved in recruitmentand specification of neural precursors seems to be conservedamong euarthropods. Furthermore, we show here that neural precursoridentity seems to be achieved in a similar way. Besides theseconserved features we found 2 characters that distinguish insects/crustaceansfrom myriapods/chelicerates. First, in insects and crustaceansthe neuroectoderm gives rise to epidermal and neural cells,whereas in chelicerates and myriapods the central area of theneuroectoderm exclusively generates neural cells. Second, neuralcells arise by stem-cell-like divisions of neuroblasts in insectsand crustaceans, whereas groups of mainly postmitotic neuralprecursors are recruited for the neural fate in cheliceratesand myriapods. We discuss whether these characteristics representa sympleisiomorphy of myriapods and chelicerates that has beenlost in the more derived Pancrustacea or whether these characteristicsare a synapomorphy of myriapods and chelicerates, providingthe first morphological support for the Myriochelata group.     

Gene expression patterns in an onychophoran reveal that regionalization predates limb segmentation in pan‐arthropods

SUMMARY In arthropods, such as Drosophila melanogaster, the leg gap genes homothorax (hth), extradenticle (exd), dachshund (dac), and Distal‐less (Dll) regionalize the legs in order to facilitate the subsequent segmentation of the legs. We have isolated homologs of all four leg gap genes from the onychophoran Euperipatoides kanangrensis and have studied their expression. We show that leg regionalization takes place in the legs of onychophorans even though they represent simple and nonsegmented appendages. This implies that leg regionalization evolved for a different function and was only later co‐opted for a role in leg segmentation. We also show that the leg gap gene patterns in onychophorans (especially of hth and exd) are similar to the patterns in crustaceans and insects, suggesting that this is the plesiomorphic state in arthropods. The reversed hth and exd patterns in chelicerates and myriapods are therefore an apomorphy for this group, the Myriochelata, lending support to the Myriochelata and Tetraconata clades in arthropod phylogeny.     

A conserved genetic mechanism specifies deutocerebral appendage identity in insects and arachnids

The segmental architecture of the arthropod head is one of the most controversial topics in the evolutionary developmental biology of arthropods. The deutocerebral (second) segment of the head is putatively homologous across Arthropoda, as inferred from the segmental distribution of the tripartite brain and the absence of Hox gene expression of this anterior-most, appendage-bearing segment. While this homology statement implies a putative common mechanism for differentiation of deutocerebral appendages across arthropods, experimental data for deutocerebral appendage fate specification are limited to winged insects. Mandibulates (hexapods, crustaceans and myriapods) bear a characteristic pair of antennae on the deutocerebral segment, whereas chelicerates (e.g. spiders, scorpions, harvestmen) bear the eponymous chelicerae. In such hexapods as the fruit fly, Drosophila melanogaster, and the cricket, Gryllus bimaculatus, cephalic appendages are differentiated from the thoracic appendages (legs) by the activity of the appendage patterning gene homothorax (hth). Here we show that embryonic RNA interference against hth in the harvestman Phalangium opilio results in homeonotic chelicera-to-leg transformations, and also in some cases pedipalp-to-leg transformations. In more strongly affected embryos, adjacent appendages undergo fusion and/or truncation, and legs display proximal defects, suggesting conservation of additional functions of hth in patterning the antero-posterior and proximo-distal appendage axes. Expression signal of anterior Hox genes labial, proboscipedia and Deformed is diminished, but not absent, in hth RNAi embryos, consistent with results previously obtained with the insect G. bimaculatus. Our results substantiate a deep homology across arthropods of the mechanism whereby cephalic appendages are differentiated from locomotory appendages.     

Gene silencing in the spider mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>: dsRNA and siRNA parental silencing of the <Emphasis Type="Italic">Distal-less</Emphasis> gene

A major prerequisite to understanding the evolution of developmental programs includes an appreciation of gene function in a comparative context. RNA interference (RNAi) represents a powerful method for reverse genetics analysis of gene function. However, RNAi protocols exist for only a handful of arthropod species. To extend functional analysis in basal arthropods, we developed a RNAi protocol for the two-spotted spider mite Tetranychus urticae focusing on Distal-less (Dll), a conserved gene involved in appendage specification in metazoans. First, we describe limb morphogenesis in T. urticae using confocal and scanning electron microscopy. Second, we examine T. urticae Dll (Tu-Dll) mRNA expression patterns and correlate its expression with appendage development. We then show that fluorescently labeled double-stranded RNA (dsRNA) and short interfering RNA (siRNA) molecules injected into the abdomen of adult females are incorporated into the oviposited eggs, suggesting that dsRNA reagents can be systemically distributed in spider mites. Injection of longer dsRNA as well as siRNA induced canonical limb truncation phenotypes as well as the fusion of leg segments. Our data suggest that Dll plays a conserved role in appendage formation in arthropods and that such conserved genes can serve as reliable starting points for the development of functional protocols in nonmodel organisms.     

Distalless expression in crustaceans and the patterning of branched limbs

 In Drosophila, Distalless (Dll) is critical in establishing the proximal/distal axis of the leg. Lack of proper Dll expression causes distal limb structures to be truncated or lost. Dll expression was examined through the course of development in the limbs of two crustaceans, Triops and Nebalia. Because the limbs of these two species are branched, they provide a comparison to the uniramous (unbranched) leg of Drosophila. In Triops and Nebalia, development of limb branches is not tightly coupled with Dll expression: in some cases, branches can arise prior to Dll expression and in others, certain branches never express Dll. These data suggest that, while Dll may indeed initiate overall limb outgrowth, limb branches are unlikely to be patterned by a simple iteration of the mechanism patterning the unbranched leg of Drosophila. Received: 14 May 1997 / Accepted: 25 September 1997     

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.     

Patterning of the branched head appendages in Schistocerca americana and Tribolium castaneum

Much of our understanding of arthropod limb development comes from studies on the leg imaginal disc of Drosophila melanogaster. The fly limb is a relatively simple unbranched (uniramous) structure extending out from the body wall. The molecular basis for this outgrowth involves the overlap of two signaling molecules, Decapentaplegic (Dpp) and Wingless (Wg), to create a single domain of distal outgrowth, clearly depicted by the expression of the Distal-less gene (Dll). The expression of wg and dpp during the development of other arthropod thoracic limbs indicates that these pathways might be conserved across arthropods for uniramous limb development. The appendages of crustaceans and the gnathal appendages of insects, however, exhibit a diverse array of morphologies, ranging from those with no distal elements, such as the mandible, to appendages with multiple distal elements. Examples of the latter group include branched appendages or those that possess multiple lobes; such complex morphologies are seen for many crustacean limbs as well as the maxillary and labial appendages of many insects. It is unclear how, if at all, the known patterning genes for making a uniramous limb might be deployed to generate these diverse appendage forms. Experiments in Drosophila have shown that by forcing ectopic overlaps of Wg and Dpp signaling it is possible to generate artificially branched legs. To test whether naturally branched appendages form in a similar manner, we detailed the expression patterns of wg, dpp, and Dll in the development of the branched gnathal appendages of the grasshopper, Schistocerca americana, and the flour beetle, Tribolium castaneum. We find that the branches of the gnathal appendages are not specified through the redeployment of the Wg-Dpp system for distal outgrowth, but our comparative studies do suggest a role for Dpp in forming furrows between tissues.     

Molecular phylogeny of the major arthropod groups indicates polyphyly of crustaceans and a new hypothesis for the origin of hexapods

A phylogeny of the arthropods was inferred from analyses of amino acid sequences derived from the nuclear genes encoding elongation factor-1 alpha and the largest subunit of RNA polymerase II using maximum- parsimony, neighbor-joining, and maximum-likelihood methods. Analyses of elongation factor-1 alpha from 17 arthropods and 4 outgroup taxa recovered many arthropod clades supported by previous morphological studies, including Diplopoda, Myriapoda, Insecta, Hexapoda, Branchiopoda (Crustacea), Araneae, Tetrapulmonata, Arachnida, Chelicerata, and Malacostraca (Crustacea). However, counter to previous studies, elongation factor-1 alpha placed Malacostraca as sister group to the other arthropods. Branchiopod crustaceans were found to be more closely related to hexapods and myriapods than to malacostracan crustaceans. Sequences for RNA polymerase II were obtained from 11 arthropod taxa and were analyzed separately and in combination with elongation factor-1 alpha. Results from these analyses were concordant with those derived from elongation factor-1 alpha alone and provided support for a Hexapoda/Branchiopoda clade, thus arguing against the monophyly of the traditionally defined Atelocerata (Hexapoda + Myriapoda).      

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.     

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.     

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.     

Velvet worm development links myriapods with chelicerates

Despite the advent of modern molecular and computational methods, the phylogeny of the four major arthropod groups (Chelicerata, Myriapoda, Crustacea and Hexapoda, including the insects) remains enigmatic. One particular challenge is the position of myriapods as either the closest relatives to chelicerates (Paradoxopoda/Myriochelata hypothesis), or to crustaceans and hexapods (Mandibulata hypothesis). While neither hypothesis receives conclusive support from molecular analyses, most morphological studies favour the Mandibulata concept, with the mandible being the most prominent feature of this group. Although no morphological evidence was initially available to support the Paradoxopoda hypothesis, a putative synapomorphy of chelicerates and myriapods has recently been put forward based on studies of neurogenesis. However, this and other morphological characters remain of limited use for phylogenetic systematics owing to the lack of data from an appropriate outgroup. Here, we show that several embryonic characters are synapomorphies uniting the chelicerates and myriapods, as revealed by an outgroup comparison with the Onychophora or velvet worms. Our findings, thus provide, to our knowledge, first morphological/embryological support for the monophyly of the Paradoxopoda and suggest that the mandible might have evolved twice within the arthropods.     

Evolution of the chelicera: a dachshund domain is retained in the deutocerebral appendage of Opiliones (Arthropoda,Chelicerata)

The proximo‐distal axis of the arthropod leg is patterned by mutually antagonistic developmental expression domains of the genes extradenticle, homothorax, dachshund, and Distal‐less. In the deutocerebral appendages (the antennae) of insects and crustaceans, the expression domain of dachshund is frequently either absent or, if present, is not required to pattern medial segments. By contrast, the dachshund domain is entirely absent in the deutocerebral appendages of spiders, the chelicerae. It is unknown whether absence of dachshund expression in the spider chelicera is associated with the two‐segmented morphology of this appendage, or whether all chelicerates lack the dachshund domain in their chelicerae. We investigated gene expression in the harvestman Phalangium opilio, which bears the plesiomorphic three‐segmented chelicera observed in “primitive” chelicerate orders. Consistent with patterns reported in spiders, in the harvestman chelicera homothorax, extradenticle, and Distal‐less have broadly overlapping developmental domains, in contrast with mutually exclusive domains in the legs and pedipalps. However, unlike in spiders, the harvestman chelicera bears a distinct expression domain of dachshund in the proximal segment, the podomere that is putatively lost in derived arachnids. These data suggest that a tripartite proximo‐distal domain structure is ancestral to all arthropod appendages, including deutocerebral appendages. As a corollary, these data also provide an intriguing putative genetic mechanism for the diversity of arachnid chelicerae: loss of developmental domains along the proximo‐distal axis.     

Hox genes and the evolution of the arthropod body plan

In recent years researchers have analyzed the expression patterns of the Hox genes in a multitude of arthropod species, with the hope of understanding the mechanisms at work in the evolution of the arthropod body plan. Now, with Hox expression data representing all four major groups of arthropods (chelicerates, myriapods, crustaceans, and insects), it seems appropriate to summarize the results and take stock of what has been learned. In this review we summarize the expression and functional data regarding the 10 arthropod Hox genes: labial proboscipedia, Hox3/zen, Deformed, Sex combs reduced, fushi tarazu, Antennapedia, Ultrabithorax, abdominal-A, and Abdominal-B. In addition, we discuss mechanisms of developmental evolutionary change thought to be important for the emergence of novel morphological features within the arthropods.     

Neurogenesis in the chilopod <Emphasis Type="Italic">Lithobius forficatus</Emphasis> suggests more similarities to chelicerates than to insects

In a recent comparative study on neurogenesis in the diplopod Glomeris marginata we have shown that the millipede and the spider share several features that cannot be found in homologous form in insects and crustaceans. The most distinctive difference is that groups of neural precursors are singled out from the neuroectoderm of the spider and the diplopod, rather than individual cells (i.e. neuroblasts) as in insects or crustacean. This observation constitutes the first morphological indication for a close myriapod/chelicerate relationship that has otherwise only been suggested by molecular phylogenetic analysis. To see whether the pattern of neurogenesis described for the diplopod is representative for myriapods, we analysed neurogenesis in the basal chilopod Lithobius forficatus. We show here that groups of cells invaginate from the chilopod neuroectoderm at strikingly similar positions as the invaginating cell groups of the diplopod and the spider. Furthermore, the expression patterns of the proneural and neurogenic genes reveal more similarities to the chelicerate and the diplopod than to insects. Thus, chelicerates and myriapods share the developmental mechanism for neurogenesis, either because they are true sister groups, or because this reflects the ancestral state of neurogenesis in arthropods.Edited by P. Simpson     

Transitions in functional morphology from “large branchiopods” to Cladocera: Video and confocal microscopic studies of Cyclestheria hislopi (Cyclestherida) and Sida crystallina (Cladocera: Ctenopoda)

Great diversity is found in morphology and functionality of arthropod appendages, both along the body axis of individual animals and between different life-cycle stages. Despite many branchiopod crustaceans being well known for displaying a relatively simple arrangement of many serially post-maxillary appendages (trunk limbs), this taxon also shows an often unappreciated large variation in appendage morphology. Diplostracan branchiopods exhibit generally a division of labor into locomotory antennae and feeding/filtratory post-maxillary appendages (trunk limbs). We here study the functionality and morphology of the swimming antennae and feeding appendages in clam shrimps and cladocerans and analyze the findings in an evolutionary context (e.g., possible progenetic origin of Cladocera). We focus on Cyclestheria hislopi (Cyclestherida), sister species to Cladocera and exhibiting many “large” branchiopod characters (e.g., many serially similar appendages), and Sida crystallina (Cladocera, Ctenopoda), which likely exhibits plesiomorphic cladoceran traits (e.g., six pairs of serially similar appendages). We combine (semi-)high-speed recordings of behavior with confocal laser scanning microscopy analyses of musculature to infer functionality and homologies of locomotory and filtratory appendages in the two groups. Our morphological study shows that the musculature in all trunk limbs (irrespective of limb size) of both C. hislopi and S. crystallina comprises overall similar muscle groups in largely corresponding arrangements. Some differences between C. hislopi and S. crystallina, such as fewer trunk limbs and antennal segments in the latter, may reflect a progenetic origin of Cladocera. Other differences seem related to the appearance of a specialized type of swimming and feeding in Cladocera, where the anterior locomotory system (antennae) and the posterior feeding system (trunk limbs) have become fully separated functionally from each other. This separation is likely one explanation for the omnipresence of cladocerans, which have conquered both freshwater and marine free water masses and a number of other habitats.