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.     

Homology of Holocene ostracode biramous appendages with those of other crustaceans: the protopod, epipod, exopod and endopod

Unambiguously biramous appendages with a proximal precoxa, well-defined coxa and basis, setose plate-like epipod originating on the precoxa, and both an endopod and exopod attached to the terminal end of the basis are described from several living Ostracoda of the order Halo-cyprida (Myodocopa). These limbs are proposed as the best choice for comparison of ostracode limbs with those of other crustaceans and fossil arthropods with preserved limbs, such as the Cambrian superficially ostracode-like Kunmingella and Hesslandona. The 2nd maxilla of Metapolycope (Cladocopina) and 1st trunk limb of Spelaeoecia, Deeveya and Thaumatoconcha (all Halocypridina) are illustrated, and clear homologies are shown between the parts of these limbs and those of some general crustacean models as well as some of the remarkable crustacean s.s. Orsten fossils. No living ostracodes exhibit only primitive morphology; all have at least some (usually many) derived characters. Few have the probably primitive attribute of trunk segmentation (two genera of halocyprid Myodocopa, one order plus one genus of Podocopa, and the problematic Manawa); unambiguously biramous limbs are limited to a few halo-cyprids. Homologies between podocopid limbs and those of the illustrated primitive myodocopid limbs are tentatively suggested. A setose plate-like extension, often attached basally to a podocopid protopod, is probably homologous to the myodocopid epipod, which was present at least as early as the Triassic. Somewhat more distal, less setose, and plate-like extensions, present on some podocopid limbs (e.g., mandible), may be homologous instead to the exopod (clearly present on myodocopid mandibles). The coxa (or precoxa) is by definition the most basal part of the limb. A molar-like tooth is present proximally on the mandibular protopod of many ostracodes; it is the coxal endite and projects medially from the coxa (or proximal protopod). The Ostracoda is probably a monophyletic crustacean group composed of Myodocopa and Podocopa. All have a unique juvenile (not a larva) initially with three or more limbs. Except that juveniles lack some setae and limbs, they are morphologially similar to the adult. Thus the following suite of characters in all instars may be considered a synapomorphy uniting all Ostracoda: (1) Each pair of limbs is uniquely different from the others. (2) The whole body is completely enclosed within a bivalved carapace that lacks growth lines. (3) No more than nine pairs of limbs are present in any instar. (4) The body shows little or no segmentation, with no more than ten dorsally defined trunk segments. No other crustaceans have this suite of characters. A probable synapomorphy uniting the Podocopa is a 2nd antenna with exopod reduced relative to the endopod.     

Expression of collier in the premandibular segment of myriapods: support for the traditional Atelocerata concept or a case of convergence?

Background  

A recent study on expression and function of the ortholog of the Drosophila collier (col) gene in various arthropods including insects, crustaceans and chelicerates suggested a de novo function of col in the development of the appendage-less intercalary segment of insects. However, this assumption was made on the background of the now widely-accepted Pancrustacea hypothesis that hexapods represent an in-group of the crustaceans. It was therefore assumed that the expression of col in myriapods would reflect the ancestral state like in crustaceans and chelicerates, i.e. absence from the premandibular/intercalary segment and hence no function in its formation.     

Evolving role of Antennapedia protein in arthropod limb patterning

Evolutional changes in homeotic gene functions have contributed to segmental diversification of arthropodan limbs, but crucial molecular changes have not been identified to date. The first leg of the crustacean Daphnia lacks a prominent ventral branch found in the second to fourth legs. We show here that this phenotype correlates with the loss of Distal-less and concomitant expression of Antennapedia in the limb primordium. Unlike its Drosophila counterpart, Daphnia Antennapedia represses Distal-less in Drosophila assays, and the protein region conferring this activity was mapped to the N terminal region of the protein. The results imply that Dapnia Antennapedia specifies leg morphology by repressing Distal-less, and this activity was acquired through a change in protein structure after separation of crustaceans and insects.     

Origin of the crustacean schizoramous limb: A re-analysis of the duplosegmentation hypothesis

Emerson and Schram's hypothesis of a duplosegmental origin of the crustacean schizoramous (biramous) limb is confronted with new data on the phylogeny of arthropods and their mechanisms of development. The hypothesis is considered to be falsified since (a) the cephalic segments of the crustaceans bear schizoramous limbs but still seem to be homologous to those of the hexapods; (b) the embryonic origin of crustacean segments, limbs, and neuromeres appears to be the same as in the hexapods; (c) no genetic mechanism allowing for duplosegmentation in the crustaceans seems to exist.     

Comparative morphology among trunk limbs of Caenestheriella gifuensis and Leptestheria kawachiensis (Crustacea: Branchiopoda: Spinicaudata)

Morphological differences among groups of the 24 trunk limbs of Caenestheriella gifuensis (Ishikawa, 1895) and differences between males and females are described and illustrated. A setose attenuate lobe located proximally near enditic lobe 1 and a discoid lobe covered with small setae proximal to enditic lobe 1 are newly described. The five ventral enditic lobes, endopod, exopod, and dorsal exite of traditional spinicaudatan morphology are redescribed. Trunk limbs 1–4 of females bear a palp on enditic lobe 5 and trunk limbs 1–15 of males bear a similar palp. A second, articulating palp is associated with the base of the endopod of trunk limbs 1–2 of males. The proximal part of trunk limbs 19–24, bearing enditic lobe 1, articulates by an arthrodial membrane with the remainder of the limb, and the exite is distal to this arthrodial membrane. Development of trunk limbs, ascertained through an examination of early juvenile instars of Leptestheria kawachiensis Uéno, 1927, includes an asetose limb followed in time by a series of setose limbs that increase in morphological complexity with age. The number of lobes on the asetose limb varies from seven (corresponding to five enditic lobes, an endopod, and an exopod) on anterior limbs to five on trunk limb 24, which lacks the lobes corresponding to enditic lobe 4 and the endopod; these two structures are added later to setose limbs. The attenuate lobe, the discoid lobe, the exite of all trunk limbs, and the palps of the anterior trunk limbs are added to the setose limbs. Development of anterior limbs is accelerated relative to that of posterior limbs, and development of the more posterior limbs is truncated relative to that of limbs immediately anterior to them. Enditic lobe 4 and the endopod of limbs like trunk limb 24 develop from, or are patterned by, enditic lobe 5; the articulating palp of male trunk limbs 1–2 also may be added in this way. A comparison of these observations with development of the copepod maxilliped suggests that the spinicaudatan trunk limb is composed of a praecoxa with three lobes, a coxa and a basis each with one lobe, and an endopod of three segments in females and four in males. This is similar to the homology scheme previously proposed by Hansen in 1925. A critique is given of attempts to homologize parts of arthropod limbs based on developmental gene expression patterns. Stenopodal to phyllopodal transformations of maxillipeds in copepods provide a model at least partly applicable to spinicaudatans, and a ‘multibranched’ interpretation of spinicaudatan (and by extension branchiopodan) limb morphology is rejected. There is nothing intrinsic to the structure of the adult trunk limbs suggesting that they are similar to the adult limbs of the ancestral branchiopod or the ancestral crustacean, but early developmental steps of more posterior limbs are good matches for the morphology of an ancestral crustacean biramal limb predicted by a hypothesis of duplication of the proximo‐distal axis. © 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 139 , 547–564. No claim to original US government works.     

Gene expression reveals evidence for EGFR‐dependent proximal‐distal limb patterning in a myriapod

Evolution of segmented limbs is one of the key innovations of Arthropoda, allowing development of functionally specific specialized head and trunk appendages, a major factor behind their unmatched evolutionary success. Proximodistal limb patterning is controlled by two regulatory networks in the vinegar fly Drosophila melanogaster, and other insects. The first is represented by the function of the morphogens Wingless (Wg) and Decapentaplegic (Dpp); the second by the EGFR‐signaling cascade. While the role of Wg and Dpp has been studied in a wide range of arthropods representing all main branches, that is, Pancrustacea (= Hexapoda + Crustacea), Myriapoda and Chelicerata, investigation of the potential role of EGFR‐signaling is restricted to insects (Hexapoda). Gene expression analysis of Egfr, its potential ligands, and putative downstream factors in the pill millipede Glomeris marginata (Myriapoda: Diplopoda), reveals that—in at least mandibulate arthropods—EGFR‐signaling is likely a conserved regulatory mechanism in proximodistal limb patterning.     

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     

Hypothesis testing in evolutionary developmental biology: a case study from insect wings

Developmental data have the potential to give novel insights into morphological evolution. Because developmental data are time-consuming to obtain, support for hypotheses often rests on data from only a few distantly related species. Similarities between these distantly related species are parsimoniously inferred to represent ancestral aspects of development. However, with limited taxon sampling, ancestral similarities in developmental patterning can be difficult to distinguish from similarities that result from convergent co-option of developmental networks, which appears to be common in developmental evolution. Using a case study from insect wings, we discuss how these competing explanations for similarity can be evaluated. Two kinds of developmental data have recently been used to support the hypothesis that insect wings evolved by modification of limb branches that were present in ancestral arthropods. This support rests on the assumption that aspects of wing development in Drosophila, including similarities to crustacean epipod patterning, are ancestral for winged insects. Testing this assumption requires comparisons of wing development in Drosophila and other winged insects. Here we review data that bear on this assumption, including new data on the functions of wingless and decapentaplegic during appendage allocation in the red flour beetle Tribolium castaneum.     

The role of wingless in the development of multibranched crustacean limbs

 Arthropods are the most diverse and speciose group of organisms on earth. A key feature in their successful radiation is the ease with which various appendages become readily adapted to new functions in novel environments. Arthropod limbs differ radically in form and function, from unbranched walking legs to multibranched swimming paddles. To uncover the developmental and genetic mechanisms underlying this diversification in form, we ask whether a three-signal model of limb growth based on Drosophila experiments is used in the development of arthropod limbs with variant shape. We cloned a Wnt-1 ortholog (Tlwnt-1) from Triops longicaudatus, a basal crustacean with a multibranched limb. We examined the mRNA in situ hybridization pattern during larval development to determine whether changes in wg expression are correlated with innovation in limb form. During larval growth and segmentation Tlwnt-1 is expressed in a segmentally reiterated pattern in the trunk. Unexpectedly, this pattern is restricted to the ventral portion of the epidermis. During early limb formation the single continuous stripe of Tlwnt-1 expression in each segment becomes ventrolaterally restricted into a series of shorter stripes. Some but not all of these shorter stripes correspond to what becomes the ventral side of a developing limb branch. We conclude that the Drosophila model of limb development cannot explain all types of arthropod proximodistal outgrowths, and that the multibranched limb of Triops develops from an early reorganization of the ventral body wall. In Triops, Tlwnt-1 plays a semiconservative role similar to that played by Drosophila wingless in segmentation and limb formation, and morphological innovation in limb form arises in part through an early modulation in the expression of the Tlwnt-1 gene. Received: 22 September 1998 / Accepted: 12 January 1999     

Homeotic duplication of the pelvic body segment in regenerating tadpole tails induced by retinoic acid

 Homeosis, the ectopic formation of a body part, is one of the key phenomena that prompted the identification of the essential selector genes controlling body organization. Shared elements of such homeotic genes exist in all studied animal classes, but homeotic transformations of the same order of magnitude as in insects, such as the duplication of the thorax in Drosophila mutants, have not been described in vertebrates. Here we investigate the capacity of retinoic acid to modify tail regeneration in amphibians. We show that retinoic acid causes the formation of an additional body segment in regenerating tails of Rana temporaria tadpoles. A second pelvic section, including vertebral elements, pelvic girdle elements and limb buds, forms at the mid-tail level. This is the first report of a homeotic duplication of a whole body segment in vertebrate axial regeneration. Received: 16 August 1996 / Accepted: 20 September 1996     

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

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

Clonal analysis of <Emphasis Type="Italic">Distal-less</Emphasis> and <Emphasis Type="Italic">engrailed</Emphasis> expression patterns during early morphogenesis of uniramous and biramous crustacean limbs

In order to investigate the correlation of cell lineage, gene expression, and morphogenesis of uniramous and biramous limbs we studied limb formation in the thorax and pleon of the amphipod Orchestia cavimana and the isopod Porcellio scaber. We took advantage of the fact that in amphipod and isopod crustaceans—both Malacostraca—uniramous limbs evolved independently in the thorax whereas ancestral biramous limbs are formed in the pleon (abdomen). The gene Distal-less is expressed in the early limb buds as in other arthropods. Accordingly, it is likely to be responsible for the development of the proximodistal axis of the appendages. Double staining of Distal-less and Engrailed proteins suggests that Distal-less in the pleon of the amphipod Orchestia might not be under the control of the Wingless protein. Additionally, we studied axis formation of the uniramous and biramous limbs. In both species investigated, biramous limbs originate exclusively by the subdivision of the original limb bud. Both distal elements continuously express Distal-less. There is flexibility in the suppression of the development of additional branches in the crustacean limb. In the amphipod O. cavimana, uniramous thoracopods are formed by downregulation of Distal-less in the area where, in biramous limbs, the exopodites would occur. In contrast, this region never expresses Distal-less in the uniramous thoracopods of the isopod P. scaber. Our results suggest that the gene expression pattern is independent of the cell division pattern. Gene expression domains and morphogenesis of limbs and segments, on the other hand, show a good correlation.Edited by D. Tautz     

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.     

The roles of wingless and decapentaplegic in axis and appendage development in the red flour beetle, Tribolium castaneum

Axis patterning and appendage development have been well studied in Drosophila melanogaster, a species in which both limb and segment morphogenesis are derived. In Drosophila, positional information from genes important in anteroposterior and dorsoventral axis formation, including wingless (wg) and decapentaplegic (dpp), is required for allocating and patterning the appendage primordia. We used RNA interference to characterize the functions of wg and dpp in the red flour beetle, Tribolium castaneum, which retains more ancestral modes of limb and segment morphogenesis. We also characterized the expression of potential targets of the WG and DPP signaling pathways in these embryos. Tribolium embryos in which dpp had been downregulated had defects in the dorsalmost body wall, but did not appear to have been globally repatterned and had normal appendages. Downregulation of wg led to the loss of segment boundaries, gnathal and thoracic appendages, and lateral head lobes, and to changes in the expression of dpp, Distal-less, and Engrailed. The functions of wg varied along both the anteroposterior and dorsoventral axes of the embryo. Phylogenetic comparisons indicate that the role of WNT signaling in segment boundary formation is evolutionarily old, but that its role in appendage allocation originated in the common ancestor of holometabolous insects.     

Evolution of distinct expression patterns for engrailed paralogues in higher crustaceans (Malacostraca)

The segment-polarity gene engrailed of Drosophila melanogaster and its homologues in other arthropods possess a highly conserved expression domain in the posterior portion of each segment. We report here that the two pan-specific antibodies, Mab4D9 and Mab4F11, reveal strikingly different accumulation patterns in both of the malacostracan crustaceans Porcellio scaber (Isopoda) and Procambarus clarkii (Decapoda), compared with insects. The signal detected with Mab4D9 resides in the posterior part of each segment, including the appendages, the ventral and lateral sides of the trunk and the CNS. However, Mab4F11 reveals a signal only in small groups of neurons in the CNS and PNS, primarily localized in the pereon. We observe similar Mab4D9 and Mab4F11 patterns in the crayfish P. clarkii, except that no Mab4F11 signal is detected in the pleon. To address the possibility of multiple engrailed paralogues, we cloned partial cDNAs of two engrailed genes, Ps-en1 and Ps-en2, from P. scaber, and studied their expression patterns using whole-mount in situ hybridization. Although the Ps-en1 and Ps-en2 patterns are comparable in early development, they become distinct in late embryogenesis. Ps-en1 is expressed in the CNS, where Mab4F11 stains, but also accumulates in the epidermis. In contrast, Ps-en2 is expressed in the lateral aspect and limbs of all segments. Phylogenetic analysis of en sequences from crustaceans and insects suggests that the two en genes from the apterygote insect Thermobia domestica (Thysanura) may be related to en1 and en2 of higher crustaceans. Received: 14 February 2000 / Accepted: 1 June 2000     

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.     

Analysis of the expression pattern of Mysidium columbiae wingless provides evidence for conserved mesodermal and retinal patterning processes among insects and crustaceans

The Wnt family includes a number of genes, such as wingless ( wg), which encode secreted glycoproteins that function in numerous developmental patterning processes. In order to gain a better understanding of crustacean pattern formation, a wg orthologue was cloned from the malacostracan crustacean Mysidium columbiae(mysid), and the expression pattern of this gene was compared with that of Drosophila wg. Although Drosophila wg is expressed in many developing tissues, such as the ventral neuroectoderm, M. columbiae wg (mcowg)expression is detected within only a subset of these tissues. mcowg is expressed in the dorsal part of each developing segment and within the developing eye, but not within the ventral neuroectoderm. Dorsal wg expression in Drosophila is required for heart and muscle development, and conservation of this dorsal wgexpression pattern suggests that mcowgmay function to pattern these tissues in mysids. Consistent with this, expression of Even-skipped (Eve) protein in heart precursor and muscle cells, which is dependent on Wg signaling in Drosophila, is also conserved in mysids. Within the developing mysid eye, mcowg is expressed in a pattern that is similar to the expression pattern of Drosophila wg in the fly eye disc. In Drosophila,Wg inhibits neural differentiation at the anterior margin of the eye disc and patterns the dorsal/ventral axis of the eye. These data indicate that mcowg may function similarly during mysid eye development. Analysis of mcowgexpression provides molecular evidence suggesting that the processes of heart, muscle, and eye patterning are likely to be conserved among insects and crustaceans.     

Crustacean biodiversity as an important factor for mosquito larval control

Newly established ponds, which are highly dynamic systems with changing levels of biological interactions among species, are common larval mosquito habitats. We investigated the impact of crustacean abundance and taxa diversity on mosquito oviposition and larval development. The effects of the biological larvicide Bacillus thuringiensis israelensis (Bti) on mosquito larvae were monitored according to fluctuations in crustacean communities. Populations of the mosquito Culex pipiens colonized artificial ponds that contained crustacean communities at different time points of colonization by crustaceans: 1) ‘no colonization’ (no crustaceans), 2) ‘simultaneous colonization’ by crustaceans and mosquitoes, and 3) ‘head‐start colonization’ by crustaceans (preceding colonization by mosquitoes). All types of ponds were treated with three concentrations of Bti (10, 100, or 1,000 µg/liter). Colonization of all ponds by Cx. pipiens (in terms of oviposition, larval abundance, and larval development) decreased significantly with increasing diversity of crustacean taxa. The total abundance of crustaceans had a minor effect on colonization by Cx. pipiens. The presence of crustaceans increased the sensitivity of Cx. pipiens larvae to Bti treatment by a factor of 10 and delayed the time of recolonization. This effect of Bti was relevant in the short term. In the long term, the presence of Cx. pipiens was determined by crustacean biodiversity.     

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.