It is arguable that the evolutionary and ecological success of insects is due in large part to the versatility of their articulated appendages. Recent advances in our understanding of appendage development in Drosophila melanogaster, as well as functional and expression studies in other insect species have begun to frame the general themes of appendage development in the insects. Here, we review current studies that provide for a comparison of limb developmental mechanisms acting at five levels: (1) the specification of ventral appendage primordia; (2) specification of the limb axes; (3) regulation and interactions of genes expressed in specific domains of the proximal-distal axis, such as Distal-less; (4) the specification of appendage identity; and (5) genetic regulation of appendage allometry.

Specification of the proximal-distal (PD) axis of insect appendages is best understood in Drosophila melanogaster, where conserved signaling molecules encoded by the genes decapentaplegic (dpp) and wingless (wg) play key roles. However, the development of appendages from imaginal discs as in Drosophila is a derived state, while more basal insects produce appendages from embryonic limb buds. Therefore, the universality of the Drosophila limb PD axis specification mechanism has been debated since dpp expression in more basal insect species differs dramatically from Drosophila. Here, we test the function of Wnt signaling in the development of the milkweed bug Oncopeltus fasciatus, a species with the basal state of appendage development from limb buds. RNA interference of wg and pangolin (pan) produce defects in the germband and eyes, but not in the appendages. Distal-less and dachshund, two genes regulated by Wg signaling in Drosophila and expressed in specific PD domains along the limbs of both species, are expressed normally in the limbs of pan-depleted Oncopeltus embryos. Despite these apparently paradoxical results, Armadillo protein, the transducer of Wnt signaling, does not accumulate properly in the nuclei of cells in the legs of pan-depleted embryos. In contrast, engrailed RNAi in Oncopeltus produces cuticular and appendage defects similar to Drosophila. Therefore, our data suggest that Wg signaling is functionally conserved in the development of the germband, while it is not essential in the specification of the limb PD axis in Oncopeltus and perhaps basal insects.

Studies in genetic model organisms such as Drosophila have demonstrated that the homeotic complex (Hox) genes impart segmental identity during embryogenesis. Comparative studies in a wide range of other insect taxa have shown that the Hox genes are expressed in largely conserved domains along the anterior-posterior body axis, but whether they are performing the same functions in different insects is an open question. Most of the Hox genes have been studied functionally in only a few holometabolous insects that undergo metamorphosis. Thus, it is unclear how the Hox genes are functioning in the majority of direct-developing insects and other arthropods. To address this question, we used a combination of RNAi and in situ hybridization to reveal the expression, functions, and regulatory interactions of the Hox genes in the milkweed bug Oncopeltus fasciatus. Our results reveal many similarities and some interesting differences compared to Drosophila. We find that the gene Antennapedia is required for the identity of all three thoracic segments, while Ultrabithorax, abdominal-A and Abdominal-B cooperate to pattern the abdomen. The three abdominal genes exhibit posterior prevalence like in Drosophila, but apparently via some post-transcriptional mechanism. The functions of the head genes proboscipedia, Deformed, and Sex combs reduced were shown previously, and here we find that the complex temporal expression of pb in the labium is like that of other insects, but its regulatory relationship with Scr is unique. Overall, our data reveal that the evolution of insect Hox genes has included many small changes within general conservation of expression and function, and that the milkweed bug provides a useful model for understanding the roles of Hox genes in a direct-developing insect.

The conservation of expression of appendage patterning genes, particularly Distal-less, has been shown in a wide taxonomic sampling of animals. However, the functional significance of this expression has been tested in only a few organisms. Here we report functional analyses of orthologues of the genes Distal-less, dachshund, and homothorax in the appendages of the milkweed bug Oncopeltus fasciatus (Hemiptera). This hemimetabolous insect has typical legs but highly derived mouthparts. Distal-less, dachshund, and homothorax are conserved in their individual expression patterns and functions in the legs of Oncopeltus, but their functions in other appendages are in some cases divergent. We find that specification of antennal identity does not require wild-type Distal-less activity in Oncopeltus as it does in Drosophila. Additionally, the mouthparts of Oncopeltus show novel patterns of gene expression and function, relative to other insects. Expression of Distal-less in the maxillary stylets of Oncopeltus does not seem necessary for proper development of this appendage, while dachshund and homothorax are crucial for formation of the mandibular and maxillary stylets. These data are used to evaluate hypotheses for the evolution of hemipteran mouthparts and the evolution of developmental mechanisms in insect appendages in general.

Serial homologs are similar structures that develop at different positions within a body plan. These structures share some, but not all, aspects of developmental patterning, and their evolution is thought to be constrained by shared, pleiotropic gene functions. Here we describe the functions of 17 developmental genes during metamorphic development of the legs in the red flour beetle, Tribolium castaneum. This study provides informative comparisons between appendage development in Drosophila melanogaster and T. castaneum, between embryonic and adult development in T. castaneum, and between the development of serially homologous appendages. The leg gap genes Distal-less and dachshund are conserved in function. Notch signaling, the zinc-finger transcription factors related to odd-skipped, and bric-à-brac have conserved functions in promoting joint development. homothorax knockdown alters the identity of proximal leg segments but does not reduce growth. Lim1 is required for intermediate leg development but not distal tarsus and pretarsus development as in D. melanogaster. Development of the tarsus requires decapentaplegic, rotund, spineless, abrupt, and bric-à-brac and the EGF ligand encoded by Keren. Metathoracic legs of T. castaneum have four tarsomeres, whereas other legs have five. Patterns of gene activity in the tarsus suggest that patterning in the middle of the tarsal region, not the proximal- or distal-most areas, is responsible for this difference in segment number. Through comparisons with other recent studies of T. castaneum appendage development, we test hypotheses for the modularity or interdependence of development during evolution of serial homologs.

Antenna structure varies widely among insects, in contrast to the well-conserved structure of legs. The adult capitate antenna of the red flour beetle, Tribolium castaneum, is composed of eleven articles, organized into four distinct morphological regions (scape, pedicel, funicle and club). Here, we report the use of RNA interference to examine the functions of 21 genes during antenna metamorphosis in T. castaneum. Genes with conserved functions relative to the developmental model species Drosophila melanogaster include Distal-less and EGF signaling (antennal growth), spineless (determination of antennal identity) and the Notch signaling pathway (antennal growth, joint formation, and sensory bristle development). However, the functions of many genes differed from those predicted from the Drosophila model. In addition to a conserved gap phenotype, depletion of dachshund transformed funicle articles toward club-like identity. Depletion of Distal-less or homothorax did not cause antenna-to-leg transformation. Lim1 was required only for development of the scape-pedicle joint. Depletion of odd-skipped-related genes led to the loss of the entire funicle, while spalt, rotund, spineless, and dachshund affected smaller regions. Growth and joint formation were linked developmentally in the funicle, but not in the club. Joint formation within the club required bric-a-brac, aristaless, apterous, and pdm. Gene functions are discussed in terms of a model of antenna development in T. castaneum. This model provides a contrast to knowledge of antenna development in D. melanogaster, insight into the likely ancestral mode of antenna development, and a framework for considering diverse antenna morphologies.

Genitalia play an important role in the life histories of insects, as in other animals. These sexually dimorphic structures evolve rapidly and derive from multiple body segments. Despite the importance of insect genitalia, descriptions of their genetic patterning have been limited to fruit flies. In this study, we report the functions, interactions and regulation of appendage patterning genes (e.g. homothorax, dachshund, and Distal-less) in two insects: the milkweed bug Oncopeltus fasciatus, and the red flour beetle Tribolium castaneum. These species differ in the anatomical complexity of their genitalia. Females of T. castaneum have a terminal ovipositor ending in short styli, while O. fasciatus have a multi-jointed subterminal ovipositor. Male O. fasciatus have a genital capsule consisting of large gonocoxopodites and claspers; T. castaneum males have relatively simple genitalia. The requirement of appendage-patterning genes in males differed between the two species: No defects were observed in T. castaneum male genitalia, and while the male claspers of O. fasciatus were affected by depletion of appendage-patterning genes, the proximal gonocoxopodite was not, suggesting a non-appendicular origin for this structure. Only the styli of the T. castaneum ovipositor were affected by RNAi depletion of appendage-patterning genes (14 genes in all). The posterior Hox genes (abdominal-A and Abdominal-B) were required for proper genital development in O. fasciatus and regulated Distal-less and homothorax similarly in both sexes. Distal-less and dachshund were regulated differently in male and female O. fasciatus. Knockdown of the sex determination gene intersex produced a partial female-to-male transformation of abdominal and genital anatomy and also resulted in abrogation of female-specific regulation of these genes. These results provide developmental genetic support for specific anatomical hypotheses of serial homology. Importantly, these gene functions and interactions describe the developmental patterning of sexually dimorphic structures that have been critical to the diversification of these species-rich insect groups.

The arthropods display a wide range of morphological diversity, varying tagmosis, as well as other aspects of the body plan, such as appendage and cuticular morphology. Here we review the roles of developmental regulatory genes in the evolution of arthropod morphology, with an emphasis on what is known from morphologically diverse species. Examination of tagmatic evolution reveals that these changes have been accompanied by changes in the expression patterns of Hox genes. In contrast, review of the modifications to wing morphology seen in insects shows that these body plan changes have generally favored alterations in downstream target genes. These and other examples are used to discuss the evolutionary implications of comparative developmental genetic data.