David R. Angelini

Genetic patterning in the adult capitate antenna of the beetle Tribolium castaneum

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.

Extent With Modification: Leg Patterning in the Beetle Tribolium castaneum and the Evolution of Serial Homologs.

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.

Sex-specific gene interactions in the patterning of insect genitalia.

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.

Patterning of the Adult Mandibulate Mouthparts in the Red Flour Beetle, Tribolium castaneum

Specialized insect mouthparts, such as those of Drosophila, are derived from an ancestral mandibulate state, but little is known about the developmental genetics of mandibulate mouthparts. Here, we study the metamorphic patterning of mandibulate mouthparts of the beetle Tribolium castaneum, using RNA interference to deplete the expression of 13 genes involved in mouthpart patterning. These data were used to test three hypotheses related to mouthpart development and evolution. First, we tested the prediction that maxillary and labial palps are patterned using conserved components of the leg-patterning network. This hypothesis was strongly supported: depletion of Distal-less and dachshund led to distal and intermediate deletions of these structures while depletion of homothorax led to homeotic transformation of the proximal maxilla and labium, joint formation required the action of Notch signaling components and odd-skipped paralogs, and distal growth and patterning required epidermal growth factor (EGF) signaling. Additionally, depletion of abrupt or pdm/nubbin caused fusions of palp segments. Second, we tested hypotheses for how adult endites, the inner branches of the maxillary and labial appendages, are formed at metamorphosis. Our data reveal that Distal-less, Notch signaling components, and odd-skipped paralogs, but not dachshund, are required for metamorphosis of the maxillary endites. Endite development thus requires components of the limb proximal–distal axis patterning and joint segmentation networks. Finally, adult mandible development is considered in light of the gnathobasic hypothesis. Interestingly, while EGF activity is required for distal, but not proximal, patterning of other appendages, it is required for normal metamorphic growth of the mandibles.

Parallel reduction in expression of the eye development gene hedgehog in separately derived cave populations of the amphipod Gammarus minus

Caves provide excellent settings to examine evolutionary questions. Subterranean environments are characterized by similar and consistent conditions. Cave‐adapted species often share characteristics such as diminished pigmentation, elongated limbs and reduced or absent eyes. Relatively little is known about the evolution and development of troglomorphic traits in invertebrates. In this study, we compare expression of the eye development genes hedgehog, pax6, sine oculis and dachshund in individuals from multiple independently derived cave populations of the amphipod Gammarus minus. hedgehog expression was significantly reduced in cave populations, compared to genetically related surface populations. Interestingly, no differences were found in pax6, sine oculis or dachshund expression. Because hedgehog‐related genes are also involved in eye reduced in Astyanax mexicanus, these genes may be consistent targets of evolution during cave adaptation. These results provide support for the hypothesis of genomic ‘hotspots’ of evolution and allow comparison of adaptive mechanisms among diverse animals in subterranean environments.

A functional genetic analysis in flour beetles (Tenebrionidae) reveals an antennal identity specification mechanism active during metamorphosis in Holometabola.

The antenna was the first arthropod ventral appendage to evolve non-leg identity. Models of antennal evolution have been based on comparisons of antennal and leg identity specification mechanisms in Drosophila melanogaster, a species in which appendages develop from highly derived imaginal discs during the larval period. We test for conservation of the Drosophila antennal identity specification mechanism at metamorphosis in Tribolium castaneum and three other flour beetle species (Tribolium confusum, Tribolium brevicornis and Latheticus oryzae) in the family Tenebrionidae. In Drosophila, loss of function of four transcription factors-homothorax, extradenticle, Distal-less, and spineless-causes large-scale transformations of the antenna to leg identity. Distal-less and spineless function similarly during metamorphosis in T. castaneum. RNA interference (RNAi) targeting homothorax (hth) or extradenticle (exd) caused transformation of the proximal antenna to distal leg identity in flour beetles, but did not affect the identity of the distal antenna. This differs from the functional domain of these genes in early instar Drosophila, where they are required for identity specification throughout the antenna, but matches their functional domain in late instar Drosophila. The similarities between antennal identity specification at metamorphosis in flour beetles and in late larval Drosophila likely reflect the conservation of an ancestral metamorphic developmental mechanism. There were two notable differences in hth/exd loss of function phenotypes between flies and beetles. Flour beetles retained all of their primary segments in both the antenna and legs, whereas flies undergo reduction and fusion of primary segments. This difference in ground state appendage morphology casts doubt on interpretations of developmental ground states as evolutionary atavisms. Additionally, adult Tribolium eyes were transformed to elytron-like structures; we provide a developmental hypothesis for this evolutionarily surprising transformation.

Metamorphic labral axis patterning in the beetle Tribolium castaneum requires multiple upstream, but few downstream, genes in the appendage patterning network.

The arthropod labrum is an anterior appendage‐like structure that forms the dorsal side of the preoral cavity. Conflicting interpretations of fossil, nervous system, and developmental data have led to a proliferation of scenarios for labral evolution. The best supported hypothesis is that the labrum is a novel structure that shares development with appendages as a result of co‐option. Here, we use RNA interference in the red flour beetle Tribolium castaneum to compare metamorphic patterning of the labrum to previously published data on ventral appendage patterning. As expected under the co‐option hypothesis, depletion of several genes resulted in similar defects in the labrum and ventral appendages. These include proximal deletions and proximal‐to‐distal transformations resulting from depletion of the leg gap genes homothorax and extradenticle, large‐scale deletions resulting from depletion of the leg gap gene Distal‐less, and smaller distal deletions resulting from knockdown of the EGF ligand Keren. However, depletion of dachshund and many of the genes that function downstream of the leg gap genes in the ventral appendages had either subtle or no effects on labral axis patterning. This pattern of partial similarity suggests that upstream genes act through different downstream targets in the labrum. We also discovered that many appendage axis patterning genes have roles in patterning the epipharyngeal sensillum array, suggesting that they have become integrated into a novel regulatory network. These genes include Notch, Delta, and decapentaplegic, and the transcription factors abrupt, bric à brac, homothorax, extradenticle and the paralogs apterous a and apterous b.