Frank W. Smith

Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade

Significance Despite fascinating scientists for over 200 years, little at the molecular level is known about tardigrades, microscopic animals resistant to extreme stresses. We present the genome of a tardigrade. Approximately one-sixth of the genes in the tardigrade genome were found to have been acquired through horizontal transfer, a proportion nearly double the proportion of previous known cases of extreme horizontal gene transfer (HGT) in animals. Foreign genes have impacted the composition of the tardigrade genome: supplementing, expanding, and replacing endogenous gene families, including those families implicated in stress tolerance. Our results extend recent findings that HGT is more prevalent in animals than previously suspected, and they suggest that organisms that survive extreme stresses might be predisposed to acquiring foreign genes. Horizontal gene transfer (HGT), or the transfer of genes between species, has been recognized recently as more pervasive than previously suspected. Here, we report evidence for an unprecedented degree of HGT into an animal genome, based on a draft genome of a tardigrade, Hypsibius dujardini. Tardigrades are microscopic eight-legged animals that are famous for their ability to survive extreme conditions. Genome sequencing, direct confirmation of physical linkage, and phylogenetic analysis revealed that a large fraction of the H. dujardini genome is derived from diverse bacteria as well as plants, fungi, and Archaea. We estimate that approximately one-sixth of tardigrade genes entered by HGT, nearly double the fraction found in the most extreme cases of HGT into animals known to date. Foreign genes have supplemented, expanded, and even replaced some metazoan gene families within the tardigrade genome. Our results demonstrate that an unexpectedly large fraction of an animal genome can be derived from foreign sources. We speculate that animals that can survive extremes may be particularly prone to acquiring foreign genes.

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.

The metameric pattern of Hypsibius dujardini(Eutardigrada) and its relationship to that of other panarthropods

IntroductionTardigrades are an ancient lineage of microinvertebrates with a unique metameric pattern consisting of a head and four lobopodal leg-bearing segments. While their close relationship to Onychophora and Arthropoda is well established, many questions remain about the structure and origin of the tardigrade metameric pattern. For example, the relationship of the tardigrade head to that of Arthropoda and Onychophora remains a contentious issue. One source of contention stems from disagreement about the structure of the tardigrade brain. The availability of developmental tools for the tardigrade Hypsibius dujardini give this species the potential to clarify questions regarding the relationship of tardigrade segmental patterns to those in Arthropoda and Onychophora. Here we investigate the nervous system, muscle system, and cuticle anatomy of H. dujardini using high-resolution microscopy methods.ResultsWe characterized nervous system anatomy of H. dujardini using a combination of anti-β -tubulin staining and DAPI staining and muscle system anatomy using phalloidin staining. We identified several brain lobes: paired outer lobes, paired inner lobes, and a single horseshoe-shaped ventral lobe. We also characterized similarities and differences in the nervous system and muscle system anatomy of the four body segments. Based on these, we detect distinct morphological identities for each segment in this species.ConclusionsBased on comparisons of our results to previous reports, we find support for an ancestral tardigrade brain exhibiting architecture similar to that of H. dujardini. Comparisons to other tardigrade species suggest that each segment in the ancestral tardigrade possessed a unique morphological identity, rather than exhibiting strictly homonomous segmentation, and thus that differentiation of anterior segment identities arose prior to the diversification of panarthropodan lineages. This hypothesis can be further tested by examination of the expression boundaries of anterior Hox genes, which differentiate anterior segments through a mechanism conserved between onychophorans and arthropods. Our investigation of H. dujardini segmental morphologies will facilitate developmental genetic studies in this species that promise to illuminate the relationship of the tardigrade metameric pattern to that of other panarthropods.

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.

Segmentation in Tardigrada and diversification of segmental patterns in Panarthropoda

The origin and diversification of segmented metazoan body plans has fascinated biologists for over a century. The superphylum Panarthropoda includes three phyla of segmented animals-Euarthropoda, Onychophora, and Tardigrada. This superphylum includes representatives with relatively simple and representatives with relatively complex segmented body plans. At one extreme of this continuum, euarthropods exhibit an incredible diversity of serially homologous segments. Furthermore, distinct tagmosis patterns are exhibited by different classes of euarthropods. At the other extreme, all tardigrades share a simple segmented body plan that consists of a head and four leg-bearing segments. The modular body plans of panarthropods make them a tractable model for understanding diversification of animal body plans more generally. Here we review results of recent morphological and developmental studies of tardigrade segmentation. These results complement investigations of segmentation processes in other panarthropods and paleontological studies to illuminate the earliest steps in the evolution of panarthropod body plans.

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.

Hox genes require homothorax and extradenticle for body wall identity specification but not for appendage identity specification during metamorphosis of Tribolium castaneum

The establishment of segment identity is a key developmental process that allows for divergence along the anteroposterior body axis in arthropods. In Drosophila, the identity of a segment is determined by the complement of Hox genes it expresses. In many contexts, Hox transcription factors require the protein products of extradenticle (exd) and homothorax (hth) as cofactors to perform their identity specification functions. In holometabolous insects, segment identity may be specified twice, during embryogenesis and metamorphosis. To glean insight into the relationship between embryonic and metamorphic segmental identity specification, we have compared these processes in the flour beetle Tribolium castaneum, which develops ventral appendages during embryogenesis that later metamorphose into adult appendages with distinct morphologies. At metamorphosis, comparisons of RNAi phenotypes indicate that Hox genes function jointly with Tc-hth and Tc-exd to specify several region-specific aspects of the adult body wall. On the other hand, Hox genes specify appendage identities along the anteroposterior axis independently of Tc-hth/Tc-exd and Tc-hth/Tc-exd specify proximal vs. distal identity within appendages independently of Hox genes during this stage. During embryogenesis, Tc-hth and Tc-exd play a broad role in the segmentation process and are required for specification of body wall identities in the thorax; however, contrasting with results from other species, we did not obtain homeotic transformations of embryonic appendages in response to Tc-hth or Tc-exd RNAi. In general, the homeotic effects of interference with the function of Hox genes and Tc-hth/Tc-exd during metamorphosis did not match predictions based on embryonic roles of these genes. Comparing metamorphic patterning in T. castaneum to embryonic and post-embryonic development in hemimetabolous insects suggests that holometabolous metamorphosis combines patterning processes of both late embryogenesis and metamorphosis of the hemimetabolous life cycle.

A Hypothesis for the Composition of the Tardigrade Brain and its Implications for Panarthropod Brain Evolution

Incredibly disparate brain types are found in Metazoa, which raises the question of how this disparity evolved. Ecdysozoa includes representatives that exhibit ring-like brains-the Cycloneuralia-and representatives that exhibit ganglionic brains-the Panarthropoda (Euarthropoda, Onychophora, and Tardigrada). The evolutionary steps leading to these distinct brain types are unclear. Phylogenomic analyses suggest that the enigmatic Tardigrada is a closely related outgroup of a Euarthropoda + Onychophora clade; as such, the brains of tardigrades may provide insight into the evolution of ecdysozoan brains. Recently, evolutionarily salient questions have arisen regarding the composition of the tardigrade brain. To address these questions, we investigated brain anatomy in four tardigrade species-Hypsibius dujardini, Milnesium n. sp., Echiniscus n. sp., and Batillipes n. sp.-that together span Tardigrada. Our results suggest that general brain morphology is conserved across Tardigrada. Based on our results we present a hypothesis that proposes direct parallels between the tardigrade brain and the segmental trunk ganglia of the tardigrade ventral nervous system. In this hypothesis, brain neuropil nearly circumscribes the tardigrade foregut. We suggest that the tardigrade brain retains aspects of an ancestral cycloneuralian brain, while exhibiting ganglionic structure characteristic of euarthropods and onychophorans.