Serial Homology and Segment Identity in the Arthropod Head.

The anterior-most unit of the crown-group arthropod body plan includes three segments, the pre-gnathal segments, that contain three neuromeres that together comprise the brain. Recent work on the development of this anterior region has shown that its three units exhibit many developmental differences to the more posterior segments, to the extent that they should not be considered serial homologs. Building on this revised understanding of the development of the pre-gnathal segments, we suggest a novel scenario for arthropod head evolution. We posit an expansion of an ancestral single-segmented head at the transition from Radiodonta to Deuteropoda in the arthropod stem group. The expanded head subdivided into three segmental units, each maintaining some of the structures of the ancestral head. This scenario is consistent with what we know of head evolution from the fossil record and helps reconcile some of the debates about early arthropod evolution.

Homologie en série et identité des segments dans la tête de l'arthropodes Oren Lev, Gregory D. Edgecombe and Ariel D. ChipmanL'unité la plus antérieure du plan corporel des arthropodes du groupe couronne comprend trois segments, les segments pré-gnathaux, qui contiennent trois neuromères qui, ensemble, constituent le cerveau. Des études récentes sur le développement de cette région antérieure ont montré que ses trois unités présentent de nombreuses différences de développement avec les segments plus postérieurs, au point qu'elles ne doivent pas être considérées comme des homologues sérielles. Basés sur cette révision de notre compréhension du développement des segments pré-gnathaux, nous proposons un nouveau scénario pour l'évolution de la tête des arthropodes. Nous postulons une expansion d'une tête ancestrale mono-segmentaire lors de la transition de Radiodonta à Deuteropoda dans le groupe souche des arthropodes. La tête élargie était divisée en trois unités segmentaires, chacune conservant certaines des structures de la tête ancestrale. Ce scénario est cohérent avec ce que nous savons de l'évolution de la tête à partir des archives fossiles et aide à concilier certains débats sur les stades premiers de l'évolution des arthropodes.

Homología serial e identidad de los segmentos en la cabeza del artrópodos Oren Lev, Gregory D. Edgecombe and Ariel D. ChipmanLa parte más anterior del plano corporal del grupo corona de los artrópodos incluye tres segmentos, los llamados segmentos pregnatales, que a su vez contienen tres neurómeros que constituyen el cerebro en su conjunto. Estudios recientes sobre el desarrollo de esta región anterior han demostrado que sus tres unidades muestran muchas diferencias de desarrollo con respecto a los segmentos posteriores, por lo que no deberían considerarse homólogos seriales. Teniendo en cuenta el desarrollo de los segmentos pregnatales, sugerimos un escenario alternativo para la evolución de la cabeza de los artrópodos. Proponemos la expansión de una cabeza ancestral con solo un segmento en la transición evolutiva de Radiodonta a Deuteropoda en el grupo troncal de los artrópodos. La cabeza expandida se subdividió en tres unidades segmentales, cada una de ellas manteniendo algunas de las estructuras de la cabeza ancestral. Este escenario es consistente con nuestro conocimiento de le evolución cefálica en el registro fósil y facilita la reconciliación de algunos de los debates sobre las etapas tempranas en la evolución de los artrópodos.

Development of the Pre-gnathal Segments in the Milkweed Bug Oncopeltus fasciatus Suggests They Are Not Serial Homologs of Trunk Segments.

The three anterior-most segments in arthropods contain the ganglia that make up the arthropod brain. These segments, the pre-gnathal segments (PGS), are known to exhibit many developmental differences to other segments, believed to reflect their divergent morphology. We have analyzed the expression and function of the genes involved in the conserved segment-polarity network, including genes from the Wnt and Hedgehog pathways, in the PGS, compared with the trunk segments, in the hemimetabolous insect Oncopeltus fasciatus. Gene function was tested by manipulating expression through RNA interference against components of the two pathways. We show that there are fundamental differences in the expression patterns of the segment polarity genes, in the timing of their expression and in the interactions among them in the process of pre-gnathal segment generation, relative to all other segments. We argue that given these differences, the PGS should not be considered serially homologous to trunk segments. This realization raises important questions about the differing evolutionary ancestry of different regions of the arthropod head.

The multiple roles of caudal in early development of the milkweed bug Oncopeltus fasciatus.

The homeobox transcription factor Caudal has conserved roles in all Bilateria in defining the posterior pole and in controlling posterior elongation. These roles are seemingly similar and are difficult to disentangle. We have carried out a detailed analysis of the expression, function and interactions of the caudal ortholog of the milkweed bug, Oncopeltus fasciatus, a hemimetabolous insect with a conservative early development process, in order to understand its different functions throughout development. In Oncopeltus, caudal is not maternally deposited, but has a sequence of roles in the posterior of the embryos throughout early development. It defines and maintains a posterior-anterior gradient in the blastoderm and modulates the activity of segmentation genes in simultaneous segmentation during the blastoderm stage. It later defines the invagination site and the posterior segment addition zone (SAZ) in the germband. It maintains the posterior SAZ cells in an undifferentiated proliferative state, while promoting dynamic expression of segmentation genes in the anterior SAZ. We show that rather than being a simple posterior determinant, Caudal is involved in several distinct regulatory networks, each with a distinct developmental role.

The evolution of the gene regulatory networks patterning the Drosophila Blastoderm.

The Drosophila blastoderm gene regulatory network is one of the best studied networks in biology. It is composed of a series of tiered sub-networks that act sequentially to generate a primary segmental pattern. Many of these sub-networks have been studied in other arthropods, allowing us to reconstruct how each of them evolved over the transition from the arthropod ancestor to the situation seen in Drosophila today. I trace the evolution of each of these networks, showing how some of them have been modified significantly in Drosophila relative to the ancestral state while others are largely conserved across evolutionary timescales. I compare the putative ancestral arthropod segmentation network with that found in Drosophila and discuss how and why it has been modified throughout evolution, and to what extent this modification is unusual.

Elongation during segmentation shows axial variability, low mitotic rates, and synchronized cell cycle domains in the crustacean, Thamnocephalus platyurus.

BACKGROUND: Segmentation in arthropods typically occurs by sequential addition of segments from a posterior growth zone. However, the amount of tissue required for growth and the cell behaviors producing posterior elongation are sparsely documented. RESULTS: Using precisely staged larvae of the crustacean, Thamnocephalus platyurus, we systematically examine cell division patterns and morphometric changes associated with posterior elongation during segmentation. We show that cell division occurs during normal elongation but that cells in the growth zone need only divide ~ 1.5 times to meet growth estimates; correspondingly, direct measures of cell division in the growth zone are low. Morphometric measurements of the growth zone and of newly formed segments suggest tagma-specific features of segment generation. Using methods for detecting two different phases in the cell cycle, we show distinct domains of synchronized cells in the posterior trunk. Borders of cell cycle domains correlate with domains of segmental gene expression, suggesting an intimate link between segment generation and cell cycle regulation. CONCLUSIONS: Emerging measures of cellular dynamics underlying posterior elongation already show a number of intriguing characteristics that may be widespread among sequentially segmenting arthropods and are likely a source of evolutionary variability. These characteristics include: the low rates of posterior mitosis, the apparently tight regulation of cell cycle at the growth zone/new segment border, and a correlation between changes in elongation and tagma boundaries.

Developing an integrated understanding of the evolution of arthropod segmentation using fossils and evo-devo.

Segmentation is fundamental to the arthropod body plan. Understanding the evolutionary steps by which arthropods became segmented is being transformed by the integration of data from evolutionary developmental biology (evo-devo), Cambrian fossils that allow the stepwise acquisition of segmental characters to be traced in the arthropod stem-group, and the incorporation of fossils into an increasingly well-supported phylogenetic framework for extant arthropods based on genomic-scale datasets. Both evo-devo and palaeontology make novel predictions about the evolution of segmentation that serve as testable hypotheses for the other, complementary data source. Fossils underpin such hypotheses as arthropodization originating in a frontal appendage and then being co-opted into other segments, and segmentation of the endodermal midgut in the arthropod stem-group. Insights from development, such as tagmatization being associated with different modes of segment generation in different body regions, and a distinct patterning of the anterior head segments, are complemented by palaeontological evidence for the pattern of tagmatization during ontogeny of exceptionally preserved fossils. Fossil and developmental data together provide evidence for a short head in stem-group arthropods and the mechanism of its formation and retention. Future breakthroughs are expected from identification of molecular signatures of developmental innovations within a phylogenetic framework, and from a focus on later developmental stages to identify the differentiation of repeated units of different systems within segmental precursors.

Molecular evolutionary trends and feeding ecology diversification in the Hemiptera, anchored by the milkweed bug genome.

BACKGROUND: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. RESULTS: The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding. CONCLUSIONS: With the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus's strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes.

Growth zone segmentation in the milkweed bug Oncopeltus fasciatus sheds light on the evolution of insect segmentation.

BACKGROUND: One of the best studied developmental processes is the Drosophila segmentation cascade. However, this cascade is generally considered to be highly derived and unusual, with segments being patterned simultaneously, rather than the ancestral sequential segmentation mode. We present a detailed analysis of the segmentation cascade of the milkweed bug Oncopletus fasciatus, an insect with a more primitive segmentation mode, as a comparison to Drosophila, with the aim of reconstructing the evolution of insect segmentation modes. RESULTS: We document the expression of 12 genes, representing different phases in the segmentation process. Using double staining we reconstruct the spatio-temporal relationships among these genes. We then show knock-down phenotypes of representative genes in order to uncover their roles and position in the cascade. CONCLUSIONS: We conclude that sequential segmentation in the Oncopeltus germband includes three slightly overlapping phases: Primary pair-rule genes generate the first segmental gene expression in the anterior growth zone. This pattern is carried anteriorly by a series of secondary pair-rule genes, expressed in the transition between the growth zone and the segmented germband. Segment polarity genes are expressed in the segmented germband with conserved relationships. Unlike most holometabolous insects, this process generates a single-segment periodicity, and does not have a double-segment pattern at any stage. We suggest that the evolutionary transition to double-segment patterning lies in mutually exclusive expression patterns of secondary pair-rule genes. The fact that many aspects of the putative Oncopeltus segmentation network are similar to those of Drosophila, is consistent with a simple transition between sequential and simultaneous segmentation.

The Evolution of Gene Regulatory Networks that Define Arthropod Body Plans.

Our understanding of the genetics of arthropod body plan development originally stems from work on Drosophila melanogaster from the late 1970s and onward. In Drosophila, there is a relatively detailed model for the network of gene interactions that proceeds in a sequential-hierarchical fashion to define the main features of the body plan. Over the years, we have a growing understanding of the networks involved in defining the body plan in an increasing number of arthropod species. It is now becoming possible to tease out the conserved aspects of these networks and to try to reconstruct their evolution. In this contribution, we focus on several key nodes of these networks, starting from early patterning in which the main axes are determined and the broad morphological domains of the embryo are defined, and on to later stage wherein the growth zone network is active in sequential addition of posterior segments. The pattern of conservation of networks is very patchy, with some key aspects being highly conserved in all arthropods and others being very labile. Many aspects of early axis patterning are highly conserved, as are some aspects of sequential segment generation. In contrast, regional patterning varies among different taxa, and some networks, such as the terminal patterning network, are only found in a limited range of taxa. The growth zone segmentation network is ancient and is probably plesiomorphic to all arthropods. In some insects, it has undergone significant modification to give rise to a more hardwired network that generates individual segments separately. In other insects and in most arthropods, the sequential segmentation network has undergone a significant amount of systems drift, wherein many of the genes have changed. However, it maintains a conserved underlying logic and function.

The Evolution of Arthropod Body Plans: Integrating Phylogeny, Fossils, and Development-An Introduction to the Symposium.

The last few years have seen a significant increase in the amount of data we have about the evolution of the arthropod body plan. This has come mainly from three separate sources: a new consensus and improved resolution of arthropod phylogeny, based largely on new phylogenomic analyses; a wealth of new early arthropod fossils from a number of Cambrian localities with excellent preservation, as well as a renewed analysis of some older fossils; and developmental data from a range of model and non-model pan-arthropod species that shed light on the developmental origins and homologies of key arthropod traits. However, there has been relatively little synthesis among these different data sources, and the three communities studying them have little overlap. The symposium "The Evolution of Arthropod Body Plans-Integrating Phylogeny, Fossils and Development" brought together leading researchers in these three disciplines and made a significant contribution to the emerging synthesis of arthropod evolution, which will help advance the field and will be useful for years to come.

Dynamics of growth zone patterning in the milkweed bug Oncopeltus fasciatus.

We describe the dynamic process of abdominal segment generation in the milkweed bug Oncopeltus fasciatus We present detailed morphological measurements of the growing germband throughout segmentation. Our data are complemented by cell division profiles and expression patterns of key genes, including invected and even-skipped as markers for different stages of segment formation. We describe morphological and mechanistic changes in the growth zone and in nascent segments during the generation of individual segments and throughout segmentation, and examine the relative contribution of newly formed versus existing tissue to segment formation. Although abdominal segment addition is primarily generated through the rearrangement of a pool of undifferentiated cells, there is nonetheless proliferation in the posterior. By correlating proliferation with gene expression in the growth zone, we propose a model for growth zone dynamics during segmentation in which the growth zone is functionally subdivided into two distinct regions: a posterior region devoted to a slow rate of growth among undifferentiated cells, and an anterior region in which segmental differentiation is initiated and proliferation inhibited.

Factors involved in early polarization of the anterior-posterior axis in the milkweed bug Oncopeltus fasciatus.

The axes of insect embryos are defined early in the blastoderm stage. Genes involved in this polarization are well known in Drosophila, but less so in other insects, such as the milkweed bug Oncopeltus fasciatus. Using quantitative PCR, we looked at differential expression of several candidate genes for early anterior-posterior patterning and found that none of them are expressed asymmetrically in the early blastoderm. We then used an RNA-Seq approach to identify novel candidate genes that might be involved in early polarization in Oncopeltus. We focused on transcription factors (TFs) as these are likely to be central players in developmental processes. Using both homology and domain based identification approaches, we were unable to find any TF encoding transcripts that are expressed asymmetrically along the anterior-posterior axis at early stages. Using a GO-term analysis of all asymmetrically expressed mRNAs, we found an enrichment of genes relating to mitochondrial function in the posterior at the earliest studied time-point. We also found a gradual enrichment of transcription related activities, giving us a putative time frame for the maternal to zygotic transition. Our dataset provides us with a list of new candidate genes in early development, which can be followed up experimentally.

Oncopeltus fasciatus as an evo-devo research organism.

The large milkweed bug Oncopeltus fasciatus was one of the main study insects for a range of biological questions throughout much of the 20th century. Its importance waned with the introduction of Drosophila melanogaster as a genetic model organism. The evo-devo revolution of the turn of the century re-introduced Oncopeltus into the scientific community, and it has proved increasingly useful, mostly within a comparative context for evolution driven research. The last few years have seen a number of significant contributions to our understanding of the evolution of developmental processes in insects, and in arthropods in general, arise from work on Oncopeltus. This review presents some of the key studies and shows how they have provided new insights into evolutionary questions. The advent of whole genome sequencing and genome editing techniques is reducing the gap between Drosophila and (re-)emerging systems such as Oncopeltus. We expect that the ease of work on Oncopeltus and its pivotal phylogenetic position will contribute to the expansion of its use within the evo-devo community and more broadly in arthropod research.

Blastoderm segmentation in Oncopeltus fasciatus and the evolution of insect segmentation mechanisms.

Segments are formed simultaneously in the blastoderm of the fly Drosophila melanogaster through a hierarchical cascade of interacting transcription factors. Conversely, in many insects and in all non-insect arthropods most segments are formed sequentially from the posterior. We have looked at segmentation in the milkweed bug Oncopeltus fasciatus. Posterior segments are formed sequentially, through what is probably the ancestral arthropod mechanism. Formation of anterior segments bears many similarities to the Drosophila segmentation mode. These segments appear nearly simultaneously in the blastoderm, via a segmentation cascade that involves orthologues of Drosophila gap genes working through a functionally similar mechanism. We suggest that simultaneous blastoderm segmentation evolved at or close to the origin of holometabolous insects, and formed the basis for the evolution of the segmentation mode seen in Drosophila We discuss the changes in segmentation mechanisms throughout insect evolution, and suggest that the appearance of simultaneous segmentation as a novel feature of holometabolous insects may have contributed to the phenomenal success of this group.

An embryological perspective on the early arthropod fossil record.

BACKGROUND: Our understanding of the early evolution of the arthropod body plan has recently improved significantly through advances in phylogeny and developmental biology and through new interpretations of the fossil record. However, there has been limited effort to synthesize data from these different sources. Bringing an embryological perspective into the fossil record is a useful way to integrate knowledge from different disciplines into a single coherent view of arthropod evolution. RESULTS: I have used current knowledge on the development of extant arthropods, together with published descriptions of fossils, to reconstruct the germband stages of a series of key taxa leading from the arthropod lower stem group to crown group taxa. These reconstruction highlight the main evolutionary transitions that have occurred during early arthropod evolution, provide new insights into the types of mechanisms that could have been active and suggest new questions and research directions. CONCLUSIONS: The reconstructions suggest several novel homology hypotheses - e.g. the lower stem group head shield and head capsules in the crown group are all hypothesized to derive from the embryonic head lobes. The homology of anterior segments in different groups is resolved consistently. The transition between "lower-stem" and "upper-stem" arthropods is highlighted as a major transition with a concentration of novelties and innovations, suggesting a gap in the fossil record. A close relationship between chelicerates and megacheirans is supported by the embryonic reconstructions, and I suggest that the depth of the mandibulate-chelicerate split should be reexamined.

The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.

Evolution of the insect terminal patterning system--insights from the milkweed bug, Oncopeltus fasciatus.

The anterior and posterior ends of the insect embryo are patterned through the terminal patterning system, which is best known from the fruitfly Drosophila melanogaster. In Drosophila, the RTK receptor Torso and its presumed co-activator Torso-like initiate a signaling cascade, which activates two terminal gap genes, tailless and huckebein. These in turn interact with various patterning genes to define terminal structures. Work on other insect species has shown that this system is poorly conserved, and not all of its components have been found in all cases studied. We place the variability of the system within a broader phylogenetic framework. We describe the expression and knock-down phenotypes of the homologues of terminal patterning genes in the hemimetabolous Oncopeltus fasciatus. We have examined the interactions among these genes and between them and other patterning genes. We demonstrate that all of these genes have different roles in Oncopeltus relative to Drosophila; torso-like is expressed in follicle cells during oogenesis and is involved in the invagination of the blastoderm to form the germ band, and possibly also in defining the growth zone; tailless is regulated by orthodenticle and has a role only in anterior determination; huckebein is expressed only in the middle of the blastoderm; finally, torso was not found in Oncopeltus and its role in terminal patterning seems novel within holometabolous insects. We then use our data, together with published data on other insects, to reconstruct the evolution of the terminal patterning gene network in insects. We suggest that the Drosophila terminal patterning network evolved recently in the lineage leading to the Diptera, and represents an example of evolutionary "tinkering", where pre-existing pathways are co-opted for a new function.

The evolution of the knirps family of transcription factors in arthropods.

The orphan nuclear receptor gene knirps and its relatives encode a small family of highly conserved proteins. We take advantage of the conservation of the family, using the recent prevalence of genomic data, to reconstruct its evolutionary history, identifying duplication events and tracing the intron-exon structure of the genes over evolution. Many arthropod species have two or three members of this family, but the orthology between members is unclear. We have analyzed the protein coding sequences of members of this family from 15 arthropod species covering all four main arthropod classes, including a total of 28 genes. All members of the family encode a highly conserved 94 amino acid core sequence, part of which is encoded by a single invariant exon. We find that many of the automated predictions of these genes contain errors, while some copies of the gene were not uncovered by automated pipelines, requiring manual corrections and curation. We use the coding sequences to present a phylogenetic analysis of the knirps family. Our analysis indicates that there was a duplication of a single ancestral gene in the lineage leading to insects, which gave rise to two paralogs, eagle and knirps-related. Descendants of this duplication can be identified by the presence or absence of a short protein-coding motif. Independent, lineage-specific duplications occurred in the two crustaceans we sampled. Within the insects, the knirps-related gene underwent further lineage-specific duplications, giving rise to--among others--the Drosophila gap gene knirps.

Diversity and biogeography of Israeli geophilomorph centipedes (Chilopoda: Geophilomorpha).

The fauna of Chilopoda Geophilomorpha of Israel has been analyzed after examining 128 new specimens from 35 localities, reinterpreting all published data including 103 records, and relating occurrence of species with major climatic parameters. A key to identification has been compiled. A total of 17 species are distinguished, of which three are reported from Israel for the first time, while five are documented by published records only. The following new synonymies are proposed and discussed: Dignathodon pachypus Verhoeff, 1943 = Dignathodon microcephalus (Lucas, 1846); Geophilus flavidus noduliger Verhoeff, 1925 = Clinopodes escherichii (Verhoeff, 1896); Pachymerium ferrugineum vosseleri Verhoeff, 1902 = P. ferrugineum (Koch, 1835). Of all the species, Bothriogaster signata and Pachymerium ferrugineum are widespread in the country, while other species occupy different climatic zones, from desert to more humid and montane.