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Background: The insect neuroendocrine system acts in the regulation of physiology, development and growth. Molecular evolution of this system hence has the potential to allow for major biological differences between insect groups. Two prohormone convertases, PC1/3 and PC2, are found in animals and both function in the processing of neuropeptide precursors in the vertebrate neurosecretory pathway. Whereas PC2-function is conserved between the fly Drosophila and vertebrates , ancestral PC1/3 was lost in the fly lineage and has not been functionally studied in any protostome. Results: In order to understand its original functions and the changes accompanying the gene loss in the fly, we investigated PC1/3 and PC2 expression and function in the beetle Tribolium castaneum. We found that PC2 is broadly expressed in the nervous system, whereas surprisingly, PC1/3 expression is restricted to specific cell groups in the posterior brain and suboesophageal ganglion. Both proteases have parallel but non-redundant functions in adult beetles’ viability and fertility. Female infertility following RNAi is caused by a failure to deposit sufficient nutritive material to the developing oocytes. Larval RNAi of both genes produced moulting defects where the larvae were not able to shed their old cuticle and became ‘locked-in’. Unexpectedly, PC1/3 RNAi larvae went through supernumerary larval moults despite minimal to zero weight gain. Conclusions: Our results indicate a yet unknown molecular mechanism for the coupling of moulting and growth in insect larvae. Conservation of the evolutionary ancient PC1/3 gene in most insect groups suggests conserved function and that its loss in dipterans may have been accompanied by major changes in the coordination of larval growth and moulting.

Arthropod brains are fascinating structures that exhibit great complexity but also contain conserved elements that can be recognized between species. There is a long tradition of research in insect neuroanatomy, cell biology, and in studying the genetics of insect brain development. Recently, the beetle Tribolium castaneum has gained attention as a model for insect head and brain development, and many anterior patterning genes have so far been characterized in beetle embryos. The outcome of embryonic anterior development is the larval and, subsequently, the adult brain. A basic requirement to understand genetic cell type diversity within these structures is the ability to localize mRNA and protein of neural genes. Here we detail our protocols for RNA in situ hybridization in combination with immunohistochemistry, optimized for dissected brains of larval and adult beetles.

The apical plate of primary marine larvae is characterized by a common set of transcription factors comprising six3 , rx , hbn , nk2.1 and FoxQ2. It harbours the apical organ, a neural and ciliary structure with neurosecretory properties. Recent studies in lophotrochozoans have found that apical organ cells form the anterior tip of the developing central nervous system. We identify an anterior medial tissue in the embryonic centipede head that shares the transcriptional profile of the apical plate of marine larvae, including nested domains of FoxQ2 and six3 expression. This domain gives rise to an anterior medial population of neural precursors distinct from those arising within the segmental neuroectoderm. These medial cells do not express achaete scute homologue in proneural clusters, but express collier , a marker for post mitotic cells committed to a neural fate, while they are still situated in the surface ectodermal layer. They then sink under the surface to form a compact cell cluster. Once internalized these cells extend axons that pioneer the primary axonal scaffold of the central nervous system. The same cells express phc2 , a neural specific prohormone convertase, which suggests that they form an early active neurosecretory centre. Some also express markers of hypothalamic neurons, including otp , vtn and vax1. These medial neurosecretory cells of the centipede are distinct from those of the pars intercerebralis , the anterior neurosecretory part of the insect brain. The pars intercerebralis derives from vsx positive placodal-like invagination sites. In the centipede, vsx expressing invaginating ectoderm is situated bilaterally adjacent to the medial pioneer cell population. Hence the pars intercerebralis is present in both insect and centipede brains, whereas no prominent anterior medial cluster of pioneer neurons is present in insects. These observations suggest that the arthropod brain retained ancestrally an anterior medial population of neurosecretory cells homologous to those of the apical plate in other invertebrate phyla, but that this cell population has been lost or greatly reduced in insects.

Background There have been few studies of head patterning in non-insect arthropods, and even in the insects, much is not yet understood. In the fly Drosophila three head gap genes, orthodenticle (otd), buttonhead (btd) and empty spiracles (ems) are essential for patterning the head. However, they do not act through the same pair-rule genes that pattern the trunk from the mandibular segment backwards. Instead they act through the downstream factors collier (col) and cap‘n’collar (cnc), and presumably other unknown factors. In the beetle Tribolium, these same gap and downstream genes are also expressed during early head development, but in more restricted domains, and some of them have been shown to be of minor functional importance. In the spider Parasteatoda tepidariorum, hedgehog (hh) and otd have been shown to play an important role in head segmentation. Results We have investigated the expression dynamics of otx (otd), SP5/btd, ems, and the downstream factors col, cnc and hh during early head development of the centipede Strigamia maritima. Our results reveal the process of head condensation and show that the anteroposterior sequence of specific gene expression is conserved with that in insects. SP5/btd and otx genes are expressed prior to and during head field formation, whereas ems is not expressed until after the initial formation of the head field, in an emerging gap between SP5/btd and otx expression. Furthermore, we observe an early domain of Strigamia hh expression in the head field that splits to produce segmental stripes in the ocular, antennal and intercalary segments. Conclusions The dynamics of early gene expression in the centipede show considerable similarity with that in the beetle, both showing more localised expression of head gap genes than occurs in the fly. This suggests that the broad overlapping domains of head gap genes observed in Drosophila are derived in this lineage. We also suggest that the splitting of the early hh segmental stripes may reflect an ancestral and conserved process in arthropod head patterning. A remarkably similar stripe splitting process has been described in a spider, and in the Drosophila head hh expression starts from a broad domain that transforms into three stripes. Electronic supplementary material The online version of this article (doi:10.1186/s13227-017-0082-x) contains supplementary material, which is available to authorized users.

Background: Cell lineage studies in amphipods have revealed an early restriction of blastomere fate. The mesendodermal cell lineage is specified with the third cleavage of the egg. We took advantage of this stereotyped mode of development by fluorescently labeling the mesodermal precursors in embryos of Orchestia cavimana and followed the morphogenesis of the mesodermal cell layer through embryonic development. Results: The mesoderm of the trunk segments is formed by a very regular and stereotypic cell division pattern of the mesoteloblasts and their segmental daughters. The head mesoderm in contrast is generated by cell movements and divisions out of a mesendodermal cell mass. Our reconstructions reveal the presence of three different domains within the trunk mesoderm of the later embryo. We distinguish a cell group median to the limbs, a major central population from which the limb mesoderm arises and a dorsolateral branch of mesodermal cells. Conclusions: Our detailed description of mesodermal development relates different precursor cell groups to distinct muscle groups of the embryo. A dorsoventral subdivision of mesoderm is prepatterned within the longitudinal mesodermal columns of the germ-band stage. This makes amphipods excellent crustacean models for studying mesodermal differentiation on a cellular and molecular level. Developmental Dynamics 241:697–717, 2012. V C 2012 Wiley Periodicals, Inc.

During early development of Eisenia andrei (Crassiclitellata), a loose arrangement of primary circular and longitudinal muscles encloses the whole embryo. Circular muscles differentiate in an anterior-posterior progression creating a segmental pattern. Primary circular muscles emerge at the segmental borders while later in development the central part of each segment is filled with circular strands. Longitudinal muscles develop in an anterio-posterior manner as well, but by continuous lengthening. Muscle growth is not restricted by segmental boundaries. The development begins with one pair of prominent longitudinal muscles differentiating ventrally along the right and the left germ band. These first muscles provide a guiding structure for the parallel organization of the afterwards differentiating longitudinal musculature. Additional primary longitudinal muscles emerge and form, together with the initial circular muscles, the primary muscle grid of the embryo. During the following development, secondary longitudinal muscle strands develop and integrate themselves into the primary grid. Meanwhile the primary circular muscles split into thin strands in a ventral to dorsal progression. Thus, a fine structured mesh of circular and longitudinal muscles is generated. Compared to other "Oligochaeta", embryonic muscle patterns in E. andrei are adapted to the development of a lecithotrophic embryo. Nevertheless, two general characteristics of annelid muscle development become evident. The first is the segmental development of the circular muscles from a set of initial muscles situated at the segment borders. Second, there is a continuous development of primary longitudinal muscles starting at the anterior pole. At least one pair of main primary longitudinal strands is characteristic in Annelida. The space between all primary strands is filled with secondary longitudinal strands during further development.

SUMMARY As a taxon of the lophotrochozoans, annelids have re-entered scientific investigations focusing on plesiomorphic bilaterian features and the evolutionary changes therein. The view of a clitellate-like plesiomorphic muscle arrangement in annelids has been challenged by recent investigations of polychaete muscle organization. However, there are few investigations of muscle formation in clitellate species that address this problem. Direct comparison of potential homologous muscles between these annelid groups is thus hampered. Somatic muscle formation during embryogenesis of two clitellates—the oligochaete Limnodrilus sp. and the hirudinean Erpobdella octoculata—occurs by distinct processes in each species, even though they share a closed outer layer of circular and an inner layer of longitudinal muscles characteristic of clitellates. In E. octoculata, the first emerging longitudinal muscles are distributed irregularly on the body surface of the embryo whereas the circular muscles appear in an orderly repetitive pattern along the anterioposterior axis. Both primary muscle types consist of fiber-bundles that branch at both their ends. This way the circular muscle bundles divide into a fine muscle-grid. The primary longitudinal muscles are incorporated into a second type of longitudinal muscles, the latter starting to differentiate adjacent to the ventral nerve cord. Those secondary muscles emerge in a ventral to dorsal manner, enclosing the embryo of E. octoculata. In Limnodrilus sp., one dorsal and one ventral bilateral pair of primary longitudinal muscles are established initially, elongating toward posterior. Initial circular muscles are emerging in a segmental pattern. Both muscle layers are completed later in development by the addition of secondary longitudinal and circular muscles. Some features of embryonic longitudinal muscle patterns in Limnodrilus sp. are comparable to structures found in adult polychaete muscle systems. Our findings show that comparative studies of body-wall muscle formation during clitellate embryogenesis are a promising approach to gain further information on annelid muscle arrangements.

As a taxon of the lophotrochozoans, annelids have re-entered scientific investigations focusing on plesiomorphic bilaterian features and the evolutionary changes therein. The view of a clitellate-like plesiomorphic muscle arrangement in annelids has been challenged by recent investigations of polychaete muscle organization. However, there are few investigations of muscle formation in clitellate species that address this problem. Direct comparison of potential homologous muscles between these annelid groups is thus hampered. Somatic muscle formation during embryogenesis of two clitellates-the oligochaete Limnodrilus sp. and the hirudinean Erpobdella octoculata-occurs by distinct processes in each species, even though they share a closed outer layer of circular and an inner layer of longitudinal muscles characteristic of clitellates. In E. octoculata, the first emerging longitudinal muscles are distributed irregularly on the body surface of the embryo whereas the circular muscles appear in an orderly repetitive pattern along the anterioposterior axis. Both primary muscle types consist of fiber-bundles that branch at both their ends. This way the circular muscle bundles divide into a fine muscle-grid. The primary longitudinal muscles are incorporated into a second type of longitudinal muscles, the latter starting to differentiate adjacent to the ventral nerve cord. Those secondary muscles emerge in a ventral to dorsal manner, enclosing the embryo of E. octoculata. In Limnodrilus sp., one dorsal and one ventral bilateral pair of primary longitudinal muscles are established initially, elongating toward posterior. Initial circular muscles are emerging in a segmental pattern. Both muscle layers are completed later in development by the addition of secondary longitudinal and circular muscles. Some features of embryonic longitudinal muscle patterns in Limnodrilus sp. are comparable to structures found in adult polychaete muscle systems. Our findings show that comparative studies of body-wall muscle formation during clitellate embryogenesis are a promising approach to gain further information on annelid muscle arrangements.

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., Author Summary Arthropods are the most abundant animals on earth. Among them, insects clearly dominate on land, whereas crustaceans hold the title for the most diverse invertebrates in the oceans. Much is known about the biology of these groups, not least because of genomic studies of the fruit fly Drosophila, the water flea Daphnia, and other species used in research. Here we report the first genome sequence from a species belonging to a lineage that has previously received very little attention—the myriapods. Myriapods were among the first arthropods to invade the land over 400 million years ago, and survive today as the herbivorous millipedes and venomous centipedes, one of which—Strigamia maritima—we have sequenced here. We find that the genome of this centipede retains more characteristics of the presumed arthropod ancestor than other sequenced insect genomes. The genome provides access to many aspects of myriapod biology that have not been studied before, suggesting, for example, that they have diversified receptors for smell that are quite different from those used by insects. In addition, it shows specific consequences of the largely subterranean life of this particular species, which seems to have lost the genes for all known light-sensing molecules, even though it still avoids light.