The impact of land-to-water transition on chemosensory genes has been explored in marine tetrapod vertebrates, with scarce data on aquatic insect lineages. Diving beetles (Dytiscidae) are predaceous freshwater insects with strictly aquatic larvae and amphibious adults. Using RNA-seq, we compared the expression of odorant receptors (ORs), gustatory receptors (GRs), ionotropic receptors (IRs), and odorant-binding proteins (OBPs) in the cephalic appendages of larval and adult Cybister lateralimarginalis . Overall, larvae expressed fewer chemosensory genes than adults but larva-specific genes displayed a unique expression pattern, not previously observed in any other holometabolous insect, with five larva-specific ORs all having a close paralogue which is adult-specific, 14 larva-specific IRs all belonging to a single gene expansion in the IR tree, and no larva-specific GR. Expression profiles across appendage types mirrored those in aerial insects, with ORs mainly in antennae, GRs in labial palps, “Antennal class” IRs in antennae, and “Divergent class” IRs in palps. This suggests that the land-to-freshwater transition in this lineage did not involve major changes in deployment of the major families of chemosensory genes among cephalic appendages. Notably, the expression of a substantial repertoire of ORs specifically in the antennae of the larva suggests that hydrophobic chemical cues are important for long-range chemodetection in freshwater, contrary to prevailing views about constraints for chemosensation within a water medium.

Sensilla on head appendages were studied in detail for the first time in a member of the relict family Hygrobiidae (squeak beetles), closely related to Dytiscidae (diving beetles). Adult and third instar larval stage specimens of Hygrobia hermanni (Fabricius, 1775) were examined using scanning electron microscopy, focusing on antennae, palps and larval mandibles. In total, 37 sensilla subtypes are described, including 22 observed in the adult (basiconica: 3; Böhm's bristles: 2; circumvallate sensilla: 2; coeloconica: 10; ovoid placodea: 3; digitiform placodea: 2) and 16 in the larva (basiconica: 4; campaniformia: 1; chaetica: 4; coeloconica: 5; trichodea: 1; unnamed: 1). Only one subtype (of sensilla coeloconica) was shared between the adult and the larva. Autapomorphies of Hygrobiidae and Dytiscidae, and putatively shared derived characters (synapomorphies) of Hygrobiidae + Dytiscidae are discussed. Among the latter, the most remarkable is the acquisition of a special sensory field, located on the apical segment of the adult maxillary palp, subapically and postero-dorsally. This sensory field is made up of ovoid multiporous sensilla placodea otherwise present on the anterior (internal) surface of antennal segments, suggesting that in a common ancestor of Hygrobiidae and Dytiscidae, maxillary palps might have taken over enhanced capacities of longe-range molecule detection.

Insects astoundingly dominate Earth’s land ecosystems and have a huge impact on human life. Almost every aspect of their life relies upon their highly efficient and adaptable chemosensory system. In the air, most chemical signals that are detected at long range are hydrophobic molecules, which insects detect using proteins encoded by multigenic families that emerged following land colonization by insect ancestors, namely the odorant-binding proteins (OBPs) and the odorant receptors (ORs). However, land-to-freshwater transitions occurred in many lineages within the insect tree of life. Whether chemosensory gene repertoires of aquatic insects remained essentially unchanged or underwent more or less drastic modifications to cope with physico-chemical constraints associated with life underwater remains virtually unknown. To address this issue, we sequenced and analyzed the transcriptome of chemosensory organs of the diving beetle Rhantus suturalis (Coleoptera, Dytiscidae). A reference transcriptome was assembled de novo using reads from five RNA-seq libraries (male and female antennae, male and female palps, and wing muscle). It contained 47,570 non-redundant unigenes encoding proteins of more than 50 amino acids. Within this reference transcriptome, we annotated sequences coding 53 OBPs, 48 ORs, 73 gustatory receptors (GRs), and 53 ionotropic receptors (IRs). Phylogenetic analyses notably revealed a large OBP gene expansion (35 paralogs in R. suturalis ) as well as a more modest OR gene expansion (9 paralogs in R. suturalis ) that may be specific to diving beetles. Interestingly, these duplicated genes tend to be expressed in palps rather than in antennae, suggesting a possible adaptation with respect to the land-to-water transition. This work provides a strong basis for further evolutionary and functional studies that will elucidate how insect chemosensory systems adapted to life underwater.

Jellyfish (medusae) are a distinctive life-cycle stage of medusozoan cnidarians. They are major marine predators, with integrated neurosensory, muscular and organ systems. The genetic foundations of this complex form are largely unknown. We report the draft genome of the hydrozoan jellyfish Clytia hemisphaerica and use multiple transcriptomes to determine gene use across life-cycle stages. Medusa, planula larva and polyp are each characterised by distinct transcriptome signatures reflecting abrupt life cycle transitions, and all deploy a mixture of phylogenetically old and new genes. Medusa specific transcription factors, including many with bilaterian orthologs, associate with diverse neurosensory structures. Compared to Clytia , the polyp-only hydrozoan Hydra has lost many of the medusa-expressed transcription factors, despite similar overall rates of gene content and sequence evolution. Absence of expression and gene loss among Clytia orthologs of genes patterning the anthozoan aboral pole, secondary axis and endomesoderm support simplification of planulae and polyps in Hydrozoa, including loss of bilateral symmetry. Consequently, although the polyp and planula are generally considered the ancestral cnidarian forms, in Clytia the medusa maximally deploys ancestral cnidarian–bilaterian transcription factor gene complexity.

The tentacular system of Clytia hemisphaerica medusa (Cnidaria, Hydrozoa) has recently emerged as a promising experimental model to tackle the developmental mechanisms that regulate cell lineage progression in an early-diverging animal phylum. From a population of proximal stem cells, the successive steps of tentacle stinging cell (nematocyte) elaboration, are spatially ordered along a “cellular conveyor belt”. Furthermore, the C. hemisphaerica tentacular system exhibits bilateral organisation, with two perpendicular polarity axes (proximo-distal and oral-aboral). We aimed to improve our knowledge of this cellular system by combining RNAseq-based differential gene expression analyses and expression studies of Wnt signalling genes. RNAseq comparisons of gene expression levels were performed (i) between the tentacular system and a control medusa deprived of all tentacles, nematogenic sites and gonads, and (ii) between three samples staggered along the cellular conveyor belt. The behaviour in these differential expression analyses of two reference gene sets (stem cell genes; nematocyte genes), as well as the relative representations of selected gene ontology categories, support the validity of the cellular conveyor belt model. Expression patterns obtained by in situ hybridisation for selected highly differentially expressed genes and for Wnt signalling genes are largely consistent with the results from RNAseq. Wnt signalling genes exhibit complex spatial deployment along both polarity axes of the tentacular system, with the Wnt/β-catenin pathway probably acting along the oral-aboral axis rather than the proximo-distal axis. These findings reinforce the idea that, despite overall radial symmetry, cnidarians have a full potential for elaboration of bilateral structures based on finely orchestrated deployment of an ancient developmental gene toolkit.

Resolving the early diversification of animal lineages has proven difficult, even using genome-scale datasets. Several phylogenomic studies have supported the classical scenario in which sponges (Porifera) are the sister group to all other animals (“Porifera-sister” hypothesis), consistent with a single origin of the gut, nerve cells, and muscle cells in the stem lineage of eumetazoans (bilaterians + ctenophores + cnidarians). In contrast, several other studies have recovered an alternative topology in which ctenophores are the sister group to all other animals (including sponges). The “Ctenophora-sister” hypothesis implies that eumetazoan-specific traits, such as neurons and muscle cells, either evolved once along the metazoan stem lineage and were then lost in sponges and placozoans or evolved at least twice independently in Ctenophora and in Cnidaria + Bilateria. Here, we report on our reconstruction of deep metazoan relationships using a 1,719-gene dataset with dense taxonomic sampling of non-bilaterian animals that was assembled using a semi-automated procedure, designed to reduce known error sources. Our dataset outperforms previous metazoan gene superalignments in terms of data quality and quantity. Analyses with a best-fitting site-heterogeneous evolutionary model provide strong statistical support for placing sponges as the sister-group to all other metazoans, with ctenophores emerging as the second-earliest branching animal lineage. Only those methodological settings that exacerbated long-branch attraction artifacts yielded Ctenophora-sister. These results show that methodological issues must be carefully addressed to tackle difficult phylogenetic questions and pave the road to a better understanding of how fundamental features of animal body plans have emerged.

Background: The Hippo pathway regulates growth rate and organ size in fly and mouse, notably through control of cell proliferation. Molecular interactions at the heart of this pathway are known to have originated in the unicellular ancestry of metazoans. They notably involve a cascade of phosphorylations triggered by the kinase Hippo, with subsequent nuclear to cytoplasmic shift of Yorkie localisation, preventing its binding to the transcription factor Scalloped, thereby silencing proliferation genes. There are few comparative expression data of Hippo pathway genes in non-model animal species and notably none in non-bilaterian phyla.Results: All core Hippo pathway genes could be retrieved from the ctenophore Pleurobrachia pileus and the hydrozoan cnidarian Clytia hemisphaerica, with the important exception of Yorkie in ctenophore. Expression study of the Hippo, Salvador and Scalloped genes in tentacle "cellular conveyor belts" of these two organisms revealed striking differences. In P. pileus, their transcripts were detected in areas where undifferentiated progenitors intensely proliferate and where expression of cyclins B and D was also seen. In C. hemisphaerica, these three genes and Yorkie are expressed not only in the proliferating but also in the differentiation zone of the tentacle bulb and in mature tentacle cells. However, using an antibody designed against the C. hemiphaerica Yorkie protein, we show in two distinct cell lineages of the medusa that Yorkie localisation is predominantly nuclear in areas of active cell proliferation and mainly cytoplasmic elsewhere.Conclusions: This is the first evidence of nucleocytoplasmic Yorkie shift in association with the arrest of cell proliferation in a cnidarian, strongly evoking the cell division-promoting role of this protein and its inhibition by the activated Hippo pathway in bilaterian models. Our results furthermore highlight important differences in terms of deployment and regulation of Hippo pathway genes between cnidarians and ctenophores.

Ctenophores are non-bilaterian metazoans of uncertain phylogenetic position, some recent studies placing them as sister-group to all other animals whereas others suggest this placement is artefactual and ctenophores are more closely allied with cnidarians and bilaterians, with which they share nerve cells, muscles and gut. Available information about developmental genes and their expression and function in ctenophores is reviewed. These data not only unveil some conserved aspects of molecular developmental mechanisms with other basal metazoan lineages, but also can be expected to enlighten the genomic and molecular bases of the evolution of ctenophore-specific traits, including their unique embryonic development, complex anatomy and high cell type diversity.

Ctenophores are a phylum of non-bilaterian marine (mostly planktonic) animals, characterised by several unique synapomorphies (e.g., comb rows, apical organ). Relationships between and within the nine recognised ctenophore orders are far from understood, notably due to a paucity of phylogenetically informative anatomical characters. Previous attempts to address ctenophore phylogeny using molecular data (18S rRNA) led to poorly resolved trees but demonstrated the paraphyly of the order Cydippida. Here we compiled an updated 18S rRNA data set, notably including a few newly sequenced species representing previously unsampled families (Lampeidae, Euryhamphaeidae), and we constructed an additional more rapidly evolving ITS1 + 5.8S rRNA + ITS2 alignment. These data sets were analysed separately and in combination under a probabilistic framework, using different methods (maximum likelihood, Bayesian inference) and models (e.g., doublet model to accommodate secondary structure; data partitioning). An important lesson from our exploration of these datasets is that the fast-evolving internal transcribed spacer (ITS) regions are useful markers for reconstructing high-level relationships within ctenophores. Our results confirm the paraphyly of the order Cydippida (and thus a "cydippid-like" ctenophore common ancestor) and suggest that the family Mertensiidae could be the sister group of all other ctenophores. The family Lampeidae (also part of the former "Cydippida") is probably the sister group of the order Platyctenida (benthic ctenophores). The order Beroida might not be monophyletic, due to the position of Beroe abyssicola outside of a clade grouping the other Beroe species and members of the "Cydippida" family Haeckeliidae. Many relationships (e.g. between Pleurobrachiidae, Beroida, Cestida, Lobata, Thalassocalycida) remain unresolved. Future progress in understanding ctenophore phylogeny will come from the use of additional rapidly evolving markers and improvement of taxonomic sampling.

Signalling through the Wnt family of secreted proteins originated in a common metazoan ancestor and greatly influenced the evolution of animal body plans. In bilaterians, Wnt signalling plays multiple fundamental roles during embryonic development and in adult tissues, notably in axial patterning, neural development and stem cell regulation. Studies in various cnidarian species have particularly highlighted the evolutionarily conserved role of the Wnt/β-catenin pathway in specification and patterning of the primary embryonic axis. However in another key non-bilaterian phylum, Ctenophora, Wnts are not involved in early establishment of the body axis during embryogenesis. We analysed the expression in the adult of the ctenophore Pleurobrachia pileus of 11 orthologues of Wnt signalling genes including all ctenophore Wnt ligands and Fz receptors and several members of the intracellular β-catenin pathway machinery. All genes are strongly expressed around the mouth margin at the oral pole, evoking the Wnt oral centre of cnidarians. This observation is consistent with primary axis polarisation by the Wnts being a universal metazoan feature, secondarily lost in ctenophores during early development but retained in the adult. In addition, local expression of Wnt signalling genes was seen in various anatomical structures of the body including in the locomotory comb rows, where their complex deployment suggests control by the Wnts of local comb polarity. Other important contexts of Wnt involvement which probably evolved before the ctenophore/cnidarian/bilaterian split include proliferating stem cells and progenitors irrespective of cell types, and developing as well as differentiated neuro-sensory structures.

Neuroglobins, previously thought to be restricted to vertebrate neurons, were detected in the brain of a photosymbiotic acoel, Symsagittifera roscoffensis, and in neurosensory cells of the jellyfish Clytia hemisphaerica. For the neuroglobin of S. roscoffensis, a member of a lineage that originated either at the base of the bilateria or of the deuterostome clade, we report the ligand binding properties, crystal structure at 2.3 (A) over circle, and brain immunocytochemical pattern. We also describe in situ hybridizations of two neuroglobins specifically expressed in differentiating nematocytes (neurosensory cells) and in statocytes (ciliated mechanosensory cells) of C. hemisphaerica, a member of the early branching animal phylum cnidaria. In silico searches using these neuroglobins as queries revealed the presence of previously unidentified neuroglobin-like sequences in most metazoan lineages. Because neural systems are almost ubiquitous in metazoa, the constitutive expression of neuroglobin-like proteins strongly supports the notion of an intimate association of neuroglobins with the evolution of animal neural systems and hints at the preservation of a vitally important function. Neuroglobins were probably recruited in the first protoneurons in early metazoans from globin precursors. Neuroglobins were identified in choanoflagellates, sponges, and placozoans and were conserved during nervous system evolution. Because the origin of neuroglobins predates the other metazoan globins, it is likely that neuroglobin gene duplication followed by co-option and subfunctionalization led to the emergence of globin families in protostomes and deuterostomes (i.e. convergent evolution).

The separation of the germ line from the soma is a classic concept in animal biology, and depending on species is thought to involve fate determination either by maternally localized germ plasm ("preformation" or "maternal inheritance") or by inductive signaling (classically termed "epigenesis" or "zygotic induction"). The latter mechanism is generally considered to operate in non-bilaterian organisms such as cnidarians and sponges, in which germ cell fate is determined at adult stages from multipotent stem cells. We have found in the hydrozoan cnidarian Clytia hemisphaerica that the multipotent "interstitial" cells (i-cells) in larvae and adult medusae, from which germ cells derive, express a set of conserved germ cell markers: Vasa, Nanos1, Piwi and PL10. In situ hybridization analyses unexpectedly revealed maternal mRNAs for all these genes highly concentrated in a germ plasm-like region at the egg animal pole and inherited by the i-cell lineage, strongly suggesting i-cell fate determination by inheritance of animal-localized factors. On the other hand, experimental tests showed that i-cells can form by epigenetic mechanisms in Clytia, since larvae derived from both animal and vegetal blastomeres separated during cleavage stages developed equivalent i-cell populations. Thus Clytia embryos appear to have maternal germ plasm inherited by i-cells but also the potential to form these cells by zygotic induction. Reassessment of available data indicates that maternally localized germ plasm molecular components were plausibly present in the common cnidarian/bilaterian ancestor, but that their role may not have been strictly deterministic.

Background Myosin II (or Myosin Heavy Chain II, MHCII) is a family of molecular motors involved in the contractile activity of animal muscle cells but also in various other cellular processes in non-muscle cells. Previous phylogenetic analyses of bilaterian MHCII genes identified two main clades associated respectively with smooth/non-muscle cells (MHCIIa) and striated muscle cells (MHCIIb). Muscle cells are generally thought to have originated only once in ancient animal history, and decisive insights about their early evolution are expected to come from expression studies of Myosin II genes in the two non-bilaterian phyla that possess muscles, the Cnidaria and Ctenophora. Results We have uncovered three MHCII paralogues in the ctenophore species Pleurobrachia pileus . Phylogenetic analyses indicate that the MHCIIa / MHCIIb duplication is more ancient than the divergence between extant metazoan lineages. The ctenophore MHCIIa gene ( PpiMHCIIa ) has an expression pattern akin to that of "stem cell markers" ( Piwi, Vasa …) and is expressed in proliferating cells. We identified two MHCIIb genes that originated from a ctenophore-specific duplication. PpiMHCIIb1 represents the exclusively muscular form of myosin II in ctenophore, while PpiMHCIIb2 is expressed in non-muscle cells of various types. In parallel, our phalloidin staining and TEM observations highlight the structural complexity of ctenophore musculature and emphasize the experimental interest of the ctenophore tentacle root, in which myogenesis is spatially ordered and strikingly similar to striated muscle formation in vertebrates. Conclusion MHCIIa expression in putative stem cells/proliferating cells probably represents an ancestral trait, while specific involvement of some MHCIIa genes in smooth muscle fibres is a uniquely derived feature of the vertebrates. That one ctenophore MHCIIb paralogue ( PpiMHCIIb2 ) has retained MHCIIa-like expression features furthermore suggests that muscular expression of the other paralogue, PpiMHCIIb1 , was the result of neofunctionalisation within the ctenophore lineage, making independent origin of ctenophore muscle cells a likely option.

Background: The Sox genes are important regulators of animal development belonging to the HMG domain-containing class of transcription factors. Studies in bilaterian models have notably highlighted their pivotal role in controlling progression along cell lineages, various Sox family members being involved at one side or the other of the critical balance between self-renewing stem cells/proliferating progenitors, and cells undergoing differentiation.Results: We have investigated the expression of 10 Sox genes in the cnidarian Clytia hemisphaerica. Our phylogenetic analyses allocated most of these Clytia genes to previously-identified Sox groups: SoxB (CheSox2, CheSox3, CheSox10, CheSox13, CheSox14), SoxC (CheSox12), SoxE (CheSox1, CheSox5) and SoxF (CheSox11), one gene (CheSox15) remaining unclassified. In the planula larva and in the medusa, the SoxF orthologue was expressed throughout the endoderm. The other genes were expressed either in stem cells/undifferentiated progenitors, or in differentiating (ed) cells with a neuro-sensory identity (nematocytes or neurons). In addition, most of them were expressed in the female germline, with their maternal transcripts either localised to the animal region of the egg, or homogeneously distributed.Conclusions: Comparison with other cnidarians, ctenophores and bilaterians suggest ancient evolutionary conservation of some aspects of gene expression/function at the Sox family level: (i) many Sox genes are expressed in stem cells and/or undifferentiated progenitors; (ii) other genes, or the same under different contexts, are associated with neuro-sensory cell differentiation; (iii) Sox genes are commonly expressed in the germline; (iv) SoxF group genes are associated with endodermal derivatives. Strikingly, total lack of correlation between a given Sox orthology group and expression/function in stem cells/progenitors vs. in differentiating cells implies that Sox genes can easily switch from one side to the other of the balance between these fundamental cellular states in the course of evolution.

Stem cells are essential for animal development and adult tissue homeostasis, and the quest for an ancestral gene fingerprint of stemness is a major challenge for evolutionary developmental biology. Recent studies have indicated that a series of genes, including the transposon silencer Piwi and the translational activator Vasa, specifically involved in germline determination and maintenance in classical bilaterian models (e.g., vertebrates, fly, nematode), are more generally expressed in adult multipotent stem cells in other animals like flatworms and hydras. Since the progeny of these multipotent stem cells includes both somatic and germinal derivatives, it remains unclear whether Vasa, Piwi, and associated genes like Bruno and PL10 were ancestrally linked to stemness, or to germinal potential. We have investigated the expression of Vasa, two Piwi paralogues, Bruno and PL10 in Pleurobrachia pileus, a member of the early-diverging phylum Ctenophora, the probable sister group of cnidarians. These genes were all expressed in the male and female germlines, and with the exception of one of the Piwi paralogues, they showed similar expression patterns within somatic territories (tentacle root, comb rows, aboral sensory complex). Cytological observations and EdU DNA-labelling and long-term retention experiments revealed concentrations of stem cells closely matching these gene expression areas. These stem cell pools are spatially restricted, and each specialised in the production of particular types of somatic cells. These data unveil important aspects of cell renewal within the ctenophore body and suggest that Piwi, Vasa, Bruno, and PL10 belong to a gene network ancestrally acting in two distinct contexts: (i) the germline and (ii) stem cells, whatever the nature of their progeny.

Ctenophores are non-bilaterian animals sharing with cnidarians and bilaterians the presence of sensory receptors, nerve cells, and synapses, absent in placozoans and sponges. Although recent immunofluorescence studies have renewed our knowledge of cnidarian neuro-anatomy, ctenophores have been much less investigated despite their importance to understanding the origin and early evolution of the nervous system. In this study, the neuro-anatomy of the ctenophore Pleurobrachia pileus (Müller, 1776) was explored by whole-mount fluorescent antibody staining using antibodies against tyrosylated -tubulin, FMRFamide, and vasopressin. We describe the morphology of nerve nets and their local specializations, and the organization of the aboral neuro-sensory complex comprising the apical organ and polar fields. Two distinct nerve nets are distinguished: a mesogleal nerve net, loosely organized throughout body mesoglea, and a much more compact “nerve net” with polygonal meshes in the ectodermal epithelium. The latter is organized as a plexus of short nerve cords. This epithelial nervous system contains distinct sub-populations of dispersed FMRFamide and vasopressin immunoreactive nerve cells. In the aboral neuro-sensory complex, our most significant observations include specialized nerve nets underlying the apical organ and polar fields, a tangential bundle of actin-rich fibers (interpreted as a muscle) within the polar fields, and distinct groups of neurons labeled by anti-FMRFamide and anti-vasopressin antibodies, within the apical organ floor. These results are discussed in a comparative perspective.

Background: The early evolution and diversification of Hox-related genes in eumetazoans has been the subject of conflicting hypotheses concerning the evolutionary conservation of their role in axial patterning and the pre-bilaterian origin of the Hox and ParaHox clusters. The diversification of Hox/ParaHox genes clearly predates the origin of bilaterians. However, the existence of a "Hox code'' predating the cnidarian-bilaterian ancestor and supporting the deep homology of axes is more controversial. This assumption was mainly based on the interpretation of Hox expression data from the sea anemone, but growing evidence from other cnidarian taxa puts into question this hypothesis.Methodology/Principal Findings: Hox, ParaHox and Hox-related genes have been investigated here by phylogenetic analysis and in situ hybridisation in Clytia hemisphaerica, an hydrozoan species with medusa and polyp stages alternating in the life cycle. Our phylogenetic analyses do not support an origin of ParaHox and Hox genes by duplication of an ancestral ProtoHox cluster, and reveal a diversification of the cnidarian HOX9-14 genes into three groups called A, B, C. Among the 7 examined genes, only those belonging to the HOX9-14 and the CDX groups exhibit a restricted expression along the oralaboral axis during development and in the planula larva, while the others are expressed in very specialised areas at the medusa stage.Conclusions/Significance: Cross species comparison reveals a strong variability of gene expression along the oral-aboral axis and during the life cycle among cnidarian lineages. The most parsimonious interpretation is that the Hox code, collinearity and conservative role along the antero-posterior axis are bilaterian innovations.