Acoel Single-Cell Transcriptomics: Cell Type Analysis of a Deep Branching Bilaterian.

Bilaterian animals display a wide variety of cell types, organized into defined anatomical structures and organ systems, which are mostly absent in prebilaterian animals. Xenacoelomorpha are an early-branching bilaterian phylum displaying an apparently relatively simple anatomical organization that have greatly diverged from other bilaterian clades. In this study, we use whole-body single-cell transcriptomics on the acoel Isodiametra pulchra to identify and characterize different cell types. Our analysis identifies the existence of ten major cell type categories in acoels all contributing to main biological functions of the organism: metabolism, locomotion and movements, behavior, defense, and development. Interestingly, although most cell clusters express core fate markers shared with other animal clades, we also describe a surprisingly large number of clade-specific marker genes, suggesting the emergence of clade-specific common molecular machineries functioning in distinct cell types. Together, these results provide novel insight into the evolution of bilaterian cell types and open the door to a better understanding of the origins of the bilaterian body plan and their constitutive cell types.

Australian spiny mountain crayfish and their temnocephalan ectosymbionts: an ancient association on the edge of coextinction?

Australian spiny mountain crayfish (Euastacus, Parastacidae) and their ecotosymbiotic temnocephalan flatworms (Temnocephalida, Platyhelminthes) may have co-occurred and interacted through deep time, during a period of major environmental change. Therefore, reconstructing the history of their association is of evolutionary, ecological, and conservation significance. Here, time-calibrated Bayesian phylogenies of Euastacus species and their temnocephalans (Temnohaswellia and Temnosewellia) indicate near-synchronous diversifications from the Cretaceous. Statistically significant cophylogeny correlations between associated clades suggest linked evolutionary histories. However, there is a stronger signal of codivergence and greater host specificity in Temnosewellia, which co-occurs with Euastacus across its range. Phylogeography and analyses of evolutionary distinctiveness (ED) suggest that regional differences in the impact of climate warming and drying had major effects both on crayfish and associated temnocephalans. In particular, Euastacus and Temnosewellia show strong latitudinal gradients in ED and, conversely, in geographical range size, with the most distinctive, northern lineages facing the greatest risk of extinction. Therefore, environmental change has, in some cases, strengthened ecological and evolutionary associations, leaving host-specific temnocephalans vulnerable to coextinction with endangered hosts. Consequently, the extinction of all Euastacus species currently endangered (75%) predicts coextinction of approximately 60% of the studied temnocephalans, with greatest loss of the most evolutionarily distinctive lineages.

NF-Y and the transcriptional activation of CCAAT promoters.

The CCAAT box promoter element and NF-Y, the transcription factor (TF) that binds to it, were among the first cis-elements and trans-acting factors identified; their interplay is required for transcriptional activation of a sizeable number of eukaryotic genes. NF-Y consists of three evolutionarily conserved subunits: a dimer of NF-YB and NF-YC which closely resembles a histone, and the "innovative" NF-YA. In this review, we will provide an update on the functional and biological features that make NF-Y a fundamental link between chromatin and transcription. The last 25 years have witnessed a spectacular increase in our knowledge of how genes are regulated: from the identification of cis-acting sequences in promoters and enhancers, and the biochemical characterization of the corresponding TFs, to the merging of chromatin studies with the investigation of enzymatic machines that regulate epigenetic states. Originally identified and studied in yeast and mammals, NF-Y - also termed CBF and CP1 - is composed of three subunits, NF-YA, NF-YB and NF-YC. The complex recognizes the CCAAT pentanucleotide and specific flanking nucleotides with high specificity (Dorn et al., 1997; Hatamochi et al., 1988; Hooft van Huijsduijnen et al, 1987; Kim & Sheffery, 1990). A compelling set of bioinformatics studies clarified that the NF-Y preferred binding site is one of the most frequent promoter elements (Suzuki et al., 2001, 2004; Elkon et al., 2003; Mariño-Ramírez et al., 2004; FitzGerald et al., 2004; Linhart et al., 2005; Zhu et al., 2005; Lee et al., 2007; Abnizova et al., 2007; Grskovic et al., 2007; Halperin et al., 2009; Häkkinen et al., 2011). The same consensus, as determined by mutagenesis and SELEX studies (Bi et al., 1997), was also retrieved in ChIP-on-chip analysis (Testa et al., 2005; Ceribelli et al., 2006; Ceribelli et al., 2008; Reed et al., 2008). Additional structural features of the CCAAT box - position, orientation, presence of multiple Transcriptional Start Sites - were previously reviewed (Dolfini et al., 2009) and will not be considered in detail here.

Acoel development indicates the independent evolution of the bilaterian mouth and anus.

Most bilaterian animals possess a through gut with a separate mouth and anus. It is commonly believed that during the transition from radial to bilateral symmetry, both openings evolved simultaneously by the lateral closure of a slit-like blastopore. Molecular phylogenies however, place the acoel flatworms, which have only one opening to their digestive system, as the sister group to all remaining Bilateria. To address how this single body opening is related to the mouth and anus of the protostomes and deuterostomes, we studied the expression of genes involved in bilaterian foregut and hindgut patterning during the development of the acoel Convolutriloba longifissura. Here we show that the genes brachyury and goosecoid are expressed in association with the acoel mouth, suggesting that this single opening is homologous to the mouth of other bilaterians. In addition, we find that the genes caudal, orthopedia and brachyury-which are expressed in various bilaterian hindguts-are expressed in a small region at the posterior end of the animal, separated from the anterior oral brachyury-expressing region by a dorsal domain of ectodermal bmp2/4 expression. These results contradict the hypothesis that the bilaterian mouth and anus evolved simultaneously from a common blastoporal opening, and suggest that a through gut might have evolved independently in different animal lineages.

The Abyssal Parasitic Flatworm Fecampia cf. abyssicola: New Records, Anatomy, and Molecular Phylogeny, with a Discussion on Its Systematic Position.

AbstractThe order Fecampiida, a group of parasitic turbellarians, has been poorly studied in terms of its species diversity, morphology, and ecology. Fecampiida is positioned within the monophyletic clade Adiaphanida, along with Tricladida and Prolecithophora, but their phylogenetic relationships are not well understood. Although the nervous and muscular systems of only two species in Fecampiida have been studied, recent research inferred morphological similarities between Fecampiida and Prolecithophora. In this study, we collected fecampiid cocoons and juveniles at depths of 1861-4438 m in Japanese waters. We identified the species on the basis of swimming juvenile specimens and by using histological and molecular methods, while we also examined its musculature and nervous system. Our study revealed a more complex nervous system than previously reported, with dorsal, lateral, and ventral pairs of longitudinal nerve cords connected through an anterior neuropile and posterior transverse commissures. While the nervous and muscular morphology suggested similarities with Prolecithophora, our phylogenetic analysis did not support a close relationship between Fecampiida and Prolecithophora.

Boule-like genes regulate male and female gametogenesis in the flatworm Macrostomum lignano.

Members of the DAZ (Deleted in AZoospermia) gene family are important players in the process of gametogenesis and their dysregulation accounts for 10% of human male infertility. Boule, the ancestor of the family, is mainly involved in male meiosis in most organisms. With the exception of Drosophila and C. elegans, nothing is known on the function of boule in non-vertebrate animals. In the present study, we report on three boule orthologues in the flatworm Macrostomum lignano. We demonstrate that macbol1 and macbol2 are expressed in testes whilst macbol3 is expressed in ovaries and developing eggs. Macbol1 RNAi blocked spermatocyte differentiation whereas macbol2 showed no effect upon RNAi treatment. Macbol3 RNAi resulted in aberrant egg maturation and led to female sterility. We further demonstrated the evolutionary functional conservation of macbol1 by introducing this gene into Drosophila bol(1) mutants. Macbol1 was able to rescue the progression of fly meiotic divisions. In summary, our findings provide evidence for an involvement of boule genes in male and female gamete development in one organism. Furthermore, boule gene function is shown here for the first time in a lophotrochozoan. Our results point to a more diverse functional assignment of boule genes. Therefore, a better understanding of boule function in flatworms can help to elucidate the molecular mechanisms of and concomitant infertility in higher organisms including humans.

How the worm got its pharynx: phylogeny, classification and Bayesian assessment of character evolution in Acoela.

Acoela are marine microscopic worms currently thought to be the sister taxon of all other bilaterians. Acoels have long been used as models in evolutionary scenarios, and generalized conclusions about acoel and bilaterian ancestral features are frequently drawn from studies of single acoel species. There is no extensive phylogenetic study of Acoela and the taxonomy of the 380 species is chaotic. Here we use two nuclear ribosomal genes and one mitochondrial gene in combination with 37 morphological characters in an analysis of 126 acoel terminals (about one-third of the described species) to estimate the phylogeny and character evolution of Acoela. We present an estimate of posterior probabilities for ancestral character states at 31 control nodes in the phylogeny. The overall reconstruction signal based on the shape of the posterior distribution of character states was computed for all morphological characters and control nodes to assess how well these were reconstructed. The body-wall musculature appears more clearly reconstructed than the reproductive organs. Posterior similarity to the root was calculated by averaging the divergence between the posterior distributions at the nodes and the root over all morphological characters. Diopisthoporidae is the sister group to all other acoels and has the highest posterior similarity to the root. Convolutidae, including several "model" acoels, is most divergent. Finally, we present a phylogenetic classification of Acoela down to the family level where six previous family level taxa are synonymized.

Molecular architecture of muscles in an acoel and its evolutionary implications.

We have characterized the homologs of an actin, a troponin I, and a tropomyosin gene in the acoel Symsagittifera roscoffensis. These genes are expressed in muscles and most likely coexpressed in at least a subset of them. In addition, and for the first time for Acoela, we have produced a species-specific muscular marker, an antibody against the tropomyosin protein. We have followed tropomyosin gene and protein expression during postembryonic development and during the posterior regeneration of amputated adults, showing that preexisting muscle fibers contribute to the wound closure. The three genes characterized in this study interact in the striated muscles of vertebrates and invertebrates, where troponin I and tropomyosin are key regulators of the contraction of the sarcomere. S. roscoffensis and all other acoels so far described have only smooth muscles, but the molecular architecture of these is the same as that of striated fibers of other bilaterians. Given the proposed basal position of acoels within the Bilateria, we suggest that sarcomeric muscles arose from a smooth muscle type, which had the molecular repertoire of striated musculature already in place. We discuss this model in a broad comparative perspective.

SMED-TLX-1 (NR2E1) is critical for tissue and body plan maintenance in Schmidtea mediterranea in fasting/feeding cycles.

Nuclear receptors (NRs), or nuclear hormone receptors (NHRs), are transcription factors that regulate development and metabolism of most if not all animal species. Their regulatory networks include conserved mechanisms that are shared in-between species as well as mechanisms that are restricted to certain phyla or even species. In search for conserved members of the NHR family in Schmidtea mediterranea, we identified a molecular signature of a class of NRs, NR2E1, in the S. mediterranea genome and cloned its complete cDNA coding sequence. The derived amino acid sequence shows a high degree of conservation of both DNA-binding domain and ligand- binding domain and a remarkably high homology to vertebrate NR2E1 and C. elegans NHR-67. Quantitative PCR detected approximately ten-fold higher expression of Smed-tlx-1 in the proximal part of the head compared to the tail region. The expression of Smed-tlx-1 is higher during fed state than during fasting. Smed-tlx-1 down-regulation by RNA interference affects the ability of the animals to maintain body plan and induces defects of brain, eyes and body shape during fasting and re-growing cycles. These results suggest that SMED-TLX-1 is critical for tissue and body plan maintenance in planaria.

Potential of Macrostomum lignano to recover from gamma-ray irradiation.

Stem cells are the only proliferating cells in flatworms and can be eliminated by irradiation with no damage to differentiated cells. We investigated the effect of fractionated irradiation schemes on Macrostomum lignano, namely, on survival, gene expression, morphology and regeneration. Proliferating cells were almost undetectable during the first week post-treatment. Cell proliferation and gene expression were restored within 1 month in a dose-dependent manner following exposure to up to 150 Gy irradiation. During recovery, stem cells did not cross the midline but were restricted within lateral compartments. An accumulated dose of 210 Gy resulted in a lethal phenotype. Our findings demonstrate that M. lignano represents a suitable model system for elucidating the effect of irradiation on the stem cell system in flatworms and for improving our understanding of the recovery potential of severely damaged stem-cell systems.

Seminal Fluid-Mediated Manipulation of Post-mating Behavior in a Simultaneous Hermaphrodite.

Seminal fluid proteins (SFPs) are uniquely positioned to mediate post-mating sexual selection and sexual conflict [1-3]. This role may be especially important in simultaneous hermaphrodites, in which individuals will often agree to receive sperm in order to be able to donate it, shifting the arena of sexual selection to post-mating reproductive interactions [4-7]. Nevertheless, as in separate-sexed organisms, identifying individual SFPs responsible for specific post-mating effects is difficult, owing to the complexity, rapid evolution, and functional redundancy of seminal fluid [8-11]. Here, we sought to identify SFPs that influence one striking post-mating behavior of the simultaneously hermaphroditic flatworm Macrostomum lignano, the so-called "suck behavior," in which worms respond to ejaculate receipt by placing their pharynx over their female genital opening and seemingly attempt to remove sperm and/or other ejaculate components [12-14]. We hypothesized that sucking is counter to the sperm donor's interests, potentially selecting for SFPs that reduce the suck propensity of mating partners. We tested this using a combination of quantitative genetics and RNA interference (RNAi) knockdown. As predicted, we found negative genetic correlations between the expression levels of six (out of 58) seminal fluid transcripts and partner suck propensity. RNAi knockdown confirmed that two of these transcripts, designated suckless-1 and suckless-2, indeed caused mating partners to suck less often. We suggest that these proteins are male counter-adaptations to recipient suck behavior, which itself is likely a female counter-adaptation in the ongoing evolutionary conflict to (re)gain control over ejaculate fate after mating in this hermaphroditic organism.

Assessing the root of bilaterian animals with scalable phylogenomic methods.

A clear picture of animal relationships is a prerequisite to understand how the morphological and ecological diversity of animals evolved over time. Among others, the placement of the acoelomorph flatworms, Acoela and Nemertodermatida, has fundamental implications for the origin and evolution of various animal organ systems. Their position, however, has been inconsistent in phylogenetic studies using one or several genes. Furthermore, Acoela has been among the least stable taxa in recent animal phylogenomic analyses, which simultaneously examine many genes from many species, while Nemertodermatida has not been sampled in any phylogenomic study. New sequence data are presented here from organisms targeted for their instability or lack of representation in prior analyses, and are analysed in combination with other publicly available data. We also designed new automated explicit methods for identifying and selecting common genes across different species, and developed highly optimized supercomputing tools to reconstruct relationships from gene sequences. The results of the work corroborate several recently established findings about animal relationships and provide new support for the placement of other groups. These new data and methods strongly uphold previous suggestions that Acoelomorpha is sister clade to all other bilaterian animals, find diminishing evidence for the placement of the enigmatic Xenoturbella within Deuterostomia, and place Cycliophora with Entoprocta and Ectoprocta. The work highlights the implications that these arrangements have for metazoan evolution and permits a clearer picture of ancestral morphologies and life histories in the deep past.

Polyploidy as a chromosomal component of stochastic noise: variable scalar multiples of the diploid chromosome complement in the invertebrate species Girardia schubarti from Brazil.

Chromosome stoichiometry, a form of genetic plasticity, specifically refers to variation in the standard diploid genomic composition of an individual or species. In the present work, freshwater planarians (Girardia schubarti) were analyzed to recognize variations in chromosomal stoichiometry especially of complete ploidal change between specimens, within specimens and between cells within specimens and any relations they might have with selected components of phenotypic plasticity. Homoploid polyploids for the group reached rational scalar multiples (e.g. tetraploids) or irrational scalar multiples (e.g. triploids). Karyotypic mosaics emerged where individual cells presented polyploid multiples in arithmetic and geometric progressions. Ploidal multiplicity, a chromosomal component of stochastic noise, had positive phenotypic effects (increased dimensions) on morphologic criteria of body length, body width and dorsal surface reflecting a significant genotypic plasticity (GP) and robust phenotypic plasticity (PP). Variable but significant association of genotypic plasticity with robust phenotypic variance suggests kinetics of phenotypic homeostasis that is species-specific permitting phenotypic adaptability to environmental variables by means of GP. That association is diminished, deactivated or lost in more advanced and more complex organisms.

New insights into the karyotype evolution of the free-living flatworm Macrostomum lignano (Platyhelminthes, Turbellaria).

The free-living flatworm Macrostomum lignano is a model organism for evolutionary and developmental biology studies. Recently, an unusual karyotypic diversity was revealed in this species. Specifically, worms are either 'normal' 2n = 8, or they are aneuploid with one or two additional large chromosome(s) (i.e. 2n = 9 or 2n = 10, respectively). Aneuploid worms did not show visible behavioral or morphological abnormalities and were successful in reproduction. In this study, we generated microdissected DNA probes from chromosome 1 (further called MLI1), chromosome 2 (MLI2), and a pair of similar-sized smaller chromosomes (MLI3, MLI4). FISH using these probes revealed that MLI1 consists of contiguous regions homologous to MLI2-MLI4, suggesting that MLI1 arose due to the whole genome duplication and subsequent fusion of one full chromosome set into one large metacentric chromosome. Therefore, one presumably full haploid genome was packed into MLI1, leading to hidden tetraploidy in the M. lignano genome. The study of Macrostomum sp. 8 - a sibling species of M. lignano - revealed that it usually has one additional pair of large chromosomes (2n = 10) showing a high homology to MLI1, thus suggesting hidden hexaploidy in its genome. Possible evolutionary scenarios for the emergence of the M. lignano and Macrostomum sp. 8 genomes are discussed.

Syndesmis aethopharynx (Umagillidae, Rhabdocoela, Platyhelminthes) from the sea urchin Paracentrotus lividus: First record from the Eastern Mediterranean, phylogenetic position and intraspecific morphological variation.

Specimens of Syndesmis aethopharynx Westervelt & Kozloff, 1990 (Umagillidae, Rhabdocoela, Platyhelminthes) were collected from the intestine of several specimens of the sea urchin Paracentrotus lividus (Lamarck, 1816) [2], Hansson, 2001 at the Greek coast. This represents the first report of a species of Syndesmis from Greece. Our study has revealed several previously-unreported morphological details and intraspecific variation, which are added to the species description. The position of S. aethopharynx within Umagillidae is confirmed for the first time through molecular data (based on nuclear 18S rDNA), using both Bayesian and maximum likelihood analyses.

Zoology: War of the Worms.

The phylogenetic affinities of Xenacoelomorpha - the phylum comprising Xenoturbella bocki and acoelomorph worms - are debated. Two recent studies conclude they represent the earliest branching bilaterally symmetrical animals, but additional tests may be needed to confirm this notion.

Surprising diversity and distribution of spliced leader RNAs in flatworms.

Trans-splicing generates the mature 5' ends of certain mRNAs through the addition of a small spliced leader (SL) exon to pre-mRNAs. To search for novel flatworm spliced leaders, degenerate oligonucleotides and 5' RACE [corrected was used to isolate and characterize the 5' terminal sequences of enolase mRNAs in diverse flatworms. Several new spliced leaders and their SL RNA genes were identified, characterized, and compared. All parasitic trematodes examined trans-splice enolase. A primitive polyclad turbellarian, Stylochus zebra, also contains a trans-spliced enolase mRNA. The S. zebra SL is the longest SL yet identified, 51 nucleotides. Comparison of flatworm SLs indicates that they vary significantly in sequence and length. This suggests that neither spliced leader exon sequence nor size is likely to be essential for trans-splicing in flatworms. Flatworm SL RNAs have unusual Sm binding sites with characteristics distinct from other known flatworm snRNA Sm binding sites. Predicted flatworm SL RNA secondary structures show variation exhibiting 2-4 stem loops. Although limited in sequence similarity, phylogenetically conserved regions within the diverse flatworm SL RNAs suggest that they are likely to be derived from a common ancestor and provide information on potentially important SL RNA elements. The identification of a SL in a primitive flatworm suggests that trans-splicing may have been an ancestral feature in the phylum. Representative species of other early and more recent clades within the phylum, however, do not trans-splice enolase, nor do they or representatives of several other flatworm groups, have an SL RNA with a phylogenetically conserved region identified in the current study.

Stage and sex specific differences in actin gene expression in Schistosoma mansoni.

We have characterized actin gene expression in Schistosoma mansoni at the RNA and protein levels. Northern blot analyses showed two size classes of actin mRNA in eggs, cercariae and adult worms of both sexes, approximately 1 900 and 1 400 bases in length. A higher abundance of actin mRNA of both size classes was demonstrated in male worms than in eggs, cercariae, and females. Using a phalloidin-rhodamine conjugate, male worms were observed to contain more actin protein than females. Southern blot-hybridization indicated that the sexual differences in actin mRNA and protein levels were not related to some S. mansoni actin genes being sex linked. In addition, two other trematodes, Schistosoma japonicum and Fasciola hepatica and a cestode, Taenia pisiformis contained two classes of actin mRNA similar in size as those in S. mansoni. In contrast, a turbellarian, Dugesia tigrina contained only a single short actin message size class approximately 1 400 bases in length.

Origin and evolution of African Polystoma (Monogenea: Polystomatidae) assessed by molecular methods.

Among Polystomatidae (Monogenea), the genus Polystoma, which mainly infests neobatrachian hosts, is the most diverse and occurs principally in Africa, from where half the species have been reported. Previous molecular phylogenetic studies have shown that this genus originated in South America, and later colonised Eurasia and Africa. No mention was made on dispersal corridors between Europe and Africa or of the origin of the African Polystoma radiation. Therefore, a molecular phylogeny was inferred from ITS1 sequences of 21 taxa comprising two species from America, seven representatives from Europe and 12 from Africa. The topology of the phylogenetic tree reveals that a single event of colonisation took place from Europe to Africa and that the putative host carrying along the ancestral polystome is to be found among ancestral pelobatids. Percentage divergences estimates suggest that some presumably distinct vesicular species in unrelated South African anurans and some neotenic forms found in several distinct hosts in Ivory Coast, could, in fact, belong to two single polystome species parasitising divergent hosts. Two main factors are identified that may explain the diversity of African polystomes: (i), we propose that following some degree of generalism, at least during the juvenile stages of both hosts and parasites, distinctive larval behaviour of polystomes engenders isolation between parasite populations that precludes sympatric speciations; (ii), cospeciation events between Ptychadena hosts and their parasites are another factor of diversification of Polystoma on the African continent. Finally, we discuss the systematic status of the Madagascan parasite Metapolystoma, as well as the colonisation of Madagascar by the host Ptychadena mascareniensis.

Molecular markers for gyrodactylids (Gyrodactylidae: Monogenea) from five fish families (Teleostei).

Thirty-one gyrodactylid species from five families of freshwater fish were examined and variable region V4 of the 18S small subunit ribosomal RNA gene and ribosomal RNA internal transcribed spacers ITS1 and ITS2 were sequenced. Both the V4 region and spacers ITS1 and ITS2 proved useful for gyrodactylid diagnosis. Sequences of these fragments exhibited interspecific variations and allowed clear determination at the species level. In some cases, the length of the ITS1 PCR fragment provided useful genetic markers. Species that yielded a short ITS1 fragment also showed distinct groupings in ITS2 and V4 sequences that were markedly different to sequences from species that contain a long ITS1. Repetitive sequences located in the ITS1 of Gyrodactylus gobii and Gyrodactylus vimbi accounted for some of the variations in length of PCR products. There was no evidence for intraspecific variation within these regions and short tandem repeats were not found in the other species studied. The number of polymorphic and intraspecific variations in nucleic acid sequences was low, therefore these variations did not affect species determination of gyrodactylids. Minor differences in the sequences between Western and Eastern European populations were detected for Gyrodactylus salaris/Gyrodactylus thymalli, Gyrodactylus teuchis and Gyrodactylus truttae, but these do not affect species diagnosis based on ribosomal DNA sequence. These results confirm the utility of both variable region V4 and the ITS as molecular markers for Gyrodactylus species.