Annelid Comparative Genomics and the Evolution of Massive Lineage-Specific Genome Rearrangement in Bilaterians.

The organization of genomes into chromosomes is critical for processes such as genetic recombination, environmental adaptation, and speciation. All animals with bilateral symmetry inherited a genome structure from their last common ancestor that has been highly conserved in some taxa but seemingly unconstrained in others. However, the evolutionary forces driving these differences and the processes by which they emerge have remained largely uncharacterized. Here, we analyze genome organization across the phylum Annelida using 23 chromosome-level annelid genomes. We find that while many annelid lineages have maintained the conserved bilaterian genome structure, the Clitellata, a group containing leeches and earthworms, possesses completely scrambled genomes. We develop a rearrangement index to quantify the extent of genome structure evolution and show that, compared to the last common ancestor of bilaterians, leeches and earthworms have among the most highly rearranged genomes of any currently sampled species. We further show that bilaterian genomes can be classified into two distinct categories-high and low rearrangement-largely influenced by the presence or absence, respectively, of chromosome fission events. Our findings demonstrate that animal genome structure can be highly variable within a phylum and reveal that genome rearrangement can occur both in a gradual, stepwise fashion, or rapid, all-encompassing changes over short evolutionary timescales.

Mitochondrial Genome Evolution in Annelida-A Systematic Study on Conservative and Variable Gene Orders and the Factors Influencing its Evolution.

The mitochondrial genomes of Bilateria are relatively conserved in their protein-coding, rRNA, and tRNA gene complement, but the order of these genes can range from very conserved to very variable depending on the taxon. The supposedly conserved gene order of Annelida has been used to support the placement of some taxa within Annelida. Recently, authors have cast doubts on the conserved nature of the annelid gene order. Various factors may influence gene order variability including, among others, increased substitution rates, base composition differences, structure of noncoding regions, parasitism, living in extreme habitats, short generation times, and biomineralization. However, these analyses were neither done systematically nor based on well-established reference trees. Several focused on only a few of these factors and biological factors were usually explored ad-hoc without rigorous testing or correlation analyses. Herein, we investigated the variability and evolution of the annelid gene order and the factors that potentially influenced its evolution, using a comprehensive and systematic approach. The analyses were based on 170 genomes, including 33 previously unrepresented species. Our analyses included 706 different molecular properties, 20 life-history and ecological traits, and a reference tree corresponding to recent improvements concerning the annelid tree. The results showed that the gene order with and without tRNAs is generally conserved. However, individual taxa exhibit higher degrees of variability. None of the analyzed life-history and ecological traits explained the observed variability across mitochondrial gene orders. In contrast, the combination and interaction of the best-predicting factors for substitution rate and base composition explained up to 30% of the observed variability. Accordingly, correlation analyses of different molecular properties of the mitochondrial genomes showed an intricate network of direct and indirect correlations between the different molecular factors. Hence, gene order evolution seems to be driven by molecular evolutionary aspects rather than by life history or ecology. On the other hand, variability of the gene order does not predict if a taxon is difficult to place in molecular phylogenetic reconstructions using sequence data or not. We also discuss the molecular properties of annelid mitochondrial genomes considering canonical views on gene evolution and potential reasons why the canonical views do not always fit to the observed patterns without making some adjustments. [Annelida; compositional biases; ecology; gene order; life history; macroevolution; mitochondrial genomes; substitution rates.].

Is there potential for estradiol receptor signaling in lophotrochozoans?

Estrogen receptors (ERs) are thought to be the ancestor of all steroid receptors and are present in most lophotrochozoans studied to date, including molluscs, annelids, and rotifers. A number of studies have investigated the functional role of estrogen receptors in invertebrate species, although most are in molluscs, where the receptor is constitutively active. In vitro experiments provided evidence for ligand-activated estrogen receptors in annelids, raising important questions about the role of estrogen signalling in lophotrochozoan lineages. Here, we review the concordant and discordant evidence of estradiol receptor signalling in lophotrochozoans, with a focus on annelids and rotifers. We explore the de novo synthesis of estrogens, the evolution and expression of estrogen receptors, and physiological responses to activation of estrogen receptors in the lophotrochozoan phyla Annelida and Rotifera. Key data are missing to determine if de novo biosynthesis of estradiol in non-molluscan lophotrochozoans is likely. For example, an ortholog for the CYP11 gene is present, but confirmation of substrate conversion and measured tissue products is lacking. Orthologs CYP17 and CYP19 are lacking, yet intermediates or products (e.g. estradiol) in tissues have been measured. Estrogen receptors are present in multiple species, and for a limited number, in vitro data show agonist binding of estradiol and/or transcriptional activation. The expression patterns of the lophotrochozoan ERs suggest developmental, reproductive, and digestive roles but are highly species dependent. E2 exposures suggest that lophotrochozoan ERs may play a role in reproduction, but no strong dose-response relationship has been established. Therefore, we expect most lophotrochozoan species, outside of perhaps platyhelminths, to have an ER but their physiological role remains elusive. Mining genomes for orthologs gene families responsible for steroidogenesis, coupled with in vitro and in vivo studies of the steroid pathway are needed to better assess whether lophotrochozoans are capable of estradiol biosynthesis. One major challenge is that much of the data are divided across a diversity of species. We propose that the polychaetes Capitella teleta or Platyneris dumerilii, and rotifer Brachionus manjavacas may be strong species choices for studies of estrogen receptor signalling, because of available genomic data, established laboratory culture techniques, and gene knockout potential.

Transcriptomic landscape of posterior regeneration in the annelid Platynereis dumerilii.

BACKGROUND: Restorative regeneration, the capacity to reform a lost body part following amputation or injury, is an important and still poorly understood process in animals. Annelids, or segmented worms, show amazing regenerative capabilities, and as such are a crucial group to investigate. Elucidating the molecular mechanisms that underpin regeneration in this major group remains a key goal. Among annelids, the nereididae Platynereis dumerilii (re)emerged recently as a front-line regeneration model. Following amputation of its posterior part, Platynereis worms can regenerate both differentiated tissues of their terminal part as well as a growth zone that contains putative stem cells. While this regeneration process follows specific and reproducible stages that have been well characterized, the transcriptomic landscape of these stages remains to be uncovered. RESULTS: We generated a high-quality de novo Reference transcriptome for the annelid Platynereis dumerilii. We produced and analyzed three RNA-sequencing datasets, encompassing five stages of posterior regeneration, along with blastema stages and non-amputated tissues as controls. We included two of these regeneration RNA-seq datasets, as well as embryonic and tissue-specific datasets from the literature to produce a Reference transcriptome. We used this Reference transcriptome to perform in depth analyzes of RNA-seq data during the course of regeneration to reveal the important dynamics of the gene expression, process with thousands of genes differentially expressed between stages, as well as unique and specific gene expression at each regeneration stage. The study of these genes highlighted the importance of the nervous system at both early and late stages of regeneration, as well as the enrichment of RNA-binding proteins (RBPs) during almost the entire regeneration process. CONCLUSIONS: In this study, we provided a high-quality de novo Reference transcriptome for the annelid Platynereis that is useful for investigating various developmental processes, including regeneration. Our extensive stage-specific transcriptional analysis during the course of posterior regeneration sheds light upon major molecular mechanisms and pathways, and will foster many specific studies in the future.

Updated phylogeny of Vestimentifera (Siboglinidae, Polychaeta, Annelida) based on mitochondrial genomes, with a new species.

Siboglinid tubeworms are found at chemosynthetic environments worldwide and the Vestimentifera clade is particularly well known for their reliance on chemoautotrophic bacterial symbionts for nutrition. The mitochondrial genomes have been published for nine vestimentiferan species to date. This study provides new complete mitochondrial genomes for ten further Vestimentifera, including the first mitochondrial genomes sequenced for Alaysia spiralis, Arcovestia ivanovi, Lamellibrachia barhami, Lamellibrachia columna, Lamellibrachia donwalshi, and unnamed species of Alaysia and Oasisia. Phylogenetic analyses combining fifteen mitochondrial genes and the nuclear 18S rRNA gene recovered Lamellibrachia as sister to the remaining Vestimentifera and Riftia pachyptila as separate from the other vent-endemic taxa. Implications and auxiliary analyses regarding differing phylogenetic tree topologies, substitution saturation, ancestral state reconstruction, and divergence estimates are also discussed. Additionally, a new species of Alaysia is described from the Manus Basin.

A transcriptome-based phylogeny for Polynoidae (Annelida: Aphroditiformia).

Polynoidae is the most diverse radiation of Aphroditiformia and one of the most successful groups of all Annelida in terms of diversity and habitats colonized. With such an unmatched diversity, phylogenetic investigations have struggled to understand their evolutionary relationships. Previous phylogenetic analyses have slowly increased taxon sampling and employed methodologies, but despite their diversity and biological importance, large genomic sampling is limited. To investigate the internal relationships within Polynoidae, we conducted the first phylogenomic analyses of the group based on 12 transcriptomes collected from species inhabiting a broad array of habitats, including shallow and deep waters, as well as hydrothermal vents, anchialine caves and the midwater. Our phylogenomic analyses of Polynoidae recovered congruent tree topologies representing the clades Polynoinae, Macellicephalinae and Lepidonotopodinae. Members of Polynoinae and Macellicephalinae clustered in well-supported and independent clades. In contrast, Lepidonotopodinae taxa were always recovered nested within Macellicephalinae. Though our sampling only covers a small proportion of the species known for Polynoidae, our results provide a robust phylogenomic framework to build from, emphasizing previously hypothesized relationships between Macellicephalinae and Lepidonotopodinae taxa, while providing new insights on the origin of enigmatic cave and pelagic lineages.

First report of the mitogenome of the invasive reef-building polychaete Ficopomatus enigmaticus (Annelida: Serpulidae) and a cryptic lineage from the Japanese Archipelago.

BACKGROUND: The mitochondrial genomes (mitogenomes) of the family Serpulidae are characterized by a high nucleotide sequence divergence and a significant number of gene order rearrangements compared with other families within the phylum Annelida. However, only two of 50 genera of serpulids have mitogenomes already sequenced. In this study, we report the first sequencing and assembly of the complete mitogenome of Ficopomatus, thus providing further knowledge on mitochondrial gene sequences of Serpulidae. METHODS AND RESULTS: A mitogenome of the invasive reef-building polychaete Ficopomatus enigmaticus was amplified by long PCR and sequenced using the Illumina MiSeq System. It comprised 15,853 bp and consisted of 12 protein-coding genes (atp8 was not found), 23 tRNA, and two rRNA genes. The AT and GC skew values were infrequent when compared to annelid mitogenomes but similar to other serpulids sequenced to date (i.e., Spirobranchus and Hydroides). The mitochondrial gene order of F. enigmaticus was highly rearranged compared to other serpulids. To amplify 16S rRNA gene sequences, we developed a 16S rRNA primer set by modifying the universal primer set 16SarL/16SbrH. We detected the 16S rRNA sequence of F. enigmaticus deposited in GenBank erroneously characterized as of serpulid origin. We reported for the first time the presence of two lineages of F. enigmaticus in Japan, which have already been identified in California, Australia, and the Mediterranean. CONCLUSIONS: The first mitochondrial genome of F. enigmaticus showed a unique gene order rearrangement, corroborating the remarkable diversity in the previously reported mitogenomes of other serpulid species. The presence of the two lineages of F. enigmaticus identified for the first time in Japan represents another case of cryptic invasion. The first 16S rRNA gene sequences of F. enigmaticus obtained in the present study can be used as reference sequences in future DNA metabarcoding studies.

Morphological Observation and Transcriptome Analysis of Ciliogenesis in Urechis unicinctus (Annelida, Echiura).

During the early development of marine invertebrates, planktic larvae usually occur, and their body surfaces often form specific types of cilia that are involved in locomotion and feeding. The echiuran worm Urechis unicinctus sequentially undergoes the formation and disappearance of different types of body surface cilia during embryonic and larval development. The morphological characteristics and molecular mechanisms involved in the process remain unclear. In this study, we found that body surface cilia in U. unicinctus embryos and larvae can be distinguished into four types: body surface short cilia, apical tufts, circumoral cilia and telotrochs. Further, distribution and genesis of the body surface cilia were characterized using light microscope and electron microscope. To better understand the molecular mechanism during ciliogenesis, we revealed the embryonic and larval transcriptome profile of the key stages of ciliogenesis in U. unicinctus using RNA-Seq technology. A total of 29,158 differentially expressed genes (DEGs) were obtained from 24 cDNA libraries by RNA-Seq. KEGG pathway enrichment results showed that Notch, Wnt and Ca2+ signaling pathways were significantly enriched during the occurrence of apical tufts and circumoral cilia. Furthermore, all DEGs were classified according to their expression pattern, and DEGs with similar expression pattern were grouped into a module. All DEG co-expression modules were correlated with traits (body surface short cilia, apical tufts, circumoral cilia and telotrochs) by WGCNA, the results showed DEGs were divided into 13 modules by gene expression patterns and that the genes in No. 7, No. 8 and No. 10 modules were to be highly correlated with the occurrence of apical tufts, circumoral cilia and telotrochs. The top 10 hub genes in the above three modules were identified to be highly correlated with ciliogenesis, including the reported cilium-related gene Cnbd2 and unreported cilium-related candidate genes FAM181B, Capsl, Chst3, TMIE and Innexin. Notably, Innexin was included in the top10 hub genes of the two modules (No. 7 and No. 8), suggesting that Innexin may play an important role in U. unicinctus apical tufts, circumoral cilia and telotrochs genesis. This study revealed the characteristics of ciliogenesis on the body surface of U. unicinctus embryos and larvae, providing basic data for exploring the molecular mechanism of ciliogenesis on the body surface.

Echiuran Hox genes provide new insights into the correspondence between Hox subcluster organization and collinearity pattern.

In many bilaterians, Hox genes are generally clustered along the chromosomes and expressed in spatial and temporal order. In vertebrates, the expression of Hox genes follows a whole-cluster spatio-temporal collinearity (WSTC) pattern, whereas in some invertebrates the expression of Hox genes exhibits a subcluster-level spatio-temporal collinearity pattern. In bilaterians, the diversity of collinearity patterns and the cause of collinearity differences in Hox gene expression remain poorly understood. Here, we investigate genomic organization and expression pattern of Hox genes in the echiuran worm Urechis unicinctus (Annelida, Echiura). Urechis unicinctus has a split cluster with four subclusters divided by non-Hox genes: first subcluster (Hox1 and Hox2), second subcluster (Hox3), third subcluster (Hox4, Hox5, Lox5, Antp and Lox4), fourth subcluster (Lox2 and Post2). The expression of U. unicinctus Hox genes shows a subcluster-based whole-cluster spatio-temporal collinearity (S-WSTC) pattern: the anterior-most genes in each subcluster are activated in a spatially and temporally colinear manner (reminiscent of WSTC), with the subsequent genes in each subcluster then being very similar to their respective anterior-most subcluster gene. Combining genomic organization and expression profiles of Hox genes in different invertebrate lineages, we propose that the spatio-temporal collinearity of invertebrate Hox is subcluster-based.

Phylogenomics resolves ambiguous relationships within Aciculata (Errantia, Annelida).

Aciculata (Eunicida + Phyllodocida) is among the largest clades of annelids, comprising almost half of the known diversity of all marine annelids. Despite the group's large size and biological importance, most phylogenomic studies on Annelida to date have had a limited sampling of this clade. The phylogenetic placement of many clades within Phyllodocida in particular has remained poorly understood. To resolve the relationships within Aciculata we conducted a large-scale phylogenomic analysis based on 24 transcriptomes (13 new), chosen to represent many family-ranked taxa that have never been included in a broad phylogenomic study. Our sampling also includes several enigmatic taxa with challenging phylogenetic placement, such as Histriobdella, Struwela, Lacydonia, Pilargis and the holopelagic worms Lopadorrhynchus, Travisiopsis and Tomopteris. Our robust phylogeny allows us to name and place some of these problematic clades and has significant implications on the systematics of the group. Within Eunicida we reinstate the names Eunicoidea and Oenonoidea. Within Phyllodocida we delineate Phyllodociformia, Glyceriformia, Nereidiformia, Nephtyiformia and Aphroditiformia. Phyllodociformia now includes: Lacydonia, Typhloscolecidae, Lopadorrhynchidae and Phyllodocidae. Nephtyiformia includes Nephtyidae and Pilargidae. We also broaden the delineation of Glyceriformia to include Sphaerodoridae, Tomopteridae and Glyceroidea (Glyceridae + Goniadidae). Furthermore, our study demonstrates and explores how conflicting, yet highly supported topologies can result from confounding signals in gene trees.

The Phylogenetic Position of Branchamphinome (Annelida, Amphinomidae) with a Description of a New Species from the North Pacific Ocean.

A new species of amphinomid polychaete, Branchamphinome kohtsukai sp. nov., is described from Japanese waters, 29-211 m in depth. The species is distinguishable from other congeners by the following features: i) branchiae with four-six filaments in midbody segments; ii) two pairs of eyes not coalescent; iii) the ventral side of the first four chaetigers broadly pigmented. This is the first record of Branchamphinome from the North Pacific Ocean. We provide a phylogenetic tree based on the sequences of four genes (COI, 16S, 18S, 28S) and discuss amphinomids' relationships.

A New Polychaete, Scolelepis (Parascolelepis) Brunnea sp. nov. (Annelida: Spionidae), from Korea.

A new spionid polychaete, Scolelepis (Parascolelepis) brunnea sp. nov., from an intertidal mud flat in Korean waters, is reported. The new species is unique among species of Scolelepis Blainville, 1828 in having conspicuously long, reddish-brown branchiae on the anteriormost chaetigers. The new species is morphologically and genetically most closely related to Scolelepis (Parascolelepis) anterobranchiata Lee and Min, 2022 from Korea. However, the new species differs from the latter by a combination of the following characteristics: presence of reddish-brown pigmentations on anteriormost body, neuropodial hooded hooks appearing from chaetigers 21 to 22, larger size of worms, and three teeth above the main fang of neuropodial hooded hooks. Detailed description and images of the new species, along with three gene regions (cytochrome c oxidase subunit I [COI], 16S ribosomal DNA [16S rDNA], and 18S rDNA), are provided.

Molecular and morphological analysis of the developing nemertean brain indicates convergent evolution of complex brains in Spiralia.

BACKGROUND: The brain anatomy in the clade Spiralia can vary from simple, commissural brains (e.g., gastrotrichs, rotifers) to rather complex, partitioned structures (e.g., in cephalopods and annelids). How often and in which lineages complex brains evolved still remains unclear. Nemerteans are a clade of worm-like spiralians, which possess a complex central nervous system (CNS) with a prominent brain, and elaborated chemosensory and neuroglandular cerebral organs, which have been previously suggested as homologs to the annelid mushroom bodies. To understand the developmental and evolutionary origins of the complex brain in nemerteans and spiralians in general, we investigated details of the neuroanatomy and gene expression in the brain and cerebral organs of the juveniles of nemertean Lineus ruber. RESULTS: In the juveniles, the CNS is already composed of all major elements present in the adults, including the brain, paired longitudinal lateral nerve cords, and an unpaired dorsal nerve cord, which suggests that further neural development is mostly related with increase in the size but not in complexity. The ultrastructure of the juvenile cerebral organ revealed that it is composed of several distinct cell types present also in the adults. The 12 transcription factors commonly used as brain cell type markers in bilaterians show region-specific expression in the nemertean brain and divide the entire organ into several molecularly distinct areas, partially overlapping with the morphological compartments. Additionally, several of the mushroom body-specific genes are expressed in the developing cerebral organs. CONCLUSIONS: The dissimilar expression of molecular brain markers between L. ruber and the annelid Platynereis dumerilii indicates that the complex brains present in those two species evolved convergently by independent expansions of non-homologous regions of a simpler brain present in their last common ancestor. Although the same genes are expressed in mushroom bodies and cerebral organs, their spatial expression within organs shows apparent differences between annelids and nemerteans, indicating convergent recruitment of the same genes into patterning of non-homologous organs or hint toward a more complicated evolutionary process, in which conserved and novel cell types contribute to the non-homologous structures.

Gotta Go Slow: Two Evolutionarily Distinct Annelids Retain a Common Hedgehog Pathway Composition, Outlining Its Pan-Bilaterian Core.

Hedgehog signaling is one of the key regulators of morphogenesis, cell differentiation, and regeneration. While the Hh pathway is present in all bilaterians, it has mainly been studied in model animals such as Drosophila and vertebrates. Despite the conservatism of its core components, mechanisms of signal transduction and additional components vary in Ecdysozoa and Deuterostomia. Vertebrates have multiple copies of the pathway members, which complicates signaling implementation, whereas model ecdysozoans appear to have lost some components due to fast evolution rates. To shed light on the ancestral state of Hh signaling, models from the third clade, Spiralia, are needed. In our research, we analyzed the transcriptomes of two spiralian animals, errantial annelid Platynereis dumerilii (Nereididae) and sedentarian annelid Pygospio elegans (Spionidae). We found that both annelids express almost all Hh pathway components present in Drosophila and mouse. We performed a phylogenetic analysis of the core pathway components and built multiple sequence alignments of the additional key members. Our results imply that the Hh pathway compositions of both annelids share more similarities with vertebrates than with the fruit fly. Possessing an almost complete set of single-copy Hh pathway members, lophotrochozoan signaling composition may reflect the ancestral features of all three bilaterian branches.

Genome-Wide Analyses of Heat Shock Protein Superfamily Provide New Insights on Adaptation to Sulfide-Rich Environments in Urechis unicinctus (Annelida, Echiura).

The intertidal zone is a transitional area of the land-sea continuum, in which physical and chemical properties vary during the tidal cycle and highly toxic sulfides are rich in sediments due to the dynamic regimes. As a typical species thriving in this habitat, Urechis unicinctus presents strong sulfide tolerance and is expected to be a model species for sulfide stress research. Heat shock proteins (HSPs) consist of a large group of highly conserved molecular chaperones, which play important roles in stress responses. In this study, we systematically analyzed the composition and expression of HSPs in U. unicinctus. A total of eighty-six HSP genes from seven families were identified, in which two families, including sHSP and HSP70, showed moderate expansion, and this variation may be related to the benthic habitat of the intertidal zone. Furthermore, expression analysis revealed that almost all the HSP genes in U. unicinctus were significantly induced under sulfide stress, suggesting that they may be involved in sulfide stress response. Weighted gene co-expression network analysis (WGCNA) showed that 12 HSPs, including 5 sHSP and 4 HSP70 family genes, were highly correlated with the sulfide stress response which was distributed in steelblue and green modules. Our data indicate that HSPs, especially sHSP and HSP70 families, may play significant roles in response to sulfide stress in U. unicinctus. This systematic analysis provides valuable information for further understanding of the function of the HSP gene family for sulfide adaptation in U. unicinctus and contributes a better understanding of the species adaptation strategies of marine benthos in the intertidal zone.

Proteomic analysis of body wall and coelomic fluid in Sipunculus nudus.

In order to make clear the protein compositions of Sipunculus nudus and investigate its immune-related proteins, proteomic analysis was performed on body wall and coelomic fluid of Sipunculus nudus. A total of 1659 proteins were identified, and 539 proteins were differentially expressed in the coelomic fluid compared to those in the body wall, of which 415 proteins were up-regulated while 124 proteins were down-regulated. Gene Ontology (GO) analysis showed that the GO terms involved in the two parts of Sipunculus nudus were similar, with metabolic processes, catalytic activity and cell occupying the top categories of biological process, molecular function and cellular component, respectively. KEGG pathway analysis showed that 49 pathways in body wall and 48 in coelomic fluid were mapped respectively, and these pathways were mainly related to cellular processes, environmental information processing, genetic information processing and metabolism. The COG analysis showed that 757 proteins from body wall and 889 from coelomic fluid were classified into 26 COG categories, respectively. Pfam annotation revealed the mainly immune-related proteins contained in Sipunculus nudus, such as insulin-like growth factor binding protein, catalase, basement membrane proteoglycan, titin. Our research provides the first proteomic information of Sipunculus nudus, which contributes to the study of functional proteins in Sipunculus nudus and is of great significance for the application of Sipunculus nudus in functional foods and medicines.

Metagenomic sequencing suggests a diversity of RNA interference-like responses to viruses across multicellular eukaryotes.

RNA interference (RNAi)-related pathways target viruses and transposable element (TE) transcripts in plants, fungi, and ecdysozoans (nematodes and arthropods), giving protection against infection and transmission. In each case, this produces abundant TE and virus-derived 20-30nt small RNAs, which provide a characteristic signature of RNAi-mediated defence. The broad phylogenetic distribution of the Argonaute and Dicer-family genes that mediate these pathways suggests that defensive RNAi is ancient, and probably shared by most animal (metazoan) phyla. Indeed, while vertebrates had been thought an exception, it has recently been argued that mammals also possess an antiviral RNAi pathway, although its immunological relevance is currently uncertain and the viral small RNAs (viRNAs) are not easily detectable. Here we use a metagenomic approach to test for the presence of viRNAs in five species from divergent animal phyla (Porifera, Cnidaria, Echinodermata, Mollusca, and Annelida), and in a brown alga-which represents an independent origin of multicellularity from plants, fungi, and animals. We use metagenomic RNA sequencing to identify around 80 virus-like contigs in these lineages, and small RNA sequencing to identify viRNAs derived from those viruses. We identified 21U small RNAs derived from an RNA virus in the brown alga, reminiscent of plant and fungal viRNAs, despite the deep divergence between these lineages. However, contrary to our expectations, we were unable to identify canonical (i.e. Drosophila- or nematode-like) viRNAs in any of the animals, despite the widespread presence of abundant micro-RNAs, and somatic transposon-derived piwi-interacting RNAs. We did identify a distinctive group of small RNAs derived from RNA viruses in the mollusc. However, unlike ecdysozoan viRNAs, these had a piRNA-like length distribution but lacked key signatures of piRNA biogenesis. We also identified primary piRNAs derived from putatively endogenous copies of DNA viruses in the cnidarian and the echinoderm, and an endogenous RNA virus in the mollusc. The absence of canonical virus-derived small RNAs from our samples may suggest that the majority of animal phyla lack an antiviral RNAi response. Alternatively, these phyla could possess an antiviral RNAi response resembling that reported for vertebrates, with cryptic viRNAs not detectable through simple metagenomic sequencing of wild-type individuals. In either case, our findings show that the antiviral RNAi responses of arthropods and nematodes, which are highly divergent from each other and from that of plants and fungi, are also highly diverged from the most likely ancestral metazoan state.

Cloning, Functional Characterization and Response to Cadmium Stress of the Thioredoxin-like Protein 1 Gene from Phascolosoma esculenta.

Cadmium (Cd) is a heavy metal toxicant and is widely distributed in aquatic environments. It can cause excessive production of reactive oxygen species (ROS) in the organism, which in turn leads to a series of oxidative damages. Thioredoxin (Trx), a highly conserved disulfide reductase, plays an important role in maintaining the intracellular redox homeostasis in eukaryotes and prokaryotes. Phascolosoma esculenta is an edible marine worm, an invertebrate that is extensively found on the mudflats of coastal China. To explore the molecular response of Trx in mudflat organisms under Cd stress, we identified a new Trx isoform (Trx-like protein 1 gene) from P. esculenta for the first time, designated as PeTrxl. Molecular and structural characterization, as well as multiple sequence and phylogenetic tree analysis, demonstrated that PeTrxl belongs to the Trx superfamily. PeTrxl transcripts were found to be ubiquitous in all tissues, and the highest expression level occurred in the coelomic fluid. Exposure to three sublethal concentrations of Cd resulted in the upregulation and then downregulation of PeTrxl expression levels over time in coelomic fluid of P. esculenta. The significant elevation of PeTrxl expression after 12 and 24 h of Cd exposure at 6 and 96 mg/L, respectively, might reflect its important role in the resistance to Cd stress. Recombinant PeTrxl (rPeTrxl) showed prominent dose-dependent insulin-reducing and ABTS free radical-scavenging abilities. After exposure to 96 mg/L Cd for 24 h, the ROS level increased significantly in the coelomic fluid, suggesting that Cd induced oxidative stress in P. esculenta. Furthermore, the injection of rPeTrxl during Cd exposure significantly reduced the ROS in the coelomic fluid. Our data suggest that PeTrxl has significant antioxidant capacity and can protect P. esculenta from Cd-induced oxidative stress.

Functional role of pax6 during eye and nervous system development in the annelid Capitella teleta.

The transcription factor Pax6 is an important regulator of early animal development. Loss of function mutations of pax6 in a range of animals result in a reduction or complete loss of the eye, a reduction of a subset of neurons, and defects in axon growth. There are no studies focusing on the role of pax6 during development of any lophotrochozoan representative, however, expression of pax6 in the developing eye and nervous system in a number of species suggest that pax6 plays a highly conserved role in eye and nervous system formation. We investigated the functional role of pax6 during development of the marine annelid Capitella teleta. Expression of pax6 transcripts in C. teleta larvae is similar to patterns found in other animals, with distinct subdomains in the brain and ventral nerve cord as well as in the larval and juvenile eye. To perturb pax6 function, two different splice-blocking morpholinos and a translation-blocking morpholino were used. Larvae resulting from microinjections with either splice-blocking morpholino show a reduction of the pax6 transcript. Development of both the larval eyes and the central nervous system architecture are highly disrupted following microinjection of each of the three morpholinos. The less severe phenotype observed when only the homeodomain is disrupted suggests that presence of the paired domain is sufficient for partial function of the Pax6 protein. Preliminary downstream target analysis confirms disruption in expression of some components of the retinal gene regulatory network, as well as disruption of genes involved in nervous system development. Results from this study, taken together with studies from other species, reveal an evolutionarily conserved role for pax6 in eye and neural specification and development.

Globins in the marine annelid Platynereis dumerilii shed new light on hemoglobin evolution in bilaterians.

BACKGROUND: How vascular systems and their respiratory pigments evolved is still debated. While many animals present a vascular system, hemoglobin exists as a blood pigment only in a few groups (vertebrates, annelids, a few arthropod and mollusk species). Hemoglobins are formed of globin sub-units, belonging to multigene families, in various multimeric assemblages. It was so far unclear whether hemoglobin families from different bilaterian groups had a common origin. RESULTS: To unravel globin evolution in bilaterians, we studied the marine annelid Platynereis dumerilii, a species with a slow evolving genome. Platynereis exhibits a closed vascular system filled with extracellular hemoglobin. Platynereis genome and transcriptomes reveal a family of 19 globins, nine of which are predicted to be extracellular. Extracellular globins are produced by specialized cells lining the vessels of the segmental appendages of the worm, serving as gills, and thus likely participate in the assembly of a previously characterized annelid-specific giant hemoglobin. Extracellular globin mRNAs are absent in smaller juveniles, accumulate considerably in growing and more active worms and peak in swarming adults, as the need for O2 culminates. Next, we conducted a metazoan-wide phylogenetic analysis of globins using data from complete genomes. We establish that five globin genes (stem globins) were present in the last common ancestor of bilaterians. Based on these results, we propose a new nomenclature of globins, with five clades. All five ancestral stem-globin clades are retained in some spiralians, while some clades disappeared early in deuterostome and ecdysozoan evolution. All known bilaterian blood globin families are grouped in a single clade (clade I) together with intracellular globins of bilaterians devoid of red blood. CONCLUSIONS: We uncover a complex "pre-blood" evolution of globins, with an early gene radiation in ancestral bilaterians. Circulating hemoglobins in various bilaterian groups evolved convergently, presumably in correlation with animal size and activity. However, all hemoglobins derive from a clade I globin, or cytoglobin, probably involved in intracellular O2 transit and regulation. The annelid Platynereis is remarkable in having a large family of extracellular blood globins, while retaining all clades of ancestral bilaterian globins.