Annelid adult cell type diversity and their pluripotent cellular origins.

Many annelids can regenerate missing body parts or reproduce asexually, generating all cell types in adult stages. However, the putative adult stem cell populations involved in these processes, and the diversity of cell types generated by them, are still unknown. To address this, we recover 75,218 single cell transcriptomes of the highly regenerative and asexually-reproducing annelid Pristina leidyi. Our results uncover a rich cell type diversity including annelid specific types as well as novel types. Moreover, we characterise transcription factors and gene networks that are expressed specifically in these populations. Finally, we uncover a broadly abundant cluster of putative stem cells with a pluripotent signature. This population expresses well-known stem cell markers such as vasa, piwi and nanos homologues, but also shows heterogeneous expression of differentiated cell markers and their transcription factors. We find conserved expression of pluripotency regulators, including multiple chromatin remodelling and epigenetic factors, in piwi+ cells. Finally, lineage reconstruction analyses reveal computational differentiation trajectories from piwi+ cells to diverse adult types. Our data reveal the cell type diversity of adult annelids by single cell transcriptomics and suggest that a piwi+ cell population with a pluripotent stem cell signature is associated with adult cell type differentiation.

Annelid adult cell type diversity and their pluripotent cellular origins.

Annelids are a broadly distributed, highly diverse, economically and environmentally important group of animals. Most species can regenerate missing body parts, and many are able to reproduce asexually. Therefore, many annelids can generate all adult cell types in adult stages. However, the putative adult stem cell populations involved in these processes, as well as the diversity of adult cell types generated by them, are still unknown. Here, we recover 75,218 single cell transcriptomes of Pristina leidyi, a highly regenerative and asexually-reproducing freshwater annelid. We characterise all major annelid adult cell types, and validate many of our observations by HCR in situ hybridisation. Our results uncover complex patterns of regionally expressed genes in the annelid gut, as well as neuronal, muscle and epidermal specific genes. We also characterise annelid-specific cell types such as the chaetal sacs and globin+ cells, and novel cell types of enigmatic affinity, including a vigilin+ cell type, a lumbrokinase+ cell type, and a diverse set of metabolic cells. Moreover, we characterise transcription factors and gene networks that are expressed specifically in these populations. Finally, we uncover a broadly abundant cluster of putative stem cells with a pluripotent signature. This population expresses well-known stem cell markers such as vasa, piwi and nanos homologues, but also shows heterogeneous expression of differentiated cell markers and their transcription factors. In these piwi+ cells, we also find conserved expression of pluripotency regulators, including multiple chromatin remodelling and epigenetic factors. Finally, lineage reconstruction analyses reveal the existence of differentiation trajectories from piwi+ cells to diverse adult types. Our data reveal the cell type diversity of adult annelids for the first time and serve as a resource for studying annelid cell types and their evolution. On the other hand, our characterisation of a piwi+ cell population with a pluripotent stem cell signature will serve as a platform for the study of annelid stem cells and their role in regeneration.

Cryptic carnivores: Intercontinental sampling reveals extensive novel diversity in a genus of freshwater annelids.

Freshwater annelids are globally widespread in aquatic ecosystems, but their diversity is severely underestimated. Obvious morphological features to define taxa are sparse, and molecular phylogenetic analyses regularly discover cryptic diversity within taxa. Despite considerable phylogenetic work on certain clades, many groups of freshwater annelids remain poorly understood. Included among these are water nymph worms of the genus Chaetogaster (Clitellata: Tubificida: Naididae: Naidinae). These worms have diverged from the detritivorous diet of most oligochaetes to become more predatory and exist as omnivores, generalist predators, parasites, or symbionts on other invertebrates. Despite their unusual trophic ecology, the true diversity of Chaetogaster and the phylogenetic relationships within the genus are uncertain. Only three species are commonly referenced in the literature (Chaetogaster diaphanus, Chaetogaster limnaei, and Chaetogaster diastrophus), but additional species have been described and prior molecular data suggests that there is cryptic diversity within named species. To clarify the phylogenetic diversity of Chaetogaster, we generated the first molecular phylogeny of the genus using mitochondrial and nuclear sequence data from 128 worms collected primarily across North America and Europe. Our phylogenetic analyses suggest that the three commonly referenced species are a complex of 24 mostly cryptic species. In our dataset, Chaetogaster "diaphanus" is represented by two species, C. "limnaei" is represented by three species, and C. "diastrophus" is represented by 19 species. North American and European sequences are largely interspersed across the phylogeny, with four pairs of clades involving distinct North American and European sister groupings. Overall, our study demonstrates that the species diversity of Chaetogaster has been underestimated and that carnivory has evolved at least twice in the genus. Chaetogaster is being used as a model for symbiotic evolution and the loss of regenerative ability, and our study indicates that researchers must be careful to identify which species of Chaetogaster they are working with in future studies.

Integrative biology of injury in animals.

Mechanical injury is a prevalent challenge in the lives of animals with myriad potential consequences for organisms, including reduced fitness and death. Research on animal injury has focused on many aspects, including the frequency and severity of wounding in wild populations, the short- and long-term consequences of injury at different biological scales, and the variation in the response to injury within or among individuals, species, ontogenies, and environmental contexts. However, relevant research is scattered across diverse biological subdisciplines, and the study of the effects of injury has lacked synthesis and coherence. Furthermore, the depth of knowledge across injury biology is highly uneven in terms of scope and taxonomic coverage: much injury research is biomedical in focus, using mammalian model systems and investigating cellular and molecular processes, while research at organismal and higher scales, research that is explicitly comparative, and research on invertebrate and non-mammalian vertebrate species is less common and often less well integrated into the core body of knowledge about injury. The current state of injury research presents an opportunity to unify conceptually work focusing on a range of relevant questions, to synthesize progress to date, and to identify fruitful avenues for future research. The central aim of this review is to synthesize research concerning the broad range of effects of mechanical injury in animals. We organize reviewed work by four broad and loosely defined levels of biological organization: molecular and cellular effects, physiological and organismal effects, behavioural effects, and ecological and evolutionary effects of injury. Throughout, we highlight the diversity of injury consequences within and among taxonomic groups while emphasizing the gaps in taxonomic coverage, causal understanding, and biological endpoints considered. We additionally discuss the importance of integrating knowledge within and across biological levels, including how initial, localized responses to injury can lead to long-term consequences at the scale of the individual animal and beyond. We also suggest important avenues for future injury biology research, including distinguishing better between related yet distinct injury phenomena, expanding the subjects of injury research to include a greater variety of species, and testing how intrinsic and extrinsic conditions affect the scope and sensitivity of injury responses. It is our hope that this review will not only strengthen understanding of animal injury but will contribute to building a foundation for a more cohesive field of 'injury biology'.

Journey beyond the embryo: The beauty of Pristina and naidine annelids for studying regeneration and agametic reproduction.

Regeneration, asexual agametic reproduction, and other forms of postembryonic development are ecologically important and widely variable across animals, yet our understanding of this important aspect of animal diversity remains limited. A significant limitation has been the dearth of adequate study systems for exploring the mechanisms and evolution of these processes. Here I describe key parts of our journey in developing naid annelids as a study system for investigating the evolution and development of regeneration and fission. Naids are small freshwater annelids that provide numerous advantages for studying postembryonic development: they are small and reproduce readily by fission, they include species with diverse regenerative abilities, and many species are easy to culture. Among the naids, Pristina leidyi is a particularly useful study species, being largely transparent and quite robust to a variety of experimental manipulations. Building on a sparse but long history of past research on these animals, we have developed this system by establishing methods and generating resources for working with them. Naids are yielding novel insights into the evolution of regeneration and fission, providing one of many examples of the value of developing new study species to enable the exploration of fundamental and understudied questions in biology. Establishing new study systems comes with challenges but is exciting and rewarding, and I provide perspectives from my own experiences with the hope of encouraging the further expansion of study systems in biology.

A phylum-wide survey reveals multiple independent gains of head regeneration in Nemertea.

Animals vary widely in their ability to regenerate, suggesting that regenerative ability has a rich evolutionary history. However, our understanding of this history remains limited because regenerative ability has only been evaluated in a tiny fraction of species. Available comparative regeneration studies have identified losses of regenerative ability, yet clear documentation of gains is lacking. We assessed ability to regenerate heads and tails either through our own experiments or from literature reports for 35 species of Nemertea spanning the diversity of the phylum, including representatives of 10 families and all three orders. We generated a phylogenetic framework using sequence data to reconstruct the evolutionary history of head and tail regenerative ability across the phylum and found that all evaluated species can remake a posterior end but surprisingly few could regenerate a complete head. Our analysis reconstructs a nemertean ancestor unable to regenerate a head and indicates independent gains of head regenerative ability in at least four separate lineages, with one of these gains taking place as recently as the last 10-15 Myr. Our study highlights nemerteans as a valuable group for studying evolution of regeneration and identifying mechanisms associated with repeated gains of regenerative ability.

Regulation of dorso-ventral polarity by the nerve cord during annelid regeneration: A review of experimental evidence.

An important goal for understanding regeneration is determining how polarity is conferred to the regenerate. Here we review findings in two groups of polychaete annelids that implicate the ventral nerve cord in assigning dorso-ventral polarity, and specifically ventral identity, to the regenerate. In nereids, surgical manipulations indicate that parapodia develop where dorsal and ventral body wall territories contact. Without a nerve cord at the wound site, the regenerate differentiates no evident polarity (with no parapodia) and only dorsal identity, while with two nerve cords the regenerate develops a twinned dorso-ventral axis (with four parapodia per segment instead of the normal two). In sabellids, a striking natural dorso-ventral inversion in parapodial morphology occurs along the body axis and this inversion is morphologically correlated with the position of the nerve cord. Parapodial inversion also occurs in segments in which the nerve cord has been removed, even without any segment amputation. Together, these data strongly support a role for the nerve cord in annelid dorso-ventral pattern regulation, with the nerve cord conferring ventral identity.

Plasticity and regeneration of gonads in the annelid Pristina leidyi.

BACKGROUND: Gonads are specialized gamete-producing structures that, despite their functional importance, are generated by diverse mechanisms across groups of animals and can be among the most plastic organs of the body. Annelids, the segmented worms, are a group in which gonads have been documented to be plastic and to be able to regenerate, but little is known about what factors influence gonad development or how these structures regenerate. In this study, we aimed to identify factors that influence the presence and size of gonads and to investigate gonad regeneration in the small asexually reproducing annelid, Pristina leidyi. RESULTS: We found that gonad presence and size in asexual adult P. leidyi are highly variable across individuals and identified several factors that influence these structures. An extrinsic factor, food availability, and two intrinsic factors, individual age and parental age, strongly influence the presence and size of gonads in P. leidyi. We also found that following head amputation in this species, gonads can develop by morphallactic regeneration in previously non-gonadal segments. We also identified a sexually mature individual from our laboratory culture that demonstrates that, although our laboratory strain reproduces only asexually, it retains the potential to become fully sexual. CONCLUSIONS: Our findings demonstrate that gonads in P. leidyi display high phenotypic plasticity and flexibility with respect to their presence, their size, and the segments in which they can form. Considering our findings along with relevant data from other species, we find that, as a group, clitellate annelids can form gonads in at least four different contexts: post-starvation refeeding, fission, morphallactic regeneration, and epimorphic regeneration. This group is thus particularly useful for investigating the mechanisms involved in gonad formation and the evolution of post-embryonic phenotypic plasticity.

A new transcriptome and transcriptome profiling of adult and larval tissue in the box jellyfish Alatina alata: an emerging model for studying venom, vision and sex.

BACKGROUND: Cubozoans (box jellyfish) are cnidarians that have evolved a number of distinguishing features. Many cubozoans have a particularly potent sting, effected by stinging structures called nematocysts; cubozoans have well-developed light sensation, possessing both image-forming lens eyes and light-sensitive eye spots; and some cubozoans have complex mating behaviors, including aggregations, copulation and internal fertilization. The cubozoan Alatina alata is emerging as a cnidarian model because it forms predictable monthly nearshore breeding aggregations in tropical to subtropical waters worldwide, making both adult and larval material reliably accessible. To develop resources for A. alata, this study generated a functionally annotated transcriptome of adult and larval tissue, applying preliminary differential expression analyses to identify candidate genes involved in nematogenesis and venom production, vision and extraocular sensory perception, and sexual reproduction, which for brevity we refer to as "venom", "vision" and "sex". RESULTS: We assembled a transcriptome de novo from RNA-Seq data pooled from multiple body parts (gastric cirri, ovaries, tentacle (with pedalium base) and rhopalium) of an adult female A. alata medusa and larval planulae. Our transcriptome comprises ~32 K transcripts, after filtering, and provides a basis for analyzing patterns of gene expression in adult and larval box jellyfish tissues. Furthermore, we annotated a large set of candidate genes putatively involved in venom, vision and sex, providing an initial molecular characterization of these complex features in cubozoans. Expression profiles and gene tree reconstruction provided a number of preliminary insights into the putative sites of nematogenesis and venom production, regions of phototransduction activity and fertilization dynamics in A. alata. CONCLUSIONS: Our Alatina alata transcriptome significantly adds to the genomic resources for this emerging cubozoan model. This study provides the first annotated transcriptome from multiple tissues of a cubozoan focusing on both the adult and larvae. Our approach of using multiple body parts and life stages to generate this transcriptome effectively identified a broad range of candidate genes for the further study of coordinated processes associated with venom, vision and sex. This new genomic resource and the candidate gene dataset are valuable for further investigating the evolution of distinctive features of cubozoans, and of cnidarians more broadly.

Developmental and molecular biology of annelid regeneration: a comparative review of recent studies.

Studies of annelid regeneration have greatly increased in frequency in recent years, providing new insights into the developmental basis and evolution of regeneration. In this review, we summarize recent findings related to regeneration in annelids, focusing on molecular and developmental studies of epimorphic (blastema-based) regeneration, morphallactic (tissue-remodeling based) regeneration, and development and regeneration of putative stem cells of the posterior growth zone and germline. Regeneration is being investigated in a broad range of annelids spanning the phylum, and comparing findings among species reveals both widely conserved features that may be ancestral for the phylum as well as features that are variable across the group.

Long-term time-lapse live imaging reveals extensive cell migration during annelid regeneration.

BACKGROUND: Time-lapse imaging has proven highly valuable for studying development, yielding data of much finer resolution than traditional "still-shot" studies and allowing direct examination of tissue and cell dynamics. A major challenge for time-lapse imaging of animals is keeping specimens immobile yet healthy for extended periods of time. Although this is often feasible for embryos, the difficulty of immobilizing typically motile juvenile and adult stages remains a persistent obstacle to time-lapse imaging of post-embryonic development. RESULTS: Here we describe a new method for long-duration time-lapse imaging of adults of the small freshwater annelid Pristina leidyi and use this method to investigate its regenerative processes. Specimens are immobilized with tetrodotoxin, resulting in irreversible paralysis yet apparently normal regeneration, and mounted in agarose surrounded by culture water or halocarbon oil, to prevent dehydration but allowing gas exchange. Using this method, worms can be imaged continuously and at high spatial-temporal resolution for up to 5 days, spanning the entire regeneration process. We performed a fine-scale analysis of regeneration growth rate and characterized cell migration dynamics during early regeneration. Our studies reveal the migration of several putative cell types, including one strongly resembling published descriptions of annelid neoblasts, a cell type suggested to be migratory based on "still-shot" studies and long hypothesized to be linked to regenerative success in annelids. CONCLUSIONS: Combining neurotoxin-based paralysis, live mounting techniques and a starvation-tolerant study system has allowed us to obtain the most extensive high-resolution longitudinal recordings of full anterior and posterior regeneration in an invertebrate, and to detect and characterize several cell types undergoing extensive migration during this process. We expect the tetrodotoxin paralysis and time-lapse imaging methods presented here to be broadly useful in studying other animals and of particular value for studying post-embryonic development.

Gonad establishment during asexual reproduction in the annelid Pristina leidyi.

Animals that can reproduce by both asexual agametic reproduction and sexual reproduction must transmit or re-establish their germ line post-embryonically. Although such a dual reproductive mode has evolved repeatedly among animals, how asexually produced individuals establish their germ line remains poorly understood in most groups. We investigated germ line development in the annelid Pristina leidyi, a species that typically reproduces asexually by paratomic fission, intercalating a new tail and head in the middle of the body followed by splitting. We found that in fissioning individuals, gonads occur in anterior segments in the anterior-most individual as well as in new heads forming within fission zones. Homologs of the germ line/multipotency genes piwi, vasa, and nanos are expressed in the gonads, as well as in proliferative tissues including the posterior growth zone, fission zone, and regeneration blastema. In fissioning animals, certain cells on the ventral nerve cord express a homolog of piwi, are abundant near fission zones, and sometimes make contact with gonads. Such cells are typically undetectable near the blastema and posterior growth zone. Time-lapse imaging provides direct evidence that cells on the ventral nerve cord migrate preferentially towards fission zones. Our findings indicate that gonads form routinely in fissioning individuals, that a population of piwi-positive cells on the ventral nerve cord is associated with fission and gonads, and that cells resembling these piwi-positive cells migrate along the ventral nerve cord. We suggest that the piwi-positive ventral cells are germ cells that transmit the germ line across asexually produced individuals via migration along the ventral nerve cord.

Fine taxonomic sampling of nervous systems within Naididae (Annelida: Clitellata) reveals evolutionary lability and revised homologies of annelid neural components.

INTRODUCTION: An important goal for understanding how animals have evolved is to reconstruct the ancestral features and evolution of the nervous system. Many inferences about nervous system evolution are weak because of sparse taxonomic sampling and deep phylogenetic distances among species compared. Increasing sampling within clades can strengthen inferences by revealing which features are conserved and which are variable within them. Among the Annelida, the segmented worms, the Clitellata are typically considered as having a largely conserved neural architecture, though this view is based on limited sampling. RESULTS: To gain better understanding of nervous system evolution within Clitellata, we used immunohistochemistry and confocal laser scanning microscopy to describe the nervous system architecture of 12 species of the basally branching family Naididae. Although we found considerable similarity in the nervous system architecture of naidids and that of other clitellate groups, our study identified a number of features that are variable within this family, including some that are variable even among relatively closely related species. Variable features include the position of the brain, the number of ciliary sense organs, the presence of septate ventral nerve cord ganglia, the distribution of serotonergic cells in the brain and ventral ganglia, and the number of peripheral segmental nerves. CONCLUSIONS: Our analysis of patterns of serotonin immunoreactive perikarya in the central nervous system indicates that segmental units are not structurally homogeneous, and preliminary homology assessments suggest that whole sets of serotonin immunoreactive cells have been gained and lost across the Clitellata. We also found that the relative position of neuroectodermal and mesodermal segmental components is surprisingly evolutionarily labile; in turn, this revealed that scoring segmental nerves by their position relative to segmental ganglia rather than to segmental septa clarifies their homologies across Annelida. We conclude that fine taxonomic sampling in comparative studies aimed at elucidating the evolution of morphological diversity is fundamental for proper assessment of trait variability.

Early events in annelid regeneration: a cellular perspective.

The ability to regenerate extensive portions of the body is widespread among the phylum Annelida and this group includes some of the most highly regenerative animals known. Knowledge of the cellular and molecular basis of regeneration in this group is thus important for understanding how regenerative processes have evolved both within the group and across animal phyla. Here, the cellular basis of annelid regeneration is reviewed, with a focus on the earliest steps of regeneration, namely wound-healing and formation of the blastema. Information from a wide range of annelids is compiled in order to identify common and variable elements. There is a large body of valuable older literature on the cellular basis of regeneration in annelids and an effort is made to review this literature in addition to more recent studies. Annelids typically seal the wound through muscular contraction and undergo some autolysis of tissue at the site of the wound. Bodily injury elicits extensive cell migration toward the wound, involving several different types of cells. Some migrating cells form a tissue-clot and phagocytize damaged tissues, whereas others are inferred to contribute to regenerated tissue, specifically mesodermal tissue. In one annelid subgroup, the clitellates, a group of mesodermal cells, sometimes referred to as neoblasts, is inferred to migrate over considerable distances, with cells moving to the wound from several segments away. Epidermis and gut epithelia severed upon amputation typically heal by fusing with like tissue, although not always. After amputation, cellular contacts with the extracellular matrix are disrupted and major changes in cell morphology and adhesion occur within tissues near the wound. Interactions of tissues at the wound appear key for initiating a blastema, with a particularly important role suggested for the ventral nerve cord, although species are variable in this regard; longer-distance effects mediated by the brain are also reported. The anterior-posterior polarity of the blastema can be mis-assigned, leading most commonly to double-headed worms, and the dorsal-ventral polarity of the blastema appears to be induced by the ventral nerve cord. The blastema is thought to arise from contributions of all three tissue layers, with each layer replacing itself in a tissue-specific manner. Blastemal cells originate mostly locally, although some long-distance migration of source-cells is suggested in clitellates. A number of important questions remain about the cellular basis of regeneration in annelids and addressing many of these would be greatly aided by developing approaches to identify and isolate specific cell types and techniques to image and trace cells in vivo.

Regeneration in spiralians: evolutionary patterns and developmental processes.

Animals differ markedly in their ability to regenerate, yet still little is known about how regeneration evolves. In recent years, important advances have been made in our understanding of animal phylogeny and these provide new insights into the phylogenetic distribution of regeneration. The developmental basis of regeneration is also being investigated in an increasing number of groups, allowing commonalities and differences across groups to become evident. Here, we focus on regeneration in the Spiralia, a group that includes several champions of animal regeneration, as well as many groups with more limited abilities. We review the phylogenetic distribution and developmental processes of regeneration in four major spiralian groups: annelids, nemerteans, platyhelminths, and molluscs. Although comparative data are still limited, this review highlights phylogenetic and developmental patterns that are emerging regarding regeneration in spiralians and identifies important avenues for future research.

Investment choices in post-embryonic development: quantifying interactions among growth, regeneration, and asexual reproduction in the annelid Pristina leidyi.

Animals capable of multiple forms of post-embryonic development, such as growth, regeneration, and asexual reproduction, must make choices about which processes to invest in. What strategies guide post-embryonic resource allocation investments? We investigated this question in the annelid Pristina leidyi, which can grow continuously, regenerates well, and reproduces asexually by fission. We found that in this species growth is concentrated in three zones: a subterminal posterior zone (forming new segments), a mid-body zone (forming fission zones), and a previously undescribed subterminal anterior zone at the base of the prostomium (which we suggest continually builds the prostomium through a "conveyor-belt" like process). Body-wide counts of proliferating cells are greater under high food than low food conditions but proliferation patterns themselves are independent of feeding level. Proliferation patterns are strongly affected by amputation, however, with proliferation rapidly shutting-down throughout the body, except at the wound site, following injury. Relative investment to fission and regeneration is highly context-dependent, being sensitive to the position of the cut and the stage of fission. Outcomes range from fission acceleration and regeneration stalling (high fission:regeneration investment) to resorption of fission zones and progression of regeneration (low fission:regeneration investment). Our findings reveal strong interactions between growth, regeneration, and fission and demonstrate a particularly important effect of injury on resource allocation patterns. Patterns of resource investment in P. leidyi show similarities to those described in two other groups that evolved fission independently (naidine annelids and catenulid flatworms), suggesting that similar developmental and physiological contexts may drive convergent evolution of resource allocation strategies.

Redescription of Alatina alata (Reynaud, 1830) (Cnidaria: Cubozoa) from Bonaire, Dutch Caribbean.

Here we establish a neotype for Alatina alata (Reynaud, 1830) from the Dutch Caribbean island of Bonaire. The species was originally described one hundred and eighty three years ago as Carybdea alata in La Centurie Zoologique-a monograph published by René Primevère Lesson during the age of worldwide scientific exploration. While monitoring monthly reproductive swarms of A. alata medusae in Bonaire, we documented the ecology and sexual reproduction of this cubozoan species. Examination of forty six A. alata specimens and additional archived multimedia material in the collections of the National Museum of Natural History, Washington, DC revealed that A. alata is found at depths ranging from surface waters to 675 m. Additional studies have reported it at depths of up to 1607 m in the tropical and subtropical Atlantic Ocean. Herein, we resolve the taxonomic confusion long associated with A. alata due to a lack of detail in the original description and conflicting statements in the scientific literature. A new cubozoan character, the velarial lappet, is described for this taxon. The complete description provided here serves to stabilize the taxonomy of the second oldest box jellyfish species, and provide a thorough redescription of the species.

Transcriptome characterization via 454 pyrosequencing of the annelid Pristina leidyi, an emerging model for studying the evolution of regeneration.

BACKGROUND: The naid annelids contain a number of species that vary in their ability to regenerate lost body parts, making them excellent candidates for evolution of regeneration studies. However, scant sequence data exists to facilitate such studies. We constructed a cDNA library from the naid Pristina leidyi, a species that is highly regenerative and also reproduces asexually by fission, using material from a range of regeneration and fission stages for our library. We then sequenced the transcriptome of P. leidyi using 454 technology. RESULTS: 454 sequencing produced 1,550,174 reads with an average read length of 376 nucleotides. Assembly of 454 sequence reads resulted in 64,522 isogroups and 46,679 singletons for a total of 111,201 unigenes in this transcriptome. We estimate that over 95% of the transcripts in our library are present in our transcriptome. 17.7% of isogroups had significant BLAST hits to the UniProt database and these include putative homologs of a number of genes relevant to regeneration research. Although many sequences are incomplete, the mean sequence length of transcripts (isotigs) is 707 nucleotides. Thus, many sequences are large enough to be immediately useful for downstream applications such as gene expression analyses. Using in situ hybridization, we show that two Wnt/ß-catenin pathway genes (homologs of frizzled and ß-catenin) present in our transcriptome are expressed in the regeneration blastema of P. leidyi, demonstrating the usefulness of this resource for regeneration research. CONCLUSIONS: 454 sequencing is a rapid and efficient approach for identifying large numbers of genes in an organism that lacks a sequenced genome. This transcriptome dataset will be a valuable resource for molecular analyses of regeneration in P. leidyi and will serve as a starting point for comparisons to non-regenerating naids. It also contributes significantly to the still limited genomic resources available for annelids and lophotrochozoans more generally.

Evolution of a novel developmental trajectory: fission is distinct from regeneration in the annelid Pristina leidyi.

Understanding how novelty arises has been a major focus of evolutionary developmental biology. While the origin of new genes, gene functions, and morphological features has been studied intensely, the origin of entire developmental trajectories, such as regeneration or agametic reproduction, remains poorly understood. Agametic reproduction by fission is a novel trajectory evolved numerous times among animal phyla, including Annelida, in which it is thought to arise by co-option of regeneration. To gain insight into how a novel trajectory may evolve, we investigated a relatively recent origin of fission. We performed a detailed comparison of morphogenesis during regeneration and fission in the annelid Pristina leidyi (Clitellata, Naididae), from the onset of these trajectories to the achievement of the final morphology. We find extensive similarities between fission and regeneration morphogenesis, and, of particular note, find evidence for a synapomorphy of fission and regeneration (apparently not shared with embryogenesis) in peripheral nervous system development, providing strong support for the hypothesis that fission is derived from regeneration. We also find important differences between fission and regeneration, during development of multiple organ systems. These are manifested by temporal shifts in developmental events and by the presence of elements unique to only one process. Differences are not obviously temporally clustered at the beginning, middle, or end of development but rather occur throughout, indicating that divergence has occurred along the entire developmental course of these trajectories.