Integrative description of a new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from Shennongjia, Central China.

A new species of the genus Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from Xiangxi River, Shennongjia Forestry District, Hubei Province, China, is described on the basis of an integrative approach, involving morphology, and molecular systematics. The new species Dugesia saccaria A-T. Wang & Sluys, sp. nov. is characterized by the following features: a dumb-bell-shaped, muscularized hump located just anterior to the knee-shaped bend in the bursal canal; a ventrally displaced ejaculatory duct, which, however, opens terminally through the dorsal portion of the blunt tip of the penis papilla; a ventrally located seminal vesicle, giving rise to a vertically running duct that eventually curves downwards to communicate with the ejaculatory duct via a small diaphragm; oviducts opening asymmetrically into the dorsal portion of the common atrium and at the knee-shaped part of the bursal canal. The phylogenetic position of the new species was determined using four molecular markers (18S rDNA; ITS-1; 28S rDNA; COI), which suggested that it groups with other species of Dugesia from the Australasian and Oriental biogeographical regions.

Exposure to polystyrene microplastics and perfluorooctane sulfonate disrupt the homeostasis of intact planarians and the growth of regenerating planarians.

Microplastics (MPs) and perfluorinated compounds (PFAS) are widespread in the global ecosystem. MPs have the ability to adsorb organic contaminants such as perfluorooctane sulfonate (PFOS), leading to combined effects. The current work aims to explore the individual and combined toxicological effects of polystyrene (PS) and PFOS on the growth and nerves of the freshwater planarian (Dugesia japonica). The results showed that PS particles could adsorb PFOS. PS and PFOS impeded the regeneration of decapitated planarians eyespots, whereas the combined treatment increased the locomotor speed of intact planarians. PS and PFOS caused significant DNA damage, while co-treatment with different PS concentrations aggravated and attenuated DNA damage, respectively. Further studies at the molecular level have shown that PS and PFOS affect the proliferation and differentiation of neoblasts in both intact and regenerating planarians, alter the expression levels of neuronal genes, and impede the development of the nervous system. PS and PFOS not only disrupted the homeostasis of intact planarians, but also inhibited the regeneration of decapitated planarians. This study is the first to assess the multiple toxicity of PS and PFOS to planarians after combined exposure. It provides a basis for the environmental and human health risks of MPs and PFAS.

Assessing the in vivo toxicity of titanium dioxide nanoparticles in Schmidtea mediterranea: uptake pathways and (neuro)developmental outcomes.

Titanium dioxide nanoparticles (TiO2-NPs) in aquatic environments, originating from urban run-off, product use and post-consumer degradation, interact with aquatic organisms through water and sediments. Thorough toxicity assessment requires comprehensive data across all ecosystem compartments especially the benthic zone, which is currently lacking. Moreover, a proper physicochemical characterization of the particles is needed before and during toxicity assessment. In the present work, we used the planarian Schmidtea mediterranea to investigate the effects of TiO2-NPs (5 mg/L and 50 mg/L). Planarians are benthic organisms that play an important role in the food chain as predators. Our study integrated particle characterization with toxicokinetic and toxicodynamic parameters and showed that the uptake of TiO2-NPs of 21 nm occurred through the epidermis and intestine. Epidermal irritation and mucus production occurred immediately after exposure, and TiO2-NPs induced stronger effects in regenerating organisms. More specifically, TiO2-NPs interfered with neuroregeneration, inducing behavioral effects. A delay in the formation of the anterior commissure between the two brain lobes after seven and nine days of exposure to 50 mg/L was observed, probably as a result of a decrease in stem cell proliferation. Our findings underscore the need to incorporate multiple exposure routes in toxicity screenings. Additionally, we highlight the vulnerability of developing organisms and recommend their inclusion in future risk assessment strategies.

A transcription factor atlas of stem cell fate in planarians.

Whole-body regeneration requires the ability to produce the full repertoire of adult cell types. The planarian Schmidtea mediterranea contains over 125 cell types, which can be regenerated from a stem cell population called neoblasts. Neoblast fate choice can be regulated by the expression of fate-specific transcription factors (FSTFs). How fate choices are made and distributed across neoblasts versus their post-mitotic progeny remains unclear. We used single-cell RNA sequencing to systematically map fate choices made in S/G2/M neoblasts and, separately, in their post-mitotic progeny that serve as progenitors for all adult cell types. We defined transcription factor expression signatures associated with all detected fates, identifying numerous new progenitor classes and FSTFs that regulate them. Our work generates an atlas of stem cell fates with associated transcription factor signatures for most cell types in a complete adult organism.

Protocol for culturing and functionally manipulating planarian neoblasts using SiR-DNA-based flow cytometry.

Neoblasts are the only cells capable of proliferation in planarians. The traditional flow cytometry protocol using Hoechst inhibits the cell cycle. Here, we present a protocol for culturing and functionally manipulating planarian neoblasts using SiR-DNA-based flow cytometry. We describe steps for cell dissociation and staining, flow cytometry, and cell collection and culture. We then detail procedures for Nanoluciferase mRNA transfection. This protocol facilitates further investigations into the pluripotency and regeneration mechanisms within neoblasts. For complete details on the use and execution of this protocol, please refer to Lei et al.1.

Mutational profile of the regenerative process and de novo genome assembly of the planarian Schmidtea polychroa.

Planarians are organisms with a unique capacity to regenerate any part of their body. New tissues are generated in a process that requires many swift cell divisions. How costly is this process to an animal in terms of mutational load remains unknown. Using whole genome sequencing, we defined the mutational profile of the process of regeneration in the planarian species Schmidtea polychroa. We assembled de novo the genome of S. polychroa and analyzed mutations in animals that have undergone regeneration. We observed a threefold increase in the number of mutations and an altered mutational spectrum. High allele frequencies of subclonal mutations in regenerated animals suggested that most of the cells in the regenerated animal were descendants of a small number of stem cells with high expansion potential. We provide, for the first time, the draft genome assembly of S. polychroa, an estimation of the germline mutation rate for a planarian species and the mutational spectrum of the regeneration process of a living organism.

Flatworm Transcriptomes Reveal Widespread Parasitism by Histophagous Ciliates.

Unicellular ciliates like Tetrahymena are best known as free-living bacteriovores, but many species are facultative or obligate parasites. These "histophages" feed on the tissues of hosts ranging from planarian flatworms to commercially important fish and the larvae of imperiled freshwater mussels. Here, we developed a novel bioinformatics pipeline incorporating the nonstandard ciliate genetic code and used it to search for Ciliophora sequences in 34 publicly available Platyhelminthes EST libraries. From 2,615,036 screened ESTs, we identified nearly 6,000 high-confidence ciliate transcripts, supporting parasitism of seven additional flatworm species. We also cultured and identified Tetrahymena from nine terrestrial and freshwater planarians, including invasive earthworm predators from the genus Bipalium and the widely studied regeneration models Dugesia japonica and Schmidtea mediterranea. A co-phylogenetic reconstruction provides strong evidence for the coevolution of histophagous Ciliophora with their Platyhelminthes hosts. We further report the antiprotozoal aminoglycoside paromomycin expels Tetrahymena from S. mediterranea, providing new opportunities to investigate the effects of this relationship on planarian biology. Together, our findings raise the possibility that invasive flatworms constitute a novel dispersal mechanism for Tetrahymena parasites and position the Platyhelminthes as an ideal model phylum for studying the ecology and evolution of histophagous ciliates.

Planarians require ced-12/elmo-1 to clear dead cells by excretion through the gut.

Cell corpse removal is a critical component of both development and homeostasis throughout the animal kingdom. Extensive research has revealed many of the mechanisms involved in corpse removal, typically involving engulfment and digestion by another cell; however, the dynamics of cell corpse clearance in adult tissues remain unclear. Here, we track cell death in the adult planarian Schmidtea mediterranea and find that, following light-induced cell death, pigment cell corpses transit to the gut and are excreted from the animal. Gut phagocytes, previously only known to phagocytose food, are required for pigment cells to enter the gut lumen. Finally, we show that the planarian ortholog of ced-12/engulfment and cell motility (ELMO) is required for corpse phagocytosis and removal through the gut. In total, we present a mechanism of cell clearance in an adult organism involving transit of dead cells to the gut, transport into the gut by phagocytes, and physical excretion of debris.

Preparation of Nanoparticles Loaded with Quercetin and Effects on Bacterial Biofilm and LPS-Induced Oxidative Stress in Dugesia japonica.

Quercetin is a kind of flavonol compound, which has been widely concerned because of its good pharmacological effects. However, its poor water solubility and poor oral absorption limit its application. To address the above problems, the optimal technological conditions for preparing quercetin-loaded chitosan sodium alginate nanoparticles (Q-CSNPs) were obtained through single-factor experiment method. Q-CSNPs were characterized by particle size analyzer, scanning electron microscope (SEM), transmission electron microscope (TEM), and Fourier transform infrared spectroscopy (FTIR). Biofilm experiment evaluated the antibacterial activity of five different concentrations of Q-CSNPs against Escherichia coli and Staphylococcus aureus. DPPH and hydroxyl radical scavenging experiments determined their antioxidant activity. The effect of Q-CSNPs labeled with FITC on the oxidative stress of planarian was determined. The results showed that quercetin was successfully encapsulated and had good antibacterial and antioxidant capacity in vitro. In vivo experiments of planarians also showed that Q-CSNPs could inhibit the oxidative stress induced by lipopolysaccharide (LPS) and especially alleviate the decrease of CAT activity and the increase of MDA content in planarians induced by LPS. After being supported by future in vivo studies, this preparation will provide research possibilities for the development of quercetin nano-drugs, quercetin dietary supplement, and so on.

A resource of single-cell gene expression profiles in a planarian Dugesia japonica.

The freshwater planarian Dugesia japonica maintains an abundant heterogeneous cell population called neoblasts, which include adult pluripotent stem cells. Thus, it is an excellent model organism for stem cell and regeneration research. Recently, many single-cell RNA sequencing (scRNA-seq) databases of several model organisms, including other planarian species, have become publicly available; these are powerful and useful resources to search for gene expression in various tissues and cells. However, the only scRNA-seq dataset for D. japonica has been limited by the number of genes detected. Herein, we collected D. japonica cells, and conducted an scRNA-seq analysis. A novel, automatic, iterative cell clustering strategy produced a dataset of 3,404 cells, which could be classified into 63 cell types based on gene expression profiles. We introduced two examples for utilizing the scRNA-seq dataset in this study using D. japonica. First, the dataset provided results consistent with previous studies as well as novel functionally relevant insights, that is, the expression of DjMTA and DjP2X-A genes in neoblasts that give rise to differentiated cells. Second, we conducted an integrative analysis of the scRNA-seq dataset and time-course bulk RNA-seq of irradiated animals, demonstrating that the dataset can help interpret differentially expressed genes captured via bulk RNA-seq. Using the R package "Seurat" and GSE223927, researchers can easily access and utilize this dataset.

MRLC controls apoptotic cell death and functions to regulate epidermal development during planarian regeneration and homeostasis.

Adult stem cells (ASCs) are pluripotent cells with the capacity to self-renew and constantly replace lost cells due to physiological turnover or injury. Understanding the molecular mechanisms of the precise coordination of stem cell proliferation and proper cell fate decision is important to regeneration and organismal homeostasis. The planarian epidermis provides a highly tractable model to study ASC complex dynamic due to the distinct spatiotemporal differentiation stages during lineage development. Here, we identified the myosin regulatory light chain (MRLC) homologue in the Dugesia japonica transcriptome. We found high expression levels of MRLC in wound region during regeneration and also expressed in late epidermal progenitors as an essential regulator of the lineage from neoblasts to mature epidermal cells. We investigated the function of MRLC using in situ hybridization, real-time polymerase chain reaction and double fluorescent and uncovered the potential mechanism. Knockdown of MRLC leads to a remarkable increase in cell death, causes severe abnormalities during regeneration and homeostasis and eventually leads to animal death. The global decrease in epidermal cell in MRLC RNAi animals induces accelerated epidermal proliferation and differentiation. Additionally, we find that MRLC is co-expressed with cdc42 and acts cooperatively to control the epidermal lineage development by affecting cell death. Our results uncover an important role of MRLC, as an inhibitor of apoptosis, involves in epidermal development.

Starvation resistance in planarians: multiple strategies to get a thrifty phenotype.

Starvation resistance is a life-saving mechanism for many organisms facing food availability fluctuation in the natural environment. Different strategies have been episodically identified for some model organisms, the first of which was the ability to suppress metabolic rate. Among the identified strategies, the ability of planarians to shrink their body under fasting conditions and revert the process after feeding (the growth-degrowth process) represents a fascinating mechanism to face long periods of fasting. The growth-degrowth process is strictly related to the capability of planarians to continuously maintain tissue homeostasis and body proportions even in challenging conditions, thanks to the presence of a population of pluripotent stem cells. Here, we take advantage of several previous studies describing the growth-degrowth process and of recent progress in the understanding of planarian homeostasis mechanisms, to identify tissue-selective transcriptional downregulation as a driving strategy for the development of a thrifty phenotype, and the p53 transcription factor as a player in adjusting tissue homeostasis in accordance with food availability.

LIM-HD transcription factors control axial patterning and specify distinct neuronal and intestinal cell identities in planarians.

Adult planarians can regenerate the gut, eyes and even a functional brain. Proper identity and patterning of the newly formed structures require signals that guide and commit their adult stem cells. During embryogenesis, LIM-homeodomain (LIM-HD) transcription factors act in a combinatorial 'LIM code' to control cell fate determination and differentiation. However, our understanding about the role these genes play during regeneration and homeostasis is limited. Here, we report the full repertoire of LIM-HD genes in Schmidtea mediterranea. We found that lim homeobox (lhx) genes appear expressed in complementary patterns along the cephalic ganglia and digestive system of the planarian, with some of them being co-expressed in the same cell types. We have identified that Smed-islet1, -lhx1/5-1, -lhx2/9-3, -lhx6/8, -lmx1a/b-2 and -lmx1a/b-3 are essential to pattern and size the planarian brain as well as for correct regeneration of specific subpopulations of dopaminergic, serotonergic, GABAergic and cholinergic neurons, while Smed-lhx1/5.2 and -lhx2/9.2 are required for the proper expression of intestinal cell type markers, specifically the goblet subtype. LIM-HD are also involved in controlling axonal pathfinding (lhx6/8), axial patterning (islet1, lhx1/5-1, lmx1a/b-3), head/body proportions (islet2) and stem cell proliferation (lhx3/4, lhx2/9-3, lmx1a/b-2, lmx1a/b-3). Altogether, our results suggest that planarians might present a combinatorial LIM code that controls axial patterning and axonal growing and specifies distinct neuronal and intestinal cell identities.

The effect of stress on rates of asexual reproduction in an invasive planarian.

Animal reproduction under stressful conditions is often reduced, with current survival and future reproduction being generally traded off against current reproductive activity. This study examines the impacts of physical and chemical stressors on the rates of asexual reproduction of the invasive planarian Girardia tigrina. 320 wild-caught planaria (mixed size class) were kept individually in Petri dishes such that their individual rates of fission through fragmentation could be easily monitored. Four treatment groups were compared, one chemical (5 mg/L ammonia) and one physical (decapitation), in comparison to a negative control (animals were starved of food) and a positive control where the animals were given an abundance of food. The two treatment groups immediately began reproducing asexually and accumulated the highest number of fissions over the course of the 12-day investigation period, while the positive control only began to fission after 7 days. We propose that the reproductive response observed here is an adaptive one to stressful conditions, whereby the likelihood of survival through numerical abundance is enhanced, although the size and vulnerability of resulting fragments may impose a balancing cost. The response may play a role in the invasiveness of G. tigrina by making it able to colonize environments where adverse conditions prevail.

Fate specification is spatially intermingled across planarian stem cells.

Regeneration requires mechanisms for producing a wide array of cell types. Neoblasts are stem cells in the planarian Schmidtea mediterranea that undergo fate specification to produce over 125 adult cell types. Fate specification in neoblasts can be regulated through expression of fate-specific transcription factors. We utilize multiplexed error-robust fluorescence in situ hybridization (MERFISH) and whole-mount FISH to characterize fate choice distribution of stem cells within planarians. Fate choices are often made distant from target tissues and in a highly intermingled manner, with neighboring neoblasts frequently making divergent fate choices for tissues of different location and function. We propose that pattern formation is driven primarily by the migratory assortment of progenitors from mixed and spatially distributed fate-specified stem cells and that fate choice involves stem-cell intrinsic processes.

CRISPR/Cas9-based depletion of 16S ribosomal RNA improves library complexity of single-cell RNA-sequencing in planarians.

BACKGROUND: Single-cell RNA-sequencing (scRNA-seq) relies on PCR amplification to retrieve information from vanishingly small amounts of starting material. To selectively enrich mRNA from abundant non-polyadenylated transcripts, poly(A) selection is a key step during library preparation. However, some transcripts, such as mitochondrial genes, can escape this elimination and overwhelm libraries. Often, these transcripts are removed in silico, but whether physical depletion improves detection of rare transcripts in single cells is unclear. RESULTS: We find that a single 16S ribosomal RNA is widely enriched in planarian scRNA-seq datasets, independent of the library preparation method. To deplete this transcript from scRNA-seq libraries, we design 30 single-guide RNAs spanning its length. To evaluate the effects of depletion, we perform a side-by-side comparison of the effects of eliminating the 16S transcript and find a substantial increase in the number of genes detected per cell, coupled with virtually complete loss of the 16S RNA. Moreover, we systematically determine that library complexity increases with a limited number of PCR cycles following CRISPR treatment. When compared to in silico depletion of 16S, physically removing it reduces dropout rates, retrieves more clusters, and reveals more differentially expressed genes. CONCLUSIONS: Our results show that abundant transcripts reduce the retrieval of informative transcripts in scRNA-seq and distort the analysis. Physical removal of these contaminants enables the detection of rare transcripts at lower sequencing depth, and also outperforms in silico depletion. Importantly, this method can be easily customized to deplete any abundant transcript from scRNA-seq libraries.

The BAF chromatin remodeling complex licenses planarian stem cells access to ectodermal and mesodermal cell fates.

BACKGROUND: The flatworm planarian, Schmidtea mediterranea, has a large population of adult stem cells (ASCs) that replace any cell type during tissue turnover or regeneration. How planarian ASCs (called neoblasts) manage self-renewal with the ability to produce daughter cells of different cell lineages (multipotency) is not well understood. Chromatin remodeling complexes ultimately control access to DNA regions of chromosomes and together with specific transcription factors determine whether a gene is transcribed in a given cell type. Previous work in planarians determined that RNAi of core components of the BAF chromatin remodeling complex, brg1 and smarcc2, caused increased ASCs and failed regeneration, but how these cellular defects arise at the level of gene regulation in neoblasts is unknown. RESULTS: Here, we perform ATAC and RNA sequencing on purified neoblasts, deficient for the BAF complex subunits brg-1 and smarcc2. The data demonstrate that the BAF complex promotes chromatin accessibility and facilitates transcription at target loci, as in other systems. Interestingly, we find that the BAF complex enables access to genes known to be required for the generation of mesoderm- and ectoderm-derived lineages, including muscle, parenchymal cathepsin, neural, and epithelial lineages. BAF complex knockdowns result in disrupted differentiation into these cell lineages and functional consequences on planarian regeneration and tissue turnover. Notably, we did not detect a role for the BAF complex in neoblasts making endodermal lineages. CONCLUSIONS: Our study provides functional insights into how the BAF complex contributes to cell fate decisions in planarian ASCs in vivo.

Evolutionary dynamics of whole-body regeneration across planarian flatworms.

Regenerative abilities vary dramatically across animals. Even amongst planarian flatworms, well-known for complete regeneration from tiny body fragments, some species have restricted regeneration abilities while others are almost entirely regeneration incompetent. Here, we assemble a diverse live collection of 40 planarian species to probe the evolution of head regeneration in the group. Combining quantification of species-specific head-regeneration abilities with a comprehensive transcriptome-based phylogeny reconstruction, we show multiple independent transitions between robust whole-body regeneration and restricted regeneration in freshwater species. RNA-mediated genetic interference inhibition of canonical Wnt signalling in RNA-mediated genetic interference-sensitive species bypassed all head-regeneration defects, suggesting that the Wnt pathway is linked to the emergence of planarian regeneration defects. Our finding that Wnt signalling has multiple roles in the reproductive system of the model species Schmidtea mediterranea raises the possibility that a trade-off between egg-laying, asexual reproduction by fission/regeneration and Wnt signalling drives regenerative trait evolution. Although quantitative comparisons of Wnt signalling levels, yolk content and reproductive strategy across our species collection remained inconclusive, they revealed divergent Wnt signalling roles in the reproductive system of planarians. Altogether, our study establishes planarians as a model taxon for comparative regeneration research and presents a framework for the mechanistic evolution of regenerative abilities.