Non-senescent species are not immortal: Stress and decline in two planaria species.

Potential immortality is observed in several species (e.g. prickly pear cactus, hydra and flatworms) and is indicative of their negligible or even negative senescence rates. Unlike in senescent species, which experience reduced individual performance with age due to physiological degradation, species with negligible or negative senescence display mortality rates that remain constant or decline with age, respectively. These rates vary across taxa and are correlated with life history traits. Yet, the extent to which variable resource availability, a key driver of variation in life history traits, impacts species that show negligible or negative senescence is currently unknown. Here, we examine whether and how variation in the quantity, quality and feeding interval of resources impact population structure, population performance and life history trait trade-offs in two long-lived planaria that do not senesce: Schmidtea mediterranea and Dugesia tahitiensis. In a full factorial design, different combinations of resource quantity (reduced intake, standard intake and high intake) and quality (high and low quality) were provided in two different feeding intervals (7-day and 14-day intervals) for 19 weeks. We show that variability in resource availability, via decreases in quantity, quality and frequency of resources, does not diminish population viability in either species but does result in suboptimal conditions of stress in S. mediterranea. The high population viability we report can be attributed to two different mechanisms: increased reproduction or increased investment into maintenance at the expense of reproduction. Moreover, which mechanism was responsible for said high population viability was context-dependent and modulated by the specific life history strategy of the two planaria species. We show that suboptimal conditions can cause stress responses that have significant impacts on non-senescent species. The context-dependent response we observe suggests that species that do not senesce but are subject to suboptimal conditions of stress may ultimately exhibit declines in performance and ultimately die. A clearer understanding of the impact of suboptimal conditions of resource availability on non-senescent species is needed to determine the extent of stress experienced and ultimately whether a species can truly be immortal.

Agelessness is possible under the disposable soma theory but system complexity makes it unlikely.

Although demographic studies have failed to find evidence of aging in certain animal species, classic evolutionary theories of aging struggle to explain how evolution could favor agelessness in such cases. Here, we develop mathematical models of the disposable soma theory to identify conditions in which agelessness would be evolutionarily favored. For any given type of damage that could accumulate and cause age-accelerating mortality risk, we find that evolution could select for its complete removal if the mortality risk it poses is severe enough and its repair does not pose too large of a penalty to reproduction. Environmental factors such as extrinsic mortality and the form of population density-dependent regulation also play a large role in determining the optimal rate of aging and whether agelessness should be evolutionarily favored. However, in a system with multiple sources of damage and multiple independent repair processes, avoiding aging is rarely evolutionarily favorable. Pleiotropic repair processes, such as those that could be present in asexual fissioning organisms, make agelessness more likely but do not guarantee it. Our results indicate that agelessness could be favored by evolution in narrow contexts but that multiple types of damage and repair make agelessness unlikely to arise in sufficiently complex organisms.

Poly (A) binding protein 2 is critical for stem cell differentiation during regeneration in the planarian Schmidtea mediterranea.

Post-transcriptional regulation has emerged as a key mechanism for regulating stem cell renewal and differentiation, which is essential for understanding tissue regeneration and homeostasis. Poly(A)-binding proteins are a family of RNA-binding proteins that play a vital role in post-transcriptional regulation by controlling mRNA stability and protein synthesis. The involvement of poly(A) binding proteins in a wide range of cellular functions is increasingly being investigated. In this study, we used the regenerative model planarian organism Schmidtea mediterranea to demonstrate the critical role of poly(A)-binding protein 2 (PABP2) in regulating neoblast maintenance and differentiation. A deficit in PABP2 blocks the transition of neoblasts toward immediate early progenitors, leading to an enhanced pool of non-committed neoblasts and a decreased progenitor population. This is reflected in variations in the transcriptome profile, providing evidence of downregulation in multiple lineages. Thus, an insufficiency of PABP2 resulted in defective formation and organization of tissue, leading to abnormal regeneration. Our study reveals the essential role of PABP2 in regulating genes that mediate stem cell commitment to early progenitors during tissue regeneration.

Schild Analysis of the Interaction between Parthenolide and Cocaine Suggests an Allosteric Relationship for Their Effects on Planarian Motility.

The freshwater planarian is an emerging animal model in neuroscience due to its centralized nervous system that closely parallels closely parallels the nervous system of vertebrates. Cocaine, an abused drug, is the 'founding member' of the local anesthetic family. Parthenolide, a sesquiterpene lactone, acts as a behavioral and physiological antagonist of cocaine in planarians and rats, respectively. Previous work from our laboratory showed that both parthenolide and cocaine reduced planarian motility and that parthenolide reversed the cocaine-induced motility decrease at concentrations where parthenolide does not affect the movement of the worms. However, the exact mechanism of the cocaine/parthenolide antagonism is unknown. Here, we report the results of a Schild analysis to explore the parthenolide/cocaine relationship in the planarian Girardia tigrina. The Schild slopes of a family of concentration-response curves of parthenolide ± a single concentration of cocaine and vice versa were -0.55 and -0.36, respectively. These slopes were not statistically different from each other. Interestingly, the slope corresponding to the parthenolide ± cocaine (but not the cocaine ± parthenolide) data set was statistically different from -1. Our data suggest an allosteric relationship between cocaine and parthenolide for their effect on planarian motility. To the best of our knowledge, this is the first study about the mechanism of action of the antagonism between cocaine and parthenolide. Further studies are needed to determine the specific nature of the parthenolide/cocaine target(s) in this organism.

Cotinine influences the effect of high and low nicotine concentrations on planarian motility differently.

Previous work from our laboratory showed that cotinine, a nicotine metabolite, reverses three nicotine-induced behavioral effects in freshwater planarians: motility decrease, seizure-like movements, and withdrawal-like behaviors. The present work explored whether cotinine, a nicotine metabolite, antagonized the nicotine-induced effects on planarian motility in a concentration-dependent manner. We found that nicotine decreased planarian motility at nicotine concentrations above 60 µM but increased planarian velocity at concentrations equal to or below 50 µM, in agreement with previous data. Cotinine did not affect planarian motility at a concentration range between 250 and 2750 µM. Furthermore, we found that cotinine alleviated the 100 µM nicotine-induced motility decrease in a concentration-dependent manner and reversed the low nicotine concentration motility increase, albeit in a concentration-independent manner. The apparent concentration-dependent alleviation of >60 µM nicotine-induced motility decrease by cotinine suggests an orthosteric relationship between nicotine and cotinine. On the other hand, the evident concentration-independent cotinine alleviation of the increase in motility induced by 50 µM nicotine suggests an allosteric relationship. Our data is consistent with the existing literature about the relationship between nicotine and cotinine in various models, reinforcing the case for the usefulness of the planarian model in pharmacological studies.

The DNA methyltransferase DMAP1 is required for tissue maintenance and planarian regeneration.

The precise regulation of transcription is required for embryonic development, adult tissue turnover, and regeneration. Epigenetic modifications play a crucial role in orchestrating and regulating the transcription of genes. These modifications are important in the transition of pluripotent stem cells and their progeny. Methylation, a key epigenetic modification, influences gene expression through changes in DNA methylation. Work in different organisms has shown that the DNA methyltransferase-1-associated protein (DMAP1) may associate with other molecules to repress transcription through DNA methylation. Thus, DMAP1 is a versatile protein implicated in a myriad of events, including pluripotency maintenance, DNA damage repair, and tumor suppression. While DMAP1 has been extensively studied in vitro, its complex regulation in the context of the adult organism remains unclear. To gain insights into the possible roles of DMAP1 at the organismal level, we used planarian flatworms that possess remarkable regenerative capabilities driven by pluripotent stem cells called neoblast. Our findings demonstrate the evolutionary conservation of DMAP1 in the planarian Schmidtea mediterranea. Functional disruption of DMAP1 through RNA interference revealed its critical role in tissue maintenance, neoblast differentiation, and regeneration in S. mediterranea. Moreover, our analysis unveiled a novel function for DMAP1 in regulating cell death in response to DNA damage and influencing the expression of axial polarity markers. Our findings provide a simplified paradigm for studying DMAP1's function in adult tissues.

Neurophysiological measurements of planarian brain activity: a unique model for neuroscience research.

Planarians are well-known model organisms for regeneration and developmental biology research due to their remarkable regenerative capacity. Here, we aim to advocate for the use of planaria as a valuable model for neurobiology, as well. Planarians have most of the major qualities of more developed organisms, including a primal brain. These traits combined with their exceptional regeneration capabilities, allow neurobiological experiments not possible in any other model organism, as we demonstrate by electrophysiological recording from planaria with two heads that control a shared body. To facilitate planarian neuroscience research, we developed an extracellular multi-unit recording procedure for the planarians fragile brain (Dugesia japonica). We created a semi-intact preparation restrained with fine dissection pins, enabling hours of reliable recording, via a suction electrode. Here, we demonstrate the feasibility and potential of planarian neurophysiological research by characterizing the neuronal activity during simple learning processes and responses to various stimuli. In addition, we examined the use of linalool as anesthetic agent to allows recordings from an intact, large worm and for fine electrophysiological approaches such as intracellular recording. The demonstrated ability for neurophysiological measurements, along with the inherent advantages of planarians, promotes this exceptional model organism for neuroscience research.

Theoretical insights, degradation, and sub-lethal toxicity of thiamethoxam to the planarian Girardia tigrina.

Advanced oxidative processes, such as Photo-Fenton, transform organic contaminants due to the attack by radicals. In this context, the lethal and sub-lethal effects of the Cruiser® 350FS (CRZ) with the active ingredient thiamethoxam (TMX) were investigated using the planarian Girardia tigrina. Degradation of thiamethoxam by the Fenton process was also assessed by using theoretical studies and the efficiency of Solar-Fenton versus Fenton. The 48 h LC50 value of CRZ for planarians was 478.6 mg L-1. The regeneration of planarians was significantly affected for concentrations ≥ 17 mg·L-1 of TMX (24 h). The Solar-Fenton showed a high degradation percentage reaching ~70%. The theoretical model showed the atoms of the TMX molecule that will suffer attacks from the formed radicals. Current results open new perspectives concerning the treatment of TMX in the aquatic environment because the 70% degradation seems to be sufficient to reach concentrations that do not induce sub-lethal effects in planarians. Further studies should determine if the by-products generated might be toxic for planaria or other organisms.

Epidermal turnover in the planarian Schmidtea mediterranea involves basal cell extrusion and intestinal digestion.

Planarian flatworms undergo continuous internal turnover, wherein old cells are replaced by the division progeny of adult pluripotent stem cells (neoblasts). How cell turnover is carried out at the organismal level remains an intriguing question in planarians and other systems. While previous studies have predominantly focused on neoblast proliferation, little is known about the processes that mediate cell loss during tissue homeostasis. Here, we use the planarian epidermis as a model to study the mechanisms of cell removal. We established a covalent dye-labeling assay and image analysis pipeline to quantify the cell turnover rate in the planarian epidermis. Our findings indicate that the ventral epidermis is highly dynamic and epidermal cells undergo internalization via basal extrusion, followed by a relocation toward the intestine and ultimately digestion by intestinal phagocytes. Overall, our study reveals a complex homeostatic process of cell clearance that may generally allow planarians to catabolize their own cells.

The DNA Methyltransferase DMAP1 is Required for Tissue Maintenance and Planarian Regeneration.

The precise regulation of transcription is required for embryonic development, adult tissue turnover, and regeneration. Epigenetic modifications play a crucial role in orchestrating and regulating the transcription of genes. These modifications are important in the transition of pluripotent stem cells and their progeny. Methylation, a key epigenetic modification, influences gene expression through changes in histone tails and direct DNA methylation. Work in different organisms has shown that the DNA methyltransferase-1-associated protein (DMAP1) may associate with other molecules to repress transcription through DNA methylation. Thus, DMAP1 is a versatile protein implicated in a myriad of events, including pluripotency maintenance, DNA damage repair, and tumor suppression. While DMAP1 has been extensively studied in vitro, its complex regulation in the context of the adult organism remains unclear. To gain insights into the possible roles of DMAP1 at the organismal level, we used planarian flatworms that possess remarkable regenerative capabilities driven by pluripotent stem cells called neoblast. Our findings demonstrate the evolutionary conservation of DMAP1 in the planarian Schmidtea mediterranea. Functional disruption of DMAP1 through RNA interference revealed its critical role in tissue maintenance, neoblast differentiation, and regeneration in S. mediterranea. Moreover, our analysis unveiled a novel function for DMAP1 in regulating cell death in response to DNA damage and influencing the expression of axial polarity markers. Our findings provide a simplified paradigm for studying DMAP1's epigenetic regulation in adult tissues.

The complexities of ligand/receptor interactions: Exploring the role of molecular vibrations and quantum tunnelling.

Molecular vibrations and quantum tunneling may link ligand binding to the function of pharmacological receptors. The well-established lock-and-key model explains a ligand's binding and recognition by a receptor; however, a general mechanism by which receptors translate binding into activation, inactivation, or modulation remains elusive. The Vibration Theory of Olfaction was proposed in the 1930s to explain this subset of receptor-mediated phenomena by correlating odorant molecular vibrations to smell, but a mechanism was lacking. In the 1990s, inelastic electron tunneling was proposed as a plausible mechanism for translating molecular vibration to odorant physiology. More recently, studies of ligands' vibrational spectra and the use of deuterated ligand analogs have provided helpful information to study this admittedly controversial hypothesis in metabotropic receptors other than olfactory receptors. In the present work, based in part on published experiments from our laboratory using planarians as an experimental organism, I will present a rationale and possible experimental approach for extending this idea to ligand-gated ion channels.

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.

mRNA Transfection of S. mediterranea for Luminescence Analysis.

Planarians have become a powerful model system for stem cell research and regeneration. While the tool kit for mechanistic investigations has been steadily expanding over the last decade, robust genetic tools for transgene expression are still lacking. We describe here methods for in vivo and in vitro mRNA transfection of the planarian species Schmidtea mediterranea. These methods utilize the commercially available TransIT-mRNA transfection reagent to efficiently deliver mRNA encoding a synthetic nanoluciferase reporter. Using a luminescent reporter overcomes the bright autofluorescent background of planarian tissues and allows quantitative measurements of protein expression levels. Collectively, our methods provide the means for heterologous reporter expression in planarian cells and the basis for future development of transgenic techniques.

Emerging roles of mitochondria in animal regeneration.

The regeneration capacity after an injury is critical to the survival of living organisms. In animals, regeneration ability can be classified into five primary types: cellular, tissue, organ, structure, and whole-body regeneration. Multiple organelles and signaling pathways are involved in the processes of initiation, progression, and completion of regeneration. Mitochondria, as intracellular signaling platforms of pleiotropic functions in animals, have recently gained attention in animal regeneration. However, most studies to date have focused on cellular and tissue regeneration. A mechanistic understanding of the mitochondrial role in large-scale regeneration is unclear. Here, we reviewed findings related to mitochondrial involvement in animal regeneration. We outlined the evidence of mitochondrial dynamics across different animal models. Moreover, we emphasized the impact of defects and perturbation in mitochondria resulting in regeneration failure. Ultimately, we discussed the regulation of aging by mitochondria in animal regeneration and recommended this for future study. We hope this review will serve as a means to advocate for more mechanistic studies of mitochondria related to animal regeneration on different scales.

Extracellular matrix-regulator MMPA is required for the orderly proliferation of neoblasts and differentiation of ectodermal progenitor cells in the planarian Dugesia japonica.

Matrix metalloproteinases (MMPs) are members of a family of zinc-dependent metallopeptidase proteins that are widely found in plants, animals, and microorganisms. As the regulators of the extracellular matrix and basement membrane, MMPs play an important role in embryogenesis, development, innate immunity, and regeneration. However, the function of MMP family in planarian, a model for regeneration research, is still ambiguous. Here, we cloned 5 MMPs genes from Dugesia japonica and found that DjMMPA was associated with the process of regeneration, neoblasts cell maintenance confusion and destruction. Loss of DjMMPA led to homeostasis confusion and eventually death, owing to neoblasts proliferation disorder. Additionally, DjMMPA RNAi-treated animals had impaired regeneration after amputation. Furthermore, knockdown of DjMMPA had noticeable defects in cell differentiation of ectoderm, especially in eyes and neural progenitor cells, possibly by inhibiting Wnt signaling. Our results suggest that extracellular matrix-regulator MMPA is required for the orderly proliferation of neoblasts and differentiation of ectodermal progenitor cells in the planarian, which provide valuable information for further explorations into the molecular mechanism of MMPS, stem cells, and regeneration.

Wnt signaling in whole-body regeneration.

Regeneration abilities are widespread among animals and select species can restore any body parts removed by wounds that sever the major body axes. This capability of whole-body regeneration as exemplified in flatworm planarians, Acoels, and Cnidarians involves initial responses to injury, the assessment of wound site polarization, determination of missing tissue and programming of blastema fate, and patterned outgrowth to restore axis content and proportionality. Wnt signaling drives many shared and conserved aspects of the biology of whole-body regeneration in the planarian species Schmidtea mediterranea and Dugesia japonica, in the Acoel Hofstenia miamia, and in Cnidarians Hydra and Nematostella. These overlapping mechanisms suggest whole-body regeneration might be an ancestral property across diverse animal taxa.

Transplantation of fragments from different planaria: A bioelectrical model for head regeneration.

Head-tail planaria morphologies are influenced by the electric potential differences across the animal's primary axis, as evidenced e.g. by voltage-sensitive dyes and functional experiments that create permanent lines of 2-headed but genetically wild-type animals. However, bioelectrical and biochemical models that make predictions on what would happen in the case of spatial chimeras made by tissue transplantation from different planaria (different species and head shapes) are lacking. Here, we use a bioelectrical model to qualitatively describe the effects of tissue transplantation on the shape of the regenerated head. To this end, we assume that the cells may have distinct sets of ion channels and ascribe the system outcome to the axial distributions of average cell potentials over morphologically relevant regions. Our rationale is that the distributions of signaling ions and molecules are spatially coupled with multicellular electric potentials. Thus, long-time downstream transcriptional events should be triggered by short-time bioelectrical processes. We show that relatively small differences between the ion channel characteristics of the cells could eventually give noticeable changes in the electric potential profiles and the expected morphological deviations, which suggests that small but timely bioelectrical actions may have significant morphological effects. Our approach is based on the observed relationships between bioelectrical regionalization and biochemical gradients in body-plan studies. Such models are relevant to regenerative, developmental, and cancer biology in which cells with distinct properties and morphogenetic target states confront each other in the same tissue.

Phylotranscriptomics interrogation uncovers a complex evolutionary history for the planarian genus Dugesia (Platyhelminthes, Tricladida) in the Western Mediterranean.

The Mediterranean is one of the most biodiverse areas of the Paleartic region. Here, basing on large data sets of single copy orthologs obtained from transcriptomic data, we investigated the evolutionary history of the genus Dugesia in the Western Mediterranean area. The results corroborated that the complex paleogeological history of the region was an important driver of diversification for the genus, speciating as microplates and islands were forming. These processes led to the differentiation of three main biogeographic clades: Iberia-Apennines-Alps, Corsica-Sardinia, and Iberia-Africa. The internal relationships of these major clades were analysed with several representative samples per species. The use of large data sets regarding the number of loci and samples, as well as state-of-the-art phylogenomic inference methods allowed us to answer different unresolved questions about the evolution of particular groups, such as the diversification path of D. subtentaculata in the Iberian Peninsula and its colonization of Africa. Additionally, our results support the differentiation of D. benazzii in two lineages which could represent two species. Finally, we analysed here for the first time a comprehensive number of samples from several asexual Iberian populations whose assignment at the species level has been an enigma through the years. The phylogenies obtained with different inference methods showed a branching topology of asexual individuals at the base of sexual clades. We hypothesize that this unexpected topology is related to long-term asexuality. This work represents the first phylotranscriptomic analysis of Tricladida, laying the first stone of the genomic era in phylogenetic studies on this taxonomic group.

A Wnt11 and Dishevelled signaling pathway acts prior to injury to control wound polarization for the onset of planarian regeneration.

Regeneration is initiated by wounding, but it is unclear how injury-induced signals precisely convey the identity of the tissues requiring replacement. In the planarian Schmidtea mediterranea, the first event in head regeneration is the asymmetric activation of the Wnt inhibitor notum in longitudinal body-wall muscle cells, preferentially at anterior-facing versus posterior-facing wound sites. However, the mechanism driving this early symmetry-breaking event is unknown. We identify a noncanonical Wnt11 and Dishevelled pathway regulating notum polarization, which opposes injury-induced notum-activating Wnt/ß-catenin signals and regulates muscle orientation. Using expression analysis and experiments to define a critical time of action, we demonstrate that Wnt11 and Dishevelled signals act prior to injury and in a growth-dependent manner to orient the polarization of notum induced by wounding. In turn, injury-induced notum dictates polarization used in the next round of regeneration. These results identify a self-reinforcing feedback system driving the polarization of blastema outgrowth and indicate that regeneration uses pre-existing tissue information to determine the outcome of wound-induced signals.

A miniaturized electrothermal array for rapid analysis of temperature preference behaviors in ecology and ecotoxicology.

Due to technical limitations, there have been minimal studies performed on thermal preferences and thermotactic behaviors of aquatic ectotherm species commonly used in ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built and miniaturized electrothermal array for rapid thermal preference behavioral tests. We applied the novel platform to define thermal preferences in multiple invertebrate and vertebrate species. Specifically, Dugesia notogaea (freshwater planarians), Chironomus tepperi (nonbiting midge larvae), Ostracoda (seed shrimp), Artemia franciscana (brine shrimp), Daphnia carinata (water flea), Austrochiltonia subtenuis (freshwater amphipod), Physa acuta (freshwater snail), Potamopyrgus antipodarum (New Zealand mud snail) and larval stage of Danio rerio (zebrafish) were tested. The Australian freshwater water fleas, amphipods, snail Physa acuta as well as zebrafish exhibited the most consistent preference to cool zones and clear avoidance of zones >27 °C out of nine species tested. Our results indicate the larval stage of zebrafish as the most responsive species highly suitable for prospective development of multidimensional behavioral test batteries. We also showcase preliminary data that environmentally relevant concentrations of pharmaceutical pollutants such as non-steroidal anti-inflammatory drug (NSAID) ibuprofen (9800 ng/L) and insecticide imidacloprid (4600 ng/L) but not anti-depressant venlafaxine (2200 ng/L) and (iv) anticonvulsant medications gabapentin (400 ng/L) can perturb thermal preference behavior of larval zebrafish. Collectively our results demonstrate the utility of simple and inexpensive thermoelectric technology in rapid exploration of thermal preference in diverse species of aquatic animals. We postulate that more broadly such technologies can also have added value in ecotoxicity testing of emerging contaminants.