The known,unknown,and unknown unknowns of cell-cell communication in planarian regeneration

Planarians represent the most primitive bilateral triploblastic animals.Most planarian species exhibit mechanisms for whole-body regeneration,exemplified by the regeneration of their cephalic ganglion after complete excision.Given their robust whole-body regeneration capacity,planarians have been model organisms in regenerative research for more than 240 years.Advancements in research tools and techniques have progressively elucidated the mechanisms underlying planarian regeneration.Accurate cell-cell communication is recognized as a fundamental requirement for regeneration.In recent decades,mechanisms associated with such communication have been revealed at the cellular level.Notably,stem cells(neoblasts)have been identified as the source of all new cells during planarian homeostasis and regeneration.The interplay between neoblasts and somatic cells affects the identities and proportions of various tissues during homeostasis and regeneration.Here,this review outlines key discoveries regarding communication between stem cell compartments and other cell types in planarians,as well as the impact of communication on planarian regeneration.Additionally,this review discusses the challenges and potential directions of future planarian research,emphasizing the sustained impact of this field on our understanding of animal regeneration.

Application of Planarian Brain Regeneration: Detection of Water Pollution

Due to over industrialisation, the environmental pollution problem is becoming increasingly serious, especially in aquatic ecosystems. Compared with traditional physical and chemical detection methods, the use of biological indicators has become popular. The freshwater planarian Dugesia japonica is distributed extensively in aquatic ecosystems and has been applied to the area of environmental toxicology for its high chemical sensitivity. Moreover, D. japonica also has a powerful regenerative capability in which the injured planarian can regenerate a new brain in 5 days and complete an adult individual remodelling in 14 days. Therefore, it has been used as a new model organism in the field of neuro-regeneration toxicology. In our past study, D. japonica can be used as a biological indicator to detect water pollution. This can provide basic data for the detection of water pollution and provide a warning system in regard to aquatic ecosystems.

Molecular actions guiding neural regeneration in planarian

Planarian is among the simplest animals that possess a centralized nervous system （CNS）, and its neural regenera- tion involves the replacement of cells lost to normal ＇wear and tear＇ （cell turnover）, and/or injury. In this review, we state and discuss the recent studies on molecular control of neural regeneration in planarians. The spatial and temporal expression patterns of genes in intact and regenerating planarian CNS have already been described relatively clearly. The bone morphogenetic protein （BMP） and Wnt signaling pathways are identified to regulate neural regeneration. During neural regeneration, conserved axon guidance mechanisms are necessary for proper wiring of the nervous system. In addition, apoptosis may play an important role in controlling cell numbers, eliminating unnecessary tissues or cells and remodeling the old tissues for regenerating CNS. The bilateral symmetry is established by determination of anterior-posterior （A-P） and dorsal-ventral （D-V） patterns. Moreover, neurons positive to dopamine, serotonin （5-HT）, and gamma-aminobutyric acid （GABA） have been detected in planarians. Therefore, planarians present us with new, experimentally accessible contexts to study the molecular actions guiding neural regeneration.

Effects of Copper on the Mobility and Regeneration of Planarian (Dugesia japonica)

[Objective] A mobility measurement and a regeneration assay were conducted to explore the behavioral effects of Cu2＋ on planarian Dugesia japonica. [Method] Dugesia japonica was respectively treated with different concentrations of copper solution for 4 and 6 d, and the blank control group was set. The mobility measurement and a regeneration assay of copper ions were determined by mIC50 and rIC50 . [Result] The mobility IC 50 for adult D. japonica at 24, 48, 72 and 96 h were 86, 3.73, 3.47 and 2.61 mg Cu2＋ /L, respectively. The mobility IC50s for newborns was 2.22, 1.64, 0.87 and 0.93 mg Cu2＋ /L, respectively. Although there was no significant difference between 24 and 48 h, and between 72 and 96 h of copper exposure, Cu2＋ decreased newborns locomotor behavior in a concentration-dependent manner at sub-toxic concentrations. The regeneration IC50 of eyespots and auricles for adult D. japonica at 6 d were calculated as 0.76, 0.78 mg Cu2＋ /L, respectively. The number of eyespots and auricles regeneration in planarian D. japonica showed a concentration-dependent manner. Results showed that newborns mobility and adult regeneration assay for D. japonica in a concentration-dependent manner. [Conclusion] The results of the present study suggest that planarians can be regarded as a useful bioindicator species for toxicological studies in the future.

Bioaccumulation and Toxicity of Neodymium in the Planarian Dugesia japonica

In this study, the bioaccumulation and toxicity of neodymium （Nd） in pla-narian Dugesia japonica were investigated. The results showed that with the in-creasing dietary Nd supplementation, the concentration of Nd in the planarian showed a significant linear increase and the balance of mineral elements was bro-ken with the decrease of Ca, Fe and Mo, and the increase of K and Mg. The content of soluble proteins and the activities of superoxide dismutase （SOD） and catalase （CAT） decreased with the increase of Nd concentration, while the content of H2O2 rose gradual y. The mortality was directly proportional to the Nd concentra-tion. The results indicated that planarian is a very sensitive aquatic animal to Nd contamination and can be an indicator organism for Nd pol ution.

Disruption of Drug Effects (Dopamine, Nicotine, Pilocarpine, κ-Opioid) in Planarians by UV Light

Based on previous work, it has been hypothesized that the energetics of ultraviolet (UV) light disrupts effects induced by receptor-binding ligands. If this hypothesis is true, then UV light should (i) disrupt a broad variety of endpoints and (ii) disrupt effects produced by ligands that bind to diverse receptor types. This was tested directly in the present study by using ligands selective for four different receptors (one ionotropic, three metabotropic) and three different behavioral endpoints. The selective dopamine D2 receptor antagonist (–)sulpiride (0.1 uM) dose-relatedly decreased spontaneous locomotor velocity, the selective nicotinic acetylcholine receptor agonist nicotine (1, 3, 5 mM) and the selective muscarinic acetylcholine receptor agonist pilocarpine (20, 30, 50 mM) induced seizure-like activity, and the selective-opioid receptor agonist U-50,488H (10 uM) produced physical dependence (manifested as abstinence-induced withdrawal) in planarian models. Each of these diverse ligand and receptor-mediated effects were attenuated by UV light (254 nm = 7.83 × 10–19 J = 4.89 eV). These findings provide further evidence that UV light disrupts ligand-receptor mediated interactions and that UV light might provide a useful tool for examining drug-receptor interactions.

Magnetic Field Intensity/Melatonin-Molarity Interactions: Experimental Support with Planarian (Dugesia sp.) Activity for a Resonance-Like Process

Synergistic interactions between specific magnetic field intensities and chemical concentrations are challenging biophysical phenomena. Planarian were exposed to one of five different concentrations of melatonin and to a “geomagnetic”—patterned 7 Hz amplitude modulated magnetic field for 6 min once per hour for 8 hr during six successive nights. The peak average strengths were within the range (50 nT) or outside the range (200 nT) derived by the equation. As predicted by a resonance equation planarian displayed highly statistically significant decreased relative activity within the 50 nT, 10–7 to 10–6 M melatonin conditions compared to lower or higher concentrations. The effect explained about 30% of the variance in these changes of activity. Activity of planarian exposed to the same melatonin concentrations but to the 200 nT field did not differ significantly from each other or from those exposed to the 50 nT field in concentrations of melatonin –7 M or >10–6 M. These results suggest the existence of non-linear, “narrow-band” mechanisms involving the numbers of molecules within a distance determined by the boundary of the organism and the intensity of naturally-patterned magnetic fields derived from energy rather than force-based resonances.

Isobolographic Method and Invertebrate (Planarian) Model for Evaluating Combinations of Waterways Pollutants

Agricultural, pharmaceutical, and other biologically active substances are emptied or leach into waterways and groundwater, where they can dose-relatedly cause pharmacologic or toxic effects on the resident or dependent animal species. Standard methods can be used to evaluate the effects of individual substances, but evaluation of combinations of substances is more difficult. The mathematically rigorous method of isobolographic analysis was coupled with a simple in vivo invertebrate model. Planarians were selected because they are the lowest extant species with a centralized nervous system. Neostigmine bromide and monopotassium phosphate (KH2PO4) were selected as representative of two types of potential pollutants. Neostigmine bromide and KH2PO4 individually produced dose-related lethality over a 60-minute observation period with LD50 values of 122 and 70 mM, respectively. The LD50 value of a 1:1 combination of the two was significantly different (p < 0.05) from the isobolographic line of additivity. We used planarians as a representative fresh-water species and joint-action (‘isobolographic’) analysis to examine possible interaction between pollutants. In the demonstrative example reported here, there was a subadditive interaction between a 1:1 fixed-ratio combination of neostigmine bromide (as a representative acetylcholinesterase inhibitor used in pesticides) and potassium phosphate (used in fertilizers and detergents).

Development of a Behavioral and Imaging Model of Feeding in Planarians

Excess weight is a major risk factor for type-2 diabetes, cardiovascular disease, and other comorbidities. Animal models of feeding provide insight into the problem and provide a means for the discovery and evaluation of pharmacotherapeutic treatment. Mammalian models are the most commonly used, but recently non-mammalian models have been utilized (e.g., C. elegans). Planarians provide an intermediate model. They are the earliest extant animal with a primitive brain-like structure and are a convenient model of mammalian behavioral endpoints and drug-induced effects. The purpose of the present study was to determine if a quantitative measure of presumptive feeding behavior could be visualized using an imaging technique. Colored food pellets were prepared and in some experiments, plasmid-delivered green fluorescent protein was added. Both visible and fluorescence microscopy displayed clear indication of internalization of the red dye and localization to the digestive system. This new methodology establishes a convenient way to study normal physiological feeding behavior as well as modifications induced by drugs or other exogenous substances.

Morphogenetic Action of Neurotransmitters on Regenerating Planarians—A Review

Planarians can be used as invertebrate bioassays to evaluate the role of neurotransmitters on regenerating cells. The influence of the nervous system is crucial to regenerate a normal complete animal. The neurotrophic action of the nervous system has been attributed to the major neurohormones present throughout the animal kingdom. The same type of transmitters found in mammals have been extensively found in many invertebrates, including planarians, but their role in regeneration is unclear. Neurotransmitters and drugs which act on neurohumoral transmission have been used to determine the role of each neurohormonc on regenerating planarians. Biochemical and pharmacological mechanisms of neurohormones on regenerative planarians are reviewed, as is their putative role on regeneration. Correlations with the roles of neurotransmitters in the central nervous system of higher organisms are also addressed.

Potential of a planarian model to study certain aspects of anti-Parkinsonism drugs

We previously created and investigated a planarian model for the study of drug action, abuse, physical dependence, receptor affinity, the toxicity of heavy metals in wastewater, and seizures. For the present pilot study, we investigated the possibility that this model might be useful for studying certain aspects of drugs used in treatment of Parkinson disease. For the first step, we were interested in finding an in vivo metric for the inhibition of L-DOPA by an inhibitor of DOPA decarboxylase. The direct clinical relevance of the endpoint was of secondary concern during this preliminary phase of model development. Two metrics were explored: L-DOPA-induced inhibition of motility (locomotor velocity) and dopamine-mediated toxicity, which was quantified using a Kaplan-Meier survival curve. L-DOPA produced both dose- and time-related toxicity. The water-soluble DOPA decarboxylase inhibitor benserazide dose-dependently inhibited the effect of L-DOPA, as manifested by a leftward shift in the Kaplan-Meier curve. Additional work was initiated using the more sensitive and a graded metric of spontaneous locomotor velocity. The encouraging results of this pilot study suggest that: 1) planarians contain DOPA decarboxylase or an equivalent enzyme, and 2) the planarian model might be useful for the study of certain aspects of anti-Parkinsonism pharmacotherapy.

Modification by Polychlorinated Biphenyls(PCBs)of Cadmium Induced Lesions in the Planarian Model,Dugesia dorotocephala

The appearance of abnormal growths on the planarian, Dugesia dorotocephala, in response to cadmium with and without pre-exposure to L-buthionine-R, S-sulfoximine (BSO) and concurrent exposure to the polychlorinated biphenyls (PCBs) Aroclor 1254, PCB 28, PCB 110 or PCB 126 is described. Pigmented rose thorn (PRT) lesions were non-invasive and appeared in response to PCBs. Post-head (PH) lesions developed in up to 100% of the animals within 6-20 days post-dosing, progressed rather rapidly and were highly invasive. Round tail tip (RTT) lesions appeared in lower frequencies within 10-30 days, but progressed extremely rapidly resulting in tail loss within 48 h. We have referred to these types of lesions as 'tumors', but they are not necessarily characteristic of vertebrate neoplasms.PCBs interacted with cadmium in a complex way, in some cases increasing total lesions and decreasing time-to-lesion and in other cases having the opposite effects. A three-factor (PCB, PCB dose, Cd dose) nested analysis of variance model was used to determine lesion rates in order to compare PCB potencies as potentiators or antagonists. The Aroclor mixture was always the least potent co-toxicant but appeared to be the most potent antagonist; the coplanar PCB 126 was the most potent co-toxicant. The complex response surfaces and the lack of stoichiometry in dose-response relationships indicate that multiple mechanisms are responsible for PH and RTT lesions in planarians. These results emphasize the complexity of PCB toxicities and suggest further studies to validate the planarian model as a screen for combinations or environmental mixtures which may have altered biological potency in other species.

Definition of Planarian Mortality in an Acute Toxicity Test:A Case Study on Dugesia japonica Exposed to 1-Octyl-3-methylimidazolium Bromide

In order to define the mortality criteria of planarian objectively,a case study of Dugesia japonica exposed to 1-octyl-3-methylimidazolium bromide([C8mim]Br)was performed followed by a recovery culture.The results showed that defining planarian mortality in light of its body disintegration was appropriate.If the disintegrated tissue of a planarian was more than 1/4 of its body length,it would completely degenerate.However,a planarian would regenerate the lost tissue and return to normal after a few days’recovery culture if the disintegrated part was less than 1/4 of body length.Therefore,we propose to use body disintegration as the endpoint of planarian mortality,i.e.,1/4 body length degenerated is the critical threshold of mortality and survival of planarians when exposed to toxicants.This criterion could be adapted in the standardization of testing protocols and comparability of acute toxicity test or other toxicological research using planarian as the test animal.

A comprehensive review:Research status and trends of planarianology in China

Planarian is an excellent model for studying several scientific issues,and planarianology has become an active research field in the world.Researchers at home and abroad have conducted a lot of in-depth studies on planarians in various areas.This paper outlined the panorama of planarian research in China and summarized the advances from three aspects of taxonomy,molecular phylogeny and phylogeography,regeneration,tissue homeostasis and immunity as well as toxicology.Furthermore,the current problems and future development direction and tendency of planarian research in China are briefly analyzed and prospected.Importantly,the catalogue of planarians in China is also presented.

中国涡虫的分类概况及分科检索表

涡虫隶属扁形动物门Platyhelminthes,作为最早出现的三胚层动物,在动物的进化中具有重要意义,也是重要的环境指示动物和再生生物学的模式生物。涡虫学已成为世界最活跃的研究领域之一。涡虫在全世界分布广泛、种类丰富,但我国的相关分类研究进展较慢。近年来,我国的涡虫研究手段和论文发表档次已达到国际领先水平,并取得了不少进展,但仍存在调查和研究范围局限、大量工作尚待开展等不足。本文总结了国内外涡虫分类的研究历史和现状,介绍了目前高级阶元的分类系统,并更新了我国涡虫种类名录,共计2亚门7目26科44属131种/亚种,首次编制了我国涡虫分目分科检索表。研究结果旨在为涡虫分类和系统发育研究服务,推动涡虫在环境调查和再生医学等相关领域的研究。

Molecular systematics, population genetics and phylogeography on planarians(Platyhelminths: Turbellaria): A brief review of molecular markers

Due to their characteristics, such as powerful regeneration capability, high susceptibility to xenobiotics, wide distribution and weak dispersion ability, planarians are used as ideal test animals in several research fields, and planarianology has become a more and more active subject. This paper reviews the research progress of planarians from the aspects of molecular phylogeny, molecular taxonomy, population genetics and phylogeography, and also summarizes the corresponding molecular markers applied. Moreover, the future direction and tendency in this field is briefly analyzed and prospected.

Neoblast-enriched zinc finger protein FIR1 triggers local proliferation during planarian regeneration

Regeneration,relying mainly on resident adult stem cells,is widespread.However,the mechanism by which stem cells initiate proliferation during this process in vivo is unclear.Using planarian as a model,we screened 46 transcripts showing potential function in the regulation of local stem cell proliferation following 48 h regeneration.By analyzing the regeneration defects and the mitotic activity of animals under administration of RNA interference(RNAi),we identified factor for initiating regeneration 1(Fir1)required for local proliferation.Our findings reveal that Fir1,enriched in neoblasts,promotes planarian regeneration in any tissue-missing context.Further,we demonstrate that DIS3 like 3-5'exoribonuclease 2(Dis3l2)is required for Fir1 phenotype.Besides,RNAi knockdown of Fir1 causes a decrease of neoblast wound response genes following amputation.These findings suggest that Fir1 recognizes regenerative signals and promotes DIS3L2 proteins to trigger neoblast proliferation following amputation and provide a mechanism critical for stem cell response to injury.

Photokinesis and Djopsin gene expression analysis during the regeneration of planarian eyes

Planarians provide the ideal model for studying eye development,with their simple eye structure and exceptionally rapid regeneration.Here,we observed the eye morphogenesis,photophobic behavior,spectral sensitivity and expression pattern of Djopsin in the freshwater planarian Dugesia japonica.The results showed that:(i)Djopsin encoding the putative protein belonged to the rhabdomeric opsins group and displayed high conservation during animal evolution;(ii)planarians displayed diverse photophobic response to different visible wavelengths and were more sensitive to light blue(495 nm)and yellow(635 nm);(iii)the morphogenesis and functional recovery of eyes were related to the expression pattern of Djopsin during head regeneration;and(iv)Djopsin gene plays a major role in functional recovery during eye regeneration and visual system maintenance in adult planarians.

Production of asexual and sexual offspring in the triploid sexual planarian Dugesia ryukyuensis

Certain freshwater planarians reproduce asexually as well as sexually,and their chromosomal ploidies include polyploidy,aneuploidy and mixoploidy.Previously,we successfully performed an experiment in which a clonal population produced by asexual reproduction of the Dugesia ryukyuensis(OH strain)switched to the sexual mode of reproduction.Worms of this strain are triploid with a pericentric inversion on Chromosome 4.The worms were switched to sexual reproduction after being fed with sexually mature Bdellocephala brunnea,which is a sexually reproducing species.The resulting sexualized OH strain produced cocoons filled with several eggs.Two putative factors,Mendelian factor(s)and chromosomal control(s),have been proposed as determining the reproductive mode.The present study demonstrated that inbreeding of the resultant sexualized worms produced the following four types of offspring through sexual reproduction:diploid asexual worms,triploid asexual worms,diploid sexual worms and triploid sexual worms.The chromosomal mutation on Chromosome 4 was inherited by these offspring independent of their reproductive mode.These results provide two important pieces of information:(i)the putative genetic factor was not necessarily inherited in a Mendelian fashion;and(ii)the reproductive mode is not regulated by chromosomal changes such as polyploidy or chromosomal mutations.This suggests that asexuality in D.ryukyuensis is regulated by an unknown factor(s)other than a Mendelian factor or a chromosomal control.

三肠亚目(扁形动物门：涡虫纲：序列目)涡虫系统发育研究进展

比较分析了基于形态特征和分子序列数据的三肠亚目涡虫的系统发育关系,总结出现今大家公认的该亚目的系统发生关系:Tricladida是一个亚目级阶元,即三肠亚目,隶属于扁形动物门、涡虫纲、序列目.该亚目下分4个次目:海栖次目、陆栖次目、沼栖次目和穴居次目,其中海栖次目是一个单系,为亚目内较原始的类群;沼栖次目是一个并系,其内的三角涡虫科和陆栖次目有最近共同祖先;陆栖次目为单系起源.三肠亚目涡虫的演化路线是由海生到淡水再到陆生,之后又有逆转现象,即个别类群(如Spathula、Romankenkius)又返回淡水生活.文中还对未来该亚目系统发育研究的焦点进行了展望和预测.