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.

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.

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.

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.

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.

Surprising Separation of Cannabinoid Physical Dependence and Withdrawal in an Invertebrate Model

Planarians have mammalian-like neurotransmitter systems and have been established as a novel in vivo model for neuropharmacology. In previous research, planarians that have been exposed to the cannabinoid receptor (CB-R) agonist WIN 55,212-2 for 1 h displayed abstinence-induced withdrawal when tested in drug-free, but not in drug-containing, water. The goals of the present study were to extend previous work and to further establish a cannabinoid behavioral model with planarians. The results showed 1) four different CB-R antagonists (AM251, AM281, SLV319 and SR144528) dose-relatedly blocked development of physical dependence induced by two different CB-R agonists (WIN 55,212-2 and JWH251);2) none of the same four antagonists (AM251, AM281, SLV319 or SR144528) precipitated withdrawal;3) short wavelength (254 nm), but not long wavelength (366 nm), ultraviolet (UV) light attenuated abstinence-induced withdrawal from WIN 55,212-2, while short wavelength UV light induced moderate withdrawal behavior. The results confirm the use of a planarian model as a simple yet robust way to study development of physical dependence to cannabinoid agonists. The effect of UV irradiation adds to the evidence that the results are receptor-related. The results also give rise to the surprising suggestion, within the limitations of the methodology, that development of cannabinoid physical dependence and antagonist-induced precipitated withdrawal might be separable phenomena in planarians.

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).

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.

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.

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.

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.

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.

A small scale expression screen identifies tissue specific markers in the Dugesia japonica strain Pek-1

Freshwater planaria has tremendous capacity to reform the missing part of the body and therefore is considered as one of the most important model organism for regeneration study. At present, Schmidtea mediterranea and Dugesia japonica are the two major species utilized for laboratory manipulations. Dugesia japonica flatworms are widely distributed in the Far East including Cherry Valley region in the north-west area of Beijing, China. We reported here the establishment of an asexual Dugesiajaponica strain Pek-1, as a suitable system for regeneration study. Using morphological, karyotypical as well as phylogenetic analyses, we confirmed that these flatworms indeed belonged to Dugesia japonica. We went on to show that the commonly used in situ probes and immunohistochemistry reagents and protocols were applicable to the Pek-1 strain. Using this strain, we carried out small scale analysis on EST, RNAi and gene expression. We identified 193 unique EST sequences and 65 of them had not been reported in planarian. By RNAi analysis, we showed that 48 genes, when down-regulated individually, had no effect on regeneration. Furthermore, we identified 3 groups of tissue specific expressing genes that were useful for cell lineage analysis. We concluded that the Dugesiajaponica Pek-1 strain could be another suitable animal model to regeneration research.

A new species of the genus Girardia(Tricladida: Dugesiidae) from China

The genus Girardia （Tricladida： Dugesiidae） was recorded in China for the first time. A new freshwater turbellarian species, G. sinensis Chen ＆ Wang, sp. nov., is described, which was collected from Xinghu Lake, Zhaoqing, Guangdong Province, China. Observation and analysis of the new species was conducted on morphology, habits, reproduction, histochemistry and molecular phylogenetics. In addition, the results indicated that morphological differences of AChE^＋ nerve structure can be used as a distinguishable character for species identification of Dugesiidae.

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.

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.