Freshwater Planarian Dugesia Research Articles

Multi-Behavioral Endpoint Testing of an 87-Chemical Compound Library in Freshwater Planarians.

There is an increased recognition in the field of toxicology of the value of medium-to-high-throughput screening methods using in vitro and alternative animal models. We have previously introduced the asexual freshwater planarian Dugesia japonica as a new alternative animal model and proposed that it is particularly well-suited for the study of developmental neurotoxicology. In this article, we discuss how we have expanded and automated our screening methodology to allow for fast screening of multiple behavioral endpoints, developmental toxicity, and mortality. Using an 87-compound library provided by the National Toxicology Program, consisting of known and suspected neurotoxicants, including drugs, flame retardants, industrial chemicals, polycyclic aromatic hydrocarbons (PAHs), pesticides, and presumptive negative controls, we further evaluate the benefits and limitations of the system for medium-throughput screening, focusing on the technical aspects of the system. We show that, in the context of this library, planarians are the most sensitive to pesticides with 16/16 compounds causing toxicity and the least sensitive to PAHs, with only 5/17 causing toxicity. Furthermore, while none of the presumptive negative controls were bioactive in adult planarians, 2/5, acetaminophen and acetylsalicylic acid, were bioactive in regenerating worms. Notably, these compounds were previously reported as developmentally toxic in mammalian studies. Through parallel screening of adults and developing animals, planarians are thus a useful model to detect such developmental-specific effects, which was observed for 13 chemicals in this library. We use the data and experience gained from this screen to propose guidelines for best practices when using planarians for toxicology screens.

Toxicity of contaminants of emerging concern to Dugesia japonica: QSTR modeling and toxicity relationship with Daphnia magna

Freshwater planarian Dugesia japonica has a critical ecological importance owing to its unique properties. This study presents for the first time an in silico approach to determine a priori the acute toxicity of contaminants of emerging concern towards D. japonica. Quantitative structure-toxicity/toxicity-toxicity relationship (QSTR/QTTR) models provided here allow producing reliable information using the existing data, thus, reducing the demand of in vivo and in vitro experiments, and contributing to the need for a more holistic approach to environmental safety assessment. Both models are promising for being notably simple and robust, meeting rigorous validation metrics and the OECD criteria. The QTTR model based on the available Daphnia magna data might also contribute to the US EPA Interspecies Correlation Estimation web application. Moreover, the proposed models were applied on hundreds of environmentally significant chemicals lacking experimental D. japonica toxicity data and predicted toxicity values were reported for the first time. The models presented here can be used as potential tools in toxicity assessment, screening and prioritization of chemicals and development of risk management measures in a scientific and regulatory frame.

Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity.

The asexual freshwater planarian Dugesia japonica has emerged as a medium-throughput alternative animal model for neurotoxicology. We have previously shown that D. japonica are sensitive to organophosphorus pesticides (OPs) and characterized the in vitro inhibition profile of planarian cholinesterase (DjChE) activity using irreversible and reversible inhibitors. We found that DjChE has intermediate features of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Here, we identify two candidate genes (Djche1 and Djche2) responsible for DjChE activity. Sequence alignment and structural homology modeling with representative vertebrate AChE and BChE sequences confirmed our structural predictions, and show that both DjChE enzymes have intermediate sized catalytic gorges and disrupted peripheral binding sites. Djche1 and Djche2 were both expressed in the planarian nervous system, as anticipated from previous activity staining, but with distinct expression profiles. To dissect how DjChE inhibition affects planarian behavior, we acutely inhibited DjChE activity by exposing animals to either an OP (diazinon) or carbamate (physostigmine) at 1µM for 4days. Both inhibitors delayed the reaction of planarians to heat stress. Simultaneous knockdown of both Djche genes by RNAi similarly resulted in a delayed heat stress response. Furthermore, chemical inhibition of DjChE activity increased the worms' ability to adhere to a substrate. However, increased substrate adhesion was not observed in Djche1/Djche2 (RNAi) animals or in inhibitor-treated day 11 regenerates, suggesting this phenotype may be modulated by other mechanisms besides ChE inhibition. Together, our study characterizes DjChE expression and function, providing the basis for future studies in this system to dissect alternative mechanisms of OP toxicity.

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.

Dynamics of Cytokine-like Activity in the Hyperplasic Ovary of Ex-fissiparous Planarians.

The origin of infertility in the hyperplasic ovary of ex-fissiparous planarians remains poorly understood. In a previous study we demonstrated that a complex process of early autophagy, followed by apoptotic processes, occurs in the hyperplasic ovary of the freshwater planarian Dugesia arabica. The present study aimed to investigate whether the mRNA expression levels of selected mRNA-like genes are altered in the hyperplasic ovary of the ex-fissiparous freshwater planarian D. arabica compared to the normal ovary. Using human cytokine-specific primers including interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), we have successfully amplified by real-time polymerase chain reaction some transcripts that could be similar to those amplified in human. The transcript levels of the human-like transcript (IL-1-like and TNF-α-like) were significantly higher, 4.89- and 3.41-fold, respectively, in the hyperplasic ovary compared to the normal ovary (P < 0.05). However, although IL-6-like levels were higher in the hyperplasic ovary than the normal ovary (2.57-fold), this difference was not significant (P > 0.05). Immunohistochemical labeling supported the quantitative real-time PCR, showing that, like their respective mRNA expression levels, IL-1, IL-6, and TNF-α-like proteins are more highly expressed in the hyperplasic ovary than in the normal ovary.

Planarian cholinesterase: in vitro characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity and reactivation

The freshwater planarian Dugesia japonica has recently emerged as an animal model for developmental neurotoxicology and found to be sensitive to organophosphorus (OP) pesticides. While previous activity staining of D. japonica, which possess a discrete cholinergic nervous system, has shown acylthiocholine catalysis, it is unknown whether this is accomplished through an acetylcholinesterase (AChE), butyrylcholinesterase (BChE), or a hybrid esterase and how OP exposure affects esterase activity. Here, we show that the majority of D. japonica cholinesterase (DjChE) activity departs from conventional AChE and BChE classifications. Inhibition by classic protonable amine and quaternary reversible inhibitors (ethopropazine, donepezil, tacrine, edrophonium, BW284c51, propidium) shows that DjChE is far less sensitive to these inhibitors than human AChE, suggesting discrete differences in active center and peripheral site recognition and structures. Additionally, we find that different OPs (chlorpyrifos oxon, paraoxon, dichlorvos, diazinon oxon, malaoxon) and carbamylating agents (carbaryl, neostigmine, physostigmine, pyridostigmine) differentially inhibit DjChE activity in vitro. DjChE was most sensitive to diazinon oxon and neostigmine and least sensitive to malaoxon and carbaryl. Diazinon oxon-inhibited DjChE could be reactivated by the quaternary oxime, pralidoxime (2-PAM), and the zwitterionic oxime, RS194B, with RS194B being significantly more potent. Sodium fluoride (NaF) reactivates OP-DjChE faster than 2-PAM. As one of the most ancient true cholinesterases, DjChE provides insight into the evolution of a hybrid enzyme before the separation into distinct AChE and BChE enzymes found in higher vertebrates. The sensitivity of DjChE to OPs and capacity for reactivation validate the use of planarians for OP toxicology studies.

Toxic effects of ionic liquid 1-octyl-3-methylimidazolium bromide on the antioxidant defense system of freshwater planarian, Dugesia japonica.

The activities of antioxidant enzymes and the levels of glutathione (GSH) and malondialdehyde (MDA) were determined when freshwater planarian Dugesia japonica was exposed to different concentrations of 1-octyl-3-methylimidazolium bromide ([C8mim]Br) for one, three, and five days. The results showed that superoxide dismutase (SOD) activity began to increase in all treated groups after three days of exposure, while catalase (CAT) activity was inhibited after the first day, but increased notably on the fifth day except for the lowest concentration group. The activity of glutathione peroxidase (GPX) was induced from the first day of exposure and increased significantly after five days in all treated groups. During the experiment, the levels of intracellular GSH in all treated groups were higher than that of the control group. Changes in MDA suggest that [C8mim]Br is toxic to D japonica and may result in lipid peroxidation in planarian. Our results also indicate that GPX as well as GSH seem to be more sensitive biomarkers of oxidative stress compared with SOD and CAT.

De Novo Transcriptome Analysis Provides Insights into Immune Related Genes and the RIG-I-Like Receptor Signaling Pathway in the Freshwater Planarian (Dugesia japonica).

BackgroundThe freshwater planarian Dugesia japonica (D. japonica) possesses extraordinary ability to regenerate lost organs or body parts. Interestingly, in the process of regeneration, there is little wound infection, suggesting that D. japonica has a formidable innate immune system. The importance of immune system prompted us to search for immune-related genes and RIG-I-like receptor signaling pathways.ResultsTranscriptome sequencing of D. japonica was performed on an IlluminaHiSeq2000 platform. A total of 27,180 transcripts were obtained by Trinity assembler. CEGMA analysis and mapping of all trimmed reads back to the assembly result showed that our transcriptome assembly covered most of the whole transcriptome. 23,888 out of 27,180 transcripts contained ORF (open reading fragment), and were highly similar to those in Schistosoma mansoni using BLASTX analysis. 8,079 transcripts (29.7%) and 8,668 (31.9%) were annotated by Blast2GO and KEGG respectively. A DYNLRB-like gene was cloned to verify its roles in the immune response. Finally, the expression patterns of 4 genes (RIG-I, TRAF3, TRAF6, P38) in the RIG-I-like receptor signaling pathway were detected, and the results showed they are very likely to be involved in planarian immune response.ConclusionRNA-Seq analysis based on the next-generation sequencing technology was an efficient approach to discover critical genes and to understand their corresponding biological functions. Through GO and KEGG analysis, several critical and conserved signaling pathways and genes related to RIG-I-like receptor signaling pathway were identified. Four candidate genes were selected to identify their expression dynamics in the process of pathogen stimulation. These annotated transcripts of D. japonica provide a useful resource for subsequent investigation of other important pathways.

Behavioural responses of freshwater planarians after short-term exposure to the insecticide chlorantraniliprole

Recent advances in video tracking technologies provide the tools for a sensitive and reproducible analysis of invertebrate activity under stressful conditions nurturing the field of behavioural ecotoxicology. This study aimed to evaluate behavioural responses of the freshwater planarian Dugesia subtentaculata exposed to a model compound, chlorantraniliprole (CAP). This compound is an anthranilic diamide insecticide and due to its neurotoxic action can, at low concentrations, impair behaviour of exposed organisms. Behavioural endpoints measured included feeding and locomotor activities. Feeding responses were based on planarian predatory behaviour using Chironomus riparius larvae as prey. Locomotion was measured by the traditional planarian locomotor velocity (pLMV) assay and additionally using an automated video tracking system using a Zebrabox® (Viewpoint, France) device. While feeding and pLMV were significantly impaired at 131.7μg/L CAP, the video tracking system showed that total distance covered by planarians was significantly reduced at concentrations as low as 26.2μg/L CAP. Our results show that more advanced automated video recording systems can be used in the development of sensitive bioassays allowing a reliable, time- and cost-effective quantification of behaviour in aquatic invertebrates. Due to their ecological relevance, behavioural responses should not be disregarded in risk assessment strategies and we advocate the suitability of planarians as suitable organisms for behavioural ecotoxicological studies.

Freshwater Planarians as an Alternative Animal Model for Neurotoxicology.

Traditional toxicology testing has relied on low-throughput, expensive mammalian studies; however, timely testing of the large number of environmental toxicants requires new invitro and invivo platforms for inexpensive medium- to high-throughput screening. Herein, we describe the suitability of the asexual freshwater planarian Dugesia japonica as a new animal model for the study of developmental neurotoxicology. As these asexual animals reproduce by binary fission, followed by regeneration of missing body structures within approximately 1week, development and regeneration occur through similar processes allowing us to induce neurodevelopment "at will" through amputation. This short time scale and the comparable sizes of full and regenerating animals enable parallel experiments in adults and developing worms to determine development-specific aspects of toxicity. Because the planarian brain, despite its simplicity, is structurally and molecularly similar to the mammalian brain, we are able to ascertain neurodevelopmental toxicity that is relevant to humans. As a proof of concept, we developed a 5-step semiautomatic screening platform to characterize the toxicity of 9 known neurotoxicants (consisting of common solvents, pesticides, and detergents) and a neutral agent, glucose, and quantified effects on viability, stimulated and unstimulated behavior, regeneration, and brain structure. Comparisons of our findings with other alternative toxicology animal models, such as zebrafish larvae and nematodes, demonstrated that planarians are comparably sensitive to the tested chemicals. In addition, we found that certain compounds induced adverse effects specifically in developing animals. We thus conclude that planarians offer new complementary opportunities for developmental neurotoxicology animal models.

Two distinct roles of the yorkie/yap gene during homeostasis in the planarian Dugesia japonica.

Adult planarians possess somatic pluripotent stem cells called neoblasts that give rise to all missing cell types during regeneration and homeostasis. Recent studies revealed that the Yorkie (Yki)/Yes-associated protein (YAP) transcriptional coactivator family plays an important role in the regulation of tissue growth during development and regeneration, and therefore we investigated the role of a planarian yki-related gene (termed Djyki) during regeneration and homeostasis of the freshwater planarian Dugesia japonica. We found that knockdown of the function of Djyki by RNA interference (RNAi) downregulated neoblast proliferation and caused regeneration defects after amputation. In addition, Djyki RNAi caused edema during homeostasis. These seemingly distinct defects induced by Djyki RNAi were rescued by simultaneous RNAi of a planarian mats-related gene (termed Djmats), suggesting an important role of Djmats in the negative regulation of Djyki, in accordance with the conservation of the functional relationship of these two genes during the course of evolution. Interestingly, Djyki RNAi did not prevent normal protonephridial structure, suggesting that Djyki RNAi induced the edema phenotype without affecting the excretory system. Further analyses revealed that increased expression of the D. japonica gene DjaquaporinA (DjaqpA), which belongs to a large gene family that encodes a water channel protein for the regulation of transcellular water flow, promoted the induction of edema, but not defects in neoblast dynamics, in Djyki(RNAi) animals. Thus, we conclude that Djyki plays two distinct roles in the regulation of active proliferation of stem cells and in osmotic water transport across the body surface in D. japonica.

Disturbances to neurotransmitter levels and their metabolic enzyme activity in a freshwater planarian exposed to cadmium

Using specific neurobehaviors as endpoints, previous studies suggested that planarian neurotransmission systems could be targets of Cd neurotoxicity. However, direct evidence for disturbed neurotransmission systems by Cd in treated planarians is still lacking. In planarians, dopamine (DA) and serotonin (5-HT) play critical roles in neuromuscular function, but little is known about their metabolic degradation. Therefore, in this study, we attempted to determine the appearances of DA, 5-HT, and their metabolic products in the freshwater planarian Dugesia japonica, characterize the activity of enzymes involved in their metabolism, and investigate the effects of Cd on planarian 5-HTergic and DAergic neurotransmission systems. Only DA, 5-HT, and 5-hydroxyindole-3-acetic acid (5-HIAA) were found in planarian tissues. Further enzymatic study revealed the activity of planarian monoamine oxidase (MAO) but not catechol-O-methyl transferase (COMT). These findings suggest that planarian MAO catalyzes the metabolism of 5-HT into 5-HIAA. However, DA metabolites from the MAO-involved metabolic pathway were not found, which might be due to a lack of COMT activity. Finally, in Cd-treated planarians, tissue levels of 5-HT and DA were decreased and MAO activity altered, suggesting that planarian neurotransmission systems are disturbed following Cd treatment.

Cloning and characterization of DjPRPS gene in freshwater planarian Dugesia japonica

Objective: To clone the phosphoribosyl pyrophosphate synthetase gene from Dugesia japonica, and to do the bioinformatics and spatial expression analysis of the gene. Methods: The phosphoribosyl pyrophosphate synthetase gene (DjPRPS) was cloned from Dugesia japonica using the rapid amplification of cDNA ends method and its sequence was analyzed with biological softwares. Spatial expression pattern of the gene was detected by whole-mount in situ hybridization. Results: The full length cDNA of DjPRPS was 1,086 bp, containing a 80 bp of 5'-untranslated region, a 55 bp of 3'- untranslated region and a 951 bp of open reading frame encoding a protein of 316 amino acids with a calculated molecular mass of 35.18 kDa and isoelectric point of 8.14. The deduced amino acid sequence shared the conserved eukaryotic PRPS family motif. Phylogenetic analysis supported the traditional concept of zoological taxonomy in large scale. Intense positive signals were detected as bilateral lines of patches in the ventral side where the ovaries and yolk glands located in the intact sexual planarians. In the regenerating planarians, the expression pattern was similar to that of the intact samples, and no intense transcipt signals were detected in the blastema. Conclusion: DjPRPS sequence was highly homologous with that of other eukaryotic phosphoribosyl pyrophosphate synthetase gene. Intense transcript signals of DjPRPS were not detected in the blastema but in ovaries and yolk glands, suggesting that its function was probably reproduction-related and maybe not involved in regeneration.

Effects of bisphenol A, two synthetic and a natural estrogens on head regeneration of the freshwater planarians, Dugesia japonica

Regeneration is a vital physiological process for survival of adult organisms. Bisphenol A (BPA), diethystilbestrol (DES), 17α-ethinylestradiol (EE2), and 17β-estradiol (E2) were examined for their effects on head formation in decapitated freshwater planarians (Dugesia japonica). The median lethal concentration of the test chemicals and the median inhibitory concentration for decapitated planarians and different regenerative endpoints, were determined for 3–7 d of exposure. For planarian head regeneration, the no-observed-effect level of BPA and DES was 0.05 mg L−1 and 1 mg L−1 for EE2 and E2. The effects of BPA, DES, and EE2 on asexual planarian regeneration did not correspond with the order of their estrogenic potencies, suggesting that the effects, at least partially, were due to general toxicity rather than their estrogenicity. In view of the published data on concentrations of xenoestrogens in water bodies versus the nominal levels tested here, this study suggests that the current environmental levels of BPA, DES, EE2, and E2 have no immediate adverse effects on freshwater planarian regeneration.

Infertility in the hyperplasic ovary of freshwater planarians: the role of programmed cell death.

Ex-fissiparous planarians produce infertile cocoons or, in very rare cases, cocoons with very low fertility. Here, we describe the features of programmed cell death (PCD) occurring in the hyperplasic ovary of the ex-fissiparous freshwater planarian Dugesia arabica that may explain this infertility. Based on TEM results, we demonstrate a novel extensive co-clustering of cytoplasmic organelles, such as lysosomes and microtubules, and their fusion with autophagosomes during the early stage of oocyte cell death occurring through an autophagic pattern. During a later stage of cell death, the generation of apoptotic vesicles in the cytoplasm can be observed. The immunohistochemical labeling supports the ultrastructural results because it has been shown that the proapoptotic protein bax was more highly expressed in the hyperplasic ovary than in the normal one, whereas the anti-apoptotic protein bcl2 was slightly more highly expressed in the normal ovary compared to the hyperplasic one. TUNEL analysis of the hyperplasic ovary confirmed that the nuclei of the majority of differentiating oocytes were TUNEL-positive, whereas the nuclei of oogonia and young oocytes were TUNEL-negative; in the normal ovary, oocytes are TUNEL-negative. Considering all of these data, we suggest that the cell death mechanism of differentiating oocytes in the hyperplasic ovary of freshwater planarians is one of the most important factors that cause ex-fissiparous planarian infertility. We propose that autophagy precedes apoptosis during oogenesis, whereas apoptotic features can be observed later.

Pharmacological assessment of methamphetamine-induced behavioral hyperactivity mediated by dopaminergic transmission in planarian Dugesia japonica

The freshwater planarian Dugesia japonica has a simple central nervous system (CNS) and can regenerate complete organs, even a functional brain. Recent studies demonstrated that there is a great variety of neuronal-related genes, specifically expressed in several domains of the planarian brain. We identified a planarian dat gene, named it D. japonica dopamine transporter (Djdat), and analyzed its expression and function. Both in situ hybridization and immunofluorescence revealed that localization of Djdat mRNA and protein was the same as that of D. japonica tyrosine hydroxylase (DjTH). Although, dopamine (DA) content in Djdat(RNAi) planarians was not altered, Djdat(RNAi) planarians showed increased spontaneous locomotion. The hyperactivity in the Djdat(RNAi) planarians was significantly suppressed by SCH23390 or sulpiride pretreatment, which are D1 or D2 receptor antagonists, respectively. These results suggest that planarians have a Djdat ortholog and the ability to regulate dopaminergic neurotransmission and association with spontaneous locomotion.

Purification, characterization and induction of a C-type lectin in the freshwater planarian Dugesia japonica

Abstract Lectins are important components of the immune defense system of invertebrates. Given their important functions, numerous investigations have been carried out on the characterization and function of lectins in invertebrates. However, lectin studies with the freshwater planarian, an evolutionarily important animal, are rare. In this paper, we demonstrate agglutination of glutaraldehyde treated erythrocytes by a lectin with preference for rabbit erythrocytes. The result of hemagglutinating activity inhibition assays with several carbohydrates showed the most potent inhibitor was maltose. A natural lectin from the crude homogenates of freshwater planarian Dugesia japonica was purified by single step affinity chromatography using amylose-coupled agarose. The purified protein appeared as one band with a molecular mass of 350 kDa in PAGE, and as one band, approximately 56 kDa, in SDS-PAGE. The purified lectin showed dependence on calcium. The activity of the purified lectin was inhibited at temperatures greater than 50°C and showed a pH optimum between 5–8. The purified lectin also has binding activity to the Gram-negative bacteria E. coli, and the Gram-positive bacteria B. subtilis. Furthermore, the purified lectin obtained from injured and bacteria-induced planarians showed increased agglutinating activity against rabbit erythrocytes. These results suggest that the purified lectin may play an important role in the innate immunity of the freshwater planarian.

Detection and characterization of phenoloxidase in the freshwater planarian Dugesia japonica

Freshwater planarians have traditionally been favored animal models in regenerative and developmental biology. Little is known about the immunological research in these animals. Here, we provide spectrophotometric, electrophoretic and histochemical evidence demonstrating the presence and characterization of phenoloxidase (EC 1.14.18.1; PO) activity in the planaria Dugenia japonica. PO activity was optimal at pH 7.5 and at a temperature of 45 degrees C. ProPO in the humoral fluid was readily activated by trypsin and zymosan, and was susceptible to phenylthiourea. Two bands of PO activity in the humoral fluid with 150 and 300 kDa by native PAGE and a single band with 150 kDa after SDS-PAGE were resolved. Enzyme histochemical staining demonstrated the presence of PO in the epidermal cell and pharyngeal epithelium, which indicate that PO could serve as a protecting agent against environmental pathogens in D. japonica.

Analysis of motor function modulated by cholinergic neurons in planarian dugesia japonica

Recent studies of the freshwater planarian Dugesia japonica have revealed fundamental mechanisms and unique aspects of neuroscience and neuroregeneration. Here, we identified the gene for planarian choline acetyltransferase (Djchat), which is essential for acetylcholine (ACh) biosynthesis. Immunofluorescence studies using anti-Dugesia japonica ChAT (DjChAT) antibody revealed that cholinergic neurons are widely distributed in the planarian nervous system, including the brain, ventral nerve cords, optic nerves, and pharyngeal nerve plexus. In order to investigate the function of cholinergic neurons in planarians, we used both pharmacological and RNA interference (RNAi) approaches. Administration of physostigmine (an acetylcholinesterase inhibitor) clearly elevated the amount of ACh, and then induced sudden muscle contraction behavior in a concentration-dependent manner. In addition, we found that pretreatment with tubocurarine (a muscle nicotinic ACh receptor antagonist) or atropine (a non-selective muscarinic ACh receptor antagonist), but not pretreatment with mecamylamine (a neural nicotinic ACh receptor antagonist), significantly extended the latency time for physostigmine-induced contraction behavior, suggesting that muscle nicotinic ACh receptors and muscarinic ACh receptors contribute to physostigmine-induced contraction behavior. We also confirmed that ACh biosynthesis ability and DjChAT-immunoreactivity were eliminated in Djchat(RNAi) planarians. Moreover, the decrease of the level of ACh induced by Djchat(RNAi) caused extension of the latency time for contraction behavior. Our findings support the possibility that the cholinergic functions of planarians are similar to those of vertebrates, suggesting that planarians are simple but useful model organisms for getting insight into the cholinergic nervous system in higher animals.

Cloning and expression analysis of hsp70 gene from freshwater planarian Dugesia japonica

Heat shock protein 70 (HSP70) is an important member of the heat shock protein family. It plays a key role in the process of protecting cells by facilitating the folding of nascent peptides as well as the cellular stress response. We isolated and sequenced a full-length HSP70 cDNA from planarian Dugesia japonica (designated Djhsp70) using rapid amplification of cDNA ends (RACE). Djhsp70 cDNA is highly homologous to other reported hsp70 family genes, and the deduced amino acid sequence shares several eukaryotic HSP70 family motifs. The length of the Djhsp70 ORF from planarian genomic DNA and cDNA was identical, which indicated the absence of introns in the Djhsp70 gene. Semi-quantitative RT-PCR was employed to detect the expression levels of Djhsp70 in response to stressors. Our results indicated that Djhsp70 was an inducible gene, expressed in response to temperature changes, amputation and starvation. Interestingly, the phylogenetic analysis of DjHSP70 supports the idea that planarians belong to a new phylogenetic position — Lophotrochozoans. This is the first molecular analysis of a heat shock protein gene in planarians.