SoxB family genes delay regeneration and cause abnormal movement in Dugesia japonica.

SoxB subfamily is an important branch of Sox family and plays a key role in animal physiological process, but little is known about their function in planarian regeneration. This study aims to evaluate the function of DjSoxB family genes in intact and regenerating planarians Dugesia japonica. Here, we amplify the full-length cDNA of DjSoxB1 and DjSoxB2 in D. japonica by rapid amplification of the cDNA ends (RACE), detect the expression of DjSoxB family genes in planarian. The results show that DjSoxBs are expressed in parenchymal tissue and the hybridization signals partially disappear after irradiation indicates DjSoxB family genes are expressed in neoblasts. After the RNA interference (RNAi) of DjSoxB1, DjSoxB2 and DjSoxB3 separately, the numbers of proliferative cells are all reduced that causes planarians show slower growth of blastema in the early stage of regeneration, and nerves of planarians are affected that the movement speed of planarians decreases in varying degrees. Specially, planarians in the DjSoxB3 RNAi group show shrinkage and twisting. Overall, this study reveals that DjSoxB family genes play a role in cell proliferation during regeneration. They also play an important role in the maintenance of normal nerve function and nerve regeneration. These results provide directions for the functional study of SoxB family genes and provide an important foundation for planarian regeneration.

Ecotoxicological evaluation and regeneration impairment of planarians by dibutyl phthalate.

Commonly utilized as a plasticizer in the food and chemical sectors, Dibutyl phthalate (DBP) poses threats to the environment and human well-being as it seeps or moves into the surroundings. Nevertheless, research on the harmfulness of DBP to aquatic organisms is limited, and its impact on stem cells and tissue regeneration remains unidentified. Planarians, recognized for their robust regenerative capabilities and sensitivity to aquatic pollutants, are emerging animal models in toxicology. This study investigated the comprehensive toxicity effects of environmentally relevant levels of DBP on planarians. It revealed potential toxicity mechanisms through the use of immunofluorescence, chromatin dispersion assay, Western blot, quantitative real-time fluorescence quantitative PCR (qRT-PCR), chromatin behavioral and histological analyses, immunofluorescence, and terminal dUTP nickel-end labeling (TUNEL). Findings illustrated that DBP caused morphological and motor abnormalities, tissue damage, regenerative inhibition, and developmental neurotoxicity. Further research revealed increased apoptosis and suppressed stem cell proliferation and differentiation, disrupting a balance of cell proliferation and death, ultimately leading to morphological defects and functional abnormalities. This was attributed to oxidative stress and DNA damage caused by excessive release of reactive oxygen species (ROS). This exploration furnishes fresh perspectives on evaluating the toxicity peril posed by DBP in aquatic organisms.

Neurotoxicity of Glyphosate to Planarian Dugesia japonica.

As one of the most widely used herbicides in agricultural industry, the residues of glyphosate (GLY) are frequent environmental pollutants. Freshwater planarian Dugesia japonica has been developed as a model for neurotoxicology. In this study, the effects of GLY on locomotion and feeding behavior, as well as neuroenzyme activities and mRNA expressions of D. japonica were determined. Additionally, histochemical localization was executed to explore the damage to the central nervous system (CNS) of planarians stressed by GLY. The results showed that the locomotor velocity, ingestion rate and the neuroenzyme activity were inhibited and the gene expressions were altered. Also, histo-architecture injury to CNS of planarians upon GLY exposure in a time-dependent manner was observed. Collectively, our results indicate that GLY can cause neurotoxicity to freshwater planarians representing as reduction in locomotor velocity and feeding rate by disturbing the neurotransmission systems and damaging the structure of CNS.

Autophagy-related DjAtg1-1 plays critical role in planarian regeneration by regulating proliferation and cell death.

Autophagy is an intracellular degradation process and plays key roles in energy recycle and homeostasis maintenance during planarian regeneration. Although planarians provide an ideal model organism for studying autophagy in vivo, the molecular mechanism of planarian autophagy is still unknown. Here, we identify three autophagy-related (Atg) gene 1 homologs from Dugesia japonica and study their roles in planarian regeneration. Both DjATG1-1 and DjATG1-2 proteins show homology to vertebrate unc-51 like autophagy activating kinase 1 (ULK1) and ULK2, DjATG1-3 shows homology to vertebrate ULK3. In contrast to the ubiquitously expressed DjAtg1-1 and DjAtg1-3, DjAtg1-2 is mainly expressed in the intestine branches and epidermis. All the three DjAtg1s can respond to planarian regeneration and starvation. Both DjAtg1-1 and DjAtg1-2 are expressed in the reproductive organs of the starved sexual worms. DjAtg1-1 or DjAtg1-3 RNAi leads to head lysis and death of starved planarians, accompanied by exhaustion of neoblasts. DjAtg1-1 RNAi causes autophagy and regeneration defects and decreases proliferation and cell death; both DjAtg1-2 and DjAtg1-3 RNAi cause no autophagy or regeneration defect but increase cell death during regeneration. Our findings uncover the roles of DjAtg1s in autophagy and regeneration of planarian and highlight the links between proliferation, cell death, and autophagy during regeneration.

PBDEs disrupt homeostasis maintenance and regeneration of planarians due to DNA damage, proliferation and apoptosis anomaly.

Polybrominated diphenyl ethers (PBDEs) are widely used as brominated flame retardants in the manufacturing industry, belonging to persistent organic pollutants in the environment. Planarians are the freshwater worms, with strong regenerative ability and extreme sensitivity to environmental toxicants. This study aimed to evaluate the potential acute comprehensive effects of PBDE-47/-209 on freshwater planarians. Methods to detect the effects include: detection of oxidative stress, observation of morphology and histology, detection of DNA fragmentation, and detection of cell proliferation and apoptosis. In the PBDE-47 treatment group, planarians showed increased oxidative stress intensity, severe tissue damage, increased DNA fragmentation level, and increased cell proliferation and apoptosis. In the PBDE-209 treatment group, planarians showed decreased oxidative stress intensity, slight tissue damage, almost unchanged DNA fragmentation level and apoptosis, proliferation increased only on the first day after treatment. In conclusion, both PBDE-47 and PBDE-209 are dangerous environmental hazardous material that can disrupt planarians homeostasis, while the toxicity of PBDE-47 is sever than PBDE-209 that PBDE-47 can lead to the death of planarians.

Antioxidant responses and lipid peroxidation can be used as sensitive indicators for the heavy metals risk assessment of the Wei River: a case study of planarian Dugesia Japonica.

PURPOSE: To verify antioxidant responses and lipid peroxidation can be used as sensitive indicators for the risk assessment of the Wei River. MATERIAL AND METHODS: We investigate the effects of the Wei River on oxidative stress of planarian Dugesia japonica by antioxidant parameters, and use ICP-MS to measure the heavy metals in the Wei River. Then, we observe the effects of three common heavy metal ions (Cr3+, Hg2+, Pb2+) on the regeneration of planarians on morphological and histological levels. RESULTS: The significant changes of antioxidant parameters (SOD, CAT, GPx, GST, T-AOC) and MDA content indicate that oxidative stress is induced after the Wei River exposure on planarians, though the heavy metals in the Wei River are not exceeding the standards. Then, the regeneration of planarians shows different degree of morphological and histological damage after Cr3+, Hg2+ and Pb2+ exposure. CONCLUSION: We speculate that the heavy metal ions in the Wei River, especially Cr3+, Hg2+ and Pb2+, may give rise to oxidative damage on planarians. These findings illustrate that planarian can serve as an indicator of aquatic ecosystem pollution, antioxidant responses and lipid peroxidation can also be used as sensitive indicators and provide an excellent opportunity for urban river risk assessment.

The negative effect of the PI3K inhibitor 3-methyladenine on planarian regeneration via the autophagy signalling pathway.

As an important PI3K (VPS34) inhibitor, 3-methyladenine (3-MA) can block the formation of autophagic vesicles in animals. Most toxicological studies using 3-MA have shown that 3-MA leads to serious disorders via autophagy suppression in mammals. However, no toxicological research on 3-MA has been performed on individuals undergoing regeneration. The freshwater planarian has powerful regenerative capability, and it can regenerate a new brain in 5 days and undergo complete adult individual remodelling in approximately 14 days. Moreover, it is also an excellent model organism for studies on environmental toxicology due to its high chemical sensitivity and extensive distribution. Here, Dugesia japonica planarians were treated with 3-MA, and the results showed that autophagy was inhibited and Djvps34 expression levels were down-regulated. After exposure to 10 mM 3-MA for 18 h, all the controls showed normal phenotypes, while one-half of the planarians treated with 3-MA showed morphological defects. In most cases, an ulcer appeared in the middle of the body, and a normal phenotype was restored 7 days following 3-MA exposure. During regeneration, disproportionate blastemas with tissue regression were observed. Furthermore, 3-MA treatment suppressed stem cell proliferation in intact and regenerating worms. These findings demonstrate that autophagy is indispensable for tissue homeostasis and regeneration in planarians and that 3-MA treatment is detrimental to planarian regeneration via its effect on the autophagy pathway.

[Functional analysis of autophagy-related gene Atg6 in planarian central nervous system regeneration].

Autophagy-related gene 6 (Atg6) plays an essential role in autophagy, and loss of its function impairs neurogenesis. Planarian is a good model for the study of the central nervous system (CNS) regeneration. It can regenerate a new head de novo in 1 week following decapitation. Therefore, functional analysis of Atg6 in planarian CNS regeneration is very important for understanding of autophagy in the regulation of neurogenesis. In this work, we reported the molecular characteristics of Atg6 in Dugesia japonica (DjAtg6) for the first time and examined its function by RNAi. The full-length cDNA of DjAtg6 is 1366 bp encoding 423 amino acids. The deduced amino sequence of DjAtg6 contains the coil-coil domain and ß-α-repeated autophagy-specific domain shared by ATG6/Beclin 1 family. Following amputation before and after the pharynx, DjAtg6 transcripts increased and were mainly distributed in the newly regenerated brain structure. RNAi-DjAtg6 delayed planarian head regeneration with a small size of brain, and decreased the expression levels of neural-related genes. In addition, our results revealed that RNAi-DjAtg6 did not affect the stem cell proliferation, but down-regulated the cell migration-related genes mmp1 and mmp2. Furthermore, RNAi-mmp1 and RNAi-mmp2 delayed planarian head regeneration. Therefore, our results suggest that DjAtg6 is important for planarian CNS regeneration. The abnormal CNS regeneration caused by RNAi-DjAtg6 may be related to cell migration, but the detailed mechanism needs to be further investigated.

Djmek is involved in planarian regeneration by regulation of cell proliferation and apoptosis.

Dugesia japonica, belonging to Platyhelminthes, plays an important role in the animal evolution and is well known for its extraordinary regenerative ability. Mitogen activated protein kinase (MAPK) pathway is an important cell signaling pathway that converts extracellular stimuli into a wide range of cellular responses. The MAP-extracellular signal-regulated kinase (MEK) is a main component of MAPK/ERK signaling, but there are few studies on mek gene in planarians. In this study, we observe the expression patterns of Djmek1 and Djmek2 in planarians, and find that both of the two genes are required for the planarian regeneration. At the same time, we also find that both Djmek1 and Djmek2 are involved in the planarian regeneration by regulation of cell proliferation and apoptosis. Together, our findings show that the functions of the two genes are similar and complementary, and they play an important role in the regeneration of planarians.

Expression and functional analysis of flotillins in Dugesia japonica.

FLOTILLIN-1 and FLOTILLIN-2 are membrane rafts associated proteins that have been implicated in insulin and growth factor signaling, endocytosis, cell migration, proliferation, differentiation, cytoskeleton remodeling and membrane trafficking. Furthermore, FLOTILLINs also play important roles in the progression of cancer and neurodegenerative diseases. In this study, the roles of flotillins are investigated in planarian Dugesia japonica. The results show that Djflotillin-1 and Djflotillin-2 play a key role in homeostasis maintenance and regeneration process by regulating the proliferation of the neoblast cells, they are not involved in the maintenance and regeneration of the central nervous system in planarians.

A novel sigma class glutathione S-transferase gene in freshwater planarian Dugesia japonica: cloning, characterization and protective effects in herbicide glyphosate stress.

As the top-selling herbicide in the world, glyphosate distributes widely in natural environment and its influence on the ecological security and human health has attracted more and more concern. Glutathione S-transferases (GSTs) are a well-characterized superfamily of isoenzymes for cellular defense against exogenous toxic substances and therefore protect organisms from injury. In this study, the complete cDNA sequence of GST gene (named as Dja-GST) in freshwater planarian Dugesia japonica was firstly cloned by means of RACE method. The full-length Dja-GST comprises of 706 nucleotides which encodes a polypeptide of 200 amino acids. Dja-GST has two representative GST domains at the N- and C-termini. The conservative GST-N domain includes G-site Y8, F9, R14, W39, K43, P52 and S64, while the variable GST-C domain contains H-site K104, V156, D159 and L161. Sequence analysis, phylogenetic tree reconstruction and multiple alignment collectively indicate that Dja-GST belongs to the Sigma class of GST superfamily. Also, GST gene expression profile, GST enzymatic activity and MDA content in response to glyphosate exposure were systematically investigated and the correlations among them were analyzed. The results suggest that glyphosate exposure modified the mRNA transcription and enzymatic activity of GST, as well as the MDA content in planarians, indicating that Dja-GST might play an important part in organisms defending against oxidative stress induced by glyphosate. This work lays a molecular foundation for further exploring the exact functions of Dja-GST and gives an important implication for evaluating the ecological environment effects of herbicide glyphosate.

Biomarkers of Planarian Dugesia japonica in Response to Herbicide Glyphosate Exposure.

As the worldwide top-selling herbicide, glyphosate is ubiquitously distributed in the natural environment, and its influence on the ecological safety and human health has being increasingly concerned. In this study, mRNA expressions of GPX and three heat shock protein genes in freshwater planarian Dugesia japonica in response to glyphosate were determined, and two oxidative stress parameters were measured. The results suggested that GPX activity can be used as a more sensitive biomarker in contrast with GPX gene expression, and mRNA expressions of Hsp70, Hsp90 genes are more sensitive than Hsp40 for planarians in response to glyphosate stress. Besides, the deduced T-AOC as well as varied GPX activity and mRNA expression levels of Hsps also indicated that glyphosate exposure would inhibit antioxidation and induce oxidative stress in D. japonica, while specific antioxidant systems and stress proteins tried to protect cells by their own regulation. The results of this study will be helpful to elucidate the stress response mechanisms of freshwater planarians to herbicide glyphosate.

The regulation of rapamycin in planarian Dugesia japonica Ichikawa & Kawakatsu, 1964 regeneration according to TOR signaling pathway.

The freshwater planarian mostly lives in the upper reaches of springs and rivers. Generally, it is realized as a suitable warning indicator of environmental toxicants. The freshwater planarian Dugesia japonica has a powerful regenerative capability and can regenerate a new individual including a complete central nervous system in one week. Rapamycin is an inhibitor of mammalian TORC1 (target of rapamycin complex-1) and used in the treatment of some diseases like cancer, cardiovascular and neurological diseases. However, the roles of rapamycin in the regulation of planarian regeneration remain to be elucidated. In present study, freshwater planarians D. japonica were firstly treated with 1 µM rapamycin for 18 h exposures and the expression patterns of Djtor was analyzed by the whole-mount in situ hybridization (WISH). Our results indicated rapamycin could strongly inhibit Djtor expression in planarian D. japonica and cause asymmetric blastemas and neuronal defects in planarians. Furthermore, knockdown of Djtor gene in planarians using RNA interference resulted in the suppression of downstream autophagy genes. These findings suggested that rapamycin might regulate freshwater planarian regeneration via Djtor signaling pathway.

Identification of a potential tissue-specific biomarker cathepsin L-like gene from the planarian Dugesia japonica: Molecular cloning, characterization, and expression in response to heavy metal exposure.

Heavy metal pollution is a global health issue affecting people worldwide, and the exploration of sensitive biomarkers to assess the toxicity of heavy metals is an important work for researchers. Cathepsin L, role as a tissue-specific biomarker to assess the biological effects of environmental pollutants, has not received much attention. In this work, the full-length cDNA of cathepsin L gene from the planarian Dugesia japonica (designated DjCatL) was cloned by rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of DjCatL is 1161 bp, which encodes a protein of 346 amino acids with a molecular weight of 39.03 kDa. Sequence analysis revealed that DjCatL contains highly conserved ERF/WNIN, GNFD, and GCXGG motifs, which are the features of the cathepsin L protein family. Whole-mount in situ hybridization (WISH) results revealed that the transcripts of DjCatL are specifically distributed in the intestinal system, suggesting that this gene is related to food digestion in planarians. Both quantitative polymerase chain reaction (qPCR) and WISH results revealed that the transcriptional levels of DjCatL are inhibited significantly by heavy metal (Cd2+, Hg2+, and Cu2+) exposure in a dose-dependent manner. Therefore, we proposed that cathepsin L can be used as a tissue-specific biomarker to assess the heavy metal pollution in the aquatic environment.

Molecular cloning, characterization, expression and enzyme activity of catalase from planarian Dugesia japonica in response to environmental pollutants.

Catalase (CAT) is an important antioxidant enzyme that protects aerobic organisms against oxidative damage by degrading hydrogen peroxide to oxygen and water. CAT mRNAs have been cloned from many species and employed as useful biomarkers of oxidative stress. In the present study, we cloned the cDNA sequence of CAT gene from freshwater planarian Dugesia japonica (designated as DjCAT) by means of RACE method. Sequence analysis and multiple alignment jointly showed that the full-length cDNA sequence consists of 1734 nucleotides, encoding 506 amino acids. Three catalytic amino acid residues of His71, Asn144 and Tyr354, two CAT family signature sequences of a proximal active site signature (60FDRERIPERVVHAKGGGA77) and a heme-ligand signature motif (350RLFSYRDTQ358) are highly conserved, suggesting that the DjCAT belongs to the NADPH and heme-binding CAT family and has similar functions. In addition, the transcriptional level of CAT gene and activity of CAT enzyme upon acute exposure of environmental pollutants glyphosate and 1-decyl-3-methylimidazolium bromide ([C10mim]Br) were investigated systematically. The variation of CAT mRNA expression in D. japonica was quantified by real-time PCR and the results indicated that it was up-regulated after exposure to glyphosate or [C10mim]Br with a dose-dependent manner but not linearly. Even though the variation trend of CAT activity upon glyphosate stress was not monotonously increased and inconsistent with that after [C10mim]Br exposure on day 1 and 3 sampling time, with the duration prolonged to day 5 they both presented a dose-dependent increase and the differences achieved extreme significance in all treated groups compared to the control. These findings suggested that DjCAT plays an important role in antioxidant defense in D. japonica, and the mRNA expression of CAT would also be used as an effective biomarker to monitor the pollution in aquatic environment just like its corresponding enzyme.

Genotoxicity Evaluation of an Urban River on Freshwater Planarian by RAPD Assay.

The aim of this study was to evaluate the genotoxic potential of an urban river - the Wei River in Xinxiang, China using randomly amplified polymorphic DNA (RAPD) assay in planarians. The results showed that the total number of polymorphic bands and varied bands in RAPD patterns of treated planarians decreased with the water sample site far away from the sewage outlet of a factory. In addition, the genome template stability of treated groups decreased and the degree of the decline was negatively related to the distance between the sample site and the sewage outlet, suggesting that the Wei River water had genotoxicity effects on planarians and strengthening the management of the Wei River was necessary. Furthermore, this work also indicated that RAPD assay in planarians was a very promising test for environmental monitoring studies.

Genotoxicity evaluation of ionic liquid 1-octyl-3-methylimidazolium bromide in freshwater planarian Dugesia japonica using RAPD assay.

The randomly amplified polymorphic DNA (RAPD) assay has been used to detect DNA alternation and mutation recently. However, the effectiveness of this method in detecting DNA damage in planarians, a model organism for assessing the toxicity of environmental pollutants is unknown. In the present study, RAPD assay was used to detect the DNA damage in planarians treated by the ionic liquid 1-octyl-3-methylimidazolium bromide ([C8mim]Br) for the first time. Among the 20 test RAPD primers, 13 primers with 60-70% GC content produced unique polymorphic band profiles. A total of 60 bands were observed in the untreated control planarians. In comparison with the control group, the [C8mim]Br-treated groups displayed differences in RAPD patterns in the band intensity, disappearance of normal bands and appearance of new bands. The variation of RAPD profiles showed both concentration- and time-effect relationships. Meanwhile, the genomic template stability (GTS) of treated planarians decreased and exhibited negative correlation to the exposure concentration and time of [C8mim]Br. Our results suggested that [C8mim]Br had genotoxic effects on planarians, and this DNA damage analysis would lay the foundation for further elucidating the toxicity mechanisms of ionic liquids on planarians. Furthermore, RAPD analysis was proved to be a highly sensitive method for the detection of DNA damage induced by environmental pollutants like toxic chemicals on planarians.

Toxicity of herbicide glyphosate to planarian Dugesia japonica and its potential molecular mechanisms.

Glyphosate (GLY) is one of the most widely used agrochemicals in the world, and its exposure has become a public health concern. The freshwater planarian is an ideal test organism for detecting the toxicity of pollutants and has been an emerging animal model in toxicological studies. Nevertheless, the underlying toxicity mechanism of GLY to planarians has not been thoroughly explored. To elucidate the toxicity effects and molecular mechanism involved in GLY exposure of planarians, we studied the acute toxicity, histological change, and transcriptional response of Dugesia japonica subjected to GLY. Significant morphological malformations and histopathological changes were observed in planarians after GLY exposure for different times. Also, a number of differentially expressed genes (DEGs) were obtained at 1, 3 and 5 d after exposure; Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of these DEGs were performed, and a global and dynamic view was obtained in planarians upon GLY exposure at the transcriptomic level. Furthermore, real-time quantitative PCR (qRT-PCR) was conducted on nine DEGs associated with detoxification, apoptosis, stress response, DNA repair, etc. The expression patterns were well consistent with the RNA sequencing (RNA-seq) results at different time points, which confirmed the reliability and accuracy of the transcriptome data. Collectively, our results established that GLY could pose adverse effects on the morphology and histo-architecture of D. japonica, and the planarians are capable of responding to the disadvantageous stress by dysregulating the related genes and pathways concerning immune response, detoxification, energy metabolism, DNA damage repair, etc. To the best of our knowledge, this is the first report of transcriptomic analyses of freshwater planarians exposed to environmental pollutants, and it provided detailed sequencing data deriving from transcriptome profiling to deepen our understanding the molecular toxicity mechanism of GLY to planarians.

Toxicity assessment of microcystin-leucine arginine in planarian Dugesia japonica.

Microcystin-leucine arginine (MC-LR), a representative cyanobacterial toxin, poses an increasing and serious threat to aquatic ecosystems. Despite investigating its toxic effects in various organisms and cells, the toxicity to tissue regeneration and stem cells in vivo still needs to be explored. Planarians are ideal regeneration and toxicology research models and have profound implications in ecotoxicology evaluation. This study conducted a systemic toxicity evaluation of MC-LR, including morphological changes, growth, regeneration, and the underlying cellular and molecular changes after MC-LR exposure, which were investigated in planarians. The results showed that exposure to MC-LR led to time- and dose-dependent lethal morphological changes, tissue damage, degrowth, and delayed regeneration in planarians. Furthermore, MC-LR exposure disturbed the activities of antioxidants, including total superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, and total antioxidant capacity, leading to oxidative stress and DNA damage, and then reduced the number of dividing neoblasts and promoted apoptosis. The results demonstrated that oxidative stress and DNA damage induced by MC-LR exposure caused apoptosis. Excessive apoptosis and suppressed neoblast activity led to severe homeostasis imbalance. This study explores the underlying mechanism of MC-LR toxicity in planarians and provides a basis for the toxicity assessment of MC-LR to aquatic organisms and ecological risk evaluation.

cDNA cloning of heat shock protein 90 gene and protein expression pattern in response to heavy metal exposure and thermal stress in planarian Dugesia japonica.

Heat shock protein 90 (HSP90) is an abundant and highly conserved molecular chaperone, playing important roles in multiple cellular stress responses. The full-length cDNA of planarian Dugesia japonica Hsp90 (designated DjHsp90) was firstly cloned using rapid amplification of cDNA ends (RACE) techniques. It is 2,354 bp, including an open reading frame (ORF) of 2,148 bp encoding a polypeptide of 715 amino acids with all five HSP90 family signatures. We sequenced the ORF sequences from genomic DNA, and found only one intron (48 bp) existed in Djhsp90 gene structure. We used western blot and immunohistochemistry to analyze the expression pattern of DjHsp90 in response to heavy metal exposure and thermal stress at the protein level. Our results show that low doses of heavy metals and elevated culture temperature induced, but high doses of heavy metals and severe heat shock inhibited DjHsp90 expression. In response to heavy metals and thermal stress, DjHsp90-positive cells only appeared in the parenchymal tissue under epidermis cells along the bilateral from head to tail. These positive cells are presumably sensor cells that can detect external environment changes. Our work provides basic data for the study of stress responses in planarians.