Freshwater Planarian Research Articles

Effects of two biopesticides and salt on behaviour, regeneration and sexual reproduction of the freshwater planarian Girardia tigrina

Microbial insecticides are being used as ecologically-friendly alternatives to traditional insecticides. However, their effects have been poorly investigated on non-target freshwater species, with exception of a few insect species. Moreover, combined effects of microbial insecticides with other environmental stressors, such as salinity, have never been investigated. Thus, our goal was to assess the effects of Bac-Control® (based in Bacillus thuringiensis - Btk) and Boveril® (based in Beauveria bassiana - Bb) with increasing salinities (NaCl) on freshwater planarian Girardia tigrina. It has been reported that increased salinity levels affect freshwater organisms compromising their survival by triggering adaptation processes to cope with osmotic stress. Our results showed delayed regeneration, decreased locomotion and feeding on planarians exposed to NaCl, whereas their sexual reproduction was not affected. Both microbial insecticides impaired feeding, locomotor activity, regeneration, and sexual reproduction of planarians. Planarians exposed to microbial insecticides compromised their progeny. Therefore, microbial insecticides might not be ecologically friendly alternatives to chemical insecticides. Interestingly, harmful effects of microbial insecticides with increasing salinities showed an inadequate response of planarians to cope with induction of their immune response and osmoregulation.

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Anticancer effects of an extract from a local planarian species on human acute myeloid leukemia HL-60 cells in vitro

Current anti-cancer drugs can cause many undesirable side effects to patients. Thus, there is a constant need to develop alternative therapeutic drugs. Bioactive compounds derived from natural products including animals, plants and microorganisms are being actively studied as sources for anticancer treatments. Freshwater planarians are important models for stem cell research and regeneration. However, to date, no studies on the biological activities of planaria extracts on cancer have been published. The aim of this study was to examine the potential antitumoral activity of the extract from planaria species-Malta (PSM) on human acute myeloid leukemia (AML) HL-60 cells. Antiproliferative activity was studied in terms of proliferation, apoptosis and differentiation. The expression of genes involved in the regulation of these important cellular processes was also analyzed using real-time PCR. PSM extract exhibited a selective cytotoxic effect on HL-60 cells when compared to normal lymphocytes. Furthermore, cell cycle analysis and Annexin V/PI assay showed that the extract induced apoptosis in HL-60 cells. The PSM extract induced myeloid differentiation with HL-60 cells showing a decreased nucleo/cytoplasmic ratio, an increase in nitroblue tetrazolium-positive cells, and CD11b- and CD14-positive cells. Finally, we also found that the PSM extract increased the expression of CEBPA, CEBPB, CEBPE, SPI1, BAX, CDKN1A and CDKN2C; whereas it reduced the expression of c-MYC and BCL2.This is the first study to reveal the antiproliferative, cytotoxic, and differentiation potential of PSM on HL-60 cells and suggests that it may have considerable potential for development as a novel natural product-based anticancer agent against AML.

Toxicity of different polycyclic aromatic hydrocarbons (PAHs) to the freshwater planarian Girardia tigrina

Freshwater planarians have been gaining relevance as experimental animals for numerous research areas given their interesting features, such as high regeneration potential, shared features with the vertebrates’ nervous system or the range of endpoints that can be easily evaluated in response to contaminants. Ecotoxicological research using these animals has been steadily increasing in the past decades, as planarians’ potentialities for this research area are being recognized. In this work, we used polycyclic aromatic hydrocarbons (PAHs) as model contaminants and evaluated effects of exposure to phenanthrene, pyrene and benzo[a]pyrene (B[a]P) in planarians. The freshwater planarian Girardia tigrina was chosen and mortality, cephalic regeneration (during and post-exposure), behavioral endpoints and presence of PAHs in tissues, were evaluated. Mortality was only observed in planarians exposed to phenanthrene, with an estimated LC50 of 830 μg L−1. Results indicate that planarian behavioral endpoints were very sensitive in response to sub-lethal concentrations of PAHs, showing a greater sensitivity towards B[a]P and pyrene. Briefly, post-exposure locomotion and post-exposure feeding were significantly impaired by sub-lethal concentrations of all compounds, whereas regeneration of photoreceptors was only significantly delayed in planarians exposed to pyrene. Moreover, levels of PAH-type compounds in planarian tissues followed a concentration-dependent increase, showing uptake of compounds from experimental solutions. The present results highlight the importance of studying alternative and complementary endpoints, such as behavior, not only because these may be able to detect effects at lower levels of contamination, but also due to their ecological relevance. The simplicity of evaluating a wide range of responses to contaminants further demonstrates the utility of freshwater planarians for ecotoxicological research.

Dugesia hepta and Dugesia benazzii (Platyhelminthes: Tricladida): two sympatric species with occasional sex?

Dugesia hepta Pala, Casu & Vacca, 1981 and Dugesia benazzii Lepori, 1951 are two freshwater planarian species from the islands of Corsica and Sardinia. Dugesia hepta is endemic of Sardinia and distributed in four northern hydrographic basins where it co-occurs with D. benazzii, which has a wider Tyrrhenian distribution. Although these species have been broadly studied—especially D. benazzii—as regards to their variety of reproductive patterns as well as for their karyological diversity, little is known about them from a molecular phylogenetic perspective. For the first time, we present a molecular phylogenetic tree of the two species and their populations based on two molecular markers—one mitochondrial, Cox1, and one nuclear, Dunuc12. Our results not only confirm that both species are molecularly distinct but also show that D. benazzii’s Corsican and Sardinian populations could belong to separate species. Furthermore, we present the first demonstration of a natural hybridization between different species in the genus Dugesia on the basis of molecular data.

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 pharyngeal nervous system orchestrates feeding behavior in planarians.

Planarians exhibit traits of cephalization but are unique among bilaterians in that they ingest food by means of goal-directed movements of a trunk-positioned pharynx, following protrusion of the pharynx out of the body, raising the question of how planarians control such a complex set of body movements for achieving robust feeding. Here, we use the freshwater planarian Dugesia japonica to show that an isolated pharynx amputated from the planarian body self-directedly executes its entire sequence of feeding functions: food sensing, approach, decisions about ingestion, and intake. Gene-specific silencing experiments by RNA interference demonstrated that the pharyngeal nervous system (PhNS) is required not only for feeding functions of the pharynx itself but also for food-localization movements of individual animals, presumably via communication with the brain. These findings reveal an unexpected central role of the PhNS in the linkage between unique morphological phenotypes and feeding behavior in planarians.

Dugesia japonica is the best suited of three planarian species for high-throughput toxicology screening

High-throughput screening (HTS) using new approach methods is revolutionizing toxicology. Asexual freshwater planarians are a promising invertebrate model for neurotoxicity HTS because their diverse behaviors can be used as quantitative readouts of neuronal function. Currently, three planarian species are commonly used in toxicology research: Dugesia japonica, Schmidtea mediterranea, and Girardia tigrina. However, only D. japonica has been demonstrated to be suitable for HTS. Here, we assess the two other species for HTS suitability by direct comparison with D. japonica. Through quantitative assessments of morphology and multiple behaviors, we assayed the effects of 4 common solvents (DMSO, ethanol, methanol, ethyl acetate) and a negative control (sorbitol) on neurodevelopment. Each chemical was screened blind at 5 concentrations at two time points over a twelve-day period. We obtained two main results: First, G. tigrina and S. mediterranea planarians showed significantly reduced movement compared to D. japonica under HTS conditions, due to decreased health over time and lack of movement under red lighting, respectively. This made it difficult to obtain meaningful readouts from these species. Second, we observed species differences in sensitivity to the solvents, suggesting that care must be taken when extrapolating chemical effects across planarian species. Overall, our data show that D. japonica is best suited for behavioral HTS given the limitations of the other species. Standardizing which planarian species is used in neurotoxicity screening will facilitate data comparisons across research groups and accelerate the application of this promising invertebrate system for first-tier chemical HTS, helping streamline toxicology testing.

Multi-gene phylogeny of Tetrahymena refreshed with three new histophagous species invading freshwater planarians

Planarians represent an insufficiently explored group of aquatic invertebrates that might serve as hosts of histophagous ciliates belonging to the hymenostome genus Tetrahymena. During our extensive research on freshwater planarians, parasitic tetrahymenas were detected in two of the eight planarian species investigated, namely, in Dugesia gonocephala and Girardia tigrina. Using the 16S and 18S rRNA genes as well as the barcoding cytochrome oxidase subunit I, one ciliate species was identified as T. scolopax and three species were recognized as new forms: T. acanthophora, T. dugesiae, and T. nigricans. Thus, 25% of the examined planarian taxa are positive for Tetrahymena species and three of them represent new taxa, indicating a large undescribed ciliate diversity in freshwater planarians. According to phylogenetic analyses, histophagous tetrahymenas show a low phylogenetic host specificity. Although T. acanthophora, T. dugesiae, and T. scolopax clustered together within the "borealis" clade, the former species has been detected exclusively in G. tigrina, while the two latter species only in D. gonocephala. Tetrahymena nigricans, which has been isolated only from G. tigrina, was classified within the "paravorax" clade along with T. glochidiophila which feeds on glochidia. The present phylogenetic reconstruction of ancestral life strategies suggested that the last common ancestor of the family Tetrahymenidae was free-living, unlike the progenitor of the subclass Hymenostomatia which was very likely parasitic. Consequently, there were at least seven independent shifts back to parasitism/histophagy within Tetrahymena: one each in the "paravorax" and "australis" clades and at least five transfers back to parasitism in the "borealis" clade.

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.

Use of species delimitation approaches to assess biodiversity in freshwater planaria (Platyhelminthes, Tricladida) from desert springs

Abstract Chihuahuan Desert springs in New Mexico and Texas harbour many endemic aquatic species. Often, these taxa share similar phenotypic characters, making it difficult to assess biodiversity accurately. Even though free‐living planaria (Platyhelminthes, Tricladida) occur in high densities, they are often omitted from biodiversity assessments of freshwater habitats owing to a lack of expertise in identifying them and lack of concern for conservation of these poorly known organisms. Using phylogenetic and species delimitation approaches, the diversity of planaria was examined from 12 springs in the northern Chihuahuan Desert. Mitochondrial and nuclear DNA sequences were used to reconstruct a phylogeny and then to delineate putative species from these springs via genetic distance and multispecies coalescent methods. The results showed that the planaria in the northern Chihuahuan Desert probably belong to four putative species; three of these are locally endemic, and the other is widely distributed across the region. These putative species may have experienced different evolutionary histories primarily because the locally endemic taxa have limited dispersal capabilities. The results suggest the need for morphological examination in order to allow the description of these putative species. This study not only uses DNA barcoding to estimate accurately the total diversity of freshwater planaria before conventional species identification, but also identifies hidden biodiversity in springs of the northern Chihuahuan Desert. These ecosystems require aggressive conservation action to protect vulnerable habitats that contain a wealth of endemic species.

Calcium ions in the aquatic environment drive planarians to food

BackgroundEven subtle changes in environmental factors can exert behavioral effects on creatures, which may alter interspecific interactions and eventually affect the ecosystem. However, how changes in environmental factors impact complex behaviors regulated by neural processes is largely unknown. The freshwater planarian Dugesia japonica, a free-living flatworm, displays distinct behavioral traits mediated by sensitive perception of environmental cues. Planarians are thus useful organisms for examining interactions between environmental changes and specific behaviors of animals.ResultsHere we found that feeding behavior was suppressed when the concentration of ions in the breeding water was low, while other behaviors were unaffected, resulting in differences in population size. Notably, the decline in feeding behavior was reversed in an ion-concentration-dependent manner soon after the planarians were moved to ion-containing water, which suggests that ions in environmental water rapidly promote feeding behavior in planarians. Moreover, the concentration of ions in the environmental water affected the feeding behavior by modulating the sensitivity of the response to foods. Finally, we found that calcium ions in the aquatic environment were required for the feeding behavior, and exposure to higher levels of calcium ions enhanced the feeding behavior, showing that there was a good correlation between the concentration of calcium ions and the responsiveness of planarians to foods.ConclusionsEnvironmental calcium ions are indispensable for and potentiate the activity level of the feeding behavior of planarians. Our findings suggest that the ions in the aquatic environment profoundly impact the growth and survival of aquatic animals via modulating their neural activities and behaviors.

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.

Analysis of beta-blocker bioconcentration in brown planaria (Girardia dorotocephala) and its effects on regeneration

Production, distribution, and disposal of pharmaceutical products, including beta-blockers, have become a global issue. Beta-blockers are known to persist in the environment months after theirrelease and may result in the disruption of the homeostatic system in non-target organisms. Here, we study the bioconcentration of three of the most commonly used beta-blockers and their effect on the regeneration of Girardia dorotocephala, a freshwater brown planarian. Acute toxicity tests determined LC50s for acebutolol, metoprolol, and propranolol to be 778 mg/L, 711 mg/L, and 111 mg/L, respectively. The quantification and analysis of beta-blocker bioconcentration during acute exposure were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After 4 days of exposure to beta-blockers, the bioconcentration drastically decreased for all three beta-blockers at all exposure levels, suggesting that an effective mechanism to reduce uptake or excrete beta-blockers could be present. Additionally, Girardia dorotocephala were cut proximal to the head and the quality of regeneration was documented from each fragment daily. No significant difference was visually observed after 2 weeks of regeneration between the brown planarians placed in beta-blocker solution and those placed in control solution.

First record of freshwater planarian Girardia tigrina (Platyhelminthes, Tricladida, Continenticola) in Eastern Europe.

The genus Girardia Ball, 1974 houses freshwater planarian species and is the second after Dugesia Girard, 1850 within Dugesiidae regarding the number of species. It contains 45 species (Tyler et al. 2006-2019) including three recently described: one from China-Girardia sinensis Chen Wang 2015 (Chen et al. 2015), and two cave-dwelling planarians from Brasil-Girardia desiderensis Souza Leal-Zanchet 2016 and Girardia pierremartini Souza Leal-Zanchet 2016 (Souza et al. 2016).

Exposure to Roundup® affects behaviour, head regeneration and reproduction of the freshwater planarian Girardia tigrina

The demand of glyphosate-based herbicides including Roundup® is rising in the tropics due to increase occurence of glyphosate-resistant weeds that require higher herbicide application rates but also because of their use associated with genetically engineered, glyphosate-tolerant crops. Consequently, there is now an excessive use of glyphosate in agricultural areas with potential adverse effects also for the surrounding aquatic environments. This study aimed to determine the sensitivity of the freshwater planarian Girardia tigrina to acute and chronic exposures of Roundup®. Planarians were exposed to a range of lethal and sub-lethal concentrations of Roundup® to determine the median lethal concentration (LC50) concerning its active ingredient glyphosate and also effects on locomotor velocity (pLMV), feeding rate, regeneration, reproductive parameters and morphological abnormalities. Regeneration endpoints included length of blastema and time for photoreceptors and auricles regeneration after decapitation, while effects on reproduction were assessed measuring fecundity (number of deposited cocoons) and fertility (number of hatchlings) over five weeks of exposure to glyphosate. The estimated 48 h LC50 of was 35.94 mg glyphosate/L. Dose dependent effects were observed for feeding, locomotion and regeneration endpoints with Lowest observed effect concentration (LOEC) values as low as 3.75 mg glyphosate/L. Chronic exposures to environmentally relevant concentrations of glyphosate significantly impaired fecundity and fertility rates of exposed planarians (median effective concentration, EC50 = 1.6 mg glyphosate/L for fecundity and fertility rates). Our results show deleterious effects of Roundup® on regeneration, behavior and reproduction of freshwater planarians and add important ecotoxicological data towards the environmental risk assessment of glyphosate-based herbicide in freshwater ecosystems.

Evaluation of a robust engineered enzyme towards organophosphorus insecticide bioremediation using planarians as biosensors

Organophosphorus compounds (OPs) are neurotoxic molecules developed as insecticides and chemical warfare nerve agents (CWNAs). They are covalent inhibitors of acetylcholinesterase (AChE), a key enzyme in central and peripheral nervous systems and are responsible for numerous poisonings worldwide. Many animal models have been studied over the years but finding a suitable in vivo model to account for both acute toxicity and long-term exposure remains a topical issue. Recently, an emerging aquatic animal model harboring a mammalian-like cholinergic nervous system, the freshwater planarian from Platyhelminthes, has been used to investigate neurotoxicity and developmental disruption.Given the tremendous toxicity of OPs, various bioremediation strategies have been considered over the years to counter their poisonous effects. Among these, enzymes have been particularly highlighted as they can degrade OPs in a fast, non toxic and environmentally friendly manner. In this article we investigated the biotechnological potential for decontaminating OPs of the previously reported variant SsoPox-αsD6 from the hyperstable enzyme SsoPox, isolated from the archaea Sulfolobus solfataricus. The capacity to hydrolyze 4 new substrates (methyl-pirimiphos, quinalphos, triazophos and dibrom) was demonstrated and the degradation products generated by enzymatic hydrolysis were characterized. We further evaluated the capacity of SsoPox-αsD6 for in vivo protection of freshwater planarians Schmidtea mediterranea (Smed). The use of SsoPox-αsD6 drastically decreased mortality and enhanced mobility of planarians. Then, an enzyme-based filtration device was developed by immobilizing intact Escherichia coli cells expressing SsoPox-αsD6 into alginate beads. The efficacy of the device was demonstrated using planarians as biosensors.

Screening for neurotoxic potential of 15 flame retardants using freshwater planarians

Asexual freshwater planarians are an attractive invertebrate model for high-throughput neurotoxicity screening, because they possess multiple quantifiable behaviors to assess distinct neuronal functions. Planarians uniquely allow direct comparisons between developing and adult animals to distinguish developmentally selective effects from general neurotoxicity. In this study, we used our automated planarian screening platform to compare the neurotoxicity of 15 flame retardants (FRs), consisting of representative phased-out brominated (BFRs) and replacement organophosphorus FRs (OPFRs). OPFRs have emerged as a proposed safer alternative to BFRs; however, limited information is available on their health effects. We found 11 of the 15 FRs (3/6 BFRs, 7/8 OPFRs, and Firemaster 550) caused adverse effects in both adult and developing planarians with similar nominal lowest-effect-levels for BFRs and OPFRs. This suggests that replacement OPFRs are comparably neurotoxic to the phased-out compounds. BFRs were primarily systemically toxic, whereas OPFRs, except Tris(2-chloroethyl) phosphate, shared a behavioral phenotype in response to noxious heat at sublethal concentrations, indicating specific neurotoxic effects. We found this behavioral phenotype was correlated with cholinesterase inhibition, thus linking behavioral outcomes to molecular targets. By directly comparing effects on adult and developing planarians, we further found that one BFR (3,3′,5,5′-Tetrabromobisphenol A) caused a developmental selective defect. Together, these results demonstrate that our planarian screening platform yields high content data from various behavioral and morphological endpoints, allowing us to distinguish selective neurotoxic effects and effects specific to the developing nervous system. Ten of these 11 bioactive FRs were previously found to be bioactive in other models, including cell culture and alternative animal models (nematodes and zebrafish). This level of concordance across different platforms emphasizes the urgent need for further evaluation of OPFRs in mammalian systems.

Two new species of marine flatworm from southern China facilitate determination of the phylogenetic position of the genus Nerpa Marcus, 1948 and the histochemical structure of the nervous system in the genus Paucumara Sluys, 1989 (Platyhelminthes, Tricladida, Maricola).

Two new species of flatworm, collected from a beach at eastern Shenzhen, China, were studied through an integrative approach by combining morphological, histological, histochemical (acetylcholinesterase, AChE), and molecular (18S r- DNA) data. These species belong to two genera of marine triclads, previously unrecorded from China, viz. Nerpa Marcus, 1948 and Paucumara Sluys, 1989. Nerpa fistulata Wang Chen, sp. nov. is characterized by: transparent body; principally pentamerous intestine with three distinct commissures; two very large, prepharyngeal testis follicles; a semi-circular lens in each eye cup; a penis papilla provided with a chitinized, pointed stylet; lateral bursae communicating with the oviduct and opening ventrally to the exterior via a duct. Phylogenetically N. fistulata groups with one member of the family Bdellouridae. This new, Chinese species of Nerpa introduces a major geographic disjunction, as the type species N. evelinae was described from the bay of Santos, Brazil, so that the genus is now known from both Atlantic as well as Pacific coasts. The species Paucumara falcata Wang Li, sp. nov. is characterized by: three distinct pale yellow transverse pigmentation bands on its dorsal side, between which some snowflake-like specks are randomly distributed, and a brown transverse band anteriorly to the eyes; 8-11 testicular follicles on either side of the body, the follicles extending from immediately behind the ovaries to half-way along the pharyngeal pocket; a musculo-parenchymatic organ with a sclerotic, curved tip projecting from the anterior wall of the male atrium, ventrally to the root of the penis papilla. Phylogenetically P. falcata groups with its congener P. trigonocephala, with the genus Paucumara forming the sister taxon of the genus Ectoplana. Comparison of the nerve structure of P. falcata, as revealed by AChE histochemistry, with that of eight other species of triclad suggested that the nervous system of marine planarians is simpler than that of species of freshwater planarians, but revealed also that the nerve structure is rather variable among species. The copulatory position exhibited by two partners in Paucumara falcata is remarkable in that they intertwine, with their heads pointing downwards and the tails pointing upwards, the entire process lasting about 10 min. Such a copulatory position has never before been reported for triclad flatworms.

Combined effects of NaCl and fluoxetine on the freshwater planarian, Schmidtea mediterranea (Platyhelminthes: Dugesiidae).

Increasing salinity levels in freshwaters due to natural and anthropogenic sources pose risk to exposed aquatic organisms. However, there is a paucity of information on how salinity may influence the effects of other chemical stressors especially psychiatric pharmaceuticals. Freshwater planarians which have been suggested as bioindicator species in aquatic habitats were used in this study to evaluate toxic effects of sodium chloride (NaCl) used here as a surrogate for increasing salinity, and its influence on the effects of the antidepressant, fluoxetine. Effects of NaCl on Schmidtea mediterranea were evaluated using survival, regeneration, locomotion, feeding, and reproduction as endpoints. Subsequently, combined effects of NaCl and fluoxetine on planarians' locomotion and reproduction were also evaluated. Result showed that exposure to increased NaCl concentrations is toxic to planarians with 48 and 96h LC50 of 9.15 and 7.55g NaCl L-1 respectively and exposure to sub-lethal concentrations led to reductions in feeding (LOEC of 0.75g NaCl L-1 or 1906μScm-1 at 20°C) and reproduction (LOEC 3.0g NaCl L-1 or 5530μScm-1 at 20°C), delayed head regeneration (LOEC of 1.5g NaCl L-1 or 3210μScm-1 at 20°C), and also slight decreases in locomotor activity. Moreover, some developmental malformations were observed in regenerating planarians, as well as delayed or inhibition of wound healing and degeneration after fissioning and during head regeneration. A significant interaction between fluoxetine and NaCl was observed for locomotor activity and unlike planarians exposed to fluoxetine alone, fissioned planarians and their pieces from the combined exposure treatments were also unable to regenerate missing portions. Results show that S. mediterranea can be highly sensitive to low NaCl concentrations and that this stressor can alter the effects of fluoxetine. The implication of these effects for planarian populations in the natural habitat is discussed as well as the need for more research on the effects of neuroactive pharmaceuticals under relevant exposure scenarios.