Behavioral and physiological responses of Girardia tigrina exposed to polyethylene microplastics.

Microplastic particles appear in great abundance and variety in freshwater ecosystems across the globe, spanning lakes and rivers, with increasingly frequent exposure of aquatic organisms. Studies on the mechanisms of any effects of plastic particles are still scarce, particularly in relation to the regenerative capacity of biota, for which there is no established model organism; however, planaria have shown sensitivity for assessing these risks to the aquatic environment. Thus, the present study aimed to investigate the behavioral and regeneration responses of the freshwater planaria Girardia tigrina exposed to polyethylene (PE) microplastics (MPs) incorporated into their food source. The greatest effect was observed on planarian regeneration, which was manifested at 10 µg/mg liver. Planaria reproduction and fertility were affected at 50 µg/mg liver; however, planaria locomotion was not affected at the concentrations evaluated. Mid-infrared absorption spectroscopy (FT-IR) was used to identify the constituent polymers, and ingestion of the polyethylene microplastic by the planaria was confirmed by infrared spectroscopy. The results highlight the potential adverse effects of exposure to polyethylene microplastic and show that the reproductive behavior and regeneration of a freshwater organism can be indicators of toxicity resulting from environmental pollution.

Molecular characterization of a flatworm Girardia isolate from Guanajuato, Mexico.

Planarian flatworms are best known for their impressive regenerative capacity, yet this trait varies across species. In addition, planarians have other features that share morphology and function with the tissues of many other animals, including an outer mucociliary epithelium that drives planarian locomotion and is very similar to the epithelial linings of the human lung and oviduct. Planarians occupy a broad range of ecological habitats and are known to be sensitive to changes in their environment. Yet, despite their potential to provide valuable insight to many different fields, very few planarian species have been developed as laboratory models for mechanism-based research. Here we describe a previously undocumented planarian isolate, Girardia sp. (Guanajuato). After collecting this isolate from a freshwater habitat in central Mexico, we characterized it at the morphological, cellular, and molecular level. We show that Girardia sp. (Guanajuato) not only shares features with animals in the Girardia genus but also possesses traits that appear unique to this isolate. By thoroughly characterizing this new planarian isolate, our work facilitates future comparisons to other flatworms and further molecular dissection of the unique and physiologically-relevant traits observed in this Girardia sp. (Guanajuato) isolate.

The sexual reproduction of the nontarget planarian Girardia tigrina is affected by ecologically relevant concentrations of difenoconazole: new sensitive tools in ecotoxicology.

The fungicide difenoconazole, widely used to reduce the negative impacts of fungi diseases on areas with intensive farming, can reach freshwater systems causing deleterious effects on nontarget organisms. The acute and chronic toxicity of a commercial formulation containing 250 g L-1 of difenoconazole (Prisma®) as the active ingredient was assessed in the freshwater planarian Girardia tigrina. The endpoints evaluated were feeding rate, locomotion, regeneration, and sexual reproduction of planarians. The estimated 48 h LC50 of the commercial formulation on planarians expressed as the concentration of the active ingredient difenoconazole was 47.5 mg a.i.L-1. A significant decrease of locomotion (LOEC = 18.56 mg a.i.L-1), delayed regeneration (LOEC = 9.28 mg a.i.L-1), and sexual reproduction impairment, i.e., decreased fecundity and fertility rates (LOEC ≤ 1.16 mg a.i.L-1) were observed on planarians exposed to sublethal concentrations of the formulation. This study demonstrated the importance of using reproductive, physiological, and behavioral parameters as more sensitive and complementary tools to assess the deleterious effects induced by a commercial formulation of difenoconazole on a nontarget freshwater organism. The added value and importance of our research work, namely, the impairment of sexual reproduction of planarians, contributes to the development of useful tools for ecotoxicology and highlights the fact that those tools should be developed as guidelines for testing of chemicals. Our results showed that the use of reproductive parameters of Girardia tigrina would help to complement and achieve a better assessment of the risk posed by triazole fungicides to freshwater ecosystems.

Two new land planarian species (Platyhelminthes: Tricladida) from the Cerrado biome in southwestern Brazil.

Most Neotropical land flatworm species are known from areas of the Atlantic Forest. Herein, we describe two new land planarian species from areas of semideciduous forest in the Cerrado biome in southwestern Brazil. Paraba aurantia Marques & Leal-Zanchet, sp. nov. can be distinguished from its congeners by the wide black median band on the dorsum, eyes occupying almost the entire dorsal surface, prostatic vesicle twisted and asymmetrical with proximal portions forked, and female atrium with an ample lumen lined by a low epithelium with stratified appearance. Pasipha liviae Marques & Leal-Zanchet, sp. nov. shows a narrow longitudinal dorsal stripe, collar-shaped pharynx, and prostatic vesicle with a pear-shaped proximal portion and a globose distal portion separated by a constriction. In addition, P. liviae can be distinguished from its congeners by molecular analyses from the cytochrome c oxidase subunit I gene (COI). Phylogenetic analyses suggest that P. liviae is closely related to P. hauseri, which occurs in areas of semidseciduous forest in southern Brazil.

Do bio-insecticides affect only insect species? Behavior, regeneration, and sexual reproduction of a non-target freshwater planarian.

Bio-insecticides have been increasingly used worldwide as ecofriendly alternatives to pesticides, but data on their effects in non-target freshwater organisms is still scarce and limited to insects. The aim of this study was to determine the lethal and sub-lethal effects of the bio-insecticides Bac Control (based on Bacillus thuringiensis kurstaki-Btk) and Boveril (based on Beauveria bassiana-Bb) on regeneration, behavioral, and reproductive endpoints of the freshwater planarian Girardia tigrina. The estimated LC50-48h were > 800 mg a.i./L for Btk and 60.74 mg a.i./L for Bb. In addition, exposure to Btk significantly decreased locomotion and feeding activities of planarians (lowest observed effect concentration (LOEC) of 12.5 mg a.i./L Btk) and fecundity rate (LOEC = 3.12 mg a.i./L Btk), whereas exposure to Bb significantly delayed regeneration (LOEC = 0.75 mg a.i./L Bb) and decreased fecundity rate (1.5 mg a.i./L Bb) of planarians. Thus, both bio-insecticides induced deleterious sub-lethal effects on a non-insect freshwater invertebrate species. However, only Bb-based formulation affected the survival, fecundity rate, and regeneration at concentrations below the maximum predicted environmental concentration (PEC = 247 mg/L). Thus, care should be taken when using such formulations as alternatives to chemical insecticides near aquatic ecosystems.

Morphology of parenchymally implanted foreign bodies indicates copulatory wounding in a planarian.

Traumatic mating, that is, copulation that involves wounding the partner's body, is a widespread phenomenon that is particularly prevalent in hermaphroditic animals. Traumatic mating is generally a collateral side effect of diverse strategies (from physical anchorage to injection of substances to manipulate the partner), but the trauma could also be adaptive by itself if it delays remating by the injured partner. In the Tricladida (the clade of planarian flatworms), reciprocal sperm transfer is often assumed to occur by means of a 'regular' nontraumatic copulation, that is, insertion of the penis through the partner's gonopore and deposition of the ejaculate into its genital atrium, with subsequent sperm migration to the oviducts. However, while studying the anatomy of Brazilian land planarians for taxonomic purposes, we found foreign bodies, reminiscent of spermatophores, implanted within the parenchyma of Choeradoplana albonigra (Riester, 1938). Herein, we describe and illustrate several lines of morphological evidence indicating that these foreign bodies likely represent a novel case of intragenital copulatory wounding (e.g., structural and histochemical similarity to land planarians spermatophores; implantation at the level of the gonopore; vestiges of rupture of the genital atrium's wall), corroborating that traumatic mating is pervasive and underreported in Metazoa. We also propose two different hypotheses to explain such copulatory wounding, viz., that it concerns (1) a regular mating strategy or (2) an accidental effect of the copulation. In any event, this land planarian may prove useful as a novel, noninsect terrestrial model organism to investigate the evolution of traumatic mating.

A brief personal account of a journey in science - an interview with Alejandro Sánchez-Alvarado.

Alejandro Sánchez Alvarado represents a younger generation of Latin American scientists that have achieved international scientific recognition. His work, together with that from other labs, has positioned the planaria Schmidtea mediterranea as a dynamic model system in which the cellular and molecular bases of regeneration in metazoans can be probed. During his professional career he has established strong ties with Latin America, hosting and training students and participating in seminars, workshops and courses throughout the region. In this interview he discusses his early scientific development and training, and his views on various issues related to the professional development of young scientists.

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.

New records of the giant planarian Polycladus gayi Blanchard, 1845 (Platyhelminthes: Geoplanidae) with notes on its conservation biology.

Land planarians (Tricladida:Geoplanidae) comprise about 910 species distributed in four subfamilies and can be found on all continents except Antarctica (Sluys Riutort 2018; Sluys 2019). The Neotropical region possesses nearly 31% of all the described terrestrial planarian species, most of them belonging to the subfamily Geoplaninae (Sluys 1999; Grau Carbayo 2010). Land planarians are mostly habitat-specialists, living in the humid soils of native forest, and predating on invertebrates like earthworms, isopods, mollusks and harvestmen, among others (Ogren 1995; Carbayo Leal-Zanchet 2003; Boll Leal-Zanchet 2016). Although most planarian species seem to be physiologically sensitive, for example to environmental moisture, a few land planarian genera like Bipalium Stimpson and Obama Carbayo et al., have successfully invaded many habitats, even in highly perturbed areas (Kawaguti 1932; Sluys 2019). Therefore, some of these invertebrate species appear to be good candidates as habitat quality bioindicators according to some authors (Sluys 1998; Gerlach et al., 2013; Negrete et al., 2014).

Cataloguing Prof. Eudóxia M. Froehlich's wet collection of land planarians (Platyhelminthes, Geoplanidae): a bounty of type specimens of 78 species.

Zoologist Prof. Eudóxia Maria Froehlich (1929*-2015†) kept an uncatalogued personal collection of land planarians in wet medium. We catalogued their contents. Glass jars contained a variable number of small vials, each with one or several labeled specimens. We transcribed the species names and field data, when available, written on card labels to a table. A total of 1772 vials in 80 jars, contained over 2700 specimens. Subsequently, we cross checked field data with that of the type specimens. As a result, we identified, with different degrees of certainty, type specimens of 78 species. These types compose a very significant potential source of additional morphological details of the species to conform with current standards in the taxonomy of the group.

Strategies of cellular energy allocation to cope with paraquat-induced oxidative stress: Chironomids vs Planarians and the importance of using different species.

Paraquat (PQ) is still used in several countries worldwide as an herbicide for weed control in agricultural production, ponds, reservoirs and irrigation canals. Thus, PQ is frequently found in surface water systems and is potentially toxic to aquatic organisms, since it can cause mitochondrial dysfunction altering in the redox state of cells. This study aimed to investigate the chronic effects of PQ to Chironomus riparius and Girardia tigrina, and compare their physiological strategies to cope with environmental stress. The mean emergence time was the most sensitive endpoint for Chironomids, with the lowest observed effect concentrations (LOEC) being 0.02 for males and 0.1 mg PQ L-1 for females. Moreover, PQ reduced the body weight of male and female imagoes, with LOECs of 0.5 and 2.5 mg PQ L-1, respectively. Paraquat also decreased the respiration rate (LOEC = 2.5 mg PQ L-1) and total glutathione (tGSH) content (LOEC = 0.5 mg PQ L-1). Thus, the aerobic production of energy was not affected and allowed chironomids to cope with oxidative stress induced by PQ, but with consequent physiological costs in terms of development rates and weight of adults. In planarians, PQ decreased the locomotion and feeding activity, and delayed photoreceptor regeneration (LOECs = 2.5 mg PQ L-1 for all endpoints). Despite increased aerobic energy production (LOEC = 0.5 mg PQ L-1), planarians were not able to cope with oxidative stress induced by the highest PQ concentrations, since lipid peroxidation levels were significantly increased (LOEC = 2.5 mg PQ L-1) concomitantly with a significant decrease of tGSH (LOEC = 2.5 mg PQ L-1). These results showed that planarians were unable to cope with oxidative stress induced by PQ with consequent impairments of behavior and regeneration despite an increased aerobic energy production.

Areas of endemism of land planarians (Platyhelminthes: Tricladida) in the Southern Atlantic Forest.

Areas of endemism (AoE) are the main study units in analytical biogeographic methods, and are often defined as an area with two or more endemic species living in them, presenting substantial congruence among their range limits. We explored the distribution of land planarians (Geoplanidae, Platyhelminthes) across the southern region of the Brazilian Atlantic forest (from the state of Rio de Janeiro, to the state of Rio Grande do Sul) utilizing DaSilva's et al. (2015) protocol. We used two methods, Endemicity Analysis (EA), and Geographical Interpolation of Endemism (GIE). We identified nine AoE of terrestrial flatworms in the Southern Atlantic forest. Performance of the methodologies is discussed. These AoE of land planarians can be explained through vicariance events combined with their physiological and ecological own limitations. Interestingly, these AoE are congruent with fine-scale approaches such as that with harvestmen. Most land planarians have revealed to present a very small distributional range evidencing their potential as a good model for fine-scale studies of AoE.

Trying to take over the world: Potential distribution of Obama nungara (Platyhelminthes: Geoplanidae), the Neotropical land planarian that has reached Europe.

Obama nungara Carbayo, Álvarez-Presas, Jones, & Riutort, 2016 is a land planarian (Platyhelminthes: Geoplanidae) native to southern South America, which has recently dispersed towards several countries of the European continent, thus becoming a threat to the native soil fauna. Its dispersion would be favoured by its wide food habit and its tendency to live linked to humans, being the plant trade its most plausible vector of dispersion. Here, we explored the potential distribution of O. nungara on a global scale by using the MaxEnt software. We used 144 records (encompassing 10 countries) from sampling campaigns, citizen science, recent literature, and material deposited in scientific collections. Our results showed that southern South America has favourable climatic conditions for O. nungara. MaxEnt also allowed predicting expansions to countries of Europe where this planarian is already established and to others not yet colonized, as well as to Asia (southern coast of the Caspian Sea, Taiwan, and south-east of mainland China) and Oceania (south-east of Australia and New Zealand). The potential distribution of O. nungara was mainly outlined by climatic factors related to temperature (annual mean temperature, mean temperature of the coldest quarter, and annual temperature range). Thus, under a global warming scenario, a significant expansion of O. nungara relative to the current prediction is expected. This information may be useful to design strategies to prevent new introductions, since the dissemination of this planarian seems to be strongly man-linked.

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.

A new genus and species of a terrestrial cavernicolan planarian from Barro Colorado Island, Panama (Platyhelminthes, Tricladida, Cavernicola).

The paper provides a taxonomic description of a new genus and species of terrestrial cavernicolan triclad flatworm from Barro Colorado Island, Panama. Molecular sequences of this species were already deposited in GenBank several years ago under the provisional taxon name Cavernicola sp. Animals have the following characteristics: absence of pigmentation and eyes; mouth opening located halfway in the pharyngeal cavity; few, ventral testes extending from a position far posterior to the ovaries to directly behind the pharyngeal cavity; strongly recurving sperm ducts, thus forming a loop; horizontally oriented cone-shaped penis papilla; common oviduct oriented perpendicular to the bursal canal and opening into the latter shortly before the canal communicates with the copulatory bursa; sac-shaped copulatory bursa lacking a distinct, single lumen and filled with a mass of syncytial cells, with interspersed nuclei.

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).

A catalogue of Eudóxia M. Froehlich's collection of planarians (Platyhelminthes) with emphasis on type specimens on histological slides.

Leading taxonomist Prof. Eudóxia Maria Froehlich (1929*-2015†) kept an uncatalogued personal collection of a large set of histological glass slides of land planarians. Many of these slides carry insufficient written information for identification purposes. 2287 slides of her collection were examined under the microscope and compared with relevant literature. We found 1633 slides bearing type material of a total of 119 nominal species, mostly collected in the Neotropical region. Identification of 148 slides (belonging to 16 species) as type material is suspected but not confirmed. Type material represents 110 triclad species. There is also type material of two Polycladida, two Rhabdocoela, three Proseriata, one Prolecitophora, and one Acoelomorpha species. The 119 nominal species were described by Ernest Marcus, Eveline du Bois Reymond-Marcus, Diva Diniz Corrêa, Cláudio Gilberto Froehlich, and Eudóxia Maria Froehlich. While keeping the slides in their original location in cupboards, the catalogue provides a quick means of retrieving desired slides. Slides were deposited in the Museu de Zoologia da Universidade de São Paulo.

Two new land planarian species (Platyhelminthes: Tricladida: Geoplanidae) from protected areas in southern Brazil.

We describe two new species of Neotropical land planarians found in protected areas of the Atlantic Forest in southern Brazil. Cratera obsidiana Amaral, Boll Leal-Zanchet, sp. nov., found in the State Park of Turvo, is the first species of the genus Cratera described from an area of deciduous seasonal forest. It can be distinguished from its congeners by the uniformly black dorsal color and the prostatic vesicle with proximal portion laterally expanded and directed upward. Luteostriata subtilis Boll, Amaral Leal-Zanchet, sp. nov., found in the National Park of Aparados da Serra, in an area covered by Araucaria moist forest, is distinguished from its congeners by only two conspicuous longitudinal stripes on the dorsal surface of the body and a large fold in the male atrium below the opening of the ejaculatory duct. We also provide an updated key for species of Cratera and the first key for species of Luteostriata.

Diet assessment of two land planarian species using high-throughput sequencing data.

Geoplanidae (Platyhelminthes: Tricladida) feed on soil invertebrates. Observations of their predatory behavior in nature are scarce, and most of the information has been obtained from food preference experiments. Although these experiments are based on a wide variety of prey, this catalog is often far from being representative of the fauna present in the natural habitat of planarians. As some geoplanid species have recently become invasive, obtaining accurate knowledge about their feeding habits is crucial for the development of plans to control and prevent their expansion. Using high throughput sequencing data, we perform a metagenomic analysis to identify the in situ diet of two endemic and codistributed species of geoplanids from the Brazilian Atlantic Forest: Imbira marcusi and Cephaloflexa bergi. We have tested four different methods of taxonomic assignment and find that phylogenetic-based assignment methods outperform those based on similarity. The results show that the diet of I. marcusi is restricted to earthworms, whereas C. bergi preys on spiders, harvestmen, woodlice, grasshoppers, Hymenoptera, Lepidoptera and possibly other geoplanids. Furthermore, both species change their feeding habits among the different sample locations. In conclusion, the integration of metagenomics with phylogenetics should be considered when establishing studies on the feeding habits of invertebrates.

Planarians as models to investigate the bioactivity of gold(I) complexes in vivo.

Gold(I)-containing complexes are used in drug discovery research for rheumatoid arthritis, cancer, and parasitic infections. In this study, we tested the bioactivity of gold(I) complexes in vivo using planarians. The planarian Schmidtea mediterranea possesses orthologues of tumor suppressor genes, such as p53, that, when silenced, cause deregulation of cell proliferation and apoptosis. In this context, we tested two triethylphosphine-gold(I) complexes (AdO and AdT) to determine if they can attenuate phenotypes that result from p53 inhibition. First, we identified the drug concentration that did not affect survival or regeneration and evaluated the drug's effect on cell division and apoptosis. We found that AdT treatment decreased the number of mitotic cells and that all drug treatments increased the number of apoptotic cells. We then performed p53(RNAi) and drug treatments concomitantly and observed the phenotype progression. Drug treatment increased survival three-fold and decreased apoptosis, which resulted in an attenuated phenotype. Our results indicate that planarians can be treated with gold(I) complexes, and that this treatment can diminish the p53(RNAi) phenotype and extend survival. In this work we show that planarians can be used as a model to study the in vivo effect of gold(I) complexes and to further investigate their mechanisms of action.