Regeneration of the flatworm Prosthiostomum siphunculus (Polycladida, Platyhelminthes).

Fueled by the discovery of head regeneration in triclads (planarians) two and a half centuries ago, flatworms have been the focus of regeneration research. But not all flatworms can regenerate equally well and to obtain a better picture of the characteristics and evolution of regeneration in flatworms other than planarians, the regeneration capacity and stem cell dynamics during regeneration in the flatworm order Polycladida are studied. Here, we show that as long as the brain remained at least partially intact, the polyclad Prosthiostomum siphunculus was able to regenerate submarginal eyes, cerebral eyes, pharynx, intestine and sucker. In the complete absence of the brain only wound closure was observed but no regeneration of missing organs. Amputated parts of the brain could not be regenerated. The overall regeneration capacity of P. siphunculus is a good fit for category III after a recently established system, in which most polyclads are currently classified. Intact animals showed proliferating cells in front of the brain which is an exception compared with most of the other free-living flatworms that have been observed so far. Proliferating cells could be found within the regeneration blastema, similar to all other flatworm taxa except triclads. No proliferation was observed in epidermis and pharynx. In pulse-chase experiments, the chased cells were found in all regenerated tissues and thereby shown to differentiate and migrate to replace the structures lost upon amputation.

Developmental scRNAseq Trajectories in Gene- and Cell-State Space-The Flatworm Example.

Single-cell RNA sequencing has become a standard technique to characterize tissue development. Hereby, cross-sectional snapshots of the diversity of cell transcriptomes were transformed into (pseudo-) longitudinal trajectories of cell differentiation using computational methods, which are based on similarity measures distinguishing cell phenotypes. Cell development is driven by alterations of transcriptional programs e.g., by differentiation from stem cells into various tissues or by adapting to micro-environmental requirements. We here complement developmental trajectories in cell-state space by trajectories in gene-state space to more clearly address this latter aspect. Such trajectories can be generated using self-organizing maps machine learning. The method transforms multidimensional gene expression patterns into two dimensional data landscapes, which resemble the metaphoric Waddington epigenetic landscape. Trajectories in this landscape visualize transcriptional programs passed by cells along their developmental paths from stem cells to differentiated tissues. In addition, we generated developmental "vector fields" using RNA-velocities to forecast changes of RNA abundance in the expression landscapes. We applied the method to tissue development of planarian as an illustrative example. Gene-state space trajectories complement our data portrayal approach by (pseudo-)temporal information about changing transcriptional programs of the cells. Future applications can be seen in the fields of tissue and cell differentiation, ageing and tumor progression and also, using other data types such as genome, methylome, and also clinical and epidemiological phenotype data.

RNA-Seq of three free-living flatworm species suggests rapid evolution of reproduction-related genes.

BACKGROUND: The genus Macrostomum consists of small free-living flatworms and contains Macrostomum lignano, which has been used in investigations of ageing, stem cell biology, bioadhesion, karyology, and sexual selection in hermaphrodites. Two types of mating behaviour occur within this genus. Some species, including M. lignano, mate via reciprocal copulation, where, in a single mating, both partners insert their male copulatory organ into the female storage organ and simultaneously donate and receive sperm. Other species mate via hypodermic insemination, where worms use a needle-like copulatory organ to inject sperm into the tissue of the partner. These contrasting mating behaviours are associated with striking differences in sperm and copulatory organ morphology. Here we expand the genomic resources within the genus to representatives of both behaviour types and investigate whether genes vary in their rate of evolution depending on their putative function. RESULTS: We present de novo assembled transcriptomes of three Macrostomum species, namely M. hystrix, a close relative of M. lignano that mates via hypodermic insemination, M. spirale, a more distantly related species that mates via reciprocal copulation, and finally M. pusillum, which represents a clade that is only distantly related to the other three species and also mates via hypodermic insemination. We infer 23,764 sets of homologous genes and annotate them using experimental evidence from M. lignano. Across the genus, we identify 521 gene families with conserved patterns of differential expression between juvenile vs. adult worms and 185 gene families with a putative expression in the testes that are restricted to the two reciprocally mating species. Further, we show that homologs of putative reproduction-related genes have a higher protein divergence across the four species than genes lacking such annotations and that they are more difficult to identify across the four species, indicating that these genes evolve more rapidly, while genes involved in neoblast function are more conserved. CONCLUSIONS: This study improves the genus Macrostomum as a model system, by providing resources for the targeted investigation of gene function in a broad range of species. And we, for the first time, show that reproduction-related genes evolve at an accelerated rate in flatworms.

New Species and Records of Anacanthorus (Monogenea: Dactylogyridae) Parasitizing Serrasalmid Fish (Characiformes) from Brazil, Including Molecular Data.

INTRODUCTION: Species of the genus Anacanthorus Mizelle & Price, 1965 represent commonly known parasites of Neotropical Characiformes. Anacanthorus is one of the most specious Neotropical monogenean genus, currently comprising 73 nominal species. MATERIALS AND METHODS: Fish were obtained from the Miranda River, Mato Grosso do Sul, from the Xingu River, Pará, from the Mogi Guaçu River, São Paulo and from the Upper Parana River, Paraná, Brazil. The monogeneans found were examined in a light microscope and partial sequences of the mitochondrial cytochrome c oxidase 1 gene were obtained for most species. RESULTS: Two new species of Anacanthorus were retrieved from Brazilian serrasalmids and are described herein. Anacanthorus paraxaniophallus n. sp. from Serrasalmus maculatus Kner has great similarity to A. xaniophallus Kritsky et al. (J Helminthol Soc Wash 59:25-51, 1992) by the general morphology of the copulatory complex, but it can be easily differentiated by a larger accessory piece with a slightly curved point, by having a basal flap in the male copulatory organ and by the morphology of hooks, with flattened thumb. Anacanthorus myleusi n. sp. from Myloplus schomburgkii (Jardine) is mainly distinguished by its unique copulatory complex, possessing an inverted J-shaped MCO with terminal flare and a hook-shaped accessory piece. Additionally, new host records are provided to A. lepyrophallus, geographical records are provided to four species of Anacanthorus (A. lepyrophallus, A. maltai, A. sciponophallus and A. thatcheri), which are being recorded in the Paraná River basin for the first time, and we also provide molecular data for six species of Anacanthorus.

Nuclear receptor NR4A is required for patterning at the ends of the planarian anterior-posterior axis.

Positional information is fundamental to animal regeneration and tissue turnover. In planarians, muscle cells express signaling molecules to promote positional identity. At the ends of the anterior-posterior (AP) axis, positional identity is determined by anterior and posterior poles, which are putative organizers. We identified a gene, nr4A, that is required for anterior- and posterior-pole localization to axis extremes. nr4A encodes a nuclear receptor expressed predominantly in planarian muscle, including strongly at AP-axis ends and the poles. nr4A RNAi causes patterning gene expression domains to retract from head and tail tips, and ectopic anterior and posterior anatomy (e.g., eyes) to iteratively appear more internally. Our study reveals a novel patterning phenotype, in which pattern-organizing cells (poles) shift from their normal locations (axis extremes), triggering abnormal tissue pattern that fails to reach equilibrium. We propose that nr4A promotes pattern at planarian AP axis ends through restriction of patterning gene expression domains.

The true colours of the flatworm: Mechanisms of pigment biosynthesis and pigment cell lineage development in planarians.

Pigment cells serve a variety of important uses across the animal kingdom, and in many species can change and regenerate throughout the lifetime of the organism. The functions of these cells, as well as their origins in both embryonic development and adult regeneration, are not fully understood. Here, we review advances in the study of pigment cells in the freshwater planarian, a model system for stem cell biology and regeneration. Freshwater planarians produce at least three pigment types to generate brown eye and body colouration: melanin, porphyrin, and ommochrome. The body pigments of planarians are produced and contained by a specialized, highly dendritic cell type located in the subepidermal parenchymal space. This cell type is specifically ablated following intense light exposure, a characteristic which has been exploited to discover the gene expression and regeneration of planarian pigment cells. Regenerating pigment cells progress through an immature state marked by upregulation of pigment synthesis genes before differentiating into mature pigment cells; these two states are dynamically regulated in homeostasis to maintain constant body pigmentation. The transcription factors Albino, FoxF-1, and Ets-1, as well as an FGFR-like molecule, are required for proper maintenance of the pigment lineage in both regeneration and homeostasis. These discoveries set the stage for research into external signals that regulate the pigment lineage, as well as possible functions for pigment cells in planarians, including the extra-ocular light response. These insights will address outstanding questions about the evolutionarily-conserved biology of pigment cells.

Composition and Diversity of Communities of Dactylogyrus spp. In Wild and Farmed Goldfish Carassius auratus.

Species composition and diversity of dactylogyrids were compared on gills of wild and cultured goldfish (silver crucian carp) Carassius auratus from 3 naturally populated lakes and 3 stocked aquaculture ponds in the Hubei province of China to examine the differences in the gill parasite community between these natural and farmed waters. Of the 7 Dactylogyrus spp. detected, all were found in lakes and 5 in ponds, with Dactylogyrus inexpectatus and Dactylogyrus anchoratus being absent from ponds. No significant correlation was found between the species richness and habitat area or host size, nor was there a significant difference in mean species richness between lakes (0.41-0.65) and ponds (0.30-0.76). Brillouin's diversity in lakes (0.049-0.067) was higher than that in ponds (0.024-0.046), but not significantly so. Although the diversity of parasite communities was higher in wild goldfish, higher mean abundance of some Dactylogyrus spp. was found in cultured goldfish. Based on Bray-Curtis similarity, it was difficult to differentiate parasite communities in lakes from those in ponds at the infracommunity level, whereas the 3 lakes and Guanqiao pond differed markedly from the remaining 2 ponds at the component community level. Although infracommunities differed among waterbodies, no effects of fish length or waterbody type were found on infracommunity or component community structure. Together, these results suggest that abundance and species richness of Dactylogyrus spp. on goldfish in lakes and farm ponds are influenced by habitat-specific environmental factors.

Predation and reproductive performance in two pelagic typhloplanid turbellarians.

We investigated feeding and reproductive performance of coexisting pelagic turbellarians from experiments on predation rates of Mesostoma ehrenbergii and M. craci as a function of food (Daphnia similis, three levels) and temperature (4 levels) during 10 days. Flatworms were collected from the pelagic of a subtropical lake in Brazil. Growth was more rapid at higher temperatures: more prey were consumed, and more eggs produced. M. craci and particularly M. ehrenbergii fitted a linear mixed-effects model and showed a type II functional response. M. craci was the more stenothermic of the two. Intrageneric predation also occurred: M. ehrenbergii fed on M. craci, but not vice versa. After a first clutch of subitaneous eggs, M. ehrenbergii produced resting eggs only. In M. craci an intermediate type of eggs hatched some time after release, survived passage through the gut of M. ehrenbergii, but did not resist drying. By primarily selecting cladoceran prey, M. ehrenbergii can make coexistence of both flatworms possible. As population density of M. ehrenbergii increases, it turns to producing resting and non-viable subitaneous eggs, thus limiting its population size. In nature, these processes structure the zooplankton community, while avoiding extinction of prey and predator.

Two new species of Annulotrema (Monogenea: Dactylogyridae) from Hydrocynus vittatus (Characiformes: Alestidae) in Lake Kariba, Zimbabwe.

Two new and one known species of Annulotrema Paperna & Thurston, 1969 are reported from the gills of the tigerfish Hydrocynus vittatus Castelnau, 1861, collected in Lake Kariba, Zimbabwe. The new species, Annulotrema pseudonili n. sp. and A. bracteatum n. sp., are described and distinguished mainly on the basis of features of the male copulatory organ (MCO). Annulotrema pseudonili n. sp. most closely resembles A. nili Paperna, 1973, but differs from it by possessing a more delicate MCO with a thin-walled base without a fibrous distal part. Annulotrema bracteatum n. sp. is most similar to Annulotrema ruahae Paperna, 1973, from which it differs by having an MCO composed of a longer copulatory tube and a leaf-shaped accessory piece enveloping the distal part of the tube. The presence of Annulotrema pikoides Guégan, Lambert & Birgi, 1988 on H. vittatus in Zimbabwe represents a new locality record for this parasite.

Supplementary taxonomic description of Demidospermus pinirampi (Monogenoidea, Dactylogyridae), with a new host record and an expansion of its distribution range / Descrição taxonômica complementar de Demidospermus pinirampi (Monogenoidea, Dactylogyridae), com registro de um novo hospedeiro e ampliação de sua área de distribuição

ABSTRACT During a survey conducted in the Lajeado Reservoir of the Tocantins River, in the state of Tocantins, Brazil, dactylogyrids were recovered from the gills of Pimelodina flavipinnis. Initial morphological analysis showed these dactylogyrids shared aspects of character with species of Demidospermus, with affinity to Demidospermus pinirampi, despite exhibiting differences with the original description by Kritsky et al. (1987) regarding the vagina, the base of the male copulatory organ and the shape and length of the bars. The analysis of the holotypes of D. pinirampi and its most morphologically close species, Demidospermus luckyi, revealed that these dactylogyrids were indeed D. pinirampi. This study therefore reports a new host and locality of occurrence, and reviews some measurements of the original description, supplementing and enhancing the morphological diagnosis of D. pinirampi.

RESUMO Durante um levantamento realizado no Reservatório de Lajeado, no Rio Tocantins, no estado de Tocantins, Brasil, dactilogirídeos foram recuperados das brânquias de Pimelodina flavipinnis. A análise morfológica inicial mostrou que esses dactilogirídeos compartilham aspectos de caráter com espécies de Demidospermus, com afinidade com Demidospermus pinirampi, apesar de apresentarem diferenças em relação à descrição original fornecida por Kritsky et al. (1987) com respeito à vagina, à base do órgão copulatório masculino e à forma e comprimento das barras. A análise dos holótipos de D. pinirampi e de sua espécie morfologicamente mais próxima, Demidospermus luckyi, revelou que estes dactilogirídeos eram realmente D. pinirampi. Este estudo, portanto, relata um novo hospedeiro e localidade de ocorrência e revisa algumas medidas da descrição original, complementando a diagnose morfológica de D. pinirampi.

Eggs hatching and oncomiracidia lifespan of Dawestrema cycloancistrium, a monogenean parasitic on Arapaima gigas.

Within the production chain of the Arapaima gigas (pirarucu), sanitary issues are still faced at the fingerling phase regarding infestations by the monogenean Dawestrema cycloancistrium. The present study had the objectives of describing the morphology and development phases of this parasite's eggs and oncomiracidia and determining the hatching time and lifespan of the oncomiracidia at different temperatures. D. cycloancistrium eggs were oval and elongated and had a single long appendage at one pole. The egg development was divided into four phases: embryonated egg, developing egg, larva appearance and ecloded egg. They were found in four forms: isolated in gill filaments; grouped in clusters that were anchored in a gill filament; grouped in clusters and entangled in part of a dead adult monogenean; and grouped in clusters in the environment, fixed to a substrate. Eclosion occurred after 72-96h, with faster development at 29°C and 32°C than at 24°C. The morphology of the oncomiracidia were rounded and elongated. They had two pairs of pigmented eye-spots as well as two ciliated areas located on each lateral part of the body and another on the anterior part of the body. The lifespan of the D. cycloancistrium oncomiracidia were 50 and 58h at 24°C and 27°C, respectively. Larvae were found in the mucus, indicating that the oncomiracidia penetrated the host integument. Understanding reproductive aspects of the D. cycloancistrium monogenean is important for developing specific prophylactic management strategies in aquaculture and providing valuable data for further studies.

Platyhelminthes.

Platyhelminthes (flatworms) have captivated the imagination of biologists for centuries. Indeed, planarian flatworms were used as experimental models decades before Caenorhabditis elegans became known as 'the worm'. Although planarians experienced a brief fall from grace, with the advent of molecular tools, planarians, such as Schmidtea mediterranea, have emerged in recent years as powerful models to study the basis of stem cell regulation and tissue regeneration. Flatworms are not just everyone's favorite experimental subjects from high school biology - they also include some of nature's most successful parasites, many of which have plagued humans throughout our history. This Primer will focus on several aspects of the remarkable biology found throughout the phylum Platyhelminthes.

Morphology, molecular and systematic analyses of Bychowskyella (Monogenea: Dactylogyridae) in siluriform fish from India.

Three species of genus Bychowskyella Akhmerov (1952), i.e. B. fossilisi Majumdar & Agarwal, 1989, B. tchangi Gusev, 1976 and B. wallagonia (Jain, 1959a) Gusev, 1961, were found to parasitize the gill filaments of siluriform fish in India. This redescription based on light microscopic examination of B. fossilisi, B. tchangi and B. wallagonia provides detailed taxonomic data for these species. We also amplified the 18S ribosomal RNA gene to evaluate the phylogenetic relationships of these three species. The morphological and genetic profiles confirmed the validation and taxonomical relationships of the above-mentioned species belonging to the genus Bychowskyella.

Evidence for Karyotype Polymorphism in the Free-Living Flatworm, Macrostomum lignano, a Model Organism for Evolutionary and Developmental Biology.

Over the past decade, the free-living flatworm Macrostomum lignano has been successfully used in many areas of biology, including embryology, stem cells, sexual selection, bioadhesion and aging. The increased use of this powerful laboratory model, including the establishment of genomic resources and tools, makes it essential to have a detailed description of the chromosome organization of this species, previously suggested to have a karyotype with 2n = 8 and one pair of large and three pairs of small metacentric chromosomes. We performed cytogenetic analyses for chromosomes of one commonly used inbred line of M. lignano (called DV1) and uncovered unexpected chromosome number variation in the form of aneuploidies of the largest chromosomes. These results prompted us to perform karyotypic studies in individual specimens of this and other lines of M. lignano reared under laboratory conditions, as well as in freshly field-collected specimens from different natural populations. Our analyses revealed a high frequency of aneuploids and in some cases other numerical and structural chromosome abnormalities in laboratory-reared lines of M. lignano, and some cases of aneuploidy were also found in freshly field-collected specimens. Moreover, karyological analyses were performed in specimens of three further species: Macrostomum sp. 8 (a close relative of M. lignano), M. spirale and M. hystrix. Macrostomum sp. 8 showed a karyotype that was similar to that of M. lignano, with tetrasomy for its largest chromosome being the most common karyotype, while the other two species showed a simpler karyotype that is more typical of the genus Macrostomum. These findings suggest that M. lignano and Macrostomum sp. 8 can be used as new models for studying processes of partial genome duplication in genome evolution.

Salinity stress from the perspective of the energy-redox axis: Lessons from a marine intertidal flatworm.

In the context of global change, there is an urgent need for researchers in conservation physiology to understand the physiological mechanisms leading to the acquisition of stress acclimation phenotypes. Intertidal organisms continuously cope with drastic changes in their environmental conditions, making them outstanding models for the study of physiological acclimation. As the implementation of such processes usually comes at a high bioenergetic cost, a mitochondrial/oxidative stress approach emerges as the most relevant approach when seeking to analyze whole-animal responses. Here we use the intertidal flatworm Macrostomum lignano to analyze the bioenergetics of salinity acclimation and its consequences in terms of reactive oxygen/nitrogen species formation and physiological response to counteract redox imbalance. Measures of water fluxes and body volume suggest that M. lignano is a hyper-/iso-regulator. Higher salinities were revealed to be the most energetically expensive conditions, with an increase in mitochondrial density accompanied by increased respiration rates. Such modifications came at the price of enhanced superoxide anion production, likely associated with a high caspase 3 upregulation. These animals nevertheless managed to live at high levels of environmental salinity through the upregulation of several mitochondrial antioxidant enzymes such as superoxide dismutase. Contrarily, animals at low salinities decreased their respiration rates, reduced their activity and increased nitric oxide formation, suggesting a certain degree of metabolic arrest. A contradictory increase in dichlorofluorescein fluorescence and an upregulation of gluthathione-S-transferase pi 1 (GSTP1) expression were observed in these individuals. If animals at low salinity are indeed facing metabolic depression, the return to seawater may result in an oxidative burst. We hypothesize that this increase in GSTP1 could be a "preparation for oxidative stress", i.e. a mechanism to counteract the production of free radicals upon returning to seawater. The results of the present study shed new light on how tolerant organisms carry out subcellular adaptations to withstand environmental change.

Myostenostomum Bulbocaudatum (Catenulida, Stenostomidae): new for Ukraine, rare for the World.

At present, the stenostomid genus Myostenostomum Luther, 1960 according to Artois et al. (2013) contains 7 species: M. Bulbocaudatum Luther (type species), M. ilmenicum Rogozin, M. lutheri Rogozin, M. marcusi Rogozin, M. vanderlandi Rogozin, M. fasciatum (Veydovsky) and M. gigerium (Kepner & Carter), although the two latter are thought to belong to the genus Stenostomum Schmidt (Tyler et al. 2006-2015). A remarkable feature of the representatives of the genus Myostenostomum is a muscular ring on their gut.

Two new species of freshwater flatworms (Platyhelminthes: Tricladida: Continenticola) from South American caves.

The diversity of freshwater triclads in the Neotropical region is considered to be low, but extensive areas of South America remain almost unexplored. Herein we describe two cave-dwelling, new species of Girardia, one from a transition zone of the Cerrado and Caatinga phytophysiognomies and the other from the Cerrado phytophysiognomy. The species from the Cerrado-Caatinga transition is a troglobite, eyeless and whitish; the species from the Cerrado area is possibly a troglophile, since it shows heavily pigmented body and eyes. Each species is easily recognized by a unique combination of features in its external morphology and copulatory apparatus. The two new species of Girardia show a restricted distribution, even the troglophile, and occur in caves without legal protection. Therefore, they must be considered as vulnerable organisms in a conservation context.

Pseudobiceros wirtzi sp. nov. (Polycladida: Cotylea) from Senegal with revision of valid species of the genus.

The genus Pseudobiceros was established based on the presence of two male gonopores, duplicated male reproductive structures, smooth dorsal surface, complex folded pseudotentacles, and simple ruffled pharynx. We describe here a new species of Pseudobiceros from the African continent that has been repeatedly reported and photographed over the years, but lacked a formal description. Pseudobiceros wirtzi sp. nov. is described based on morphological and histological characters. Our bibliographical revision of the genus considers 39 species to be valid. We applied the diagnostic characters of Pseudobiceros to Pseudoceros species listed before the split between these genera, and to species listed as incertae sedis. Pseudobiceros punctatus (Laidlaw 1902) nov. comb. shows typical features of Pseudobiceros. We also discuss other species with historically conflictive generic placements or problematic synonymy and summarize distributions and species characteristics in a comparative table. Most Pseudobiceros species still need re-descriptions to fill gaps regarding internal and pharynx details. Further sampling on the Eastern Africa coast and on mid-Atlantic Islands should prove useful in getting a general view of polyclad biodiversity and biogeography in the Tropical and South Atlantic.

Light-sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development.

BACKGROUND: Selective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratory's own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri - a non-model animal. RESULTS: Our OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm M. crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of M. crozieri with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope. CONCLUSIONS: We conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our experience gained during this project will help future OpenSPIM users with similar ambitions.

Land flatworms of the genus Pasipha (Platyhelminthes, Geoplanidae) in Argentina, with description of three new species.

The genus Pasipha Ogren & Kawakatsu, 1990 currently includes 22 species, most of them recorded in the Brazilian Atlantic Forest. Recently, Pasipha hauseri was documented in north-eastern Argentina, thus extending its distribution range. This paper reports new records of the genus Pasipha in the Interior Atlantic Forest ecoregion in Argentina, with the description of three new species: Pasipha atla sp. nov., Pasipha johnsoni sp. nov., and Pasipha mbya sp. nov. These new species exhibit similarities in internal anatomy, such as pharynx cylindrical, extrabulbar prostatic vesicle with folded walls and proximally forked, male atrium highly folded, being at least two times longer than the female atrium and with small folds in their proximal portion. However, they can be distinguished from each other mainly by the secretion types discharged into the prostatic vesicle as well as into the male and female atria. In addition, they have different colour patterns on the dorsal surface.