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Acoel flatworms have played a relevant role in classical (and current) discussions on the evolutionary origin of bilaterian animals. This is mostly derived from the apparent simplicity of their body architectures. This tenet has been challenged over the last couple of decades, mostly because detailed studies of their morphology and the introduction of multiple genomic technologies have unveiled a complexity of cell types, tissular arrangements and patterning mechanisms that were hidden below this 'superficial' simplicity. One tissue that has received a particular attention has been the nervous system (NS). The combination of ultrastructural and single cell methodologies has revealed unique cellular diversity and developmental trajectories for most of their neurons and associated sensory systems. Moreover, the great diversity in NS architectures shown by different acoels offers us with a unique group of animals where to study key aspects of neurogenesis and diversification od neural systems over evolutionary time.In this review we revisit some recent developments in the characterization of the acoel nervous system structure and the regulatory mechanisms that contribute to their embryological development. We end up by suggesting some promising avenues to better understand how this tissue is organized in its finest cellular details and how to achieve a deeper knowledge of the functional roles that genes and gene networks play in its construction., (© 2024. The Author(s).)

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.

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.

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.

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.

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., Competing Interests: DL, CM, PR No competing interests declared, (© 2019, Li et al.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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.

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.

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.

Using parasites as sentinel organisms has received increased attention due to their responses toward environmental degradation. In some sections of the Vaal River, South Africa, water quality is altered and biota distribution affected. The aims of this study were to assess and compare infection variables of Paradipolozoon ichthyoxanthonAvenant-Oldewage, 2014 in Avenant- Oldewage et al. (2014) at two sites along the Vaal River and compare the water quality and trace element levels at the sites. Infection variables of the parasite, physico-chemical variables, and sediment and water samples for trace element analysis were collected at each site. Conductivity and trace element concentrations were higher at the Vaal River site than the Vaal Dam. Temporal variances in water quality and parasite prevalence, mean intensity and mean abundance, at intervals over a 14 year period occurred, P. ichthyoxanthon was absent at the Vaal River site though the host fish are present. Prevalence peaks in summer and winter. Comparison of infection data to water quality and trace elements indicated that conductivity and trace element levels negatively affected/correlated with the infection variables. Higher trace elements concentration and conductivity at the Vaal River site are thought to be driving factors responsible for absence of the parasite.

Trematodes of the genus Alaria develop into an arrested stage, known as mesocercariae, within their amphibian second intermediate host. The mesocercariae are frequently transmitted to a non-obligate paratenic host before reaching a definitive host where further development and reproduction can occur. Snakes are common paratenic hosts for Alaria spp. with the mesocercariae often aggregating in the host's tail. In the current study, we used morphological examination and molecular analyses based on partial sequences of nuclear large ribosomal subunit gene and mitochondrial cytochrome C oxidase subunit 1 gene to identify larvae in the tails of red-sided garter snakes (Thamnophis sirtalis parietalis) as mesocercariae of Alaria marcianae, Alaria mustelae, and Alaria sp. as well as metacercariae of Diplostomidae sp. of unknown generic affiliation. We assessed infection prevalence, absolute and relative intensity, and associated pathological changes in these snakes. Infection prevalence was 100 % for both male and female snakes. Infection intensity ranged from 11 to more than 2000 mesocercariae per snake tail but did not differ between the sexes. Gross pathological changes included tail swelling while histopathological changes included mild inflammation and the presence of mucus-filled pseudocysts surrounding mesocercariae, as well as the compression and degeneration of muscle fibers. Our results indicate that mesocercariae can lead to extensive muscle damage and loss in both sexes which likely increases the fragility of the tail making it more prone to breakage. As tail loss in garter snakes can affect both survival and reproduction, infection by Alaria mesocercariae clearly has serious fitness implications for these snakes.

Six taxa of limnoterrestrial rhabdocoels are discussed, two of them, both belonging to Typhloplanidae Graff, 1905, are new to science. Adenoplea reisingeri n. sp. can be distinguished from its congeners by the absence of a separate seminal receptacle, the presence of a copulatory bursa and the fact that it has an unarmed copulatory organ. Carcharodopharynx schlitzensis n. sp. can be distinguished from C. arcanus (Reisinger, 1924) Poche, 1926, the only other species within the genus, by the fact that the cirrus bears spines. For four other taxa: Adenoplea perigraptopera Reisinger, 1924, A. meridionalis Kolasa, 1981, C. arcanus (Reisinger, 1924) Poche, 1926 and Archivortex silvestris Reisinger, 1924, new data on morphology and distribution are provided. Neotypes are designated for A. perigraptopera and C. arcanus.

Phenotypic variation results from the balance between sources of variation and counteracting regulatory mechanisms. Canalization and developmental stability are two such mechanisms, acting at two different levels of regulation. The issue of whether or not they act concurrently as a common developmental buffering capacity has been subject to debate. We used geometric morphometrics to quantify the mechanisms that guarantee phenotypic constancy in the haptoral anchors of Ligophorus cephali. Canalization and developmental stability were appraised by estimating inter- and intra-individual variation, respectively, in size and shape of dorsal and ventral anchors. The latter variation was estimated as fluctuating asymmetry (FA) between anchor pairs. The general-buffering-capacity hypothesis was tested by two different methods based on correlations and Principal Components Analyses of the different components of size and shape variation. Evidence for FA in the dorsal and ventral anchors in both shape and size was found. Our analyses supported the hypothesis of a general developmental buffering capacity. The evidence was more compelling for shape than for size and, particularly, for the ventral anchors than for the dorsal ones. These results are in line with previous studies of dactylogyrids suggesting that ventral anchors secure a firmer, more permanent attachment, whereas dorsal anchors are more mobile. Because fixation to the host is crucial for survival in ectoparasites, we suggest that homeostatic development of the ventral anchors has been promoted to ensure the morphological constancy required for efficient attachment. Geometric morphometrics can be readily applied to other host-monogenean models, affording not only to disentangle the effects of canalization and developmental stability, as shown herein, but to further partition the environmental and genetic components of the former.

Polyclad species diversity, although generally well known for European North Atlantic waters, is nearly unknown for the Iberian Peninsula. The "Ría de Arousa", located on the Atlantic coast of Galicia (Spain), is a place where many positive biological factors for species biodiversity converge. Therefore, it is an ideal location to study polyclad diversity. This research, which describes new records and new species, contributes to the knowledge of the distribution of Polycladida (Platyhelminthes), particularly of the suborder Acotylea, in the Atlantic waters of the Iberian Peninsula. The new records include the re-descriptions of Cryptocelis compacta Lang, 1884, Stylochus neapolitanus (Delle Chiaje, 1841-1844) and Discocelis tigrina (Blanchard, 1847), while the two newly described species are Hoploplana elisabelloi n. sp. and Armatoplana celta n. sp.

In the present work eleven polyclad species of Lizard Island are studied. Seven of them are new records for this locality of the Australian coral reef and one is new to science, Lurymare clavocapitata n. sp. (Family Prosthiostomidae). The remaining recorded species belong to the genera Pseudoceros (P. bimarginatus, P. jebborum, P. stimpsoni, P. zebra, P. paralaticlavus and P. prudhoei) and Pseudobiceros (Pb. hancockanus, Pb. hymanae, Pb. flowersi and Pb. uniarborensis). Regardless of the different distribution patterns, all pseudocerotid species show brilliant colours, but similar internal morphology. Furthermore, differences in the form and size of the stylet are characteristic, because it is a sclerotic structure that is not affected during fixation. In Pseudoceros, the distance between the sucker and the female pore also differs among species. These features do not vary enough to be considered as diagnostic, but they provide information that can help to disentangle similarly coloured species complexes. A key of the genera Pseudoceros and Pseudobiceros of the Indo-Pacific region is provided, in order to facilitate the identification of species from this area.

Two previously unknown species of Haplogonaria (Acoela), H. schillingi sp. nov. and H. baki sp. nov., are described from the coastline of Maine, USA. The two species are morphologically similar to each other but H. schillingi can be distinguished from H. baki by its red pigmentation, its possession of a large genital atrium that branches posteriorly to the seminal vesicle and anteriorly to the vagina, a seminal vesicle that is more ellipsoid-shaped than spherical, and a well-defined wall in the seminal bursa. We provide a description of the new species using live observation, light microscopy of serial sagittal sections, and confocal microscopy imaging of F-actin. We compare the morphology of the new species with other members of the genus and discuss the phylogenetic position of H. schillingi in light of conflicting morphological and molecular data.

Macrostomum is a diverse genus of turbellarians with more than 180 species described from around the world. However, the Macrostomum fauna in China is poorly known. In this study, three new species of freshwater Macrostomum were described from southern China based on morphology of the penis stylet, an important character for species identification in this genus. In M. heyuanensis n. sp., the penis stylet bends 108° leftwards at its 1/2 length then backwards besides the distal opening, and the terminal region is thicker than other parts of penis stylet. In M. dongyuanensis n. sp., the penis stylet is J-shaped, with the opening at the tail end. In M. bicaudatum n. sp., the penis stylet is C-shaped, with the upper margin of the distal end longer but slimmer than the lower margin. In addition, molecular phylogenetic analyses were conducted to aid the classification of the novel species. Finally, their habitat and taxonomic status are compared and discussed.

Helminth parasites of veterinary importance cause huge revenue losses to agrarian economy worldwide. With the emergence of drug resistance against the current formulations, there is a need to focus on the alternative approaches in order to control this menace. In the present study, biocompatible zinc oxide nanoparticles (ZnO NPs) were used to see their in vitro effect on the biliary amphistomes, Gigantocotyle explanatum, infecting Bubalus bubalis because these nanoparticles are involved in generation of free radicals that induce oxidative stress, resulting in disruption of cellular machinery. The ZnO NPs were synthesized by using egg albumin as a biotemplate and subsequently characterized by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction and Spectrophotometrical, which showed that ZnO NPs were highly purified wurtzite type polycrystals, with a mean size of 16.7 nm. When the parasites were treated with lower concentrations (0.004% and 0.008%) of the ZnO NPs, the worms mounted a protective response by stimulating the antioxidant system but the treatment of G. explanatum with 0.012% ZnO NPs produced significant inhibition of the antioxidant enzymes like superoxide dismutase (SOD) (p< 0.05) and glutathione S- transferase (GST) (p<0.01), while the level of malondialdehyde (MDA), a lipid peroxidation marker, was significantly (p< 0.01) elevated. SEM and histopathology revealed pronounced tegumental damage showing the disruption of surface papillae and the annulations, particularly in the posterior region near acetabulum. The under expression of a number of polypeptides, loss of worm motility in a time dependent manner, further reflect strong anthelmintic potential of ZnO NPs. It can be concluded that the anthelmintic effect might be due to the production of reactive oxygen species that target a variety of macromolecules such as nucleic acid, protein and lipids which are involved in different cellular processes.

Effective parasite management can be achieved through strategically timed treatments that break the life cycle. We examined the effects of temperature (2 °C increments from 22 to 34 °C) and salinity (0, 11, 22, 35, 40‰) on the life cycle (embryonation period, hatching success, oncomiracidia (larvae) longevity, infection success, and time to sexual maturity) of Neobenedenia sp. (Monogenea: Capsalidae), a harmful ectoparasite of farmed marine fishes. Experiments were conducted in controlled conditions in the laboratory. The life cycle was faster in warm, high saline conditions compared to cooler conditions (10-13 days between 26-32 °C, 40‰; 15-16 days between 22-24 °C at 40‰). Warm seawater and high saline conditions (24-32 °C, 35-40‰) improved egg hatching success, reduced time to sexual maturity, and resulted in parasites reaching sexual maturity at a larger size (at 30-32 °C) compared to cooler conditions (22 °C). In contrast, cool, hypersaline conditions (22 °C, 40‰) increased oncomiracidia longevity and infection success. Linear and quantile regression models were used to construct an interactive, online parasite management interface to enable strategic treatment of parasites in aquaculture corresponding to observed temperature and salinity variation on farms in the tropics. It was recommended that farmers treat their stock more frequently during summer (27-31 °C) when parasites can complete their life cycle more quickly. Nevertheless, farmers should be aware of the potential for increased Neobenedenia sp. infections during winter months (21-26 °C) due to increased infection success.

Six species of dactylogyrid monogenoideans were collected at the upper São Francisco River, State of Minas Gerais, Brazil, from the gills of Salminus franciscanus. Anacanthorus adkruidenieri sp. n. and Anacanthorus paradouradensis sp. n. are described and compared with the other species of Anacanthorus. Anacanthorus adkruidenieri sp. n. is more similar to A. kruidenieri and A. parakruidenieri, but it can be distinguished from both by the rod-shaped accessory piece, which is articulated with the base of the male copulatory organ and is coiled around it, and by the shank of the hook, with expanded proximal bulbous portion bearing a central, elliptical foramen. Anacanthorus paradouradensis sp. n. resembles A. douradensis, but differs in the accessory piece, which is approximately half the length of the copulatory organ. Salminus franciscanus is a new host record and the São Francisco Basin is a new locality record for Anacanthorus daulometrus, Annulotrematoides glossophallus, Jainus iocensis and Tereancistrum arcuatus.

Background: The cutaneous monostome trematode Collyriclum faba (Bremser in Schmalz, 1831) is a bird parasite with a hitherto unknown life cycle and highly focal occurrence across the Holarctic and Neotropic ecozones., Methods: Representative specimens of benthic organisms were sampled at multiple sites and dates within the known foci of C. faba occurrence in Slovakia. A combined approach involving detailed morphological examination and sequencing of two independent DNA loci was used for their analysis., Results: We elucidated the complete life cycle of C. faba, which we determined to include the aquatic gastropod mollusk Bythinella austriaca (Frauenfeld, 1857) as the first intermediate host, the mayflies of the family Heptageniidae, Ecdyonurus venosus (Fabricius, 1775) and Rhithrogena picteti Sowa, 1971 x iridina (Kolenati, 1839), as the second intermediate hosts, and birds (primarily but not exclusively passeriform birds) as the definitive hosts. Bythinella austriaca occurs focally in the springs of tributaries of the Danube in the Alpine-Carpathian region. The restricted distribution of B. austriaca explains the highly focal distribution of C. faba noticed previously in spite of the broad distribution of its second intermediate and definitive host species. Utilization of both larval and adult Ephemeroptera spp. as the second intermediate hosts explains the known spectrum of the definitive host species, with the highest prevalence in species feeding on larvae of Ephemeroptera, such as Cinclus cinclus (Linnaeus, 1758) and Motacilla cinerea Tunstall, 1771, or adults of Ephemeroptera, such as Sylvia atricapilla (Linnaeus, 1758) and Regulus regulus (Linnaeus, 1758). In this study, we also determine the prevalence and DNA sequences of other immature trematode specimens found in the examined benthic organisms (particularly the families Microphallidae, Troglotrematidae and Nanophyetidae and Euryhelmis zelleri Grabda-Kazubska, 1980, Heterophyidae), and describe cercariae of C. faba., Conclusions: We determined the full life cycle of the Central European populations of C. faba. We speculate that other species of Bythinella and the closely related genus Amnicola may serve as first intermediate hosts in other parts of the distribution range of C. faba. Similarly, other Ephemeroptera of the family Heptageniidae may serve as the second intermediate hosts of C. faba in the Americas.

Neobenedenia melleni is a significant monogenean pathogen of fish in aquaculture facilities and public aquaria. Immunity after exposure to live N. melleni is well established, but the mechanisms of immunity remain unclear. In this study, tilapia (Oreochromis mossambicus) were continuously exposed to N. melleni over a four-month period and assessed for immunity as determined by a reduction in the number of parasites dislodged from the experimental animals during freshwater immersion. Specific mucosal and systemic antibody levels were by determined via enzyme-linked immunosorbent assay. At 45 days postexposure (DPE), fish displayed high parasite loads and baseline levels of mucosal antibodies. At 102 and 120 DPE parasite loads were significantly decreased, and antibody levels were significantly increased for mucus and plasma samples. The correlation between immunity (reduction in parasite load) and an increased humoral antibody response suggests a key role of antibody in the immune response. This is the first report of immunity against N. melleni that is associated with specific mucosal or systemic antibodies.