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Marine microturbellarians are an assemblage of meiofaunal flatworms abundant in sediments and on seaweeds around the world. The diversity and distribution of these animals in Japan are poorly understood. Here, we provide an overview of all recorded species in Japan and characterize two new species of the rhabdocoel genus Reinhardorhynchus based on morphological features and a molecular phylogeny inferred from 18S and 28S rDNA sequences. Reinhardorhynchus ryukyuensis sp. nov. can be distinguished from other species in the genus by the lack of an armed cirrus and by the presence of two larger opposing hooks and five smaller interconnected hooks in its male copulatory organ. Reinhardorhynchus sagamianus sp. nov. differs from its congeners because its male copulatory organ combines a bipartite cirrus armed with a belt of overlapping scale-like spines, an unarmed accessory cirrus, and two large distal accessory hooks. Our molecular phylogenetic analyses show that R. ryukyuensis sp. nov. and R. sagamianus sp. nov. form a clade with all the other species of Reinhardorhynchus for which DNA sequence data are available. Within this clade, R. sagamianus sp. nov. is in a clade that also includes R. riegeri and R. anamariae. The discovery of these new species highlights the importance of uncovering and documenting the hidden biodiversity along Japan's coastal margin. [ABSTRACT FROM AUTHOR]

In the course of Digenea evolution the transitions to a passive strategy of infection of the first intermediate host occurred multiple times. In these cases, miracidia – ciliated larvae – relinquished their role as active seekers for a suitable snail host and began relying on the chance of being accidently ingested by it. The structural changes resulting from these transitions are diverse, but the majority of them are reductive. In this study we present our ultrastructural survey on the opisthorchioid Cryptocotyle lingua miracidium. The transition to passivity led to extensive reduction in almost all somatic elements of the larva: only four epithelial plates cover its body; just a single nervous cell, a single protonephridium and a single longitudinally-oriented muscle cell have been found in this miracidium. Several undifferentiated cells comprise the germinal material of the larva. The greatest part of its body is occupied by three glandular elements. The position of these glands is consistent within Opisthorchioidea and strikingly corresponds to that of some of the optically studied miracidia of Plagiorchioidea. This similarity is suspicious, because the C. lingua larva appeared to differ vastly from other ultrastructurally described passive miracidia. Here we create a basis for the future comparison of the miracidia of Opisthorchioidea and Plagiorchioidea, expecting the ultrastructural data on the latter to be published someday. [ABSTRACT FROM AUTHOR]

Purpose: Studies on monogeneans parasitizing representatives of Siluriformes in Africa remain scarce and fragmentary. In order to contribute to fill this gap, we screened Heterobranchus longifilis from the Lindi River in the Democratic Republic of the Congo. Methods: Heterobranchus longifilis was purchased from fishermen in DR Congo, province of Tshopo, in Kisangani town, the Lindi River. The monogeneans were individually isolated from the gill and transferred directly onto a slide in a drop of Hoyer's medium. Results: Quadriacanthus lindiensis n. sp. is morphologically similar to Quadriacanthus longifilisi. However, it is distinguished from the latter by its non-sclerotized vagina, its dorsal cunei shape and the size of its bars. Conclusion: Only Q. lindiensis n. sp. was found on the gills of the single individual of H. longifilis screened. In the future, a representative sampling should enable us to verify if Q. lindiensis n. sp. co-occurs with other species known to parasitize H. longifilis. [ABSTRACT FROM AUTHOR]

In the Tropical Eastern Pacific (TEP), while knowledge of the order Polycladida is longstanding, at the moment no study has synthesized how many and which species are known from this region. In this work, a new species of polyclad from the Oaxaca coast, Callioplana marianae sp. nov., is described. It is characterized by a beige background with light brown and white pigmentation, nipple-shaped nuchal tentacles, cerebral and tentacular eyes, an oval seminal vesicle, a penis papilla armed with a filiform stylet, and a tripartite Y-shaped Lang’s vesicle. An exhaustive review of polyclad studies in the TEP over 173 years (1851–2024) yielded a checklist of 82 species belonging to 53 genera and 22 families have been recorded, with 53 species (66.25%) first described in the region. The countries with the highest number of recorded species are Mexico (45 spp.), Ecuador (18 spp.), Costa Rica (10 spp.), Panama (10 spp.), and Peru (7 spp.). The family with the most species in the region was Leptoplanidae (9 spp.). The best represented genera were Leptoplana (5 spp.) and Paraplanocera (4 spp.). Planocera pellucida, Paraplanocera oligoglena, and Pseudobiceros splendidus are species with pantropical distribution. Koinostylochus burchami and Armatoplana panamensis have the deepest known distributions at 70 and 73 meters, respectively.

[Display omitted] • Strong mitonuclear discordance in phylogeny and evolutionary rates were found in Neodermata. • Polyopisthocotylea may have the fastest-evolving mtDNA sequences in Bilateria. • Polyopisthocotylea exhibits a long stem branch in the mtDNA, but not in the nDNA topology. • We found weak evidence of mitonuclear co-evolution and none for compensatory evolution. • A large (≈6.3 kb) numt in Diplorchis sp. exhibits signs of ncRNA expression. The genomic evolution of Polyopisthocotylea remains poorly understood in comparison to the remaining three classes of Neodermata: Monopisthocotylea, Cestoda, and Trematoda. Moreover, the evolutionary sequence of major events in the phylogeny of Neodermata remains unresolved. Herein we sequenced the mitogenome and transcriptome of the polyopisthocotylean Diplorchis sp., and conducted comparative evolutionary analyses using nuclear (nDNA) and mitochondrial (mtDNA) genomic datasets of Neodermata. We found strong mitonuclear discordance in the phylogeny of Neodermata. Polyopisthocotylea exhibited striking mitonuclear discordance in relative evolutionary rates: the fastest-evolving mtDNA in Neodermata and a comparatively slowly-evolving nDNA genome. This was largely attributable to its very long stem branch in mtDNA topologies, not exhibited by the nDNA data. We found indications that the fast evolution of mitochondrial genomes of Polyopisthocotylea may be driven both by relaxed purifying selection pressures and elevated levels of directional selection. We identified mitochondria-associated genes encoded in the nuclear genome: they exhibited unique evolutionary rates, but not correlated with the evolutionary rate of mtDNA, and there is no evidence for compensatory evolution (they evolved slower than the rest of the genome). Finally, there appears to exist an exceptionally large (≈6.3 kb) nuclear mitochondrial DNA segment (numt) in the nuclear genome of newly sequenced Diplorchis sp. A 3′-end segment of the 16S rRNA gene encoded by the numt was expressed, suggesting that this gene acquired novel, regulatory functions after the transposition to the nuclear genome. In conclusion, Polyopisthocotylea appears to be the lineage with the fastest-evolving mtDNA sequences among all of Bilateria, but most of the substitutions were accumulated deep in the evolutionary history of this lineage. As the nuclear genome does not exhibit a similar pattern, the circumstances underpinning this evolutionary phenomenon remain a mystery. [ABSTRACT FROM AUTHOR]

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Abstract Background Previous flatworm phylogenetic research has been carried out analysing 18S and 28S DNA markers. Through this methodology, Mariplanellinae subfamily has been recently re-classified as Mariplanellida status novus. This new classification implied that 3 genera belonged to Mariplanellida: Mariplanella, Lonchoplanella and Poseidoplanella. In this study, we aim to clarify some of the relationships within Rhabdocoela analysing 18S and 28S DNA markers of a total of 91 species through Maximum Likelihood and Bayesian Inference methodologies. A total of 11 species and genera, including Lonchoplanella, from the island of Sylt are included and had not previously been involved in any molecular phylogenetic analyses. Results Our phylogenetic results support Mariplanellida as an independent group within Rhabdocoela and its status as an infraorder. Our study suggests that Lonchoplanella axi belongs to Mariplanellida. Within Rhabdocoela, Haloplanella longatuba is nested within Thalassotyphloplanida, instead of Limnotyphloplanida. Within Kalyptorhynchia, the taxon Eukalyptorhynchia turned out to be paraphyletic including members of Schizorhynchia. These results also support the position of the genus Toia separate from Cicerinidae. Conclusions Lonchoplanella axi belongs to Mariplanellida, whose status as infraorder is herein confirmed. The genus Toia belongs separate from Cicerinidae. Further research is needed to clarify the phylogenetic relationships of Hoploplanella. Most of the species, genera and families included in this study with more than one terminal are monophyletic and well supported. Adding gene markers and complementary morphological studies will help to clarify those relationships that remain uncertain.

The Temnocephalidae sensu Van Steenkiste et al., 2021 is a diverse group of rhabdocoel flatworms that are ectosymbiotic mostly on freshwater crustaceans. The family is found on all inhabited continents except for N. America (north of Mexico) and Africa. Here, we explore the systematic implications of a molecular phylogeny inferred from partial sequences of the nuclear 18S and 28S ribosomal RNA genes. In agreement with previous views based on morphology, the earliest diverging group within the family was exemplified by the genus Scutariella (subfamily Scutariellinae) followed by the genus Didymorchis (Didymorchinae). The genus Dactylocephala from Madagascar, previously thought to be in the Temnocephalinae, then diverged as sister to the remaining temnocephalids. A new subfamily, Dactylocephalinae, is proposed to contain this genus. Remaining subfamilies were, in turn, Diceratocephalinae, Actinodactylellinae and Temnocephalinae. Further subdivision was apparent within the Temnocephalinae. One well-supported clade included the genera Temnohaswellia (eastern Australia and New Zealand), Temnomonticellia (Tasmania) and Temnocephala (South America). The widespread genus Temnosewellia (= Ts.) was not supported as monophyletic. One clade of Temnosewellia sensu lato included numerous species known primarily from crayfish of the genus Euastacus, found in eastern and south-eastern Australia. Two other clades circumscribed the taxa Ts. minor and Ts. dendyi. Identities and membership of these two clades are clouded by frequent misidentifications, the reasons for which are discussed here. A fourth clade of Temnosewellia s.l. included Ts. rouxi and other taxa from northern Australia and Asia (ranging at least from Japan to India). The diversity of temnocephalids in Australia, and especially in Asia, is greatly underappreciated. Some general comments are made about geographic distribution of temnocephalids, but detailed biogeographic analysis requires broader sampling. [ABSTRACT FROM AUTHOR]

Background: Previous flatworm phylogenetic research has been carried out analysing 18S and 28S DNA markers. Through this methodology, Mariplanellinae subfamily has been recently re-classified as Mariplanellida status novus. This new classification implied that 3 genera belonged to Mariplanellida: Mariplanella, Lonchoplanella and Poseidoplanella. In this study, we aim to clarify some of the relationships within Rhabdocoela analysing 18S and 28S DNA markers of a total of 91 species through Maximum Likelihood and Bayesian Inference methodologies. A total of 11 species and genera, including Lonchoplanella, from the island of Sylt are included and had not previously been involved in any molecular phylogenetic analyses. Results: Our phylogenetic results support Mariplanellida as an independent group within Rhabdocoela and its status as an infraorder. Our study suggests that Lonchoplanella axi belongs to Mariplanellida. Within Rhabdocoela, Haloplanella longatuba is nested within Thalassotyphloplanida, instead of Limnotyphloplanida. Within Kalyptorhynchia, the taxon Eukalyptorhynchia turned out to be paraphyletic including members of Schizorhynchia. These results also support the position of the genus Toia separate from Cicerinidae. Conclusions: Lonchoplanella axi belongs to Mariplanellida, whose status as infraorder is herein confirmed. The genus Toia belongs separate from Cicerinidae. Further research is needed to clarify the phylogenetic relationships of Hoploplanella. Most of the species, genera and families included in this study with more than one terminal are monophyletic and well supported. Adding gene markers and complementary morphological studies will help to clarify those relationships that remain uncertain. [ABSTRACT FROM AUTHOR]

Information on temporal and spatial trends with regard to the introduction of non-indigenous species (NIS) is often sparsely available. These trends may potentially help improve the design and focus of monitoring programs, give insights into new pathways and hotspots, and facilitate horizon scanning. We provide an overview of 215 marine and brackish water NIS recorded in The Netherlands. Temporal trends over the most recent three decades for taxonomic groups, species origin, introduction vectors, and water systems were analysed. We attempt to explain the observed patterns and discuss factors that hamper their explanation. A shift in the region of origin from Pacific to W Atlantic can potentially be linked to legislation prohibiting Pacific oyster imports, whereas a subsequent shift backwards cannot. Case studies illustrate that NIS may not be first detected in the water systems where they were originally introduced. Additionally, it is shown that changes in allegedly native species' distribution or seasonal pattern should be linked to an introduced cryptic NIS instead. We also discuss the shortcomings of monitoring programs that were originally not focused on NIS, the importance of naturalists' observations, and the added value of a more recent network that is focused on NIS detection in the coastal waters of The Netherlands. [ABSTRACT FROM AUTHOR]

Schistosome helminths infect over 200 million people across 78 countries and are responsible for nearly 300,000 deaths annually. However, our understanding of basic genetic pathways crucial for schistosome development is limited. The sex determining region Y-box 2 (Sox2) protein is a Sox B type transcriptional activator that is expressed prior to blastulation in mammals and is necessary for embryogenesis. Sox expression is associated with pluripotency and stem cells, neuronal differentiation, gut development, and cancer. Schistosomes express a Sox-like gene expressed in the schistosomula after infecting a mammalian host when schistosomes have about 900 cells. Here, we characterized and named this Sox-like gene SmSOXS1. SmSoxS1 protein is a developmentally regulated activator that localizes to the anterior and posterior ends of the schistosomula and binds to Sox-specific DNA elements. In addition to SmSoxS1, we have also identified an additional six Sox genes in schistosomes, two Sox B, one SoxC, and three Sox genes that may establish a flatworm-specific class of Sox genes with planarians. These data identify novel Sox genes in schistosomes to expand the potential functional roles for Sox2 and may provide interesting insights into early multicellular development of flatworms. [ABSTRACT FROM AUTHOR]

Cilia and flagella are evolutionarily conserved organelles that form protrusions on the surface of many growth-arrested or differentiated eukaryotic cells. Due to the structural and functional differences, cilia can be roughly classified as motile and non-motile (primary). Genetically determined dysfunction of motile cilia is the basis of primary ciliary dyskinesia (PCD), a heterogeneous ciliopathy affecting respiratory airways, fertility, and laterality. In the face of the still incomplete knowledge of PCD genetics and phenotype-genotype relations in PCD and the spectrum of PCD-like diseases, a continuous search for new causative genes is required. The use of model organisms has been a great part of the advances in understanding molecular mechanisms and the genetic basis of human diseases; the PCD spectrum is not different in this respect. The planarian model (Schmidtea mediterranea) has been intensely used to study regeneration processes, and—in the context of cilia—their evolution, assembly, and role in cell signaling. However, relatively little attention has been paid to the use of this simple and accessible model for studying the genetics of PCD and related diseases. The recent rapid development of the available planarian databases with detailed genomic and functional annotations prompted us to review the potential of the S. mediterranea model for studying human motile ciliopathies. [ABSTRACT FROM AUTHOR]

Live imaging of regenerative processes can reveal how animals restore their bodies after injury through a cascade of dynamic cellular events. Here, we present a comprehensive toolkit for live imaging of tissue regeneration in the flatworm Macrostomum lignano , including a high-throughput cloning pipeline, targeted cellular ablation, and advanced microscopy solutions. Using tissue-specific reporter expression, we examine how various structures regenerate. Enabled by a custom luminescence/fluorescence microscope, we overcome intense stress-induced autofluorescence to demonstrate genetic cellular ablation and reveal the limited regenerative capacity of neurons and their essential role during wound healing, contrasting muscle cells' rapid regeneration after ablation. Finally, we build an open-source tracking microscope to continuously image freely moving animals throughout the week-long process of regeneration, quantifying kinetics of wound healing, nerve cord repair, body regeneration, growth, and behavioral recovery. Our findings suggest that nerve cord reconnection is highly robust and proceeds independently of regeneration. [Display omitted] • A toolbox enables live imaging and tissue ablation in the flatworm Macrostomum lignano • Neurons are essential for wound healing and regeneration but do not regenerate after ablation • Severed nerve cords extend at a linear rate inversely proportional to their remaining length • Nerve cord reconnection is separate from Wnt/β-catenin-dependent posterior regeneration Hall et al. present an advanced toolkit for manipulating and monitoring regeneration in the flatworm Macrostomum lignano , enabling tissue-specific labeling, ablation, and long-term tracking. These methods reveal critical insights into neural repair and regeneration, opening avenues for regeneration research and the functional characterization of marine meiofauna. [ABSTRACT FROM AUTHOR]

Platyhelminthes (flatworms) are a diverse invertebrate phylum useful for exploring life-history evolution. Within Platyhelminthes, only two clades develop through a larval stage: free-living polyclads and parasitic neodermatans. Neodermatan larvae are considered evolutionarily derived, whereas polyclad larvae are hypothesized to be ancestral due to ciliary band similarities among polyclad and other spiralian larvae. However, larval evolution has been challenging to investigate within polyclads due to low support for deeper phylogenetic relationships. To investigate polyclad life-history evolution, we generated transcriptomic data for 21 species of polyclads to build a well-supported phylogeny for the group. The resulting tree provides strong support for deeper nodes, and we recover a new monophyletic clade of early branching cotyleans. We then used ancestral state reconstructions to investigate ancestral modes of development within Polycladida and more broadly within flatworms. In polyclads, we were unable to reconstruct the ancestral state of deeper nodes with significant support because early branching clades show diverse modes of development. This suggests a complex history of larval evolution in polyclads that likely includes multiple losses and/or multiple gains. However, our ancestral state reconstruction across a previously published platyhelminth phylogeny supports a direct developing prorhynchid/polyclad ancestor, which suggests that a larval stage in the life cycle evolved along the polyclad stem lineage or within polyclads.

Four new species of marine polycystidid microturbellarians (Platyhelminthes, Kalyptorhynchia) are described from Cuba and Panama. These species are Brachyrhynchoides ortizi sp. nov., Djeziraia adriani sp. nov., Phonorhynchoides lalanai sp. nov., and Sabulirhynchus ibarrae sp. nov. All species occur in Cuba, and P. lalanai sp. nov. was also retrieved from the Pacific coast of Panama. From the latter locality, we also report Sabulirhynchus axi Artois & Schockaert, 2000. All species are readily distinguished from their congeners by the morphology of the sclerotised structures of the male genital system. The prostatic stylet of B. ortizi sp. nov. is the shortest and widest known for Brachyrhynchoides. Djeziraia adriani sp. nov. can be recognized by its paired seminal vesicles and S-shaped prostate stylet. The prostatic stylet of P. lalanai sp. nov. is proximally twisted and the length proportion between its accessory and prostatic stylet is the largest among all known species of Phonorhynchoides. Sabulirhynchus axi and Sabulirhynchus ibarrae sp. nov. are morphologically similar, yet the latter species is unique due to the characteristic ridge in the middle of the proximally tubular prostatic stylet. With the data available today, a possible explanation of rhabdocoel biogeographic patterns across the Isthmus of Panama remains largely speculative.

In a free-living flatworm, Macrostomum lignano, an S-phase kinase-associated protein 1 (SKP1) homologous gene was identified as enriched in proliferating cells, suggesting that it can function in the regulation of stem cells or germline cells since these are the only two types of proliferating cells in flatworms. SKP1 is a conserved protein that plays a role in ubiquitination processes as a part of the Skp1-Cullin 1-F-box (SCF) ubiquitin ligase complex. However, the exact role of Mlig-SKP1 in M. lignano was not established. Here, we demonstrate that Mlig-SKP1 is neither involved in stem cell regulation during homeostasis, nor in regeneration, but is required for spermatogenesis. Mlig-SKP1(RNAi) animals have increased testes size and decreased fertility as a result of the aberrant maturation of sperm cells. Our findings reinforce the role of ubiquitination pathways in germ cell regulation and demonstrate the conserved role of SKP1 in spermatogenesis. [ABSTRACT FROM AUTHOR]

Transgenesis is an important and often irreplaceable method to study numerous processes of animal life. To create animal transgenic lines, it is necessary to have a suitable model organism that has necessary traits for efficient and affordable transgenesis. The concise review characterizes the existing model organisms of different taxa for which an efficient transgenesis protocol has been developed. Special attention is paid to flatworms and, in particular, Macrostomum lignano as a promising model organism for studying aging, regeneration, and carcinogenesis. [ABSTRACT FROM AUTHOR]

The purpose of this application, under Articles 55.3 and 81.2.1 of the Code, is to resolve the homonymy of the family-group name MecynostomatidaeKukalová, 1969 (Insecta, Paleodictyoptera) by suppression of its senior homonym MecynostomatidaeLameere, 1917 (Insecta, Paleodictyoptera) for the purposes of both the Principle of Priority and the Principle of Homonymy. [ABSTRACT FROM AUTHOR]

Many species‐rich ecological communities emerge from adaptive radiation events. Yet the effects of adaptive radiation on community assembly remain poorly understood. Here, we explore the well‐documented radiations of African cichlid fishes and their interactions with the flatworm gill parasites Cichlidogyrus spp., including 10,529 reported infections and 477 different host–parasite combinations collected through a survey of peer‐reviewed literature. We assess how evolutionary, ecological, and morphological parameters determine host–parasite meta‐communities affected by adaptive radiation events through network metrics, host repertoire measures, and network link prediction. The hosts' evolutionary history mostly determined host repertoires of the parasites. Ecological and evolutionary parameters predicted host–parasite interactions. Generally, ecological opportunity and fitting have shaped cichlid‐Cichlidogyrus meta‐communities suggesting an invasive potential for hosts used in aquaculture. Meta‐communities affected by adaptive radiations are increasingly specialised with higher environmental stability. These trends should be verified across other systems to infer generalities in the evolution of species‐rich host–parasite networks. [ABSTRACT FROM AUTHOR]

More than a billion people are infected with parasitic worms, including nematodes, such as hookworms, and flatworms, such as blood flukes. Few drugs are available to treat worm infections, but high-throughput screening approaches hold promise to identify novel drug candidates. One problem for researchers who find an interesting ‘hit’ from a high-throughput screen is to identify whether that compound, or a similar compound has previously been published as having anthelmintic or anti-parasitic activity. Here, we present (i) data sets of 2,828 anthelmintic compounds, and 1,269 specific anti-schistosomal compounds, manually curated from scientific papers and books, and (ii) a data set of 24,335 potential anthelmintic and anti-parasitic compounds identified by text-mining PubMed abstracts. We provide their structures in simplified molecular-input line-entry system (SMILES) format so that researchers can easily compare ‘hits’ from their screens to these anthelmintic compounds and anti-parasitic compounds and find previous literature on them to support/halt their progression in drug discovery pipelines.

Schistosome helminths infect over 200 million people across 78 countries and are responsible for nearly 300,000 deaths annually. However, our understanding of basic genetic pathways crucial for schistosome development is limited. The sex determining region Y-box 2 (Sox2) protein is a Sox B type transcriptional activator that is expressed prior to blastulation in mammals and is necessary for embryogenesis. Sox expression is associated with pluripotency and stem cells, neuronal differentiation, gut development, and cancer. Schistosomes express a Sox-like gene expressed in the schistosomula after infecting a mammalian host when schistosomes have about 900 cells. Here, we characterized and named this Sox-like gene SmSOXS1. SmSoxS1 protein is a developmentally regulated activator that localizes to the anterior and posterior ends of the schistosomula and binds to Sox-specific DNA elements. In addition to SmSoxS1, we have also identified an additional six Sox genes in schistosomes, two Sox B, one SoxC, and three Sox genes that may establish a flatworm-specific class of Sox genes with planarians. These data identify novel Sox genes in schistosomes to expand the potential functional roles for Sox2 and may provide interesting insights into early multicellular development of flatworms.

The Chilean land planarian genus Gusana (Platyhelminthes, Tricladida, Geoplaninae) currently comprises three species that were described in the 19th century. Four new species of the genus are described herein, namely G. hualpensis Carbayo, sp. nov., G. lujanae Almeida & Carbayo, sp. nov., G. melipeucensis Almeida & Carbayo, sp. nov. and G. purensis Bolonhezi, Almeida & Carbayo, sp. nov. An integrative taxonomy approach was adopted by combining morphological (anatomy, histology) and molecular (COI and 28S genes) information. Additionally, the monophyletic status of Gusana is investigated and the species are delimited by a recently introduced molecular delimitation method based on pairwise genetic distances. All Gusana species are very similar in attributes such as body shape, dorsal colour pattern and internal organs, namely the pharynx and copulatory apparatus and only differ in the details. Gusana is retrieved as monophyletic and the molecular delimitation method recovered the same species recognised morphologically. A re-diagnosis of the genus and replacement of G. lata into Pseudogeoplana are also proposed. A new role of the penis papilla is also suggested based on the spermatophore found in Gusana lujanae. Only one species of the Chilean land planarian genus Gusana (Geoplanidae) is relatively well known under the current taxonomic standards. Four new species from the genus are described by combining morphological and molecular (DNA) information. Additionally, a recently introduced independent test based on pairwise genetic distances is applied. Morphological and molecular results are congruent with each other and support the recognition of the four new species. [ABSTRACT FROM AUTHOR]

Hundreds of millions of people worldwide are infected by various species of parasitic flatworms. Without treatment, acute and chronical infections frequently lead to the development of severe pathologies and even death. Emerging data on a decreasing eff iciency of some important anthelmintic compounds and the emergence of resistance to them force the search for alternative drugs. Parasitic flatworms have complex life cycles, are laborious and expensive in culturing, and have a range of anatomic and physiological adaptations that complicate the application of standard molecular-biological methods. On the other hand, free-living flatworm species, evolutionarily close to parasitic flatworms, do not have the abovementioned diff iculties, which makes them potential alternative models to search for and study homologous genes. In this review, we describe the use of the basal free-living flatworm Macrostomum lignano as such a model. M. lignano has a number of convenient biological and experimental properties, such as fast reproduction, easy and non-expensive laboratory culturing, optical body transparency, obligatory sexual reproduction, annotated genome and transcriptome assemblies, and the availability of modern molecular methods, including transgenesis, gene knockdown by RNA interference, and in situ hybridization. All this makes M. lignano amenable to the most modern approaches of forward and reverse genetics, such as transposon insertional mutagenesis and methods of targeted genome editing by the CRISPR/Cas9 system. Due to the availability of an increasing number of genome and transcriptome assemblies of different parasitic flatworm species, new knowledge generated by studying M. lignano can be easily translated to parasitic f latworms with the help of modern bioinformatic methods of comparative genomics and transcriptomics. In support of this, we provide the results of our bioinformatics search and analysis of genes homologous between M. lignano and parasitic flatworms, which predicts a list of promising gene targets for subsequent research.

In eukaryotes, trans-splicing is a process of nuclear pre-mRNA maturation where two different RNA molecules are joined together by the spliceosomal machinery utilizing mechanisms similar to cis-splicing. In diverse taxa of lower eukaryotes, spliced leader (SL) trans-splicing is the most frequent type of trans-splicing, when the same sequence derived from short small nuclear RNA molecules, called SL RNAs, is attached to the 5’ ends of different non-processed pre-mRNAs. One of the functions of SL trans-splicing is processing polycistronic pre-mRNA molecules transcribed from operons, when several genes are transcribed as one pre-mRNA molecule. However, only a fraction of trans-spliced genes reside in operons, suggesting that SL trans-splicing must also have some other, less understood functions. Regenerative flatworms are informative model organisms which hold the keys to understand the mechanism of stem cell regulation and specialization during regeneration and homeostasis. Their ability to regenerate is fueled by the division and differentiation of the adult somatic stem cell population called neoblasts. Macrostomum lignano is a flatworm model organism where substantial technological advances have been achieved in recent years, including the development of transgenesis. Although a large fraction of genes in M. lignano were estimated to be SL trans-spliced, SL trans-splicing was not studied in detail in M. lignano before. Here, we performed the first comprehensive study of SL trans-splicing in M. lignano. By reanalyzing the existing genome and transcriptome data of M. lignano, we estimate that 30 % of its genes are SL trans-spliced, 15 % are organized in operons, and almost 40 % are both SL trans-spliced and in operons. We annotated and characterized the sequence of SL RNA and characterized conserved cis- and SL transsplicing motifs. Finally, we found that a majority of SL trans-spliced genes are evolutionarily conserved and signif icantly over-represented in neoblast-specific genes. Our findings suggest an important role of SL trans-splicing in the regulation and maintenance of neoblasts in M. lignano.

Background: Planarian flatworms are popular invertebrate models for basic research on stem cell biology and regeneration. These animals are commonly maintained on a diet of homogenized calf liver or boiled egg yolk in the laboratory, introducing a source of uncontrolled experimental variability. Results: Here, we report the development of defined diets, prepared entirely from standardized, commercially sourced ingredients, for the freshwater species Schmidtea mediterranea, Dugesia japonica, and Girardia dorotocephala. These food sources provide an opportunity to test the effects of specific nutritional variables on biological phenomena of interest. Defined diet consumption was not sufficient for growth and only partially induced the increase in stem cell division that normally accompanies feeding, suggesting these responses are not solely determined by caloric intake. Our defined diet formulations enable delivery of double‐stranded RNA for gene knockdown in a manner that provides unique advantages in some experimental contexts. We also present a new approach for preserving tissue integrity during hydrogen peroxide bleaching of liver‐fed animals. Conclusions: These tools will empower research on the connections between diet, metabolism, and stem cell biology in the experimentally tractable planarian system. Key Findings: Development of standardized laboratory diets for freshwater planarians.Defined diets provide new avenues for experimental manipulation of nutritional variables.Defined diets offer unique advantages for introduction of dsRNA in RNAi experiments.Organismal growth and feeding‐induced stem cell division are not determined solely by caloric intake. [ABSTRACT FROM AUTHOR]

Abstract Background Chromosome-level assemblies are indispensable for accurate gene prediction, synteny assessment, and understanding higher-order genome architecture. Reference and draft genomes of key helminth species have been published, but little is yet known about the biology of their chromosomes. Here, we present the complete genome of the tapeworm Hymenolepis microstoma, providing a reference quality, end-to-end assembly that represents the first fully assembled genome of a spiralian/lophotrochozoan, revealing new insights into chromosome evolution. Results Long-read sequencing and optical mapping data were added to previous short-read data enabling complete re-assembly into six chromosomes, consistent with karyology. Small genome size (169 Mb) and lack of haploid variation (1 SNP/3.2 Mb) contributed to exceptionally high contiguity with only 85 gaps remaining in regions of low complexity sequence. Resolution of repeat regions reveals novel gene expansions, micro-exon genes, and spliced leader trans-splicing, and illuminates the landscape of transposable elements, explaining observed length differences in sister chromatids. Syntenic comparison with other parasitic flatworms shows conserved ancestral linkage groups indicating that the H. microstoma karyotype evolved through fusion events. Strikingly, the assembly reveals that the chromosomes terminate in centromeric arrays, indicating that these motifs play a role not only in segregation, but also in protecting the linear integrity and full lengths of chromosomes. Conclusions Despite strong conservation of canonical telomeres, our results show that they can be substituted by more complex, species-specific sequences, as represented by centromeres. The assembly provides a robust platform for investigations that require complete genome representation.

Abstract Macrostomum lignano is a free-living flatworm that is emerging as an attractive experimental animal for research on a broad range of biological questions. One feature setting it apart from other flatworms is the successful establishment of transgenesis methods, facilitated by a steady supply of eggs in the form of single-cell zygotes that can be readily manipulated. This, in combination with the transparency of the animal and its small size, creates practical advantages for imaging and fluorescence-activated cell sorting in studies related to stem cell biology and regeneration. M. lignano can regenerate most of its body parts, including the germline, thanks to the neoblasts, which represent the flatworm stem cell system. Interestingly, neoblasts seem to have a high capacity of cellular maintenance, as M. lignano can survive up to 210 Gy of γ-irradiation, and partially offset the negative consequence of ageing. As a non-self-fertilizing simultaneous hermaphrodite that reproduces in a sexual manner, M. lignano is also used to study sexual selection and other evolutionary aspects of sexual reproduction. Work over the past several years has led to the development of molecular resources and tools, including high-quality genome and transcriptome assemblies, transcriptional profiling of the germline and somatic neoblasts, gene knockdown, and in situ hybridization. The increasingly detailed characterization of this animal has also resulted in novel research questions, such as bio-adhesion based on its adhesion-release glands and genome evolution due to its recent whole-genome duplication.

Four new species of limnoterrestrial rhabdocoels (‘Typhloplanidae’ Graff, 1905) are described. One of these – Faunulus nielsi Houben, Proesmans & Artois gen. et sp. nov. – could not be unambiguously placed within an existing genus. Faunulus nielsi most closely resembles species of the genus Adenocerca Reisinger, 1924 but can be clearly distinguished by the position of the testes. The three other new species described are Bryoplana belgica Houben, Proesmans & Artois sp. nov., Hoplopera isis Houben, Proesmans & Artois sp. nov., and Protoplanella leiae Houben, Proesmans & Artois sp. nov. All three belong to the subfamily ‘Protoplanellinae’ Reisinger, 1924 and are distinguished based on a detailed description of the reproductive system. Finally, new data are provided for nine other, known typhloplanids: Adenocerca minima Kolasa, 1981; Chorizogynopora italica Kolasa, 1981; Hoplopera opaca Reisinger, 1924; K. subterranea Reisinger, 1933; Krumbachia virginiana (Kepner & Carter, 1931) Ruebush, 1938; Olisthanellinella rotundula Reisinger, 1924; Prorhynchella minuta Ruebush, 1939; Protoplanella simplex Reisinger, 1924; and Ventrociliella romanae Kolasa, 1977. A detailed comparison of our material of V. romanae to what is described for Bockia deses Reisinger, 1924, leads us to consider the latter a nomen dubium.

Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. The free-living flatworm Macrostomum lignano is currently the only flatworm where stable transgenesis is available, and as such it offers a powerful experimental platform to address questions that were previously difficult to answer. The published transgenesis approach relies on random integration of DNA constructs into the genome. Despite its efficiency, there is room and need for further improvement and diversification of transgenesis methods in M. lignano. Transposon-mediated transgenesis is an alternative approach, enabling easy mapping of the integration sites and the possibility of insertional mutagenesis studies. Here, we report for the first time that transposon-mediated transgenesis using piggyBac can be performed in M. lignano to create stable transgenic lines with single-copy transgene insertions. [ABSTRACT FROM AUTHOR]

The monotypic genus of land planarians Timyma (Rhynchodeminae, Geoplanidae) has been suggested to be a relict that could reflect a pre‐Tertiary Antarctic connection between South America and Australia and New Zealand. Two species belonging to this genus, Timyma juliae E. M. Froehlich, 1978 and Timyma olmuensis Almeida & Carbayo sp. n., are (re)described herein. Both species present ventral testes and a semilunate headplate, the same as the members of the Oriental subfamily Bipaliinae. Timyma olmuensis Almeida & Carbayo sp. n. is studied by means of an integrative approach. Two nuclear gene fragments (28S, EF) and a mitochondrial gene fragment (COI) were sequenced. Phylogenetic analyses pointed out that Timyma is the sister–group of the Neotropical Geoplaninae and is phylogenetically distant from Bipaliinae, contradicting the hypothesis of Timyma as a South American relict of a Gondwanan taxon. These results indicate that the headplate evolved independently in Timyma and Bipaliinae. Morphological data reinforce this view. Accordingly, Timyminae subfam. n. is proposed for Timyma and a new diagnosis for the genus is proposed. [ABSTRACT FROM AUTHOR]

Abstract Background Genomes of eukaryotes are inhabited by myriads of mobile genetic elements (MGEs) – transposons and retrotransposons - which play a great role in genome plasticity and evolution. A lot of computational tools were developed to annotate them either in genomic assemblies or raw reads using de novo or homology-based approaches. But there has been no pipeline enabling users to get coding and flanking sequences of MGEs suitable for a downstream analysis from genome assemblies. Results We developed a new pipeline, MGERT (Mobile Genetic Elements Retrieving Tool), that automates all the steps necessary to obtain protein-coding sequences of mobile genetic elements from genomic assemblies even if no previous knowledge on MGE content of a particular genome is available. Conclusions Using MGERT, researchers can easily find MGEs, their coding and flanking sequences in the genome of interest. Thus, this pipeline helps researchers to focus on the biological analysis of MGEs rather than excessive scripting and pipelining.

Abstract Background The free-living marine flatworm Macrostomum lignano is a powerful model organism for use in studying mechanisms of regeneration and stem cell regulation due to its combination of biological and experimental properties, including the availability of transgenesis methods, which is unique among flatworm models. However, due to its relatively recent introduction in research, many aspects of this animal’s biology remain unknown. One such question is the influence of culture temperature on Macrostomum biology. Results We systematically investigated how different culture temperatures affect development time, reproduction rate, regeneration, heat shock response, and gene knockdown efficiency by RNA interference (RNAi) in M. lignano. We used marker transgenic lines to accurately measure the regeneration endpoint, and to establish the stress response threshold for temperature shock. We found that compared to the culture temperature of 20 °C commonly used for M. lignano, temperatures of 25 °C–30 °C substantially increase the speed of development and regeneration, lead to faster manifestation of RNAi phenotypes, and increase reproduction rate without detectable negative consequences for the animal, while temperatures above 30 °C elicit a heat shock response. Conclusions We show that altering temperature conditions can be used to reduce the time required to establish M. lignano cultures, perform RNAi experiments, store important lines, and optimize microinjection procedures for transgenesis. These findings will help to optimize the design of experiments in M. lignano, and thus facilitate future research using this model organism.

Nuclear receptors (NRs), a series of key transcription factors that are mostly activated by endogenous ligands or environmental xenobiotics, are reportedly good phylogenetic markers of animal genome evolution. As the early diverging class of bilaterians, however, a comprehensive view of the NR family in a marine free-living flatworm Macrostomum lignano and comparative information in flatworms are still lacking, which is of significance to address the evolutionary diversification of the NR family and imply the adaptive evolution in the early diverging Bilateria. Herein, a total of 51, 26, and 23 putative NR genes were identified in M. lignano, Sparganum proliferum, and Clonorchis sinensis, respectively, which were classified into eight subfamilies, implying an extensive expansion of the NR family in M. lignano. It is presumed that the extensive expansion was mainly attributed to the M. lignano-specific hidden polyploidy, segmental, and tandem duplication events. The duplicated NR pairs in M. lignano and the NR orthologs in flatworms all experienced the purifying selection. Phylogenetic analyses indicated the presence of NR3-like genes in M. lignano, which is first reported in flatworms. Intron loss and reduced intron size were mainly contributed to the structural divergence of NR genes in flatworms. The combined data provide indispensable information for a better understanding of the complexity and the adaptive evolution of the NR gene family in metazoans.

Platyhelminthes comprise one of the major phyla of invertebrate animals, inhabiting a wide range of ecosystems, and one of the most successful in adapting to parasitic life. Small non-coding RNAs have been implicated in regulating complex developmental transitions in model parasitic species. Notably, parasitic flatworms have lost Piwi RNA pathways but gained a novel Argonaute gene. Herein, we analyzed, contrasted and compared the conservation of small RNA pathways among several free-living species (a paraphyletic group traditionally known as 'turbellarians') and parasitic species (organized in the monophyletic clade Neodermata) to disentangle possible adaptations during the transition to parasitism. Our findings showed that complete miRNA and RNAi pathways are present in all analyzed free-living flatworms. Remarkably, whilst all 'turbellarians' have Piwi proteins, these were lost in parasitic Neodermantans. Moreover, two clusters of Piwi class Argonaute genes are present in all 'turbellarians'. Interestingly, we identified a divergent Piwi class Argonaute in free living flatworms exclusively, which we named 'Fliwi'. In addition, other key proteins of the Piwi pathways were conserved in 'turbellarians', while none of them were detected in Neodermatans. Besides Piwi and the canonical Argonaute proteins, a flatworm-specific class of Argonautes (FL-Ago) was identified in the analyzed species confirming its ancestrallity to all Platyhelminthes. Remarkably, this clade was expanded in parasitic Neodermatans, but not in free-living species. These phyla-specific Argonautes showed lower sequence conservation compared to other Argonaute proteins, suggesting that they might have been subjected to high evolutionary rates. However, key residues involved in the interaction with the small RNA and mRNA cleavage in the canonical Argonautes were more conserved in the FL-Agos than in the Piwi Argonautes. Whether this is related to specialized functions and adaptations to parasitism in Neodermatans remains unclear. In conclusion, differences detected in gene conservation, sequence and structure of the Argonaute family suggest tentative biological and evolutionary diversifications that are unique to Platyhelminthes. The remarkable divergencies in the small RNA pathways between free-living and parasitic flatworms indicate that they may have been involved in the adaptation to parasitism of Neodermatans. [ABSTRACT FROM AUTHOR]

Species of rhabdocoels within the family Dalyelliidae (Platyhelminthes) generally inhabit fresh water and are rare in marine and brackish environments. Here we describe a new species of marine dalyelliid, Grappleria corona gen. et sp. nov., from the Pacific coast of British Columbia. Based on the distinctive morphology of its male copulatory organ and a thorough comparison of traits in all known species of marine and brackish water dalyelliids, we place this new species in a genus of its own. Phylogenetic analyses inferred from rRNA gene sequences derived from G. corona gen. et sp. nov., other representatives of the Dalyelliidae and the closely related Temnocephalida, provide justification for an updated taxonomic classification of these groups. In light of these molecular phylogenetic data, we establish the new family Jenseniidae fam. nov. to encompass genera previously considered dalyelliids but are found only in marine and brackish environments; the composition of the Dalyelliidae sensu novo now contains only freshwater genera. We also classify all temnocephalids into one family and into several subfamilies in order to reflect our improved understanding of rhabdocoel phylogeny. [ABSTRACT FROM AUTHOR]

The systematics and distribution of the order Polycladida within the Macaronesian archipelagos are analysed. New species (Marcusia alba sp. nov., Prostheceraeus crisostomum sp. nov., Parviplana sodade sp. nov., Euplana claridade sp. nov., Stylochus salis sp. nov. and Distylochus fundae sp. nov.), new variety (Pseudoceros rawlinsonae var. galaxy), new records and records of shared species among different archipelagos are studied to compare the marine flatworm biodiversity of each island. The complex of archipelagos known as Macaronesia (including Madeira, Selvagens Islands, Canary Islands, Azores and Cape Verde) share a volcanic origin and European political influence. The five archipelagos are located along the eastern coast of the Atlantic Ocean and are subject to similar trade winds, streams (like the Gulf Stream) and cold currents. The term Macaronesia has suffered several changes throughout the years and it still is a topic of discussion in present times. The new delimitation of Macaronesia is mainly based on systematic studies on the invertebrate fauna of the islands. The resulting analyses shed new light on the differences and similarities among these archipelagos. In addition, molecular analyses employing 28S nuclear gene sequences are compared to verify relationships among anatomically similar species of marine polyclads.

Living representatives of the Neotropical genus Choeradoplana Graff, 1896 (Geoplaninae, Tricladida, Platyhelminthes) are easily recognized by the typical shape of the head which is laterally expanded, rolled-up, and ventrally provided with two glandular cushions. In this study, the morphology and phylogeny (cytochrome C oxidase subunit I gene) of several species of land planarians are taxonomically investigated. Four of the six species studied are new to science, namely: Ch. eudoxiae Silva & Carbayo, sp. nov., Ch. claudioi Lago-Barcia & Carbayo, sp. nov., Ch. onae Lago-Barcia & Carbayo, sp. nov., and Ch. riutortae Lago- Barcia & Carbayo, sp. nov. The species Choeradoplana albonigra and Ch. eudoxiae deviate from the usual body shape pattern in that the head does not present lateral expansions nor glandular cushions, becoming indistinguishable from its sister genus Cephaloflexa. Pseudogeoplana tristriata (Schultze & Müller, 1857) is also redescribed from a newly collected specimen and was discovered to be a member of Choeradoplana. Graff (1899) also studied another specimen that was considered to be conspecific with P. tristriata; however, in this new it is concluded that it is not conspecific but rather a new species. The name Pseudogeoplana aevipandemiae Lago-Barcia & Carbayo, sp. nov. is suggested for Graff's specimen. [ABSTRACT FROM AUTHOR]

Background Cratera is a genus of land planarians endemic to the Brazilian Atlantic forest. The species of this genus are distinguished from each other by a series of external and internal characters, nonetheless they represent a challenging taxonomic issue due to the extreme alikeness of the species analysed in the present work. To resolve these difficulties, we have performed morphological analyses and used three nuclear markers (ribosomal 18S and 28S, Elongation Factor, a new anonymous marker named Tnuc813) and two mitochondrial fragments (Cytochrome c oxidase subunit I gene, and a fragment encompasing NADH deshydrogenase subunit 4 gene, trnF and the beginning of the Cytochrome c oxidase I gene) in an integrative taxonomic study. Methods To unveil cryptic species, we applied a molecular species delimitation approach based on molecular discovery methods, followed by a validation method. The putative species so delimited were then validated on the basis of diagnostic morphological features. Results We discovered and described four new species, namely Cratera assu, C. tui, C. boja, and C. imbiri. A fifth new species, C. paraitinga was not highly supported by molecular evidence, but was described because its morphological attributes are unique. Our study documents for the genus Cratera the presence of a number of highly similar species, a situation that is present also in other genera of land planarians. The high number of poorly differentiated and presumably recent speciation events might be explained by the recent geological history of the area.

Abstract Background Universal stress proteins (USPs) are present in all domains of life. Their expression is upregulated in response to a large variety of stress conditions. The functional diversity found in this protein family, paired with the sequence degeneration of the characteristic ATP-binding motif, suggests a complex evolutionary pattern for the paralogous USP-encoding genes. In this work, we investigated the origin, genomic organization, expression patterns and evolutionary history of the USP gene family in species of the phylum Platyhelminthes. Results Our data showed a cluster organization, a lineage-specific distribution, and the presence of several pseudogenes among the USP gene copies identified. The absence of a well conserved -CCAATCA- motif in the promoter region was positively correlated with low or null levels of gene expression, and with amino acid changes within the ligand binding motifs. Despite evidence of the pseudogenization of various USP genes, we detected an important functional divergence at several residues, mostly located near sites that are critical for ligand interaction. Conclusions Our results provide a broad framework for the evolution of the USP gene family, based on the emergence of new paralogs that face very contrasting fates, including pseudogenization, subfunctionalization or neofunctionalization. This framework aims to explain the sequence and functional diversity of this gene family, providing a foundation for future studies in other taxa in which USPs occur.

Background. Cratera is a genus of land planarians endemic to the Brazilian Atlantic forest. The species of this genus are distinguished from each other by a series of external and internal characters, nonetheless they represent a challenging taxonomic issue due to the extreme alikeness of the species analysed in the present work. To resolve these difficulties, we have performed morphological analyses and used three nuclear markers (ribosomal 18S and 28S, Elongation Factor, a new anonymous marker named Tnuc813) and two mitochondrial fragments (Cytochrome c oxidase subunit I gene, and a fragment encompasing NADH deshydrogenase subunit 4 gene, trnF and the beginning of the Cytochrome c oxidase I gene) in an integrative taxonomic study. Methods. To unveil cryptic species, we applied a molecular species delimitation approach based on molecular discovery methods, followed by a validation method. The putative species so delimited were then validated on the basis of diagnostic morphological features. Results. We discovered and described four new species, namely Cratera assu, C. tui, C. boja, and C. imbiri. A fifth new species, C. paraitinga was not highly supported by molecular evidence, but was described because its morphological attributes are unique. Our study documents for the genus Cratera the presence of a number of highly similar species, a situation that is present also in other genera of land planarians. The high number of poorly differentiated and presumably recent speciation events might be explained by the recent geological history of the area. [ABSTRACT FROM AUTHOR]

Macrostomum lignano is a free-living flatworm that is emerging as an attractive experimental animal for research on a broad range of biological questions. One feature setting it apart from other flatworms is the successful establishment of transgenesis methods, facilitated by a steady supply of eggs in the form of single-cell zygotes that can be readily manipulated. This, in combination with the transparency of the animal and its small size, creates practical advantages for imaging and fluorescence-activated cell sorting in studies related to stem cell biology and regeneration. M. lignano can regenerate most of its body parts, including the germline, thanks to the neoblasts, which represent the flatworm stem cell system. Interestingly, neoblasts seem to have a high capacity of cellular maintenance, as M. lignano can survive up to 210 Gy of γ-irradiation, and partially offset the negative consequence of ageing. As a non-self-fertilizing simultaneous hermaphrodite that reproduces in a sexual manner, M. lignano is also used to study sexual selection and other evolutionary aspects of sexual reproduction. Work over the past several years has led to the development of molecular resources and tools, including high-quality genome and transcriptome assemblies, transcriptional profiling of the germline and somatic neoblasts, gene knockdown, and in situ hybridization. The increasingly detailed characterization of this animal has also resulted in novel research questions, such as bio-adhesion based on its adhesion-release glands and genome evolution due to its recent whole-genome duplication. [ABSTRACT FROM AUTHOR]

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

The genus Macrostomum represents a diverse group of rhabditophoran flatworms with >200 species occurring around the world. Earlier we uncovered karyotype instability linked to hidden polyploidy in both M. lignano (2n = 8) and its sibling species M. janickei (2n = 10), prompting interest in the karyotype organization of close relatives. In this study, we investigated chromosome organization in two recently described and closely related Macrostomum species, M. mirumnovem and M. cliftonensis, and explored karyotype instability in laboratory lines and cultures of M. lignano (DV1/10, 2n = 10) and M. janickei in more detail. We revealed that three of the four studied species are characterized by karyotype instability, while M. cliftonensis showed a stable 2n = 6 karyotype. Next, we performed comparative cytogenetics of these species using fluorescent in situ hybridization (FISH) with a set of DNA probes (including microdissected DNA probes generated from M. lignano chromosomes, rDNA, and telomeric DNA). To explore the chromosome organization of the unusual 2n = 9 karyotype discovered in M. mirumnovem, we then generated chromosome-specific DNA probes for all chromosomes of this species. Similar to M. lignano and M. janickei, our findings suggest that M. mirumnovem arose via whole genome duplication (WGD) followed by considerable chromosome reshuffing. We discuss possible evolutionary scenarios for the emergence and reorganization of the karyotypes of these Macrostomum species and consider their suitability as promising animal models for studying the mechanisms and regularities of karyotype and genome evolution after a recent WGD. [ABSTRACT FROM AUTHOR]

Antecedentes: los Opisthorchiidae son una familia de parásitos trematodos‚ que infectan principalmente a vertebrados‚ incluidos humanos‚ y se encuentran en diferentes partes del mundo‚ especialmente en regiones donde se consumen pescados crudos o poco cocidos. En Colombia ha sido comprobada la presencia de Opisthorchiidos asociados a animales domésticos y humanos sin establecerse rutas de atención o investigación‚ demostrando que su impacto está subvalorado. Objetivo: detección y caracterización molecular y taxonómica de Opisthorchiidos asociados a cultivos piscícolas de economía campesina en el Urabá antioqueño Métodos: se evaluaron diez cultivos piscícolas de economía campesina con presencia de animales domésticos y sus afluentes. De cada cultivo se tomaron 20 peces y muestras de coprológicos de los animales domésticos. Se hizo una evaluación preliminar de ojos y aletas bajo estereoscopio y luego se utilizó un protocolo para digestión peptídica de la musculatura de los peces‚ para la búsqueda de morfos usando microscopio óptico. Las muestras fueron enviadas en alcohol al laboratorio para extracción de DNA. Se realizó PCR con siente pares de cebadores. Proyecto aprobado en acta N°131 por Comité de Ética para la Experimentación Animal de la UdeA. Resultados: se analizaron un total de 321 peces pertenecientes en su mayoría a las especies de Oreochromis niloticus (tilapia) (200/321)‚ otras (Poecilia reticulata y Colossoma macropomum)‚ 14 coprológicos‚ todos negativos. En total se han encontrado doce metacercarias de Diplostomidae‚ Echinostomatidae‚ Microphallidae y Clinostomum y Acanthocephalo peces de las especies P. reticulata y O. niloticus. Se logró estandarizar y optimizar la PCR para los siete pares de cebadores. Conclusiones: hasta el momento se ha logrado estandarizar y optimizar las PCRs para la detención de Opisthorchiidos así como comprobar su presencia en la región del Urabá. Se espera que los hallazgos de estos parásitos asociados a animales domésticos e incluso a humanos permitirá comunicar a las comunidades y los sistemas de salud médico y veterinario sobre su presencia e importancia‚ contribuyendo al aviso de alertas tempranas y riesgos en la zona de estudio. [ABSTRACT FROM AUTHOR]

The study is dedicated to the investigation of serotonin (5-hydroxytryptamine, 5-HT) and 5-HT7 type serotonin receptor of localisation in larvae of two parasitic flatworms Opisthorchis felineus (Rivolta, 1884) Blanchard, 1895 and Hymenolepis diminuta Rudolphi, 1819, performed using the immunocytochemical method and confocal laser scanning microscopy (CLSM). Using whole mount preparations and specific antibodies, a microscopic analysis of the spatial distribution of 5-HT7-immunoreactivity(-IR) was revealed in worm tissue. In metacercariae of O. felineus 5-HT7-IR was observed in the main nerve cords and in the head commissure connecting the head ganglia. The presence of 5-HT7-IR was also found in several structures located on the oral sucker. 5-HT7-IR was evident in the round glandular cells scattered throughout the larva body. In cysticercoids of H. diminuta immunostaining to 5-HT7 was found in flame cells of the excretory system. Weak staining to 5-HT7 was observed along the longitudinal and transverse muscle fibres comprising the body wall and musculature of suckers, in thin longitudinal nerve cords and a connective commissure of the central nervous system. Available publications on serotonin action in flatworms and serotonin receptors identification were reviewed. Own results and the published data indicate that the muscular structures of flatworms are deeply supplied by 5-HT7-IR elements. It suggests that the 5-HT7 type receptor can mediate the serotonin action in the investigated species and is an important component of the flatworm motor control system. The study of the neurochemical basis of parasitic flatworms can play an important role in the solution of fundamental problems in early development of the nervous system and the evolution of neuronal signalling components.

Abstract Background Small non-coding RNAs, including miRNAs, and gene silencing mediated by RNA interference have been described in free-living and parasitic lineages of flatworms, but only few key factors of the small RNA pathways have been exhaustively investigated in a limited number of species. The availability of flatworm draft genomes and predicted proteomes allowed us to perform an extended survey of the genes involved in small non-coding RNA pathways in this phylum. Results Overall, findings show that the small non-coding RNA pathways are conserved in all the analyzed flatworm linages; however notable peculiarities were identified. While Piwi genes are amplified in free-living worms they are completely absent in all parasitic species. Remarkably all flatworms share a specific Argonaute family (FL-Ago) that has been independently amplified in different lineages. Other key factors such as Dicer are also duplicated, with Dicer-2 showing structural differences between trematodes, cestodes and free-living flatworms. Similarly, a very divergent GW182 Argonaute interacting protein was identified in all flatworm linages. Contrasting to this, genes involved in the amplification of the RNAi interfering signal were detected only in the ancestral free living species Macrostomum lignano. We here described all the putative small RNA pathways present in both free living and parasitic flatworm lineages. Conclusion These findings highlight innovations specifically evolved in platyhelminths presumably associated with novel mechanisms of gene expression regulation mediated by small RNA pathways that differ to what has been classically described in model organisms. Understanding these phylum-specific innovations and the differences between free living and parasitic species might provide clues to adaptations to parasitism, and would be relevant for gene-silencing technology development for parasitic flatworms that infect hundreds of million people worldwide.

Abstract Background To date, mitochondrial genomes of more than one hundred flatworms (Platyhelminthes) have been sequenced. They show a high degree of similarity and a strong taxonomic bias towards parasitic lineages. The mitochondrial gene atp8 has not been confidently annotated in any flatworm sequenced to date. However, sampling of free-living flatworm lineages is incomplete. We addressed this by sequencing the mitochondrial genomes of the two small-bodied (about 1 mm in length) free-living flatworms Stenostomum sthenum and Macrostomum lignano as the first representatives of the earliest branching flatworm taxa Catenulida and Macrostomorpha respectively. Results We have used high-throughput DNA and RNA sequence data and PCR to establish the mitochondrial genome sequences and gene orders of S. sthenum and M. lignano. The mitochondrial genome of S. sthenum is 16,944 bp long and includes a 1,884 bp long inverted repeat region containing the complete sequences of nad3, rrnS, and nine tRNA genes. The model flatworm M. lignano has the smallest known mitochondrial genome among free-living flatworms, with a length of 14,193 bp. The mitochondrial genome of M. lignano lacks duplicated genes, however, tandem repeats were detected in a non-coding region. Mitochondrial gene order is poorly conserved in flatworms, only a single pair of adjacent ribosomal or protein-coding genes – nad4l-nad4 – was found in S. sthenum and M. lignano that also occurs in other published flatworm mitochondrial genomes. Unexpectedly, we unambiguously identified the full metazoan mitochondrial protein-coding gene complement including atp8 in S. sthenum and M. lignano. A subsequent search detected atp8 in all mitochondrial genomes of polyclad flatworms published to date, although the gene wasn’t previously annotated in these species. Conclusions Manual, but not automated genome annotation revealed the presence of atp8 in basally branching free-living flatworms, signifying both the importance of manual data curation and of diverse taxon sampling. We conclude that the loss of atp8 within flatworms is restricted to the parasitic taxon Neodermata.

In the present work, we performed a comparative genome-wide analysis of 22 species representative of the main clades and lifestyles of the phylum Platyhelminthes. We selected a set of 700 orthologous genes conserved in all species, measuring changes in GC content, codon, and amino acid usage in orthologous positions. Values of 3rd codon position GC spanned over a wide range, allowing to discriminate two distinctive clusters within freshwater turbellarians, Cestodes and Trematodes respectively. Furthermore, a hierarchical clustering of codon usage data differs remarkably from the phylogenetic tree. Additionally, we detected a synonymous codon usage bias that was more dramatic in extreme GC-poor or GC-rich genomes, i.e., GC-poor Schistosomes preferred to use AT-rich terminated synonymous codons, while GC-rich M. lignano showed the opposite behavior. Interestingly, these biases impacted the amino acidic usage, with preferred amino acids encoded by codons following the GC content trend. These are associated with non-synonymous substitutions at orthologous positions. The detailed analysis of the synonymous and non-synonymous changes provides evidence for a two-hit mechanism where both mutation and selection forces drive the diverse coding strategies of flatworms.

As coral aquaculture is increasing around the world for reef restoration and trade, mitigating the impact of coral predators, pathogens and parasites is necessary for optimal growth. The Acropora coral-eating flatworm (AEFW), Prosthiostomum acroporae (Platyhelminthes: Polycladida: Prosthiostomidae) feeds on wild and cultivated Acropora species and its inadvertent introduction into reef tanks can lead to the rapid death of coral colonies. To guide the treatment of infested corals we investigated the flatworm’s life cycle parameters at a range of temperatures that represent those found in reef tanks, coral aquaculture facilities and seasonal fluctuations in the wild. We utilized P. acroporae from a long-term in vivo culture on Acropora species to examine the effects of temperature (3°C increments from 21 to 30°C) on flatworm embryonation period, hatching success, hatchling longevity, and time to sexual maturity. Our findings show that warmer seawater shortened generation times; at 27°C it took, on average, 11 days for eggs to hatch, and 35 days for flatworms to reach sexual maturity, giving a minimum generation time of 38 days, whereas at 24°C the generation time was 64 days. Warmer seawater (24–30°C) also increased egg hatching success compared to cooler conditions (21°C). These results indicate that warmer temperatures lead to higher population densities of P. acroporae. Temperature significantly increased the growth rate of P. acroporae, with individuals reaching a larger size at sexual maturity in warmer temperatures, but it did not influence hatchling longevity. Hatchlings, which can swim as well as crawl, can survive between 0.25 and 9 days in the absence of Acropora, and could therefore disperse between coral colonies and inter-connected aquaria. We used our data to predict embryonation duration and time to sexual maturity at 21–30°C, and discuss how to optimize current treatments to disrupt the flatworm’s life cycle in captivity.