Natural variation in the binding pocket of a parasitic flatworm TRPM channel resolves the basis for praziquantel sensitivity.

The drug praziquantel (PZQ) is the key clinical therapy for treating schistosomiasis and other infections caused by parasitic flatworms. A schistosome target for PZQ was recently identified- a transient receptor potential ion channel in the melastatin subfamily (TRPMPZQ)-however, little is known about the properties of TRPMPZQ in other parasitic flatworms. Here, TRPMPZQ orthologs were scrutinized from all currently available parasitic flatworm genomes. TRPMPZQ is present in all parasitic flatworms, and the consensus PZQ binding site was well conserved. Functional profiling of trematode, cestode, and a free-living flatworm TRPMPZQ ortholog revealed differing sensitives (~300-fold) of these TRPMPZQ channels toward PZQ, which matched the varied sensitivities of these different flatworms to PZQ. Three loci of variation were defined across the parasitic flatworm TRPMPZQ pocketome with the identity of an acidic residue in the TRP domain acting as a gatekeeper residue impacting PZQ residency within the TRPMPZQ ligand binding pocket. In trematodes and cyclophyllidean cestodes, which display high sensitivity to PZQ, this TRP domain residue is an aspartic acid which is permissive for potent activation by PZQ. However, the presence of a glutamic acid residue found in other parasitic and free-living flatworm TRPMPZQ was associated with lower sensitivity to PZQ. The definition of these different binding pocket architectures explains why PZQ shows high therapeutic effectiveness against specific fluke and tapeworm infections and will help the development of better tailored therapies toward other parasitic infections of humans, livestock, and fish.

Planarian LDB and SSDP proteins scaffold transcriptional complexes for regeneration and patterning.

Sequence-specific transcription factors often function as components of large regulatory complexes. LIM-domain binding protein (LDB) and single-stranded DNA-binding protein (SSDP) function as core scaffolds of transcriptional complexes in animals and plants. Little is known about potential partners and functions for LDB/SSDP complexes in the context of tissue regeneration. In this work, we find that planarian LDB1 and SSDP2 promote tissue regeneration, with a particular function in anterior regeneration and mediolateral polarity reestablishment. We find that LDB1 and SSDP2 interact with one another and with characterized planarian LIM-HD proteins Arrowhead, Islet1, and Lhx1/5-1. We also show that SSDP2 and LDB1 function with islet1 in polarity reestablishment and with lhx1/5-1 in serotonergic neuron maturation. Finally, we find new roles for LDB1 and SSDP2 in regulating gene expression in the planarian intestine and parenchyma; these functions are likely LIM-HD-independent. Together, our work provides insight into LDB/SSDP complexes in a highly regenerative organism. Further, our work provides a strong starting point for identifying and characterizing potential binding partners of LDB1 and SSDP2 and for exploring roles for these proteins in diverse aspects of planarian physiology.

The anthelmintic meclonazepam activates a schistosome transient receptor potential channel.

Parasitic flatworms cause various clinical and veterinary infections that impart a huge burden worldwide. The most clinically impactful infection is schistosomiasis, a neglected tropical disease caused by parasitic blood flukes. Schistosomiasis is treated with praziquantel (PZQ), an old drug introduced over 40 years ago. New drugs are urgently needed, as while PZQ is broadly effective it suffers from several limitations including poor efficacy against juvenile worms, which may prevent it from being completely curative. An old compound that retains efficacy against juvenile worms is the benzodiazepine meclonazepam (MCLZ). However, host side effects caused by benzodiazepines preclude development of MCLZ as a drug and MCLZ lacks an identified parasite target to catalyze rational drug design for engineering out human host activity. Here, we identify a transient receptor potential ion channel of the melastatin subfamily, named TRPMMCLZ, as a parasite target of MCLZ. MCLZ potently activates Schistosoma mansoni TRPMMCLZ through engagement of a binding pocket within the voltage-sensor-like domain of the ion channel to cause worm paralysis, tissue depolarization, and surface damage. TRPMMCLZ reproduces all known features of MCLZ action on schistosomes, including a lower activity versus Schistosoma japonicum, which is explained by a polymorphism within this voltage-sensor-like domain-binding pocket. TRPMMCLZ is distinct from the TRP channel targeted by PZQ (TRPMPZQ), with both anthelmintic chemotypes targeting unique parasite TRPM paralogs. This advances TRPMMCLZ as a novel druggable target that could circumvent any target-based resistance emerging in response to current mass drug administration campaigns centered on PZQ.

The effect of stress on rates of asexual reproduction in an invasive planarian.

Animal reproduction under stressful conditions is often reduced, with current survival and future reproduction being generally traded off against current reproductive activity. This study examines the impacts of physical and chemical stressors on the rates of asexual reproduction of the invasive planarian Girardia tigrina. 320 wild-caught planaria (mixed size class) were kept individually in Petri dishes such that their individual rates of fission through fragmentation could be easily monitored. Four treatment groups were compared, one chemical (5 mg/L ammonia) and one physical (decapitation), in comparison to a negative control (animals were starved of food) and a positive control where the animals were given an abundance of food. The two treatment groups immediately began reproducing asexually and accumulated the highest number of fissions over the course of the 12-day investigation period, while the positive control only began to fission after 7 days. We propose that the reproductive response observed here is an adaptive one to stressful conditions, whereby the likelihood of survival through numerical abundance is enhanced, although the size and vulnerability of resulting fragments may impose a balancing cost. The response may play a role in the invasiveness of G. tigrina by making it able to colonize environments where adverse conditions prevail.

First record of Cathaemasia hians (Trematoda: Cathaemasiidae) in a new bird host, the Eastern Imperial Eagle (Aquila heliaca).

An injured young individual of the Eastern Imperial Eagle (Aquila heliaca; Accipitridae) from the Protected Bird Area "Medzibodrozie" in the south-eastern Slovakia was subjected to the complete clinical examination at the Clinic for Birds and Exotic Animals of the University of Veterinary Medicine and Pharmacy. Adult trematodes were isolated from the pharynx of the eagle after oesophagoscopy. The morphological and molecular identification of the flukes confirmed a trematode Cathaemasia hians (Cathaemasiidae), the obligate parasite of black storks (Ciconia nigra) and white storks (Ciconia ciconia). This finding represents the first documented case of C. hians in new bird host species and indicates broader spectrum of definitive hosts of the fluke.

CBP/p300 homologs CBP2 and CBP3 play distinct roles in planarian stem cell function.

Chromatin modifications function as critical regulators of gene expression and cellular identity, especially in the regulation and maintenance of the pluripotent state. However, many studies of chromatin modification in stem cells-and pluripotent stem cells in particular-are performed in mammalian stem cell culture, an in vitro condition mimicking a very transient state during mammalian development. Thus, new models for studying pluripotent stem cells in vivo could be helpful for understanding the roles of chromatin modification, for confirming prior in vitro studies, and for exploring evolution of the pluripotent state. The freshwater flatworm, Schmidtea mediterranea, is an excellent model for studying adult pluripotent stem cells, particularly in the context of robust, whole-body regeneration. To identify chromatin modifying and remodeling enzymes critical for planarian regeneration and stem cell maintenance, we took a candidate approach and screened planarian homologs of 25 genes known to regulate chromatin biology in other organisms. Through our study, we identified six genes with novel functions in planarian homeostasis, regeneration, and behavior. Of the list of genes characterized, we identified five planarian homologs of the mammalian CREB-Binding Protein (CBP) and p300 family of histone acetyltransferases, representing an expansion of this family in planarians. We find that two planarian CBP family members are required for planarian survival, with knockdown of Smed-CBP2 and Smed-CBP3 causing distinct defects in stem cell maintenance or function. Loss of CBP2 causes a quick, dramatic loss of stem cells, while knockdown of CBP3 affects stem cells more narrowly, influencing differentiation of several cell types that include neuronal subtypes and cells of the eye. Further, we find that Smed-CBP1 is required for planarian fissioning behavior. We propose that the division of labor among a diversified CBP family in planarians presents an opportunity to dissect specific functions of a broadly important histone acetyltransferase family.

Glycomic and glycotranscriptomic profiling of mucin-type O-glycans in planarian Schmidtea mediterranea.

O-Glycans on cell surfaces play important roles in cell-cell, cell-matrix and receptor-ligand interaction. Therefore, glycan-based interactions are important for tissue regeneration and homeostasis. Free-living flatworm Schmidtea mediterranea, because of its robust regenerative potential, is of great interest in the field of stem cell biology and tissue regeneration. Nevertheless, information on the composition and structure of O-glycans in planaria is unknown. Using mass spectrometry and in silico approaches, we characterized the glycome and the related transcriptome of mucin-type O-glycans of planarian S. mediterranea. Mucin-type O-glycans were composed of multiple isomeric, methylated, and unusually extended mono- and disubstituted O-N-acetylgalactosamine structures. Extensions made of hexoses and 3-O-methyl hexoses were the glycoforms observed. From glycotranscriptomic analysis, 60 genes belonging to five distinct enzyme classes were identified to be involved in mucin-type O-glycan biosynthesis. These genes shared homology with those in other invertebrate systems. Although a majority of the genes involved in mucin-type O-glycan biosynthesis were highly expressed during organogenesis and in differentiated cells, a few select genes in each enzyme class were specifically enriched during early embryogenesis. Our results indicate a unique temporal and spatial role for mucin-type O-glycans during embryogenesis and organogenesis and in adulthood. In summary, this is the first report on O-glycans in planaria. This study expands the structural and biosynthetic possibilities in cellular glycosylation in the invertebrate glycome and provides a framework towards understanding the biological role of mucin-type O-glycans in tissue regeneration using planarians.

Pulmonary Paragonimiasis in Native Community, Esmeraldas Province, Ecuador, 2022.

Paragonimiasis is a food-borne infection caused by several species of the Paragonimus fluke. Clinical manifestations can mimic tuberculosis and contribute to diagnostic delay. We report a cluster of paragonimiasis in a community in Ecuador, where active surveillance was set up after detection of the first 2 cases.

A New Species and New Record of Freshwater Turbellarians of the Genus Gieysztoria (Platyhelminthes: Rhabdocoela) from China.

A new species of Dalyelliidae, Gieysztoria pellucida Wang and You, is described based on material collected in southern China through an integrative approach combining morphological, histological, and molecular (18S and 28S rDNA) data. Gieysztoria pellucida sp. nov. is morphologically characterized by a fan-shaped (about 270° when pressed) stylet, consisting of 13 similar distal spines and a broad girdle without fenestrae region. This stylet is distinct from that of any other similar species in the Aequales group to which this species belongs. In addition, specimens identifiable as Gieysztoria garudae Van Steenkiste, Van Mulken, and Artois, 2012 were discovered from the same location as G. pellucida sp. nov. Gieysztoria garudae has previously been known only from India; the present study thus represents the first record of the species from China.

Structural Characterization and Spatial Mapping of Tetrodotoxins in Australian Polyclads.

Tetrodotoxin (TTX) is a potent marine neurotoxin that occurs in several Australian phyla, including pufferfish, toadfish, gobies, and the blue-ringed octopus. These animals are partially immune, and TTX is known to bioaccumulate and subject to trophic transfer. As such, it could be more ubiquitously distributed in animals than is currently known. Flatworms of the order Polycladida are commonly occurring invertebrates in intertidal ecosystems and are especially diverse in Australian waters. While TTX has been identified in polyclads from Japan and New Zealand, Australian species have yet to be tested. In this study, several eastern Australian polyclad flatworm species from the suborders Cotylea and Acotylea were tested for TTX and analogs by HILIC-HRMS to understand the distribution of this toxin within these suborders. Herein, we report the detection of TTX and some known analogs in polyclad species, one of which is a pest to shellfish aquaculture. We also report, for the first time, the application of MALDI mass spectrometry imaging utilized to map TTX spatially within the intestinal system of polyclads. The identification of TTX and its analogs in Australian flatworms illustrates a broader range of toxic flatworms and highlights that analogs are important to consider when studying the distributions of toxins in animals.

It is not all about regeneration: Planarians striking power to stand starvation.

All living forms, prokaryotes as eukaryotes, have some means of adaptation to food scarcity, which extends the survival chances under extreme environmental conditions. Nowadays we know that dietary interventions, including fasting, extends lifespan of many organisms and can also protect against age-related diseases including in humans. Therefore, the capacity of adapting to periods of food scarcity may have evolved billions of years ago not only to allow immediate organismal survival but also to be able to extend organismal lifespan or at least to lead to a healthier remaining lifespan. Planarians have been the center of attention since more than two centuries because of their astonishing power of full body regeneration that relies on a large amount of adult stem cells or neoblasts. However, they also present an often-overlooked characteristic. They are able to stand long time starvation. Planarians have adapted to periods of fasting by shrinking or degrowing. Here we will review the published data about starvation in planarians and conclude with the possibility of starvation being one of the processes that rejuvenate the planarian, thus explaining the historical notion of non-ageing planarians.

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

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

Molecular insights into symbiosis-mapping sterols in a marine flatworm-algae-system using high spatial resolution MALDI-2-MS imaging with ion mobility separation.

Waminoa sp. acoel flatworms hosting Symbiodiniaceae and the related Amphidinium dinoflagellate algae are an interesting model system for symbiosis in marine environments. While the host provides a microhabitat and safety, the algae power the system by photosynthesis and supply the worm with nutrients. Among these nutrients are sterols, including cholesterol and numerous phytosterols. While it is widely accepted that these compounds are produced by the symbiotic dinoflagellates, their transfer to and fate within the sterol-auxotrophic Waminoa worm host as well as their role in its metabolism are unknown. Here we used matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging combined with laser-induced post-ionization and trapped ion mobility spectrometry (MALDI-2-TIMS-MSI) to map the spatial distribution of over 30 different sterol species in sections of the symbiotic system. The use of laser post-ionization crucially increased ion yields and allowed the recording of images with a pixel size of 5 µm. Trapped ion mobility spectrometry (TIMS) helped with the tentative assignment of over 30 sterol species. Correlation with anatomical features of the worm, revealed by host-derived phospholipid signals, and the location of the dinoflagellates, revealed by chlorophyll a signal, disclosed peculiar differences in the distribution of different sterol species (e.g. of cholesterol versus stigmasterol) within the receiving host. These findings point to sterol species-specific roles in the metabolism of Waminoa beyond a mere source of energy. They also underline the value of the MALDI-2-TIMS-MSI method to future research in the spatially resolved analysis of sterols.

Two New Brackish-Water Species of Macrostomum (Platyhelminthes: Macrostomorpha) from China and Their Phylogenetic Positions.

In this paper, two new brackish-water species of the macrostomid turbellarian genus Macrostomum, Macrostomum pseudosinense sp. nov. and Macrostomum taurinum sp. nov., collected from coastal water at Shenzhen, Guangdong Province, China, are described based on morphological, histological, and molecular phylogenetic analyses. Macrostomum pseudosinense sp. nov. differs from similar species within the genus in the length of the stylet (152 ± 15.0 µm), diameter of stylet opening (20 ± 4.0 µm proximally; 7 ± 0.5 µm distally), two bends of the stylet, and the non-spiral end of the stylet. Macrostomum taurinum sp. nov. differs from its congeners in the length of the stylet (81 ± 7.4 µm), the stylet bending position and angle (50% and 60°), diameter of stylet proximal opening (15 ± 3.0 µm), sperm with bristles and brush, and the smooth-walled ovaries. Phylogenetic analyses inferred from nuclear 18S and 28S rRNA genes support the establishments of these two new species. In addition, reciprocal mating behavior of M. pseudosinense sp. nov. was observed and documented.

The anthelmintic drug praziquantel activates a schistosome transient receptor potential channel.

The anthelmintic drug praziquantel (PZQ) is used to treat schistosomiasis, a neglected tropical disease that affects over 200 million people worldwide. PZQ causes Ca2+ influx and spastic paralysis of adult worms and rapid vacuolization of the worm surface. However, the mechanism of action of PZQ remains unknown even after 40 years of clinical use. Here, we demonstrate that PZQ activates a schistosome transient receptor potential (TRP) channel, christened SmTRPMPZQ, present in parasitic schistosomes and other PZQ-sensitive parasites. Several properties of SmTRPMPZQ were consistent with known effects of PZQ on schistosomes, including (i) nanomolar sensitivity to PZQ; (ii) stereoselectivity toward (R)-PZQ; (iii) mediation of sustained Ca2+ signals in response to PZQ; and (iv) a pharmacological profile that mirrors the well-known effects of PZQ on muscle contraction and tegumental disruption. We anticipate that these findings will spur development of novel therapeutic interventions to manage schistosome infections and broader interest in PZQ, which is finally unmasked as a potent flatworm TRP channel activator.

No head regeneration here: regeneration capacity and stem cell dynamics of Theama mediterranea (Polycladida, Platyhelminthes).

Research on the regeneration potential of flatworms (Platyhelminthes) has been mainly undertaken with planarians (Tricladida), where most species can regenerate a head and no proliferation takes place in the blastema, i.e. the early undifferentiated regenerative tissue. Only few studies are available for an early-branching group within the Platyhelminthes, the Polycladida. Head regeneration in polyclads is not possible, with a single exception from a study performed more than 100 years ago: Cestoplana was reported to be able to regenerate a head if cut a short distance behind the brain. Here, we show that 'Cestoplana' was misdetermined and most likely was the small interstitial polyclad Theama mediterranea. We revisited regeneration capacity and dynamics of T. mediterranea with live observations and stainings of musculature, nervous system, and proliferating and differentiating stem cells. In our experiments, after transversal amputation, only animals retaining more than half of the brain could fully restore the head including the brain. If completely removed, the brain was never found to regenerate to any extent. Different from planarians, but comparable to other free-living flatworms we detected cell proliferation within the posterior regeneration blastema in T. mediterranea. Similar to other free-living flatworms, proliferation did not occur within, but only outside, the differentiating organ primordia. Our results strongly imply that brain regeneration in the absence of the latter is not possible in any polyclad studied so far. Also, it appears that proliferation of stem cells within the regeneration blastema is a plesiomorphy in flatworms and that planarians are derived in this character.

Molecular Phylogenetic Analysis of Acotylea (Platyhelminthes: Polycladida).

Acotylea is a suborder of Polycladida (Rhabditophora, Platyhelminthes) characterized by lack of a cotyl (sucker-like structure) on the ventral surface of the body. We newly determined partial sequences of two mitochondrial (16S ribosomal RNA and cytochrome c oxidase subunit I) and two nuclear (18S and 28S ribosomal RNA) genes from 24 acotylean species (12 families and 14 genera). Based on these sequences in addition to those available in public databases, we inferred the phylogeny of 16 families and 27 genera of Acotylea from molecular phylogenetic analyses (maximum likelihood and Bayesian inference) based on concatenated gene sequences. Our analyses supported three clades corresponding to Discoceloidea, Leptoplanoidea, and Stylochoidea. The phylogenetic position of Callioplanidae remains unclear. Among family- or genus-level taxa, Gnesiocerotidae, Stylochoplanidae, and Comoplana were not monophyletic. We discuss the validities of Notocomplanidae and Koinostylochus, and the family-level assignment of Mirostylochus.

Echinococcus canadensis G8 Tapeworm Infection in a Sheep, China, 2018.

We report a sheep infected with Echinococcus canadensis G8 tapeworm in China in 2018. This pathogen was previously detected in moose, elk, muskox, and mule deer in Europe and North America; our findings suggest a wider host range and geographic distribution. Surveillance for the G8 tapeworm should be conducted in China.

Distribution of the newly invasive New Guinea flatworm Platydemus manokwari (Platyhelminthes: Geoplanidae) in Thailand and its potential role as a paratenic host carrying Angiostrongylus malaysiensis larvae.

Invasive species constitute one of the most serious threats to biodiversity and ecosystems, and they potentially cause economic problems and impact human health. The globally invasive New Guinea flatworm, Platydemus manokwari (Platyhelminthes: Geoplanidae), has been identified as a threat to terrestrial biodiversity, particularly soil-dwelling native species (e.g. molluscs, annelids and other land planarians), and is listed among 100 of the world's worst invasive alien species. We report here, for the first time, P. manokwari occurrences in many locations throughout Thailand, using voluntary digital public participation from the social network portals associated with the Thailand Biodiversity Conservation Group and collections of living flatworm specimens. Mitochondrial cytochrome c oxidase subunit I (COI) sequences confirmed that all collected flatworms were P. manokwari and placed them in the "world haplotype" clade alongside other previously reported specimens from France, Florida (USA), Puerto Rico, Singapore, French Polynesia, New Caledonia, and the Solomon Islands. In addition, infective stage larvae (L3) of the nematode Angiostrongylus malaysiensis were found in the flatworm specimens, with a 12.4% infection rate (15/121 specimens examined). Platydemus manokwari occurrence in Thailand and its capacity to carry L3 of Angiostrongylus should be of concern to biodiversity conservation and human health practitioners, because this invasive flatworm species may be involved in the life cycle of angiostrongylid worms in Thailand.

A targeted in situ hybridization screen identifies putative seminal fluid proteins in a simultaneously hermaphroditic flatworm.

BACKGROUND: Along with sperm, in many taxa ejaculates also contain large numbers of seminal fluid proteins (SFPs). SFPs and sperm are transferred to the mating partner, where they are thought to play key roles in mediating post-mating sexual selection. They modulate the partner's behavior and physiology in ways that influence the reproductive success of both partners, thus potentially leading to sexual conflict. Despite the presumed general functional and evolutionary significance of SFPs, their identification and characterization has to date focused on just a few animal groups, predominantly insects and mammals. Moreover, until now seminal fluid profiling has mainly focused on species with separate sexes. Here we report a comprehensive screen for putative SFPs in the simultaneously hermaphroditic flatworm Macrostomum lignano. RESULTS: Based on existing transcriptomic data, we selected 150 transcripts known to be (a) predominantly expressed in the tail region of the worms, where the seminal fluid-producing prostate gland cells are located, and (b) differentially expressed in social environments differing in sperm competition level, strongly implying that they represent a phenotypically plastic aspect of male reproductive allocation in this species. For these SFP candidates, we then performed whole-mount in situ hybridization (ISH) experiments to characterize tissue-specific expression. In total, we identified 98 transcripts that exhibited prostate-specific expression, 76 of which we found to be expressed exclusively in the prostate gland cells; additional sites of expression for the remaining 22 included the testis or other gland cells. Bioinformatics analyses of the prostate-limited candidates revealed that at least 64 are predicted to be secretory proteins, making these especially strong candidates to be SFPs that are transferred during copulation. CONCLUSIONS: Our study represents a first comprehensive analysis using a combination of transcriptomic and ISH screen data to identify SFPs based on transcript expression in seminal fluid-producing tissues. We thereby extend the range of taxa for which seminal fluid has been characterized to a flatworm species with a sequenced genome and for which several methods such as antibody staining, transgenesis and RNA interference have been established. Our data provide a basis for testing the functional and evolutionary significance of SFPs.