The Acoel nervous system: morphology and development.

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

Proteomic analysis of Neobenedenia sp. and Rhabdosynochus viridisi (Monogenea, Monopisthocotylea): Insights into potential vaccine targets and diagnostic markers for finfish aquaculture.

Monogeneans are parasitic flatworms that represent a significant threat to the aquaculture industry. Species like Neobenedenia melleni (Capsalidae) and Rhabdosynochus viridisi (Diplectanidae) have been identified as causing diseases in farmed fish. In the past years, molecular research on monogeneans of the subclass Monopisthocotylea has focused on the generation of genomic and transcriptomic information and the identification in silico of some protein families of veterinary interest. Proteomic analysis has been suggested as a powerful tool to investigate proteins in parasites and identify potential targets for vaccine development and diagnosis. To date, the proteomic dataset for monogeneans has been restricted to a species of the subclass Polyopisthocotylea, while in monopisthocotyleans there is no proteomic data. In this study, we present the first proteomic data on two monopisthocotylean species, Neobenedenia sp. and R. viridisi, obtained from three distinct sample types: tissue, excretory-secretory products (ESPs), and eggs. A total of 1691 and 1846 expressed proteins were identified in Neobenedenia sp. and R. viridisi, respectively. The actin family was the largest protein family, followed by the tubulin family and the heat shock protein 70 (HSP70) family. We focused mainly on ESPs because they are important to modulate the host immune system. We identified proteins of the actin, tubulin, HSP70 and HSP90 families in both tissue and ESPs, which have been recognized for their antigenic activities in parasitic flatworms. Furthermore, our study uncovered the presence of proteins within ESPs, such as annexin, calcium-binding protein, fructose bisphosphate aldolase, glutamate dehydrogenase, myoferlin, and paramyosin, that are targets for immunodiagnostic and vaccine development and hold paramount relevance in veterinary medicine. This study expands our knowledge of monogeneans and identified proteins that, in other platyhelminths are potential targets for vaccines and drug discovery.

Sperm competition favours intermediate sperm size in a hermaphrodite1.

Sperm competition is a potent mechanism of postcopulatory sexual selection that has been found to shape reproductive morphologies and behaviours in promiscuous animals. Especially sperm size has been argued to evolve in response to sperm competition through its effect on sperm longevity, sperm motility, the ability to displace competing sperm, and ultimately fertilization success. Additionally, sperm size has been observed to co-evolve with female reproductive morphology. Theoretical work predicts that sperm competition may select for longer sperm but may also favour shorter sperm if sperm size trades-off with number. In this study, we studied the relationship between sperm size and postmating success in the free-living flatworm, Macrostomum lignano. Specifically, we used inbred isolines of M. lignano that varied in sperm size to investigate how sperm size translated into the ability of worms to transfer and deposit sperm in a mating partner. Our results revealed a hump-shaped relationship with individuals producing sperm of intermediate size having the highest sperm competitiveness. This finding broadens our understanding of the evolution of sperm morphology by providing empirical support for stabilizing selection on sperm size under sperm competition.

Phylogenetic position of Ancyrocephalus (sensu lato) curtus Achmerov, 1952 (Monopisthocotylea, Dactylogyridae), a parasite of fish Perccottus glenii Dybowski, 1877(Gobiiformes: Odontobutidae).

The genus Ancyrocephalus sensu lato is a large assemblage of species of dactylogyrid monopisthocotyleans without clear taxonomic boundaries. Despite an urgent need for revision, only three representatives of this taxon have been molecularly characterised so far. We found specimens of Ancyrocephalus curtus, a previously non-genotyped species, in gills of Perccottus glenii caught in the River Syumnyur, Amur Basin, Russia. The aim of this study was to assess the phylogenetic position of this parasite using partial sequences of 28S rRNA gene. In the phylogenetic tree, A. curtus appeared as a sister taxon to the dactylogyrine genus Gobioecetes. The new molecular evidence supports the hypothesis about the non-monophyletic status of Ancyrocephalus sensu lato.

Volume X-Ray Micro-Computed Tomography Analysis of the Early Cephalized Central Nervous System in a Marine Flatworm, Stylochoplana pusilla.

Platyhelminthes are a phylum of simple bilaterian invertebrates with prototypic body systems. Compared with non-bilaterians such as cnidarians, the bilaterians are likely to exhibit integrated free-moving behaviors, which require a concentrated nervous system "brain" rather than the distributed nervous system of radiatans. Marine flatworms have an early cephalized 'central' nervous system compared not only with non-bilaterians but also with parasitic flatworms or freshwater planarians. In this study, we used the marine flatworm Stylochoplana pusilla as an excellent model organism in Platyhelminthes because of the early cephalized central nervous system. Here, we investigated the three-dimensional structures of the flatworm central nervous system by the use of X-ray micro-computed tomography (micro-CT) in a synchrotron radiation facility. We found that the obtained tomographic images were sufficient to discriminate some characteristic structures of the nervous system, including nerve cords around the cephalic ganglion, mushroom body-like structures, and putative optic nerves forming an optic commissure-like structure. Through the micro-CT imaging, we could obtain undistorted serial section images, permitting us to visualize precise spatial relationships of neuronal subpopulations and nerve tracts. 3-D micro-CT is very effective in the volume analysis of the nervous system at the cellular level; the methodology is straightforward and could be applied to many other non-model organisms.

Japanese Planocerid Flatworms: Difference in Composition of Tetrodotoxin and Its Analogs and the Effects of Ingestion by Toxin-Bearing Fishes in the Ryukyu Islands, Japan.

Tetrodotoxin (TTX), known as pufferfish toxin, is a potent neurotoxin blocking sodium channels in muscle and nerve tissues. TTX has been detected in various taxa other than pufferfish, including marine polyclad flatworms, suggesting that pufferfish toxin accumulates in fish bodies via food webs. The composition of TTX and its analogs in the flatworm Planocera multitentaculata was identical to those in wild grass puffer Takifugu alboplumbeus. Previously, Planocera sp. from Okinawa Island, Japan, were reported to possess high level of TTX, but no information was available on TTX analogs in this species. Here we identified TTX and analogs in the planocerid flatworm using high-resolution liquid chromatography-mass spectrometry, and compared the composition of TTX and analogs with those of another toxic and non-toxic planocerid species. We show that the composition of TTX and several analogs, such as 5,6,11-trideoxyTTX, dideoxyTTXs, deoxyTTXs, and 11-norTTX-6(S)-ol, of Planocera sp. was identical to those of toxic species, but not to its non-toxic counterpart. The difference in the toxin composition was reflected in the phylogenetic relationship based on the mitochondrial genome sequence. A toxification experiment using predatory fish and egg plates of P. multitentaculata demonstrated that the composition of TTX and analogs in wild T. alboplumbeus juveniles was reproduced in artificially toxified pufferfish. Additionally, feeding on the flatworm egg plates enhanced the signal intensities of all TTX compounds in Chelonodon patoca and that of deoxyTTXs in Yongeichthys criniger.

An inside out journey: biogenesis, ultrastructure and proteomic characterisation of the ectoparasitic flatworm Sparicotyle chrysophrii extracellular vesicles.

BACKGROUND: Helminth extracellular vesicles (EVs) are known to have a three-way communication function among parasitic helminths, their host and the host-associated microbiota. They are considered biological containers that may carry virulence factors, being therefore appealing as therapeutic and prophylactic target candidates. This study aims to describe and characterise EVs secreted by Sparicotyle chrysophrii (Polyopisthocotyla: Microcotylidae), a blood-feeding gill parasite of gilthead seabream (Sparus aurata), causing significant economic losses in Mediterranean aquaculture. METHODS: To identify proteins involved in extracellular vesicle biogenesis, genomic datasets from S. chrysophrii were mined in silico using known protein sequences from Clonorchis spp., Echinococcus spp., Fasciola spp., Fasciolopsis spp., Opisthorchis spp., Paragonimus spp. and Schistosoma spp. The location and ultrastructure of EVs were visualised by transmission electron microscopy after fixing adult S. chrysophrii specimens by high-pressure freezing and freeze substitution. EVs were isolated and purified from adult S. chrysophrii (n = 200) using a newly developed ultracentrifugation-size-exclusion chromatography protocol for Polyopisthocotyla, and EVs were characterised via nanoparticle tracking analysis and tandem mass spectrometry. RESULTS: Fifty-nine proteins involved in EV biogenesis were identified in S. chrysophrii, and EVs compatible with ectosomes were observed in the syncytial layer of the haptoral region lining the clamps. The isolated and purified nanoparticles had a mean size of 251.8 nm and yielded 1.71 × 108 particles · mL-1. The protein composition analysis identified proteins related to peptide hydrolases, GTPases, EF-hand domain proteins, aerobic energy metabolism, anticoagulant/lipid-binding, haem detoxification, iron transport, EV biogenesis-related, vesicle-trafficking and other cytoskeletal-related proteins. Several identified proteins, such as leucyl and alanyl aminopeptidases, calpain, ferritin, dynein light chain, 14-3-3, heat shock protein 70, annexin, tubulin, glutathione S-transferase, superoxide dismutase, enolase and fructose-bisphosphate aldolase, have already been proposed as target candidates for therapeutic or prophylactic purposes. CONCLUSIONS: We have unambiguously demonstrated for the first time to our knowledge the secretion of EVs by an ectoparasitic flatworm, inferring their biogenesis machinery at a genomic and transcriptomic level, and by identifying their location and protein composition. The identification of multiple therapeutic targets among EVs' protein repertoire provides opportunities for target-based drug discovery and vaccine development for the first time in Polyopisthocotyla (sensu Monogenea), and in a fish-ectoparasite model.

The invasive land flatworm Arthurdendyus triangulatus has repeated sequences in the mitogenome, extra-long cox2 gene and paralogous nuclear rRNA clusters.

Using a combination of short- and long-reads sequencing, we were able to sequence the complete mitochondrial genome of the invasive 'New Zealand flatworm' Arthurdendyus triangulatus (Geoplanidae, Rhynchodeminae, Caenoplanini) and its two complete paralogous nuclear rRNA gene clusters. The mitogenome has a total length of 20,309 bp and contains repetitions that includes two types of tandem-repeats that could not be solved by short-reads sequencing. We also sequenced for the first time the mitogenomes of four species of Caenoplana (Caenoplanini). A maximum likelihood phylogeny associated A. triangulatus with the other Caenoplanini but Parakontikia ventrolineata and Australopacifica atrata were rejected from the Caenoplanini and associated instead with the Rhynchodemini, with Platydemus manokwari. It was found that the mitogenomes of all species of the subfamily Rhynchodeminae share several unusual structural features, including a very long cox2 gene. This is the first time that the complete paralogous rRNA clusters, which differ in length, sequence and seemingly number of copies, were obtained for a Geoplanidae.

Two new Neotetraonchus species (Dactylogyridea, Dactylogyridae) parasitising the Peruvian sea catfish Galeichthys peruvianus (Siluriformes, Ariidae), including molecular data.

As part of a parasitological survey, several specimens of two new monopisthocotylean species, Neotetraonchus celsomanueli sp. nov. and N.peruvianus sp. nov. (Dactylogyridea, Dactylogyridae), were collected from the gill filaments of the Peruvian sea catfish Galeichthys peruvianus (Siluriformes, Ariidae) off Puerto Pizarro, Tumbes region, Peru. Neotetraonchus celsomanueli sp. nov. is characterised by an MCO with a T-shaped distal end and an accessory piece that is ribbed and expanded proximally with a worm-shaped termination. Neotetraonchus peruvianus sp. nov. is typified by its MCO, which has a sledgehammer-shaped distal end and an accessory piece with a claw-shaped distal end. Additionally, N.peruvianus sp. nov. is characterised by its jellyfish-shaped onchium. A partial 28S rDNA sequence was obtained from N.celsomanueli sp. nov., and a phylogenetic analysis was conducted. This analysis revealed the phylogenetic position of Neotetraonchus celsomanueli sp. nov. within a clade comprising monopisthocotylean parasites of diadromous and marine ariid catfishes, including Hamatopeduncularia spp., Chauhanellus spp., Thysanotohaptor Kritsky, Shameem, Kumari & Krishnaveni, , and Neocalceostomoides spinivaginalis Lim, 1995. This finding brings the number of known Neotetraonchus species to seven and represents the first described Neotetraonchus species infecting marine catfishes from Peru.

Description of three new species of Gyrodactylus von Nordmann, 1832 (Monogenea: Gyrodactylidae) on bitterling fishes (Acheilognathinae) from China.

Three new species of Gyrodactylus are described from three species of bitterling in Donghu Lake, China: Gyrodactylus ocellorhodei n. sp. from Rhodeus ocellatus; G. sinenorhodei n. sp. from Rhodeus sinensis; and G. acheilorhodei n. sp. from Acheilognathus macropterus. All the three new species showed similar opisthaptor morphology, especially the marginal hooks: all had a slender and perpendicular sickle shaft, and flat sickle base with distinct heel and inner arch which was different from the G. rhodei-group species parasitic on bitterling. Multivariate analyses based on hamulus and marginal hooks suggested that these three new species cannot be completely distinguished, despite some morphology divergence observed in certain less reliable morphometric features, such as hamulus root length, ventral bar total length and process shape. These three new species shared an identical 18S ribosomal RNA gene sequence, while the variation in the Internal Transcribed Spacers (ITS1-ITS2) sequence among them (8.4-11.2%, K2P) far exceeded the 1% ITS sequence difference that had been suggested as a threshold for species delimitation of Gyrodactylus. Phylogenetic analysis based on ITS1-ITS2 showed that all these sequenced Gyrodactylus spp. parasitic on the subfamily Acheilognathinae host formed a monophyletic group. However, a clear differentiation (18.9-20.9%, K2P of ITS1-ITS2) could be found between the subgroup from China (G. ocellorhodei n. sp., G. sinenorhodei n. sp. and G. acheilorhodei n. sp.) and that from Europe (G. rhodei).

Genome-wide identification of histone lysine methyltransferases and their implications in the epigenetic regulation of eggshell formation-related genes in a trematode parasite Clonorchis sinensis.

Epigenetic writers including DNA and histone lysine methyltransferases (DNMT and HKMT, respectively) play an initiative role in the differentiation and development of eukaryotic organisms through the spatiotemporal regulation of functional gene expressions. However, the epigenetic mechanisms have long been suspected in helminth parasites lacking the major DNA methyltransferases DNMT1 and DNMT3a/3b. Very little information on the evolutionary status of the epigenetic tools and their role in regulating chromosomal genes is currently available in the parasitic trematodes. We previously suggested the probable role of a DNMT2-like protein (CsDNMT2) as a genuine epigenetic writer in a trematode parasite Clonorchis sinensis. Here, we analyzed the phylogeny of HKMT subfamily members in the liver fluke and other platyhelminth species. The platyhelminth genomes examined conserved genes for the most of SET domain-containing HKMT and Disruptor of Telomeric Silencing 1 subfamilies, while some genes were expanded specifically in certain platyhelminth genomes. Related to the high gene dosages for HKMT activities covering differential but somewhat overlapping substrate specificities, variously methylated histones were recognized throughout the tissues/organs of C. sinensis adults. The temporal expressions of genes involved in eggshell formation were gradually decreased to their lowest levels proportionally to aging, whereas those of some epigenetic tool genes were re-boosted in the later adult stages of the parasite. Furthermore, these expression levels were significantly affected by treatment with DNMT and HKMT inhibitors. Our data strongly suggest that methylated histones are potent epigenetic markers that modulate the spatiotemporal expressions of C. sinensis genes, especially those involved in sexual reproduction.

Biocontrol efficiency and characterization of insecticidal protein from sugarcane endophytic Serratia marcescens (SM) against oriental armyworm Mythimna separata (Walker).

The plant endophytic bacteria are a great source of nature insecticides. However, no such endophytic bacteria have been found in sugarcane, to address this gap, we isolated and identified a strain of Serratia marcescens with moderate insecticidal activity from sugarcane. Taken armyworm Mythimna separata as example, the mortality rates of oral infection and injection infection were 47.06 % and 91 %, respectively. The SM has significant negative affect on the growth, development, and reproduction of M. separata. After determining that these insecticidal substances, 33 potential virulence proteins were screened through the identification and prediction of bacterial proteins. Later we confirmed serralysin was a vital toxic protein from SM that caused M. separata death by prokaryotic expression. In addition, we also found that the intestinal tissue cells infected with SM or serralysin were severely diseased, which may be a major factor in M. separata demise. Finally, through gene expression level, protein molecular docking, we found the aminopeptidase-N would be one of the potential receptors of serralysin. Taken together, our findings indicate that sugarcane endophyte S. marcescens may be beneficial for pest control in sugarcane and explain its insecticidal mechanism. This study provides new ideas and materials for the biological control of pests.

Description of two new species of dactylogirids (Monogenoidea: Dactylogyridae) from Mylossoma albiscopum (Cope, 1872) (Characiformes: Serrasalmidae) from the Peruvian Amazonia.

INTRODUCTION: Two new dactylogyrid monogeneans, Anacanthorus cultro n. sp. and Notozothecium palometae n. sp., are described based on specimens collected from the gill filaments of Mylossoma albiscopum (Cope, 1872) (Characiformes: Serrasalmidae), a pelagic and herbivore teleost collected in the Tigre river, Loreto, Peru. MATERIALS AND METHODS: Some monogeneans were stained with Gomori's trichrome and mounted in Canada Balsam to determine internal soft structures. Others were cleared in Hoyer's medium for the study of sclerotized structures. Drawings were made using a drawing tube and a microprojector. RESULTS: Anacanthorus cultro n. sp. is characterized by the presence of a rod-shaped male copulatory organ (MCO) lacking featherlike structures, and a knife-shaped accessory piece with a submedial knob. Notozothecium palometae n. sp. can be distinguished from all congeners by its MCO with a subbasal spur and an accessory piece with inverted hammer shaped. CONCLUSIONS: This is the first data on the parasites of M. albiscopum, a popular fish in local markets. Anacanthorus cultro n. sp. is the twenty-fifth documented species of the genus in Peru, while N. palometae n. sp. represents the seventh species of the genus known to infect freshwater fish species in Peru.

Optimized protocols for RNA interference in Macrostomum lignano.

Macrostomum lignano, a marine free-living flatworm, has emerged as a potent invertebrate model in developmental biology for studying stem cells, germline, and regeneration processes. In recent years, many tools have been developed to manipulate this worm and to facilitate genetic modification. RNA interference is currently the most accessible and direct technique to investigate gene functions. It is obtained by soaking worms in artificial seawater containing dsRNA targeting the gene of interest. Although easy to perform, the original protocol calls for daily exchange of dsRNA solutions, usually until phenotypes are observed, which is both time- and cost-consuming. In this work, we have evaluated alternative dsRNA delivery techniques, such as electroporation and osmotic shock, to facilitate the experiments with improved time and cost efficiency. During our investigation to optimize RNAi, we demonstrated that, in the absence of diatoms, regular single soaking in artificial seawater containing dsRNA directly produced in bacteria or synthesized in vitro is, in most cases, sufficient to induce a potent gene knockdown for several days with a single soaking step. Therefore, this new and highly simplified method allows a very significant reduction of dsRNA consumption and lab work. In addition, it enables performing experiments on a larger number of worms at minimal cost.

Structure of putative epidermal sensory receptors in an acoel flatworm, Praesagittifera naikaiensis.

Acoel flatworms possess epidermal sensory-receptor cells on their body surfaces and exhibit behavioral repertoires such as geotaxis and phototaxis. Acoel epidermal sensory receptors should be mechanical and/or chemical receptors; however, the mechanisms of their sensory reception have not been elucidated. We examined the three-dimensional relationship between epidermal sensory receptors and their innervation in an acoel flatworm, Praesagittifera naikaiensis. The distribution of the sensory receptors was different between the ventral and dorsal sides of worms. The nervous system was mainly composed of a peripheral nerve net, an anterior brain, and three pairs of longitudinal nerve cords. The nerve net was located closer to the body surface than the brain and the nerve cords. The sensory receptors have neural connections with the nerve net in the entire body of worms. We identified five homologs of polycystic kidney disease (PKD): PKD1-1, PKD1-2, PKD1-3, PKD1-4, and, PKD2, from the P. naikaiensis genome. All of these PKD genes were implied to be expressed in the epidermal sensory receptors of P. naikaiensis. PKD1-1 and PKD2 were dispersed across the entire body of worms. PKD1-2, PKD1-3, and PKD1-4 were expressed in the anterior region of worms. PKD1-4 was also expressed around the mouth opening. Our results indicated that P. naikaiensis possessed several types of epidermal sensory receptors to convert various environmental stimuli into electrical signals via the PKD channels and transmit the signals to afferent nerve and/or effector cells.

Geography, phylogeny and host switch drive the coevolution of parasitic Gyrodactylus flatworms and their hosts.

BACKGROUND: Gyrodactylus is a lineage of monogenean flatworm ectoparasites exhibiting many features that make them a suitable model to study the host-parasite coevolutionary dynamics. Previous coevolutionary studies of this lineage mainly relied on low-power datasets (a small number of samples and a single molecular marker) and (now) outdated algorithms. METHODS: To investigate the coevolutionary relationship of gyrodactylids and their fish hosts in high resolution, we used complete mitogenomes (including two newly sequenced Gyrodactylus species), a large number of species in the single-gene dataset, and four different coevolutionary algorithms. RESULTS: The overall coevolutionary fit between the parasites and hosts was consistently significant. Multiple indicators confirmed that gyrodactylids are generally highly host-specific parasites, but several species could parasitize either multiple (more than 5) or phylogenetically distant fish hosts. The molecular dating results indicated that gyrodactylids tend to evolve towards high host specificity. Speciation by host switch was identified as a more important speciation mode than co-speciation. Assuming that the ancestral host belonged to Cypriniformes, we inferred four major host switch events to non-Cypriniformes hosts (mostly Salmoniformes), all of which occurred deep in the evolutionary history. Despite their relative rarity, these events had strong macroevolutionary consequences for gyrodactylid diversity. For example, in our dataset, 57.28% of all studied gyrodactylids parasitized only non-Cypriniformes hosts, which implies that the evolutionary history of more than half of all included lineages could be traced back to these major host switch events. The geographical co-occurrence of fishes and gyrodactylids determined the host use by these gyrodactylids, and geography accounted for most of the phylogenetic signal in host use. CONCLUSIONS: Our findings suggest that the coevolution of Gyrodactylus flatworms and their hosts is largely driven by geography, phylogeny, and host switches.

Flatworm cocoons in the abyss: same plan under pressure.

While knowledge of early ontogeny in abyssal animals is highly limited in general, it was completely lacking for abyssal, free-living platyhelminths. We discovered flatworm egg capsules (or 'cocoons') on rocks collected at depths of 6176-6200 m on the abyssal slope of the Kuril-Kamchatka Trench, northwestern Pacific. The egg capsules were black and spherical, around 3 mm in diameter, and contained three to seven individuals (n = 4) at the same developmental stage, either the spherical (putative early embryo) or vermiform (putative late embryo) stages. A molecular phylogenetic analysis based on 18S and 28S rRNA sequences revealed that the flatworms belong in suborder Maricola in Tricladida and suggested that they may have colonized from shallow to deep waters. This study provides the deepest record for free-living flatworms and the first information on their early life stages in the abyssal zone, which were very similar to those in shallow-water forms. This similarity in development between the relatively benign shallow-water and the extreme abyssal environments suggests that triclads adapting to the latter faced primarily physiological and/or ecological adaptive challenges rather than developmental ones.

The ribosomal transcription units of five echinostomes and their taxonomic implications for the suborder Echinostomata (Trematoda: Platyhelminthes).

Five newly obtained nuclear ribosomal transcription unit (rTU) sequences from Echinostomatidae and Echinochasmidae are presented. The inter- and intrafamilial relationships of these and other families in the suborder Echinostomata are also analyzed. The sequences obtained are the complete rTU of Artyfechinostomum malayanum (9,499 bp), the near-complete rTU of Hypoderaeum conoideum (8,076 bp), and the coding regions (from 5'-terminus of 18S to 3'-terminus of 28S rRNA gene) in Echinostoma revolutum (6,856 bp), Echinostoma miyagawai (6,854 bp), and Echinochasmus japonicus (7,150 bp). Except for the longer first internal transcribed spacer (ITS1) in Echinochasmus japonicus, all genes and spacers were almost identical in length. Comprehensive maximum-likelihood phylogenies were constructed using the PhyML software package. The datasets were either the concatenated 28S + 18S rDNA sequences (5.7-5.8 kb) from 60 complete rTUs of 19 families or complete 28S sequences only (about 3.8-3.9 kb) from 70 strains or species of 22 families. The phylogenetic trees confirmed Echinostomatoidea as monophyletic. Furthermore, a detailed phylogeny constructed from alignments of 169 28S D1-D3 rDNA sequences (1.1-1.3 kb) from 98 species of 50 genera of 10 families, including 154 echinostomatoid sequences (85 species/42 genera), clearly indicated known generic relationships within Echinostomatidae and Echinochasmidae and relationships of families within Echinostomata and several other suborders. Within Echinostomatidae, Echinostoma, Artyfechinostomum, and Hypoderaeum appeared as monophyletic, while Echinochasmus (Echinochasmidae) was polyphyletic. The Echinochasmidae are a sister group to the Psilostomidae. The datasets provided here will be useful for taxonomic reappraisal as well as studies of evolutionary and population genetics in the superfamily Echinostomatoidea, the sole superfamily in the suborder Echinostomata.

piRNA generation is associated with the pioneer round of translation in stem cells.

Much insight has been gained on how stem cells maintain genomic integrity, but less attention has been paid to how they maintain their transcriptome. Here, we report that the PIWI protein SMEDWI-1 plays a role in the filtering of dysfunctional transcripts from the transcriptome of planarian stem cells. SMEDWI-1 accomplishes this through association with the ribosomes during the pioneer round of translation, and processing of poorly translated transcripts into piRNAs. This results in the removal of such transcripts from the cytoplasmic pool and at the same time creates a dynamic pool of small RNAs for post-transcriptional surveillance through the piRNA pathway. Loss of SMEDWI-1 results in elevated levels of several non-coding transcripts, including rRNAs, snRNAs and pseudogene mRNAs, while reducing levels of several coding transcripts. In the absence of SMEDWI-1, stem cell colonies are delayed in their expansion and a higher fraction of descendants exit the stem cell state, indicating that this transcriptomic sanitation mediated by SMEDWI-1 is essential to maintain stem cell health. This study presents a new model for the function of PIWI proteins in stem cell maintenance, that complements their role in transposon repression, and proposes a new biogenesis pathway for piRNAs in stem cells.

Contrasting the form and strength of pre- and postcopulatory sexual selection in a flatworm.

Sexual traits may be selected during multiple consecutive episodes of selection, occurring before, during, or after copulation. The overall strength and form of selection acting on traits may thus be determined by how selection (co-)varies along different episodes. However, it is challenging to measure pre- and postcopulatory phenotypic traits alongside variation in fitness components at each different episode. Here, we used a transgenic line of the transparent flatworm Macrostomum lignano expressing green fluorescent protein (GFP) in all cell types, including sperm cells, enabling in vivo sperm tracking. We assessed the mating success, sperm-transfer efficiency, and sperm fertilizing efficiency of GFP(+) focal worms in which we measured 13 morphological traits. We found linear selection on sperm production rate arising from pre- and postcopulatory components and on copulatory organ shape arising from sperm fertilizing efficiency. We further found nonlinear (mostly concave) selection on combinations of copulatory organ and sperm morphology traits arising mostly from sperm-transfer efficiency and sperm fertilizing efficiency. Our study provides a fine-scale quantification of sexual selection, showing that both the form and strength of selection can change across fitness components. Quantifying how sexual selection builds up along episodes of selection allows us to better understand the evolution of sexually selected traits.