Comprehensive analysis of miRNA profiling in Schistosoma mekongi across life cycle stages.

Schistosoma mekongi, a significant schistosome parasite, has various life stages, including egg, cercaria, female, and male, that play crucial roles in the complex life cycle. This study aimed to explore the microRNA (miRNA) profiles across these developmental stages to understand their potential functions and evolutionary significance, which have not been studied. Pre-processed sequencing reads of small RNA (sRNA) were obtained, and annotations were performed against the S. japonicum reference miRNA database. Results indicated marked variations in miRNA profiles across different life stages, with notable similarities observed between female and male S. mekongi. Principal Coordinate Analysis (PCoA) and unsupervised clustering revealed distinct miRNA signatures for each stage. Gene ontology (GO) analysis unveiled the potential roles of these miRNAs in various biological processes. The differential expression of specific miRNAs was prominent across stages, suggesting their involvement in crucial developmental processes. Furthermore, orthologous miRNA analysis against various worm species revealed distinct presence-absence patterns, providing insights into the evolutionary relationships of these miRNAs. In conclusion, this comprehensive investigation into the miRNA profiles of S. mekongi offers valuable insights into the functional and evolutionary aspects of miRNAs in schistosome biology.

A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex.

Spliced leader trans-splicing is essential for gene expression in many eukaryotes. To elucidate the molecular mechanism of this process, we characterise the molecules associated with the Caenorhabditis elegans major spliced leader snRNP (SL1 snRNP), which donates the spliced leader that replaces the 5' untranslated region of most pre-mRNAs. Using a GFP-tagged version of the SL1 snRNP protein SNA-1 created by CRISPR-mediated genome engineering, we immunoprecipitate and identify RNAs and protein components by RIP-Seq and mass spectrometry. This reveals the composition of the SL1 snRNP and identifies associations with spliceosome components PRP-8 and PRP-19. Significantly, we identify a novel, nematode-specific protein required for SL1 trans-splicing, which we designate SNA-3. SNA-3 is an essential, nuclear protein with three NADAR domains whose function is unknown. Mutation of key residues in NADAR domains inactivates the protein, indicating that domain function is required for activity. SNA-3 interacts with the CBC-ARS2 complex and other factors involved in RNA metabolism, including SUT-1 protein, through RNA or protein-mediated contacts revealed by yeast two-hybrid assays, localisation studies and immunoprecipitations. Our data are compatible with a role for SNA-3 in coordinating trans-splicing with target pre-mRNA transcription or in the processing of the Y-branch product of the trans-splicing reaction.

MicroRNAs in Helminth Parasites: A Systematic Review.

BACKGROUND: MicroRNAs (miRNAs) are about 22-nucleotide, small, noncoding RNAs that control gene expression post-transcriptionally. Helminth parasites usually express a unique repertoire of genes, including miRNAs, across different developmental stages with subtle regulatory mechanisms. OBJECTIVE: There is a necessity to investigate the involvement of miRNAs in the development of parasites, host-parasite interaction, immune evasion and their abilities to govern infection in hosts. MiRNAs present in helminth parasites have been summarized in the current systematic review (SR). METHODS: Electronic databases, including PubMed, Scopus, ProQuest, Embase, and Google Scholar search engine, were searched to identify helminth miRNA studies published from February 1993 till December 2019. Only the published articles in English were included in the study. RESULTS: A total of 1769 articles were preliminarily recorded. Following the strict inclusion and exclusion criteria, 105 studies were included in this SR. Most of these studies focused on the identification of miRNAs in helminth parasites and/or probing of differentially expressed host miRNA profiles in specific relevant tissues, while 12 studies aimed to detect parasite-derived miRNAs in host circulating system and 15 studies characterized extracellular vesicles (EV)-derived miRNAs secreted by parasites. CONCLUSION: In the current SR, information regarding all miRNAs expressed in helminth parasites has been comprehensively provided and the utility of helminth parasitesderived miRNAs in diagnosis and control of parasitic infections has been discussed. Furthermore, functional studies on helminth-derived miRNAs have also been presented.

An unknown species of Leucochloridium (Trematoda: Leucochloridiidae) from northern Honshu, Japan.

Pulsating broodsacs of Leucochloridium sp. (Trematoda: Leucochloridiidae) were found from amber snails (Succinea lauta) in Iwate, the northern part of Honshu, Japan. A pattern with red-brown vertical stripes was characteristic of the broodsac. Very similar broodsacs were already detected from Okinawa Islands, the southern archipelago of Japan, and tentatively identified as Leucochloridium cf. passeri. A phylogenetic analysis based on DNA sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) showed that Leucochloridium sp. is different at species level from L. cf. passeri and that both species are related to Leucochloridium vogtianum from Europe. In this study the definitive identification of larval Leucochloridium sp. was impossible, but the resulting phylogeny confirmed that at least 4 species of Leucochloridium are distributed in Japan, depending on locality and climate. The DNA barcode generated in this study will be useful in detecting the adult stage of Leucochloridium sp. from birds.

Global Run-On sequencing to measure nascent transcription in C. elegans.

Global Run-On sequencing (GRO-seq) is one of the most sensitive techniques to detect nascent transcription from RNA polymerase (Pol) at a genome-wide level. The protocol incorporates labeled ribonucleotides into nascent RNAs from Pol I, II, and III. We have adapted the GRO-seq protocol to the nematode Caenorhabditis elegans to measure transcription from embryos and adult worms. Here, we provide a detailed overview of the protocol highlighting the critical steps for generating successful libraries. For complete details on the use and execution of this protocol, please refer to Quarato et al. (2021).

Dityrosine Crosslinking of Collagen and Amyloid-ß Peptides Is Formed by Vitamin B12 Deficiency-Generated Oxidative Stress in Caenorhabditis elegans.

(1) Background: Vitamin B12 deficiency in Caenorhabditis elegans results in severe oxidative stress and induces morphological abnormality in mutants due to disordered cuticle collagen biosynthesis. We clarified the underlying mechanism leading to such mutant worms due to vitamin B12 deficiency. (2) Results: The deficient worms exhibited decreased collagen levels of up to approximately 59% compared with the control. Although vitamin B12 deficiency did not affect the mRNA expression of prolyl 4-hydroxylase, which catalyzes the formation of 4-hydroxyproline involved in intercellular collagen biosynthesis, the level of ascorbic acid, a prolyl 4-hydroxylase coenzyme, was markedly decreased. Dityrosine crosslinking is involved in the extracellular maturation of worm collagen. The dityrosine level of collagen significantly increased in the deficient worms compared with the control. However, vitamin B12 deficiency hardly affected the mRNA expression levels of bli-3 and mlt-7, which are encoding crosslinking-related enzymes, suggesting that deficiency-induced oxidative stress leads to dityrosine crosslinking. Moreover, using GMC101 mutant worms that express the full-length human amyloid ß, we found that vitamin B12 deficiency did not affect the gene and protein expressions of amyloid ß but increased the formation of dityrosine crosslinking in the amyloid ß protein. (3) Conclusions: Vitamin B12-deficient wild-type worms showed motility dysfunction due to decreased collagen levels and the formation of highly tyrosine-crosslinked collagen, potentially reducing their flexibility. In GMC101 mutant worms, vitamin B12 deficiency-induced oxidative stress triggers dityrosine-crosslinked amyloid ß formation, which might promote its stabilization and toxic oligomerization.

Dual roles for piRNAs in promoting and preventing gene silencing in C. elegans.

Piwi-interacting RNAs (piRNAs) regulate many biological processes through mechanisms that are not fully understood. In Caenorhabditis elegans, piRNAs intersect the endogenous RNA interference (RNAi) pathway, involving a distinct class of small RNAs called 22G-RNAs, to regulate gene expression in the germline. In the absence of piRNAs, 22G-RNA production from many genes is reduced, pointing to a role for piRNAs in facilitating endogenous RNAi. Here, however, we show that many genes gain, rather than lose, 22G-RNAs in the absence of piRNAs, which is in some instances coincident with RNA silencing. Aberrant 22G-RNA production is somewhat stochastic but once established can occur within a population for at least 50 generations. Thus, piRNAs both promote and suppress 22G-RNA production and gene silencing. rRNAs and histones are hypersusceptible to aberrant silencing, but we do not find evidence that their misexpression is the primary cause of the transgenerational sterility observed in piRNA-defective mutants.

Diagnosis of Taenia solium infections based on "mail order" RNA-sequencing of single tapeworm egg isolates from stool samples.

Combined community health programs aiming at health education, preventive anti-parasitic chemotherapy, and vaccination of pigs have proven their potential to regionally reduce and even eliminate Taenia solium infections that are associated with a high risk of neurological disease through ingestion of T. solium eggs. Yet it remains challenging to target T. solium endemic regions precisely or to make exact diagnoses in individual patients. One major reason is that the widely available stool microscopy may identify Taenia ssp. eggs in stool samples as such, but fails to distinguish between invasive (T. solium) and less invasive Taenia (T. saginata, T. asiatica, and T. hydatigena) species. The identification of Taenia ssp. eggs in routine stool samples often prompts a time-consuming and frequently unsuccessful epidemiologic workup in remote villages far away from a diagnostic laboratory. Here we present "mail order" single egg RNA-sequencing, a new method allowing the identification of the exact Taenia ssp. based on a few eggs found in routine diagnostic stool samples. We provide first T. solium transcriptome data, which show extremely high mitochondrial DNA (mtDNA) transcript counts that can be used for subspecies classification. "Mail order" RNA-sequencing can be administered by health personnel equipped with basic laboratory tools such as a microscope, a Bunsen burner, and access to an international post office for shipment of samples to a next generation sequencing facility. Our suggested workflow combines traditional stool microscopy, RNA-extraction from single Taenia eggs with mitochondrial RNA-sequencing, followed by bioinformatic processing with a basic laptop computer. The workflow could help to better target preventive healthcare measures and improve diagnostic specificity in individual patients based on incidental findings of Taenia ssp. eggs in diagnostic laboratories with limited resources.

The RNA modification in Echinococcus granulosus cysts revealed by mass spectrometry.

RNA modifications, as one of epigenetic mechanisms, are important and conserved mechanisms for maintaining the homeostasis of organisms. Little is known about RNA modifications in Echinococcus granulosus, an obligate parasite that inhabits mammals and gives rise to a huge public health and economic impact. Here, we focused on the RNA modification characteristics of E. granulosus for the first time by using mass spectrometry (UPLC-MS/MS) to qualitatively and quantitatively analyze 47 types of RNA modifications in E. granulosus total RNA. Furthermore, the E. granulosus homologs of writer enzymes preforming RNA modifications were identified, and their gene expression pattern at different developmental stages were analyzed by bioinformatics analysis. Finally, 23 types of RNA modifications were found in E. granulosus cysts total RNA, of which m1A, Ψ and m5C are the most abundant. The homologs of writer enzymes involved in these modifications were identified in the E. granulosus genome, with the dynamic gene expression during the different parasitic developmental stages. This work confirms that E. granulosus retains the conserved RNA modification mechanism during evolution, suggesting the important role of RNA modification in regulating its development and parasitic process. Moreover, the differences of amino acid sequences of RNA modification writer enzymes between parasite and host make it possible to use these enzymes as the candidate drug targets in the follow-up in-depth researches.

Screening by deep sequencing reveals mediators of microRNA tailing in C. elegans.

microRNAs are frequently modified by addition of untemplated nucleotides to the 3' end, but the role of this tailing is often unclear. Here we characterize the prevalence and functional consequences of microRNA tailing in vivo, using Caenorhabditis elegans. MicroRNA tailing in C. elegans consists mostly of mono-uridylation of mature microRNA species, with rarer mono-adenylation which is likely added to microRNA precursors. Through a targeted RNAi screen, we discover that the TUT4/TUT7 gene family member CID-1/CDE-1/PUP-1 is required for uridylation, whereas the GLD2 gene family member F31C3.2-here named GLD-2-related 2 (GLDR-2)-is required for adenylation. Thus, the TUT4/TUT7 and GLD2 gene families have broadly conserved roles in miRNA modification. We specifically examine the role of tailing in microRNA turnover. We determine half-lives of microRNAs after acute inactivation of microRNA biogenesis, revealing that half-lives are generally long (median = 20.7 h), as observed in other systems. Although we observe that the proportion of tailed species increases over time after biogenesis, disrupting tailing does not alter microRNA decay. Thus, tailing is not a global regulator of decay in C. elegans. Nonetheless, by identifying the responsible enzymes, this study lays the groundwork to explore whether tailing plays more specialized context- or miRNA-specific regulatory roles.

RNA editing restricts hyperactive ciliary kinases.

Protein kinase activity must be precisely regulated, but how a cell governs hyperactive kinases remains unclear. In this study, we generated a constitutively active mitogen-activated protein kinase DYF-5 (DYF-5CA) in Caenorhabditis elegans that disrupted sensory cilia. Genetic suppressor screens identified that mutations of ADR-2, an RNA adenosine deaminase, rescued ciliary phenotypes of dyf-5CA We found that dyf-5CA animals abnormally transcribed antisense RNAs that pair with dyf-5CA messenger RNA (mRNA) to form double-stranded RNA, recruiting ADR-2 to edit the region ectopically. RNA editing impaired dyf-5CA mRNA splicing, and the resultant intron retentions blocked DYF-5CA protein translation and activated nonsense-mediated dyf-5CA mRNA decay. The kinase RNA editing requires kinase hyperactivity. The similar RNA editing-dependent feedback regulation restricted the other ciliary kinases NEKL-4/NEK10 and DYF-18/CCRK, which suggests a widespread mechanism that underlies kinase regulation.

DESCRIPTION AND PHYLOGENETIC AFFINITIES OF A NEW SPECIES OF NEOPSILOTREMA (DIGENEA: PSILOSTOMIDAE) FROM LESSER SCAUP, AYTHYA AFFINIS (ANSERIFORMES: ANATIDAE).

Neopsilotrema is a small genus of psilostomid digeneans parasitic in the intestine of birds in the Palearctic and Nearctic. At present, the genus includes 4 species: Neopsilotrema lisitsynae from the Palearctic and Neopsilotrema affine, Neopsilotrema lakotae, and Neopsilotrema marilae from the Nearctic. Herein, we describe a new species, Neopsilotrema itascae n. sp., from lesser scaup Aythya affinis collected in Minnesota. The species can be distinguished from congeners on the basis of the ventral sucker:oral sucker width ratio, body width:length ratio, and cirrus sac size, along with other characters. We generated new 28S ribosomal deoxyribonucleic acid (DNA) and NADH dehydrogenase (ND1) mitochondrial DNA sequence data of a variety of psilostomids from the Palearctic and Nearctic along with sequences of the ribosomal internal transcribed spacer (ITS) region (ITS1 + 5.8S + ITS2) from 3 Neopsilotrema species. The molecular phylogenetic affinities of a variety of psilostomid taxa were studied using 28S sequence data. The 28S sequences of psilostomids demonstrated 1-7.9% intergeneric divergence, whereas the sequences of ND1 had 17.7-34.1% intergeneric divergence. The interspecific divergence among members of Neopsilotrema was somewhat lower (0.2-0.5% in 28S; 0.3-0.4% in ITS; 12-15.7% in ND1). Our comparison of DNA sequences along with morphologic study suggests Holarctic distribution of N. lisitsynae.

Small RNAs and chromatin in the multigenerational epigenetic landscape of Caenorhabditis elegans.

For decades, it was thought that the only heritable information transmitted from one individual to another was that encoded in the DNA sequence. However, it has become increasingly clear that this is not the case and that the transmission of molecules from within the cytoplasm of the gamete also plays a significant role in heritability. The roundworm, Caenorhabditis elegans, has emerged as one of the leading model organisms in which to study the mechanisms of transgenerational epigenetic inheritance (TEI). Collaborative efforts over the past few years have revealed that RNA molecules play a critical role in transmitting transgenerational responses, but precisely how they do so is as yet uncertain. In addition, the role of histone modifications in epigenetic inheritance is increasingly apparent, and RNA and histones interact in a way that we do not yet fully understand. Furthermore, both exogenous and endogenous RNA molecules, as well as other environmental triggers, are able to induce heritable epigenetic changes that affect transcription across the genome. In most cases, these epigenetic changes last only for a handful of generations, but occasionally can be maintained much longer: perhaps indefinitely. In this review, we discuss the current understanding of the role of RNA and histones in TEI, as well as making clear the gaps in our knowledge. We also speculate on the evolutionary implications of epigenetic inheritance, particularly in the context of a short-lived, clonally propagating species. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

Protease-mediated processing of Argonaute proteins controls small RNA association.

Small RNA pathways defend the germlines of animals against selfish genetic elements, yet pathway activities need to be contained to prevent silencing of self genes. Here, we reveal a proteolytic mechanism that controls endogenous small interfering (22G) RNA activity in the Caenorhabditis elegans germline to protect genome integrity and maintain fertility. We find that DPF-3, a P-granule-localized N-terminal dipeptidase orthologous to mammalian dipeptidyl peptidase (DPP) 8/9, processes the unusually proline-rich N termini of WAGO-1 and WAGO-3 Argonaute (Ago) proteins. Without DPF-3 activity, these WAGO proteins lose their proper complement of 22G RNAs. Desilencing of repeat-containing and transposon-derived transcripts, DNA damage, and acute sterility ensue. These phenotypes are recapitulated when WAGO-1 and WAGO-3 are rendered resistant to DPF-3-mediated processing, identifying them as critical substrates of DPF-3. We conclude that N-terminal processing of Ago proteins regulates their activity and promotes silencing of selfish genetic elements by ensuring Ago association with appropriate small RNAs.

Urocleidus sayani n. sp. (Monogenea: Dactylogyridae) from Gills of Pirate Perch (Aphredoderus sayanus).

Urocleidus sayani n. sp. is described from the gills of pirate perch (Aphredoderus sayanus) in the Wisconsin backwaters of the upper Mississippi River and was found in samples from the Southeastern United States. Urocleidus sayani n. sp. is the second monogenean described from the pirate perch and the first for this host within Dactylogyridae. The description includes a partial 18S rRNA gene sequence (623 bp), filling a void in sequence data from North American monogeneans.

Stress resets ancestral heritable small RNA responses.

Transgenerational inheritance of small RNAs challenges basic concepts of heredity. In Caenorhabditis elegans nematodes, small RNAs are transmitted across generations to establish a transgenerational memory trace of ancestral environments and distinguish self-genes from non-self-elements. Carryover of aberrant heritable small RNA responses was shown to be maladaptive and to lead to sterility. Here, we show that various types of stress (starvation, high temperatures, and high osmolarity) induce resetting of ancestral small RNA responses and a genome-wide reduction in heritable small RNA levels. We found that mutants that are defective in various stress pathways exhibit irregular RNAi inheritance dynamics even in the absence of stress. Moreover, we discovered that resetting of ancestral RNAi responses is specifically orchestrated by factors that function in the p38 MAPK pathway and the transcription factor SKN-1/Nrf2. Stress-dependent termination of small RNA inheritance could protect from run-on of environment-irrelevant heritable gene regulation.

Novel high-performance detection of Raillietina echinobothrida, Raillietina tetragona, and Raillietina cesticillus using loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD).

Cestodes belonging to the genus Raillietina are a major veterinary health problem in the poultry industry, especially in chickens (Gallus gallus domesticus) and ducks (Anas playtrhynchos domesticus). In this study, loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay was established and validated for the detection of R. echinobothrida, R. tetragona, and R. cesticillus in one reaction. The LAMP-LFD assay can be completed in 75 min under isothermal conditions at 66 °C and the results can be obtained by observation with the naked eye. This assay was very specific and had no cross-amplification with other closely related parasites (Cotugnia sp., Diorchis formosensis, Fimbriaria sp., Echinostoma sp., E. miyagawai, Hypoderaeum conoideum, Prosthogonimus cuneatus, and Ascaridia galli) or their definitive hosts (G. g. domesticus, A. p. domesticus). The sensitivity of the LAMP-LFD assay was detected with three Raillietina species at 0.5 ng, which was enough for gravid proglottid DNA detection. The accuracy test showed that the LAMP-LFD assay demonstrated accurate verification results when compared to morphological results. This is a novel LAMP-LFD assay that is highly specific and sensitive for the detection of Raillietina species. It can be applied to detection for epidemiological investigations, monitoring programs, surveillance, control, and to solve veterinary health problems for the poultry industry in Raillietina endemic areas.

Hidden diversity of the most basal tapeworms (Cestoda, Gyrocotylidea), the enigmatic parasites of holocephalans (Chimaeriformes).

Gyrocotylideans are evolutionary ancient parasitic flatworms, and like their hosts-a relict group of holocephalan fishes (Chimaeriformes)-they are considered to be "living fossils" of a vanished past. However, the species diversity, host associations and biogeography of these most basal tapeworms are poorly known. Herein, we provide evidence of a conspicuous contrast between the genetic and morphological data based on an examination of newly collected and properly processed Gyrocotyle specimens (hologenophores) isolated from holocephalans off Taiwan and Argentina. Our molecular data, inferred from three genes (COI, 28S rRNA, 18S rRNA), showed unexpected genetic interrelationships among isolates of the genus Gyrocotyle, because each of the four genotypes from Taiwan clustered with isolates of distinct gyrocotylideans from the North Atlantic. Three genotypes of Gyrocotyle from Taiwan were morphologically almost indistinguishable from each other but represented distinct genetic lineages; a single specimen of Gyrocotyle sp. genotype 4 exhibited a clear genetic and morphological distinctness, though its formal description as a new species would be premature. Additionally, specimens of Gyrocotyle rugosa Diesing, 1850, from the type host Callorhinchus callorynchus from Argentina, provided the first genetic data on the type species of the genus and enabled us to characterise it, which is necessary for future taxonomic studies. The finding of some specimens of Gyrocotyle sp. genotype 3 in Chimaera phantasma, and another one in C. cf. argiloba, together with the putative conspecificity of an unidentified gyrocotylidean from Callorhinchus milii off Australia and G. rugosa from C. callorynchus off Argentina, represent evidence that one gyrocotylidean species may parasitise more than one holocephalan host species. Existing taxonomic problems and conflicts between morphological and molecular data on species of Gyrocotyle can only be resolved if hologenophores from type hosts and localities of nominal taxa are properly characterised genetically and morphologically.

Dracunculiasis in a domestic dog in Brazil.

We report and discuss the surprising encounter of a dog naturally infected by Dracunculus sp. in Brazil, a brief clinical history of the animal and a procedure for removing the nematode. We also present details on the morphology of the fragments collected from the nematode and a phylogenetic comparison of the partial sequences of the mitochondrial 18S rRNA and cytochrome c oxidase subunit I (COI) genes, deposited with others in GenBank. The samples were an independent lineage forming a well-supported monophyletic assemblage with D. medinensis. We thus conclude that this species has not yet been sequenced or even described and will only be elucidated by more information because only two species of Dracunculus have been reported in Brazil, D. fuelleborni and D. brasiliensis.

Characterization of the complete mitochondrial genome of Notocotylus sp. (Trematoda, Notocotylidae) and its phylogenetic implications.

The parasite genus Notocotylus comprises at least 50 species colonizing mainly aquatic birds and to a lesser extent some mammals, particularly rodents. Here trematode specimens isolated from a wild black swan were characterized and identified to belong to the genus Notocotylus via morphological and molecular analyses. Phylogenetic position of the isolate among other trematodes was determined based on the ribosomal internal transcribed spacer (ITS) 1 and 2. The complete mitochondrial (mt) genome of the isolate was amplified, sequenced, assembled, analyzed, and annotated. The isolate has an AT-rich mt genome (14,317 bp in length) that comprises 12 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA genes. The Notocotylus isolate identified in this study has relatively high mt genome sequence identity and identical gene content and arrangement to a known Notocotylidae species, Ogmocotyle sikae. The isolate formed a genetic clade with O. sikae in phylogenetic analysis of the concatenated PCG amino acid sequences. Compared to the ITS, the trematode mt genome appears more informative for resolving high-level phylogenies. To the best of our knowledge, this is the first study exploring the complete mt genome for the genus Notocotylus, and it offers a novel genomic resource that has important implications for trematode phylogenetics.