RNAscope: a novel method for the detection of Heterobilharzia americana ova in canine liver.

Canine schistosomiasis caused by Heterobilharzia americana can lead to severe morbidity and eventual mortality, in part due to the deposition of fluke ova in the liver and gastrointestinal tract, which promotes an influx of peri-ova inflammatory cells. Although fluke eggs can be identified in H&E-stained histologic sections, cases exist in which only fragments of the ova persist, or the egg is obscured by inflammatory infiltrates, which can confound definitive histologic diagnosis. Unfortunately, antibodies specific to Heterobilharzia are not commercially available for immunohistochemical labeling. Therefore, we aimed to use an RNA in situ hybridization strategy to fluorescently label Heterobilharzia ova. Using the H. americana 18S rRNA sequence, we developed an RNA probe and validated its performance on archival formalin-fixed, paraffin-embedded canine tissue. A positive signal was observed for all identifiable ova, fragmented and whole. Use of this methodology could aid understanding of the pathogenesis of H. americana infection in dogs. This technique augments standard diagnostic methodology, enabling spatial colocalization of fluke ova and inflammatory infiltrates when using fluorescent techniques.

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).

Integrative taxonomy of Mesocriconema onoense (Tylenchida: Criconematidae) from Vietnam highly suggests the synonymization of Mesocriconema brevistylus and related species.

The genus Mesocriconema is one of the most diverse genera within the family Criconematidae, known as ring nematodes, with more than 90 species. Although species in this genus usually show distinct morphological characterizations, the identification based only on morphology can lead to misidentification in many studies resulted in a number of synonymizations in the genus over time. In this study, an integrated approach has been applied in characterizing Mesocriconema onoense from Vietnam. The molecular data of 28S rRNA, ITS, 18S rRNA regions were analyzed and discussed to confirm the correct names on GenBank. Besides, phylogenetic analyses of 28S rRNA, ITS, and 18S rRNA regions of Mesocriconema species revealed that Mesocriconema brevistylus should be considered as a junior synonym of M. onoense. Consequently, M. helicus, M. onostris, and M. paronostris should also be considered as the synonyms of M. onoense.

Morphological and molecular characterization of the marine-teleost parasitizing acanthocephalan Echinorhynchus hexagrammi (Syndermata: Palaeacanthocephala) from a new host, Liparis sp. (Actinopterygii: Scorpaeniformes).

Originally described from the masked greenling Hexagrammos octogrammus (Pallas, 1814), the palaeacanthocephalan Echinorhynchus hexagrammi Baeva, 1965 has so far been known from seven species in six families of marine teleosts distributed in the Sea of Okhotsk off Sakhalin and in the Northwestern Pacific off Hokkaido, Japan. In this study, we examined the phylogenetic position of E. hexagrammi based on material obtained from the intestine of an unidentified snailfish, Liparis sp., dredged in Akkeshi Bay, Hokkaido, Japan. We performed an analysis using two gene markers, the mitochondrial cytochrome c oxidase subunit I and the nuclear 28S rRNA, along with other sequences available in public databases. In the resulting tree, E. hexagrammi was more closely related to two species complexes, the E. bothniensis Zdzitowiecki and Valtonen, 1987 complex and the E. gadi Zoega in Müller, 1776 complex, rather than to E. brayi Wayland, Sommerville, and Gibson, 1999, E. cinctulus (Porta, 1905), E. salmonis Müller, 1784, and E. truttae Schrank, 1788. The morphology of the examined material herein identified as E. hexagrammi is briefly described. This study represents the first host record of E. hexagrammi from the snailfish family Liparidae.

Phylogenetic position of Tremiorchis ranarum Mehra and Negi, 1926 (Trematoda: Plagiorchiidae) with remark on this monotypic genus.

Tremiorchis is a monotypic genus of digenetic trematode (Plagiorchiidae: Plagiorchiinae), infecting the frogs Rana tigrina (Hoplobatrachus tigerinus) and R. cyanophlyctis (Euphlyctis cyanophlyctis). Metacercaria use to infect Rana tigrina (Hoplobatrachus tigerinus) and R. cyanophlyctis (Euphlyctis cyanophlyctis) as intermediate hosts, while the cercaria stage found from apple snail, Pila virens. Adults of T. ranarum harbor mature frogs of H. tigerinus and E. cyanophlyctis. Besides the frequent infection of Tremiorchis, no DNA sequence data are currently available for this monotypic genus. The present communication, deals with the sequence data for nuclear ribosomal genes, 18S, small internal transcribed spacers (ITS1-5.8S-ITS2) and 28S to molecularly characterize T. ranarum. Besides this, phylogenetic relationship among the members of the Plagiorchiida is also discussed in detail. An attempt has also been made to provide detailed molecular affinities of T. ranarum with other trematode genera.

Systematics of Crepidostomum species from the Russian Far East and northern Japan, with description of a new species and validation of the genus Stephanophiala.

Current article touched upon the issue of the complicated taxonomic status of some species from the genus Crepidostomum collected from the freshwater fish in the rivers of Primorsky region, Sakhalin, and Hokkaido Islands. Primary morphological analyses showed affiliation of the worms to the species C. farionis (Müller, 1784) Lühe, 1909; C. metoecus Braun, 1900b; C. chaenogobii Yamaguti and Matsumura, 1942; C. nemachilus Krotov, 1959. We described the new species Crepidostomum achmerovi sp. nov. that is a sibling species of C. nemachilus. Molecular-genetic investigation have shown that C. nemachilus and C. achmerovi sp. nov. are closely related to C. metoecus in both 28S rDNA and cox1 mtDNA markers. Crepidostomum nemachilus forms a separate branch within the C. metoecus clade on the 28S BI tree with strong statistical support and separate clade in relation to C. metoecus clade on the cox1 BI tree. Values of p-distances between Crepidostomum species were at intergeneric level. Crepidostomum metoecus species complex including five species (C. metoecus, C. nemachilus, C. oschmarini, C. brinkmanni, and C. achmerovi sp. nov.) was reconsidered as independent genus Crepidostomum sensu stricto. Minimum Spanning Network showed that C. nemachilus, C. metoecus and C. achmerovi sp. nov. were separated by large number of mutational events and represent independent phyletic lines. An amended diagnosis is provided for the subfamily Crepidostomatinae, the genera Crepidostomum s. str. and Stephanophiala Nicoll, 1909, along with keys to species of both genera.

Detection and molecular characteristics of Pyelosomum cochlear (Digenea: Pronocephalidae) in the urinary bladder of the green sea turtle (Chelonia mydas) in the Northwest Pacific Ocean.

The genus Pyelosomum consists of parasitic flukes occurring primarily in marine turtles; Pyelosomum cochlear Looss 1899 is the only species of this genus that parasitizes the urinary bladder. In this study, we detected flukes in the urinary bladders of 20 of 88 green sea turtles (Chelonia mydas) harvested in the Ogasawara Islands, in the Northwest Pacific Ocean. We identified the flukes as P. cochlear based on detailed morphological observations and comparisons of morphometric measurements of the species reported previously. Nucleotide sequences of nuclear ribosomal 18S and 28S regions and the mitochondrial cytochrome c oxidase subunit 1 (COI) region were determined for the flukes. The 18S and 28S phylogenetic trees revealed that the species of the superfamily Pronocephaloidea, including P. cochlear, constituted a single clade, but the species of the family Pronocephalidae did not constitute a single taxon. These findings suggest that Pronocephalidae is a paraphyletic group. The COI sequences of P. cochlear exhibited high genetic diversity, suggesting that they would be useful markers to understand the genetic structure of the parasite and its evolutionary relationship with the host turtle populations. This is the first study to provide the nucleotide sequences of Pyelosomum species; these data will be available for further molecular studies of this genus and its related taxa.

Morphological and molecular characterization of Quinqueserialis (Digenea: Notocotylidae) species diversity in North America.

Estimates of trematode diversity are inaccurate due to unrecognized cryptic species and phenotypic plasticity within species. Integrative taxonomy (genetics, morphology and host use) increases the clarity of species delineation and improves knowledge of parasite biology. In this study, we used this approach to resolve taxonomic issues and test hypotheses of cryptic species in a genus of trematode, Quinqueserialis. Specimens from throughout North America were field collected from hosts and obtained from museums. We found three morphologically distinct groups and successfully sequenced specimens from two of these groups. DNA sequencing at the 28S and CO1 gene regions revealed that two of the three groups were genetically distinct. One genetic group included two morphological clusters demonstrating host-induced phenotypic plasticity within Quinqueserialis quinqueserialis. The other unique genetic group is a novel species, Quinqueserialis kinsellai n. sp., which is described herein. Our study illustrates the importance of integrating multiple sources of evidence when investigating trematode diversity to account for the influence of cryptic species or phenotypic plasticity. However, further sampling is needed to understand Quinqueserialis spp. diversity as some species have no genetic information associated with them.

Morphological observation and first molecular characterization of Grassenema procaviae Petter, 1959 (Cosmocercoidea: Atractidae) in the stomach of Cape hyrax (Procavia capensis) raised in a zoo in Japan.

The superfamily Cosmocercoidea comprises three families: Cosmocericidae, Kathlaniidae and Atractidae. Information on the nucleotide sequences of the Cosmocercoidea is quite limited, and the molecular classification of the whole superfamily has been slow to progress. The genus Grassenema of the family Atractidae is a parasitic nematode group that occurs in the digestive tract of hyraxes and includes three species: Grassenema procaviae, G. dendrohyraci, and G. hyracis. The type species of the genus, G. procaviae, was isolated from the digestive tract of Cape hyraxes (Procavia capensis) and has the potential to cause gastric ulcers. Although G. procaviae is a common parasite of Cape hyraxes, no genetic information for the parasite is currently available. In this study, we obtained the first genomic sequences of G. procaviae and performed detailed morphological observations. Furthermore, molecular phylogenetic analysis was performed, and the taxonomic position of the parasite was evaluated using 18S and 28S rDNA sequences. Those data will be useful for molecular identification of G. procaviae and future phylogenetic analysis within the Atractidae.

Morphological and molecular characterization of Udonella brasiliensis n. sp. (Monogenoidea), an epibiont on Caligus sp. parasite of Ariidae from the southeastern coast of Brazil.

The present study describes Udonella brasiliensis n. sp., an epibiont found on Caligus sp., a parasite the ariids Genidens barbus (Lacepède) and Aspistor luniscutis (Valenciennes), caught on the coast of the state of São Paulo, Brazil. Morphological and molecular analyses (partial 18S rDNA) were carried out. The morphological data showed that U. brasiliensis n. sp. can be distinguished from current valid species by its morphometric attributes (e.g., body, pharynx, ovary and testis), while the molecular information supports the proposal of a new species. The 18S rDNA phylogenetic analysis shows a close relationship between the new species and Udonella australis Carvajal & Sepulveda, in a subclade formed of species that parasitize South American fish. Finally, this study also discusses a scenario of initial irradiation for udonellids.

Abdominal dioctophymosis in a domestic cat from the Peruvian rainforest confirmed morphologically and molecularly.

A case of abdominal dioctophymosis in a domestic cat was found in San Juan Bautista district, the Peruvian rainforest, in the Loreto department of Peru. The pet went to a veterinary clinic for a routine ovariohysterectomy during which a large nematode was found in the abdominal cavity. The nematode was morphologically identified as an adult female of Dioctophyme sp. A few morphological parameters, such as the vagina distance from the anterior part and the egg size, were different than D. renale. Partial sequences of the cytochrome c oxidase subunit I (cox1) and the small subunit 18S ribosomal RNA genes were compared with the references from public sequence database and showed a genetic identifies of 89.25% and 99.65% with D. renale, respectively. This is the first mitochondrial molecular analysis of a Dioctophyme specimen from South America and the results showed up to 12.5% nucleotide sequence variation in cox 1 gene of D. renale.

Trematode diversity in freshwater snails from a stopover point for migratory waterfowls in Hokkaido, Japan: An assessment by molecular phylogenetic and population genetic analyses.

The cryptic diversity of trematodes was evaluated in the Nagayama-Shinkawa River, an artificial canal of the Ishikari River System of Hokkaido, Japan. Numerous migratory waterfowls use the canal as a stopover point in every spring season. The lymnaeid snail, Radix auricularia, and the semisulcospirid snail, Semisulcospira libertina, colonize the static and flowing water areas, respectively. The trematode fauna of the two snails was assessed by molecular phylogenetic and population genetic analyses. Each of distinctive clades in mitochondrial DNA trees was arbitrarily set as a species. In total, 14 species of the families Diplostomidae, Echinostomatidae, Notocotylidae, Plagiorchiidae, and Strigeidae occurred in R. auricularia, wherease S. libertina harbored 10 species of the families Echinochasmidae, Heterophyidae, Notocotylidae, and Lecithodendridae and Cercaria creta, an unclassified species whose adult stage is still unknown. The species diversity of the larval trematodes could be recognized as a "hot spot", suggesting that the seasonal visit of waterfowls is very important to spread trematodes and to keep their diversity. A high intraspecific genetic diversity was observed in the echinostomatid, notocotylid, echinochasmid, and heterophyid species, whose definitive hosts include birds. It seems likely that each of the parasite populations is always disturbed by repeated visits of waterfowls.

Notocotylus ikutai n. sp. (Digenea: Notocotylidae) from lymnaeid snails and anatid birds in Hokkaido, Japan.

An unknown species of the genus Notocotylus (Digenea: Notocotylidae) was found as the larval stage from the lymnaeid snail, Radix auricularia, in a static water area of the Chubetsu River, Hokkaido, the northernmost island of Japan. A DNA barcoding identification system was applied to detect the adult stage. Through the inspection of anatid game birds in Hokkaido, Anas crecca, Anas platyrhynchos, Anas zonorhyncha, and Mareca penelope were demonstrated to serve as the definitive hosts. The detailed morphological features of the species were characterized using adults raised experimentally in immunosuppressed mice and naturally developed larvae in R. auricularia. Although the species is morphologically similar to Notocotylus attenuatus and Notocotylus magniovatus in both adult and larval stages, its taxonomic independence was confirmed by a comprehensive study based on molecular phylogeny, morphology, and ecology. Here we propose Notocotylus ikutai n. sp. for this species. The migratory behavior of the anatid hosts and the North-Eurasian distribution of R. auricularia suggest that the new species is widely distributed in the northern Far East.

Molecular cytogenetic analysis of a triploid population of the human broad tapeworm, Dibothriocephalus latus (Diphyllobothriidea).

The large-sized tapeworm Dibothriocephalus latus is known as the broad or fish-borne cestode of mammals that is capable to infect humans and cause diphyllobothriosis. Recently, molecular data on D. latus has been accumulating in the literature and a complete genome sequence has been published; however, little is known about the karyotype and chromosome architecture. In this study, an in-depth karyological analysis of 2 D. latus specimens was carried out. The plerocercoids originated from a perch caught in subalpine Lake Iseo (Italy) and the tapeworms were reared in hamsters. Both specimens contained cells with a highly variable number of chromosomes ranging from18 to 27. Nevertheless, the largest portion of mitotic figures (47%) showed a number corresponding to the triploid set, 3n = 27. Accordingly, the karyotype of the analyzed specimens consisted of 9 triplets of metacentric chromosomes. Fluorescence in situ hybridization (FISH) with the 18S rDNA probe clearly demonstrated the presence of 3 clusters of hybridization signals on the triplet of chromosome 7, thus confirming the triploid status of the specimens. FISH with a telomeric (TTAGGG)n probe confined hybridization signals exclusively to the terminal chromosomal regions, supporting the earlier findings that this repetitive motif is a conserved feature of tapeworm telomeres.

First report on Mermithidae (Mermithida) infection in Ligidium sp. (Isopoda, Ligiidae).

Specimens of Ligidium sp. (Crustacea, Isopoda, Oniscidea) collected from Kanagawa Prefecture in Japan were found to be infected with parasitic nematodes. We have obtained two Mermithidae (Nematoda) by dissecting the fresh specimen and from the dead specimen of Ligidium, which marks the second known discovery of a mermithid infection in the order Isopoda. Herein, a report on the nuclear 28S rDNA and 18S rDNA sequence of the isolated Mermithidae nematodes and on the morphology of the juveniles is provided.

Crossing barriers of zoogeographical regions: Molecular evidence of vicariance of the only cestode parasite of loaches (Cobitidae) in the Indomalayan region, Paracaryophyllaeus lepidocephali (Cestoda: Caryophyllidea), and its redescription.

Tapeworms of the genus Paracaryophyllaeus Kulakovskaya, 1961 (Cestoda: Caryophyllidea) are specific parasites of loaches (Cypriniformes: Cobitoidea) and occur almost exclusively in the Palaearctic region. The only exception and example of vicariance over the borders of two zoogeographical regions is Paracaryophyllaeus lepidocephali (Kundu, 1985), an insufficiently known species described from the Indomalayan region, with uncertain generic allocation. In the present paper, the species is redescribed based on new material collected from the type host, Lepidocephalichthys guntea, in West Bengal, India. Molecular data reveal this species as a member of Paracaryophyllaeus, within which it is the most closely related to P. cf. gotoi from Misgurnus anguillicaudatus in China and Japan on the basis of large subunit of ribosomal nuclear DNA (28S rDNA). Generic position of P. lepidocephali examined herein is also confirmed by morphology including cross sections, in particular, by a small, cylindrical body, medullary testes with testicular fields crossing the anterior margin of the cirrus sac, medullary vitelline follicles, with some follicles paramuscular, a shallow common genital atrium, short vagina and uterus not extending anteriorly beyond the cirrus sac. It differs from all Palaearctic congeners but Paracaryophyllaeus vladkae Scholz, Oros and Aydogdu, 2014 by the testicular field crossing the anterior margin of the cirrus sac. It differs from P. vladkae by more anterior position of the first vitelline follicles compared to the first testes. This species is a unique example of a fish tapeworm crossing the borders of the Palaearctic and Indomalayan zoogeographical regions.

Hamatopeduncularia Yamaguti, 1953 (Monogenea: Ancylodiscoididae) from catfish off Peninsular Malaysia: Description of two new species and insights on the genus.

Hamatopeduncularia longiangusticirrata sp. nov. and H. petalumvaginata sp. nov. were collected from Arius maculatus and Nemapteryx caelata, respectively from Tanjung Karang, Peninsular Malaysia. Morphological and molecular investigations were carried out to ascertain the identity of the new species. The two new species differ from previously described Hamatopeduncularia species in the morphology of the male and female reproductive organs. Hamatopeduncularia longiangusticirrata sp. nov. possesses a long penis similar to H. elongata, H. longicopulatrix, H. brisbanensis, H. major and H. petalumvaginata sp. nov., but differs in having a thread-like tapering distal end and can be distinguished from H. brisbanensis and H. major in not having an accessory piece. Hamatopeduncularia longiangusticirrata sp. nov. is also unique in having an ornamented penis initial and a vaginal tube surrounded by fine hair-like structures. Hamatopeduncularia petalumvaginata sp. nov. possesses a simple penis without an accessory piece and a petaloid vaginal opening that resembles the arrangement of petals on a flower. Maximum likelihood trees were constructed from partial 28S and 18S rDNA sequences of the two new species and other ancylodiscoidids to reveal a strongly supported monophyletic branch consisting of the two new species for both markers. According to Lim's classification in 1996 of Hamatopeduncularia species penis type, H. petalumvaginata sp. nov. has been classified within the elegans-type and H. longiangusticirrata sp. nov. is proposed as the longiangusticirrata-type.

First report of Melanoides tuberculata (Mollusca: Thiaridae) harboring a xiphidiocercaria in Brazil: A new parasite introduced in the Americas?

Xiphidiocercariae were found in the invasive snail Melanoides tuberculata collected during a malacological survey in Ceará-Mirim, State of Rio Grande do Norte, Northeastern Brazil in November 2018 and submitted to morphological and molecular analyses. The morphology revealed similarities between the larvae here reported for the first time in M. tuberculata from Brazil and other xiphidiocercariae described in thiarid snails from Asia and Africa. Phylogenetic analyses based on 28S and ITS-2 sequences revealed that the larvae correspond to an unidentified species of the family Lecithodendriidae. Aspects related to the morphology and taxonomy of xiphidiocercariae found in M. tuberculata are briefly discussed. It is possible that the parasite here reported is a newly introduced species transmitted by M. tuberculata in the American continent.

An integrative taxonomic study of Susanlimocotyle narina n. gen. n. sp. (Monogenoidea, Dactylogyridae) from the nasal cavities of a marine catfish (Siluriformes, Ariidae) from the Atlantic Amazon Coast of Brazil and new molecular data of Chauhanellus spp.

Based on a taxonomic approach, combining morphological characters with DNA sequences (i.e.,18S rDNA, ITS1, 5.8S rDNA and ITS2), Susanlimocotyle n. gen. is proposed to accommodates Susanlimocotyle narina n. sp. from the nostrils of the ariid Sciades herzbergii (Bloch) from the coast of the state of Pará, Brazil. Susanlimocotyle n. gen. is characterized by species possessing: an intestinal ceca confluent posteriorly; a male copulatory organ, comprising a variable tube, articulated with the accessory piece; a sclerotized vagina, vaginal aperture dextro-ventral; an onchium; a robust ventral bar; two dorsal bars; a ventral anchor with elongated shaft and a dorsal anchor with deep root expanding into wings. In addition, new molecular data of Chauhanellus spp. are also provided and used for the evaluation of the phylogenetic relationships among monogenoids parasitizing siluriforms. Susanlimocotyle n. gen. exhibited a higher genetic divergence level for 18S rDNA (4.6 to 7.2% [83-130 bp]) with respect to Chauhanellus spp. despite sharing S. herzbergii as a host, than Hamatopeduncularia spp., (4.1 to 5.8% [75-110 bp]) from Oriental ariids. For the 18S rDNA, 5.8S rDNA, ITS1 and ITS2 regions, C. boegeri and C. susamlimae were observed to have the smallest interspecific distances, and C. velum was revealed to be the most genetically distant species to Chauhanellus. The proposal for Susanlimocotyle n. gen. is also supported by phylogenetic analysis based on the 18S rDNA gene, which supports the close relationship between the new genus and Hamatopeduncularia and Chauhanellus from ariids from the South America and Oriental regions. Moreover, the patterns towards the shared diversification between monogenoids and their ariid hosts were addressed.

Characterization of microRNAs expressed in the cystic legion of the liver of Mus musculus perorally infected with Echinococcus multilocularis Nemuro strain.

Alveolar echinococcosis (AE) is a zoonosis caused by the metacestode of Echinococcus multilocularis. The published genome of E. multilocularis showed that approximately 86% of its genome is non-coding. Micro RNAs (miRNAs) are small non-coding regulatory RNAs, and recent studies on parasitic helminths expect miRNAs as a promising target for drug development and diagnostic markers. Prior to this study, only a few studies reported the E. multilocularis miRNA profiles in the intermediate host. The primary objective of this study was to characterize miRNA profiles via small RNA-seq in E. multilocularis Nemuro strain, a laboratory strain of Asian genotype, using mice perorally infected with the parasite eggs. The data were then compared with two previously published small RNA-seq data. We identified 44 mature miRNAs as E. multilocularis origin out of the 68 mature miRNA sequences registered in the miRNA database miRbase. The highest quantities of miRNAs detected were miR-10-5p, followed by bantam-3p, let-7-5p, miR-61-3p, and miR-71-5p. The top two most abundant miRNAs (miR-10-5p and bantam-3p) accounted for approximately 80.9% of the total parasite miRNAs. The highly expressed miRNA repertoire is mostly comparable to that obtained from the previous experiment using secondary echinococcosis created by an intraperitoneal administration of metacestodes. A detailed characterization and functional annotations of these shared miRNAs will lead to a better understanding of parasitic dynamics, which could provide a basis for the development of novel diagnostic and treatment methods for AE.