A new ring nematode, Xenocriconemella costaricense sp. nov., (Nematoda: Criconematidae) from Costa Rica.

During nematode surveys of natural vegetation in forests of La Cima de Copey de Dota, San José, San José province, Costa Rica, a Xenocriconemella species closely resembling X. macrodora and related species was found. Integrative taxonomical approaches demonstrated that it is a new species described herein as X. costaricense sp. nov. The new species is parthenogenetic (only females have been detected) and characterised by a short body (276-404 µm); lip region with two annuli, not offset, not separated from body contour; first lip annulus partially covering the second lip annulus. Stylet thin, very long (113-133 µm) and flexible, occupying 30.5-47.8% of body length. Excretory pore located from one or two annuli anterior to one or two annuli posterior to level of stylet knobs, at 42 (37-45) µm from anterior end. Female genital tract monodelphic, prodelphic, outstretched, and occupying 35-45% of body length, with vagina slightly ventrally curved (14-18 µm long). Anus located 6-11 annuli from the tail terminus. Tail conoid and bluntly rounded terminus, the last 2-3 annuli oriented dorsally. Results of molecular characterisation and phylogenetic analyses of D2-D3 expansion segments of 28S rRNA, ITS, and partial 18S rRNA, as well as cytochrome oxidase c subunit 1 gene sequences further characterised the new species and clearly separated it from X. macrodora and other related species (X. iberica, X. paraiberica, and X. pradense).

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.

A set of nematode rRNA cistron databases and a primer assessment tool to enable more flexible and comprehensive metabarcoding.

The ITS-2-rRNA has been particularly useful for nematode metabarcoding but does not resolve all phylogenetic relationships, and reference sequences are not available for many nematode species. This is a particular issue when metabarcoding complex communities such as wildlife parasites or terrestrial and aquatic free-living nematode communities. We have used markerDB to produce four databases of distinct regions of the rRNA cistron: the 18S rRNA gene, the 28S rRNA gene, the ITS-1 intergenic spacer and the region spanning ITS-1_5.8S_ITS-2. These databases comprise 2645, 254, 13,461 and 10,107 unique full-length sequences representing 1391, 204, 1837 and 1322 nematode species, respectively. The comparative analysis illustrates the complementary value but also reveals a better representation of Clade III, IV and V than Clade I and Clade II nematodes in each case. Although the ITS-1 database includes the largest number of unique full-length sequences, the 18S rRNA database provides the widest taxonomic coverage. We also developed PrimerTC, a tool to assess primer sequence conservation across any reference sequence database, and have applied it to evaluate a large number of previously published rRNA cistron primers. We identified sets of primers that currently provide the broadest taxonomic coverage for each rRNA marker across the nematode phylum. These new resources will facilitate more comprehensive metabarcoding of nematode communities using either short-read or long-read sequencing platforms. Further, PrimerTC is available as a simple WebApp to guide or assess PCR primer design for any genetic marker and/or taxonomic group beyond the nematode phylum.

Phylogeny and morphology of some European cyathocotylid digeneans (Trematoda: Diplostomoidea).

The Cyathocotylidae Mühling, 1898 is a family of primitive diplostomoid trematodes important for understanding the evolution of the superfamily Diplostomoidea. However, cyathocotylids remain poorly studied with the use of molecular techniques. In this study we sequenced the 5.8S + ITS2 region, 28S rRNA, and cox1 genes of two cyathocotylid species and obtained new morphological data on them. We propose Georduboisia nom. nov. instead of the preoccupied name Duboisia Szidat, 1936 (junior homonym of Duboisia Stremme, 1911). Adults of Georduboisia cf. teganuma (Ishii, 1935) and Paracoenogonimus ovatus Katsurada, 1914 were collected from fish-eating birds in the south of the European part of Russia. Georduboisia cf. teganuma was very similar to G.teganuma but differed from it in the shape of the testes. The 28S rRNA gene dataset provided the best-resolved phylogeny of the Cyathocotylidae to date. In the phylogram based on partial sequences of this gene, P. ovatus was close to members of Holostephanoides Dubois, 1983, Neogogatea Chandler & Rausch, 1947 and Gogatea Szidat, 1936. Georduboisia cf. teganuma clustered with members of Cyathocotyle Mühling, 1896 and Holostephanus Szidat, 1936. Phylogenetic analysis based on the 5.8S + ITS2 dataset showed that adults of P. ovatus examined in our study were conspecific with the metacercariae from the musculature of fish collected in Hungary and Italy. It also revealed probable misidentifications of larvae and adults of cyathocotylids whose sequences are deposited in GenBank NCBI.

Identification of Lepidapedon oregonense as the current world's deepest trematode.

The deepest recorded depth for trematodes currently stands at approximately 6200 m. This depth record was achieved solely through sequence datasets of Lepidapedon sp. obtained from a gastropod. Given that trematodes of this genus typically use fish as definitive hosts, the origin of the trematode sequence was thought to be larval stages. However, the specific species remained unclear owing to the absence of reported adult-stage sequences. In the present study, we definitively identified the deepest trematode as Lepidapedon oregonense by comparing 28S ribosomal DNA sequences from adult worms from the macrourid fish Coelorinchus gilberti with data from the gastropod in the previous study.

Morphological and molecular data on Pseudozoogonoides ugui Shimazu, 1974 (Digenea: Microphalloidea: Zoogonidae) ex Pseudaspius hakonensis (Günther, 1877) and taxonomic problems in Zoogoninae genera.

New morphological and molecular data were generated for trematodes recovered from the intestines of the fish Pseudaspius hakonensis from two locations in the south of the Russian Far East. Morphologically, these trematodes are identical to Pseudozoogonoides ugui (Microphalloidea: Zoogonidae) from Japan. According to results of phylogenetic analysis based on 28S rDNA sequence data, P. ugui was closely related to Zoogonoides viviparus, and P. subaequiporus appears as a sister taxon to these two species. Genetic distance values, calculated based on both 28S rDNA and ITS2 rDNA, between P. ugui and Z. viviparus represents an interspecific differentiation level. Our results have an ambiguous explanation, indicating that the implication of the presence of one or two compact vitellarial aggregations for the differentiation of Zoogonoides and Pseudozoogonoides should be reconsidered or that our results open up the question of the taxonomical status of trematodes previously denoted as Z. viviparus and P. subaequiporus.

Extant interspecific hybridization among trematodes within the Schistosoma haematobium species complex in Nigeria.

BACKGROUND: Natural interspecific hybridization between the human parasite (Schistosoma haematobium [Sh]) and bovine parasites (Schistosoma bovis [Sb], Schistosoma curassoni [Sc]) is increasingly reported in Africa. We developed a multi-locus PCR DNA-Seq strategy that amplifies two unlinked nuclear (transITS, BF) and two linked organellar genome markers (CO1, ND5) to genotype S. haematobium eggs collected from infected people in Ile Oluji/Oke Igbo, Ondo State (an agrarian community) and Kachi, Jigawa State (a pastoral community) in Southwestern and Northern Nigeria, respectively. PRINCIPAL FINDINGS: Out of a total of 219 urine samples collected, 57 were positive for schistosomes. All patients from Jigawa state possessed an Sh mitochondrial genome and were infected with a genetic profile consistent with an Sh x Sb hybrid based on sequences obtained at CO1, ND5, transITS and BF nuclear markers. Whereas samples collected from Ondo state were more varied. Mitonuclear discordance was observed in all 17 patients, worms possessed an Sb mitochondrial genome but one of four different genetic profiles at the nuclear markers, either admixed (heterozygous between Sh x Sc or Sh x Sb) at both markers (n = 10), Sh at BF and admixed at transITS (Sh x Sc) (n = 5), admixed (Sh x Sc) at BF and homozygous Sc at transITS (n = 1) or homozygous Sh at BF and homozygous Sc at transITS (n = 1). SIGNIFICANCE: Previous work suggested that zoonotic transmission of S. bovis in pastoral communities, where humans and animals share a common water source, is a driving factor facilitating interspecific hybridization. However, our data showed that all samples were hybrids, with greater diversity identified in Southwestern Nigeria, a non-pastoral site. Further, one patient possessed an S. bovis mitochondrial genome but was homozygous for S. haematobium at BF and homozygous for S. curassoni at transITS supporting at least two separate backcrosses in its origin, suggesting that interspecific hybridization may be an ongoing process.

Validation of deep amplicon sequencing of Dicrocoelium in small ruminants from Northern regions of Pakistan.

Dicrocoelium lancet flukes cause significant production loss in ruminant livestock. Although co-infection with multiple Dicrocoelium species within a host is common, techniques for studying the composition of these complex parasite communities are lacking. The pathogenicity, epidemiology, and therapeutic susceptibility of different helminth species vary, and little is known about the interactions that take place between co-infecting species and their hosts. Here, we describe the first applicationof metabarcoding deep amplicon sequencing method to studythe Dicrocoelium species in sheep and goats. First, rDNA ITS-2 sequences of four Dicrocoelium species (Dicrocoelium dendriticum, Dicrocoelium hospes, Dicrocoelium orientalis, and Dicrocoelium chinensis) were extracted from the NCBI public database. Phylogenetic analysis revealed separate clades of Dicrocoelium species; hence, molecular differentiation between each species is possible in co-infections. Second, 202 flukes belonging to seventeen host populations (morphologically verified as belonging to the Dicrocoelium genus) were evaluated to determine the deep amplicon sequencing read threshold of an individual fluke for each of the four species. The accuracy of the method in proportional quantification of samples collected from single hosts was further assessed. Overall, 198 (98.01%) flukes were confirmed as D. dendriticum and 1.98% produced no reads. The comparison of genetic distances between rDNA ITS-2 revealed 86% to 98% identity between the Dicrocoelium species. Phylogenetic analysis demonstrated a distinct clustering of species, apart from D. orientalis and D. chinensis, which sit very close to each other in a single large clade whereas D. hospes and D. dendriticum are separated into their own clade. In conclusion each sample was identified as D. dendriticum based on the proportion of MiSeq reads and validated the presence of this group of parasites in the Gilgit Baltistan and Khyber Pakhtunkhwa provinces of Pakistan. The metabarcoding deep amplicon sequencing technology and bioinformatics pathway have several potential applications, including species interactions during co-infections, identifying the host and geographical distribution of Dicrocoelium in livestock, drug therapy response evaluation and understanding of the emergence and spread of drug resistance.

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.

Dirofilaria immitis and Onchocercidae spp. in wild felids from Brazil.

Among the species described within the Onchocercidae family, Dirofilaria immitis is regarded as the most common worldwide, causing severe and often fatal conditions in dogs, cats, and occasionally humans. Dirofilaria spp. are vectored by mosquitoes, simulids, and culicoids, with their epidemiology dependent on the geographical distribution of competent vectors. Eight species of Dirofilaria have been reported so far in Brazil, of which six parasitize non-human primates, deer, procyonids, and marsupials. Here, we investigated the occurrence of Onchocercidae in wild felids (i.e., Panthera onca, Puma concolor, Herpailurus yagouaroundi, Leopardus geoffroyi, Leopardus guttulus, Leopardus pardalis, Leopardus wiedii, Leopardus munoai) from different locations in Brazil. Overall, 82 samples (n = 63 blood; n = 19 tissues) were molecularly screened for cytochrome c oxidase subunit-1 (cox1) gene. Four (i.e., 4.8%) wild felid samples were positive, and at BLAST analysis, the obtained sequences showed varying percentage of nucleotide identity with the genera Brugia (i.e., 87-88%), Setaria (i.e., 89%), and D. immitis (i.e., 94.4%). Phylogenetic analyses clustered sequences obtained into three distinct clades, one with D. immitis and the remaining two with other Onchocercidae spp. Data herein obtained highlight the need for a more comprehensive understanding of the diversity and biology of Onchocercidae in South America in order to assess the potential impact that these species may have for domestic and wild animals, as well as humans.

Chromosome fusion and programmed DNA elimination shape karyotypes of nematodes.

An increasing number of metazoans undergo programmed DNA elimination (PDE), where a significant amount of DNA is selectively lost from the somatic genome during development. In some nematodes, PDE leads to the removal and remodeling of the ends of all germline chromosomes. In several species, PDE also generates internal breaks that lead to sequence loss and increased numbers of somatic chromosomes. The biological significance of these karyotype changes associated with PDE and the origin and evolution of nematode PDE remain largely unknown. Here, we assembled the single germline chromosome of the nematode Parascaris univalens and compared the karyotypes, chromosomal gene organization, and PDE features among other nematodes. We show that PDE in Parascaris converts an XX/XY sex-determination system in the germline into an XX/XO system in the somatic cells. Comparisons of Ascaris, Parascaris, and Baylisascaris ascarid chromosomes suggest that PDE existed in the ancestor of these nematodes, and their current distinct germline karyotypes were derived from fusion events of smaller ancestral chromosomes. The DNA breaks involved in PDE resolve these fused germline chromosomes into their pre-fusion karyotypes. These karyotype changes may lead to alterations in genome architecture and gene expression in the somatic cells. Cytological and genomic analyses further suggest that satellite DNA and the heterochromatic chromosome arms are dynamic and may play a role during meiosis. Overall, our results show that chromosome fusion and PDE have been harnessed in these ascarids to sculpt their karyotypes, altering the genome organization and serving specific functions in the germline and somatic cells.

Epidemiology of rumen fluke infection in selected buffalo farms in perak, malaysia: prevalence, molecular species identification, and associated risk factors.

Rumen flukes cause heavy economic losses in the ruminant industry worldwide, especially in tropical and subtropical countries. This study estimated the prevalence of rumen flukes in buffaloes, identified the species diversity, and determined risk factors associated with rumen fluke prevalence in Perak, Peninsular Malaysia. A cross-sectional study was conducted, and 321 faecal samples were collected from six buffalo farms. A structured questionnaire was developed, and farmers were interviewed to obtain information regarding risk factors associated with rumen fluke infection. The faecal samples were examined using sedimentation and Flukefinder® techniques. Genomic DNA was extracted from the fluke eggs recovered using the Flukefinder® method, and the internal transcribed spacer 2 (ITS2) fragment was amplified and sequenced to facilitate species identification. The results showed that the overall prevalence of rumen fluke across the sampled farms was 40.2% (129/321). Three rumen fluke species were identified, namely, Fischoederius elongatus, F. cobboldi, and Orthocoelium streptocoelium. Several management factors had a significant association (P < 0.05) with rumen fluke prevalence, including production type, cleaning of the stable, drinking water system, flooding around the farm, grazing system, pasture sharing with other livestock, and deworming program. This work constitutes the first attempt to understand the epidemiology of rumen fluke infection in the region and suggests that good farm management, pasture management, choosing appropriate drugs, and proper husbandry practices may improve buffalo health and production in areas where rumen flukes are prevalent.

Morphology, genetic characterization and phylogeny of Moniliformis tupaia n. sp. (Acanthocephala: Moniliformidae) from the northern tree shrew Tupaia belangeri chinensis Anderson (Mammalia: Scandentia).

A new species of Moniliformis, M. tupaia n. sp. is described using integrated morphological methods (light and scanning electron microscopy) and molecular techniques (sequencing and analysing the nuclear 18S, ITS, 28S regions and mitochondrial cox1 and cox2 genes), based on specimens collected from the intestine of the northern tree shrew Tupaia belangeri chinensis Anderson (Scandentia: Tupaiidae) in China. Phylogenetic analyses show that M. tupaia n. sp. is a sister to M. moniliformis in the genus Moniliformis, and also challenge the systematic status of Nephridiacanthus major. Moniliformis tupaia n. sp. represents the third Moniliformis species reported from China.

Gastropod invasions in anthropogenically impacted impoundments in South Africa: Tracing their origins and exploring field evidence of parasite spillback and amplification.

Invasive snails are associated with ecological problems in freshwater bodies worldwide. However, their impact on the transmission of digenean infections remain underreported. In the present study, 1708 specimens representing four snail species were sampled from four impoundments in the Limpopo River system in South Africa. Gyraulus chinensis (Planorbidae), Physella acuta (Physidae) and Pseudosuccinea columella (Lymnaeidae), which are invasive, were found in all the sampling sites. In contrast, the native lymnaeid Radix natalensis occurred at only one study site. Digeneans were observed only from R. natalensis (prevalence = 49%) and Ps. columella (prevalence = 23%). Morphological and genetic analyses revealed four digeneans: Fasciola nyanzae, Orientocreadium sp., Petasiger sp. and Patagifer vioscai. Pseudosuccinea columella was infected by the four digeneans while R. natalensis harboured only Orientocreadium sp. and Petasiger sp. Partial sequences of Orientocreadium sp. from the current study differed from congeners whose DNA data are available on GenBank, by p-distances of at least 1.84 and 2.2% for 28S and the internal transcribed spacer (ITS) rDNA, respectively. Phylogenetic analyses demonstrated that the present species is sister to Orientocreadium batrachoides. Genetic and phylogenetic data based on 28S and ITS rDNA suggested that Petasiger sp. from the present study and isolates of three unidentified Petasiger spp. from Kenya, Hungary and Australia, were representatives of the same species. This is the first known report of Orientocreadium, Petasiger and Patagifer from Ps. columella. The occurrence of F. nyanzae in Ps. columella indicates spillback from R. natalensis. These findings echo the concerns raised in previous studies about the potential role of Ps. columella in the amplification of digenean diseases in its introduced range. Phylogenetic analyses of partial sequences of the cytochrome c oxidase subunit 1 mitochondrial gene (cox1) showed multiple lineages of Ps. columella in North and South America. Pseudosuccinea columella specimens from the present study belong to an invasive genotype that has spread globally and has been reported from Zimbabwe, Egypt, Portugal, Australia, Argentina, Colombia and New Mexico (USA). Physella acuta from the current study had a stronger genetic relationship with isolates from Canada and Iceland, than with isolates from other parts of Africa, suggesting several invasion routes into Africa. This is the first known DNA characterisation of G. chinensis from Africa. Phylogenetic reconstruction indicated multiple exit events of G. chinensis from Asia into Europe and Africa. South African isolates clustered in a recent branch containing isolates from the Czech Republic and Hong Kong, China. Considering the presence of invasive snails in all the sampling sites in the present study, it is necessary to investigate the factors that enhance their establishment and to monitor their effects on the native snail populations.

Occurrence and First Molecular Characterization of Spinitectus notopteri Karve et Naik, 1951, Infected Bronze Featherback (Notopterus notopterus) in India.

PURPOSE: The nematode genus Spinitectus Fourment, 1883, comprises species that are mainly parasitic on freshwater and marine fishes. However, our knowledge of the distribution and molecular identification of Spinitectus spp. in the Indian region is rather limited. This study aims to fill this gap in our knowledge using molecular data as evidence for Spinitectus species characterization. METHODS: Bronze featherback were obtained opportunistically from the fish markets of district Muzaffarnagar (29.4727° N, 77.7085° E), Uttar Pradesh, India. Nematode species collected from the gastrointestinal tract were characterized morphologically and molecularly. Partial sequences of the ribosomal 18S rRNA gene were used for molecular characterization of the present specimens. RESULTS: The current study represented molecular analysis that determined the presence of the species Spinitectus notopteri Karve et Naik, 1951. The sequences obtained were closely related to representatives of the family Rhabdochonidae. CONCLUSION: This first molecular exploration of S. notopteri Karve et Naik, 1951, in the GenBank database and for any species of Spinitectus from India indicates a lack of genetic data for parasitic nematodes.

Application of Nested-qPCR-High Resolution Melting (HRM) Technology on Strongyloides stercoralis Isolates from Iran.

PURPOSE: Strongyloides stercoralis is a parasite with special characteristics presenting it as a unique nematode. Iran is an endemic area for S. stercoralis. In this study, nested-qPCR-high resolution melting (HRM) technology was applied on some human isolates of S. stercoralis from this country by focusing on evolutionary genetics analysis. METHODS: Twelve human isolates of S. stercoralis were collected from four endemic provinces of Iran. Genomic DNA was extracted from a single filariform larva for every isolate. Using specific primers targeting partial regions in cox1 gene, nested-qPCR-HRM was performed and melting-curve profiles were analyzed alongside the evaluation of genetic proximity and phylogenetic analysis using MEGA7 and DnaSP5 software. RESULTS: The melting temperature (Tm) values of the isolates were 77.9 °C-78.3 °C. All isolates from Guilan, Mazandaran, and Khouzestan Provinces shared Tm values of 78.2 °C to 78.3 °C, while the isolates from Hormozgan Province showed Tm values of 77.9 °C, 78.0 °C, and 78.1 °C. The phylogenetic tree illustrated that the sequences of the current study included nine haplotypes. Tajima's D index analyses showed that cox1 gene in S. stercoralis isolates was negative (Tajima's D = - 0.27). CONCLUSION: The isolates were divided into five temperature groups. Although HRM assay compared to PCR sequencing identified more limited genetic changes, it revealed that the mean of Tm of the isolates from Hormozgan Province was lower than those of other provinces and represented specific haplotypes for this geographical region on the phylogenetic tree.

Integrative Taxonomy of Prosogonotrema bilabiatum Vigueras, 1940 (Digenea: Sclerodistomidae): A Parasite in Atlantic Spadefish Chaetodipterus faber (Broussonet, 1782) (Acanthuriformes: Ephippidae) from Brazil.

OBJECTIVES: The present work aims to expand the knowledge of the digenean species Prosogonotrema bilabiatum (Sclerodistomidae), a parasite of Chaetodipterus faber (Acanthuriformes) from Brazil, with an integrative taxonomic approach, using light microscopy, scanning electron microscopy, histology, and molecular biology. METHODS: Forty-one digenean specimens were stained with hydrochloric carmine for morphological studies. Eleven parasites were dehydrated through a graded ethanol series, critical point dried with carbon dioxide, and coated with gold for scanning electron microscopy analysis. Four specimens were processed following histological routine and stained with hematoxylin and eosin and Gomori trichrome. DNA extracted was amplified using 28S partial primer D1-D3. Maximum likelihood and Bayesian inference were performed for phylogenetic analysis. RESULTS: Morphometric and morphological data of the specimens studied ranged in accordance as observed in previous descriptions of the species. Observations from scanning electron microscopy and histology corroborated with those observed in stained whole mounts. Molecular analysis showed that specimens of P. bilabiatum from Brazil clustered with another two sequences of this species from different hosts and localities, with a high node support value. CONCLUSIONS: The integrative taxonomic approach allowed to record and describe new characteristics of P. bilabiatum related to the tegument, the structure and the arrangement of its tissues. The use of molecular markers confirmed that specimens identified as P. bilabiatum from different hosts and localities are all conspecific. Further studies, mainly molecular with less conserved genetic markers, should be carried out to better understand the phylogenetic relationships of Prosogonotrema with Hemiuroidea.

Laboratory and field validation of the recombinase polymerase amplification assay targeting the Schistosoma mansoni mitochondrial minisatellite region (SmMIT-RPA) for snail xenomonitoring for schistosomiasis.

Improvements in diagnostics for schistosomiasis in both humans and snail hosts are priorities to be able to reach the World Health Organization (WHO) goal of eliminating the disease as a public health problem by 2030. In this context, molecular isothermal amplification tests, such as Recombinase Polymerase Amplification (RPA), are promising for use in endemic areas at the point-of-need for their accuracy, robustness, simplicity, and time-effectiveness. The developed recombinase polymerase amplification assay targeting the Schistosoma mansoni mitochondrial minisatellite region (SmMIT-RPA) was used to detect S. mansoni DNA from both laboratory and field Biomphalaria snails. Laboratory snails were experimentally infected and used at one, seven, and 28 days post-exposure (dpe) to 10 S. mansoni miracidia to provide samples in the early pre-patent infection stage. Field samples of Biomphalaria spp. were collected from the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil, which are endemic for S. mansoni. The sensitivity and specificity of the SmMIT-RPA assay were analysed and compared with existing loop-mediated isothermal amplification (LAMP), PCR-based methods, parasitological examination of the snails, and nucleotide sequencing. The SmMIT-RPA assay was able to detect S. mansoni DNA in the experimentally infected Biomphalaria glabrata as early as one dpe to 10 miracidia. It also detected S. mansoni infections (55.5% prevalence) in the field samples with the highest accuracy (100% sensitivity and specificity) compared with the other molecular tests used as the reference. Results from this study indicate that the SmMIT-RPA assay is a good alternative test to be used for snail xenomonitoring of S. mansoni due to its high sensitivity, accuracy, and the possibility of detecting early pre-patent infection. Its simplicity and portability also make it a suitable methodology in low-resource settings.

Secondary Structure Analysis of Fasciola from Semi-wild Ruminants of Northeast India.

PURPOSE: The objective of this study is to study the secondary structure analysis of Fasciola flukes from a rare mithun host from Manipur. Fascioliasis, a neglected tropical trematodiasis, is poorly studied in India and is widely believed to be predominantly caused by F. gigantica. Through this study, we want to assess the flukes from the rare semi-wild ruminants of Northeast India. This study is important as the mithun population is semi-wild and its population is declining in Manipur. METHODS: Sample collected from the difficult and challenging terrain of Northeast India. The sample was collected from mithun and observed under the microscope. DNA was isolated, sequenced, and analyzed using various bioinformatics tools. The secondary structure analysis of the Internal Transcribed Spacer 2 (ITS2) region was also performed. RESULTS: The secondary structure species tree corroborated the Bayesian inference and, hence, strengthened the phylogeny reconstructed. The annotated ITS2 sequence and RNA secondary of the Manipur isolate displayed the typical four-helix or four-domain model. Helix III reveals the presence of the UGGU motif with other deviations like UGG and GGU. CONCLUSION: This is an in-depth analysis of the secondary structure of Fasciola species. The present study has demonstrated the usefulness of ITS2 and its secondary structures for characterizing parasites. The information on fascioliasis in the mithun's population presents itself useful with regards to their conservation strategy as their populations in both Manipur and Nagaland are dwindling.

Sequence and Haplotype Analyses of Ligula intestinalis in Acanthobrama marmid (Cyprinidae) in Turkey.

PURPOSE: Ligulosis caused by Ligula intestinalis adversely affects the fisheries carried out in the lakes and ponds, causing economic losses in the fish industry. In this study, it was aimed to reveal the molecular characterization of L. intestinalis isolates obtained from woodfish (Acanthobrama marmid) in Keban Dam Lake in Elazig province of Turkey by using mt-CO1 gene sequences and to determine the genetic differences and haplotypes between the isolates. METHODS: In the examination made in terms of L. intestinalis, the intestine of the fish was opened with the help of fine-tipped scissors, the contents were allowed to come out, and the parasites were taken into a petri dish containing phosphate buffered saline (PBS). Then, L. intestinalis plerocercoids were taken into 15 ml falcon tubes containing 70% ethanol and stored at - 20 °C until further analysis. From each isolate, total gDNA was extracted from the plerocercoids. A partial (480 bp) mt-CO1 gene was amplified by PCR and sequenced unidirectionally. The final size of the trimmed sequences was 392 bp for 43 sequences. Sequence and haplotype analyses were performed, followed by phylogenetic analyses. RESULTS: All isolates were confirmed as L. intestinalis by BLAST analysis. In addition, 87 nucleotide mutation positions were determined among 43 CO1 gene sequences. As a result of the haplotype network performed for the mt-CO1 gene region of L. intestinalis isolates; arranged in a star-like configuration with the main haplotype (Hap05), separated from other haplotypes by 1-6 mutation steps, and 29 haplotypes were identified, covering 13.9% (6/43) of the total isolates. Also, 75 variable (polymorphic) sites were determined, 52 of which were parsimony informative sites. CONCLUSIONS: The molecular characterization of L. intestinalis in woodfish (A. marmid) was identified for the first time in Turkey.