Ultra-structure, genetic characterization and Immunological approach of fish borne zoonotic trematodes (Family: Heterophyidae) of a redbelly tilapia.

Fish-borne zoonotic trematodes (FBZT) are highly significant zoonotic trematodes that can infect humans by eating raw or undercooked fish harboring active metacercaria. In this investigation, FBZT was found in samples of widely cultivated redbelly tilapia (Tilapia zillii) obtained from the Fayum governorate. Encysted metacercaria (EMC) infection was identified in fish belonging to the heterophyid family morphologically. The prevalence of heterophyid EMC was 30.5%. EMC was identified and implemented in a subsequent study on domestic pigeons (Columba livia domestica) carried out to allow adult flukes of Pygidiopsis (P.) genata; P. summa; and Ascocotyle (A.) pindoramensis species in their small intestine. This study presents the first report that combines ultra-structure, molecular approach of three species of heterophyid flukes, ultra-structure using transmission electron microscope in P. genata, and the study of host immunological responses and associated cytokines during Pygidiopsis species infection of pigeons in Egypt. Using Quantitative Real-time PCR (qRT- PCR), the gene expression levels of six cytokines (IL-1, IL-2, IL-6, IL-10, IFN-γ and TGF-ß3) were assessed. The molecular confirmation of P. genata, P. summa, and A. pindoramensis have a registration in the GenBank under accession number MT672308.1, OR083433.1, and OR083431.1, respectively. Throughout the infection, the gut produced cytokines in considerably variable amounts. As a result of the Pygidiopsis species infection in pigeons, our data showed distinctive cytokine alterations, which could aid in figuring out the immunological pathogenesis and host defense mechanism against this infection. This study focused on different types of fish-borne trematodes, particularly the zoonotically important ones.

Scaphanocephalus spp. (Trematoda: Opisthorchiidae) in intermediate and definitive hosts of the Yucatán Peninsula, Mexico, with a re-description of Scaphanocephalus expansus.

Scaphanocephalus is a small trematode genus belonging to the family Opistorchiidae. The genus currently contains only three species associated with marine fish as intermediate hosts and fish-eating birds as definitive hosts. Here, specimens of Scaphanocephalus were collected from the Osprey, Pandion haliaetus, and the White mullet, Mugil curema in the Yucatán Peninsula, Mexico. We report for the first-time DNA sequences of adult specimens of Scaphanocephalus, particularly S. expansus, as well as a sequence of a different species sampled as metacercaria. Morphological comparisons of Scaphanocephalus expansus confirmed the identity of the adult specimens, with minor morphological variations; Scanning electron photomicrographs were included, and the species was re-described. Phylogenetic analysis based on 28S rDNA sequences showed that Scaphanocephalus is monophyletic within Opisthorchiidae and consists of three independent lineages. Sequences of adults are identical to those of S. expansus. Instead, the sequence of the metacercaria sampled from the mesentery of Mugil curema nested with specimens reported as Scaphanocephalus sp. from a labrid fish in the Mediterranean Sea, herein named it as Scaphanocephalus sp. 2.

Molecular evidence of the absence of Metagonimus yokogawai (Katsurada, 1912) in Europe: report of Metagonimus sp. in cyprinoid fish from the River Danube in Hungary.

Trematodes of the genus Metagonimus Katsurada, 1912 (Digenea: Heterophyidae) are zoonotic parasites that cause infections in humans, with most cases reported in Southeast Asia. Larvae from the second intermediate host, called metacercariae, of one of human-infecting species, M. yokogawai (Katsurada, 1912), have been reported from cyprinoid fish in Europe. In the present study, we provided DNA-based evidence that metacercariae of Metagonimus, which are commonly found in the scales of various cyprinoids in Central Europe (Danube River in Hungary) do not belong to M. yokogawai. Sequence analysis of the ITS region, 28S rDNA, and cox1 genes showed that this species is clearly distinct from all Asian species, including M. yokogawai, which probably does not occur in Europe. Metacercariae from cyprinoids might belong to Metagonimus romanicus (Ciurea, 1915), an insufficiently known species described from Romania.

Assessing the population structure of trematode Metagonimus suifunensis using three mitochondrial markers.

In this work, for the first time, the genetic variability of the Metagonimus suifunensis population in the Russian southern Far East was estimated based on the full-length sequences of the nad1 gene of mitochondrial DNA. In addition, for a sample of the same size, the sequences of cox1 and cytb genes, previously used for population studies for M. suifunensis, were reanalysed. Three markers were combined to a common sequence, and the obtained data were studied. Despite the higher level of variability, nad1 and cox1 mtDNA genes did not reveal subdivisions within the population. The combined dataset made it possible to determine that the sample from the Odyr River was the centre of the species' range formation and clarified the continental migration route of the parasite from south to north. According to the data obtained, it was presumed that piscivorous birds participate in the life cycle of the parasite. The subdivision within population revealed that using all three mitochondrial markers is consistent with the features of differentiation within populations of related species, but the reasons for its formation remain unclear due to the insufficient amount of data and the use of different markers in studies of different species.

Ancient DNA of Metagonimus yokogawai Recovered from Joseon Period Human Remains Newly Discovered at Goryeong County in South Korea.

PURPOSE: Metagonimiasis, commonly seen in East Asian countries, is a parasitic disorder caused by definitive hosts' ingestion of undercooked freshwater fishes. Recently, genetic analysis has proved 28S rRNA and cytochrome c oxidase subunit I (COI) mtDNA gene to be a successful marker differentiating species of the genus Metagonimus. In the present study, using specimens from the newly discovered Joseon Dynasty human remains of Goryeong, we obtained updated genetic data on genus Metagonimus, which was also prevalent during the Joseon period. METHODS: The ancient DNA (aDNA) was retrieved from the coprolite sample of the seventeenth century, half-mummified individual discovered at Goryeong Country, South Korea. Cloning and sequencing were performed on PCR-amplified amplicons for M. yokogawai 28S rRNA and COI mtDNA gene. The consensus sequences were used for species identification and phylogenetic analysis using NCBI/BLAST and MEGA X software. RESULTS: Based on the COI mtDNA gene region, the Goryeong sequence was confirmed as belonging to M. yokogawai, as it was shown to form a separate cluster with other M. yokogawai taxa that are distinct also from M. takahashii and M. miyatai. CONCLUSION: In a series of our genetic analyses on genus Metagonimus using samples retrieved from Joseon-period cases, aDNA sequences of genus Metagonimus revealed in South Korea thus far are those of M. yokogawai, but not of M. miyatai or M. takahashii yet.

A review of molecular identification tools for the opisthorchioidea.

The superfamily Opisthorchioidea encompasses the families Cryptogonimidae, Opisthorchiidae and Heterophyidae. These parasites depend on the aquatic environment and include marine and freshwater species. Some species, such as Clonorchis sinensis and Opisthorchis viverrini, have a high impact on public health with millions of infected people worldwide and have thus been the object of many studies and tool developments. However, for many species, tools for identification and detection are scarce. Although morphological descriptions have been used and are still important, they are often not efficient on the immature stages of these parasites. Thus, during the past few decades, molecular approaches for parasite identification have become commonplace. These approaches are efficient, quick and reliable. Nonetheless, for some parasites of the superfamily Opisthorchioidea, reference genomic data are limited. This study reviews available genetic data and molecular tools for the identification and/or the detection of this superfamily. Molecular data on this superfamily are mostly based on mitochondrial and ribosomal gene sequence analyses, especially on the cytochrome c oxidase subunit 1 gene and internal transcribed spacer regions respectively.

Cytochrome b as a more promising marker for analysing the distribution vector for Metagonimus suifunensis (Trematoda: Heterophyidae).

In this study of Metagonimus suifunensis (M. suifunensis) in the Russian Southern Far East, the variability of the full-length sequences of the cytochrome b (cytb) mtDNA gene was assessed for the first time. In addition, the cox1 mtDNA gene sequences were also obtained for this species from new localities. In total, 87 and 81 sequences of the cytb and cox1 genes, respectively, were used in the current study. The cytb gene proved more promising and revealed two haplogroups that are associated with the spatial distribution of the species: geographical isolation caused the fixation of differences between northern and southern populations. In addition, the results obtained for the cytb gene opened up new perspectives in the analysis of sequences of the cox1 gene, which was not sufficiently effective as a sole marker. Based on data for both mitochondrial genes, molecular processes influencing the formation of the modern population were analysed for M. suifunensis. The new data confirmed the previously expressed opinion that this species colonized the study territory from north to south and will form the basis for determining possible ways of its further expansion, which is important for predicting the emergence of new foci of metagonimosis.

Ascocotyle longa (Digenea: Heterophyidae) infecting dolphins from the Atlantic Ocean.

We report for the first time the infection of dolphins with Ascocotyle longa found in the intestines of three different species, Sotalia guianensis, Steno bredanensis, and Tursiops truncatus gephyreus, which were found washed ashore along the southeastern and southern Brazilian coast. The worms were identified based on morphological and molecular data using the 28S rDNA gene and the COI gene. Specimens of A. longa from the pinniped Otaria flavescens were also analyzed. As the first isolation of A. longa from cetaceans, the present study increases the distribution area and range of definitive hosts of this trematode, and provides new molecular data to complement the phylogeny of the group in future studies, thus contributing to the scientific knowledge of this potentially zoonotic parasite.

Metagonimus yokogawai Ancient DNA Recovered from 16th- to 17th-Century Korean Mummy Feces of the Joseon Dynasty.

Metagonimiasis is foodborne intestinal parasitism occurring by the definitive hosts' ingestion of raw or undercooked fish, mostly commonly sweetfish. Most Metagonimus infection is caused by Metagonimus yokogawai but also rarely by Metagonimus takahashii as well as Metagonimus miyatai. Despite recent molecular work on Metagonimus spp., there are still insufficient data to reveal the genetic characteristics of ancient M. yokogawai in a wide geo-historical scope. In this study, we were successful in the analysis of M. yokogawai ancient DNA (aDNA) using coprolite samples retrieved from 16th- to 17th-century Korean mummies. In BLAST and phylogenetic analyses, M. yokogawai 28S rDNA of Korean mummies were clustered along with the 28S rDNA taxa of M. takahashii and M. miyatai in GenBank. Conversely, the cytochrome c oxidase subunit I (COI) of M. yokogawai aDNA from Korean mummies was distinctly clustered apart from M. takahashii and M. miyatai sequences. This study is the first report of its kind to identify M. yokogawai aDNA retrieved from the archaeological specimens and confirms the usefulness of COI in molecular diagnosis of M. yokogawai. Considering the rarity of reports on the genetics of genus Metagonimus spp., our study will be fundamental for the future study of M. yokogawai paleogenetics.

Current Knowledge of Small Flukes (Digenea: Heterophyidae) from South America.

Fish-borne heterophyid trematodes are known to have a zoonotic potential, since at least 30 species are able to infect humans worldwide, with a global infection of around 7 million people. In this paper, a 'state-of-the-art' review of the South American heterophyid species is provided, including classical and molecular taxonomy, parasite ecology, host-parasite interaction studies and a list of species and their hosts. There is still a lack of information on human infections in South America with undetected or unreported infections probably due to the information shortage and little attention by physicians to these small intestinal flukes. Molecular tools for specific diagnoses of South American heterophyid species are still to be defined. Additional new sequences of Pygidiopsis macrostomum, Ascocotyle pindoramensis and Ascocotyle longa from Brazil are also provided.

Proteomic analysis of Ascocotyle longa (Trematoda: Heterophyidae) metacercariae.

Ascocotyle longa is parasitic trematode with wide distribution throughout America, Europe, Africa, and Middle East. Despite the fact that this fish-borne pathogen has been considered an agent of human heterophyiasis in Brazil, the molecules involved in the host-parasite interaction remain unknown. The present study reports the proteome profile of A. longa metacercariae collected from the fish Mugil liza from Brazil. This infective stage for humans, mammals and birds was analyzed using nLC-MS/MS approach. We identified a large repertoire of proteins, which are mainly involved in energy metabolism and cell structure. Peptidases and immunogenic proteins were also identified, which might play roles in host-parasite interface. Our data provided unprecedented insights into the biology of A. longa and represent a first step to understand the natural host-parasite interaction. Moreover, as the first proteome characterized in this trematode, it will provide an important resource for future studies.

Haplorchis pumilio (Trematoda: Heterophyidae) as a new fish-borne zoonotic agent transmitted by Melanoides tuberculata (Mollusca: Thiaridae) in Brazil: A morphological and molecular study.

The fishborne zoonotic trematode Haplorchis pumilio (Looss, 1896) is here reported for the first time in Brazil based on morphological, experimental and molecular studies. Pleurolophocercous cercariae emerged from the invasive snail Melanoides tuberculata collected in the municipality of Ceará-Mirim, state of Rio Grande do Norte, Northeast region of Brazil, in November 2018, were used for experimental infection of guppies, Poecilia reticulata. Metacercariae were extracted from the caudal musculature of these fishes and were used to infect mice. Adult parasites recovered in the small intestine of the mice were morphologically identified as H. pumilio. Molecular sequences were obtained for the Brazilian and Peruvian isolates of H. pumilio, and were compared with data available in GenBank. Analyses of fragments of the nuclear genes 28S (1219 bp) and ITS-2 (290 bp) revealed 98.48-100% similarity between the South American and Asian isolates of H. pumilio. Moreover, new sequences of the mitochondrial gene cox-1 obtained for the Brazilian (797 bp) and Peruvian (646 pb) isolates were 100% similar to a Mexican isolate of this species and 97.54% similar to an isolate from Thailand. This finding reveals the potential for occurrence of human haplorchiasis in Brazil, especially because of the increasing popularity of raw fish dishes in the country.

A New Species of Cryptocotyle (Digenea: Heterophyidae) Infecting Kelp Gull and a Galaxiid Fish in Patagonian Freshwater Environments: Morphological and Molecular Analyses.

Adults of a species of Cryptocotyle were found infecting the intestine of the kelp gull, Larus dominicanus Lichtenstein, 1823, and metacercariae were found in the fins and muscle of the galaxiid fish, Galaxias platei Steindachner, 1898 (local name "puyen grande"), in Nahuel Huapi National Park (Patagonia). Morphometrics and genetic markers of mitochondrial cytochrome oxidase (COI) and ribosomal internal transcribed spacer 1 (ITS1) confirmed that adults and metacercariae were conspecific and represent a new species, Cryptocotyle dominicana n. sp. The only congener known from South America (Cryptocotyle thapari McIntosh, 1953) matures in river otters (Lutra spp. and Pteronura spp.) in Brazil and Bolivia and differs from the new species in the arrangement of the testes, which are located in tandem in the species from otters. Adults of the new species resemble Cryptocotyle lingua (Creplin, 1825) Fischoeder, 1903 from marine environments of the Northern Hemisphere, both species being characterized by having a linguiform body, oblique testes, and vitelline follicles that extend posteriorly to the level of the ventral sucker. Considering the morphological findings that differentiate the new species from other Cryptocotyle, as well as the molecular analysis that shows significant differences from C. lingua, we conclude that these specimens represent a new species, the first of the genus Cryptocotyle to be described from birds in South America.

First description of genetic diversity for the genus Metagonimus using the complete cox1 gene sequence.

The complete cox1 gene sequence was analysed for Metagonimus suifunensis from eight localities in the Russian southern Far East, and the level of variability was compared with that of Clonorchis sinensis from the same territory of Russia. These species belong to the superfamily Opisthorchioidea, have a similar distribution in the Russian southern Far East and share second intermediate and definitive hosts, but are distinguished by their first intermediate hosts belonging to different orders of caenogastropods. The data obtained showed that the nucleotide sequence variability of the M. suifunensis cox1 gene was significantly lower. This fact is considered in connection with a recent bottleneck passage for the M. suifunensis population, in contrast to C. sinensis, which could be due to the features of the Metagonimus life cycle under seasonal freezing temperatures, as well as historical geological and climatic changes in the Russian Far East. These factors could influence the microevolutionary processes and lead to a decrease in the level of variability in the M. suifunensis population. Based on the combination of genetic data and historical geo-processes in the region, the probable route of M. suifunensis expansion from the northern part of its current area in the Amur River basin to the southern territories of the Russian Far East is justified.

Galactosomum otepotiense n. sp. (Trematoda: Heterophyidae) infecting four different species of fish-eating birds in New Zealand: genetically identical but morphologically variable.

Trematodes of the genus Galactosomum are cosmopolitan parasites that infect the intestines of fish-eating birds and mammals. Adults of named Galactosomum species have not been recorded from bird hosts in New Zealand, despite their cercarial stage being known from various studies of the first intermediate host, Zeacumantus subcarinatus. Here we describe a new species of Galactosomum infecting four different piscivorous birds in New Zealand: Caspian terns, red-billed and black-backed gulls and little blue penguins. Specimens from each of these hosts are genetically identical in the genes sequenced, but show considerable morphological variability. Galactosomum otepotiense n. sp. is distinguished from most other members of the 'bearupi-group' in having a single circle of spines on the ventral sucker, and spines, as opposed to scales, over most of the body. It is most similar to G. bearupi and G. angelae, both from Caspian terns in Australia, but differs in the relative sizes of the reproductive organs and in the possession of a very long forebody. Molecular data confirm that G. otepotiense is not conspecific with G. bearupi, but 28S and ITS2 phylogenies show its close relationship to G. bearupi and other Australian species. We use the cox1 sequence to confirm identity with the larval stage infecting Z. subcarinatus, as previously described in the literature. We discuss briefly the relationships between Australian and New Zealand Galactosomum spp. and their hosts, variability between genetically identical specimens found in different hosts and their potential for harm to mariculture economy.

Morphological and Molecular Identification of Stellantchasmus dermogenysi n. sp. (Digenea: Heterophyidae) in Thailand.

We tried a series of morphological and molecular approaches to identify a new species of Stellantchasmus (Digenea: Heterophyidae) originating from the wrestling half-beaked fish, Dermogenys pusillus of Thailand. Adult worm samples of the new species were recovered from hamsters experimentally infected with the metacercariae from D. pusillus in Thailand. Two isolates (Thai and Korean) of Stellantchasmus falcatus were used as comparative control groups. Worm samples of 3 Stellantchasmus groups were morphologically observed and molecularly analyzed with the mitochondrial cytochrome c oxidase 1 gene. The morphological characteristics of S. dermogenysi n. sp. are similar to S. falcatus originating from brackish water fish, but minor difference was noted including the absence of the prepharynx, position of the ovary near the ceca end, smaller body size, and shorter esophageal length. A phylogenetic tree derived from neighborjoining and maximum-likelihood methods suggests that S. dermogenysi n. sp. is separated from S. falcatus supported by high bootstrap values. The relative divergences persist between these host-specific trematodes, which we suggest should be recognized as 2 distinct species. Comparisons of S. dermogenysi n. sp. with S. falcatus isolated from mullets in Thailand and Korea indicate a genetic divergence of mitochondrial DNA of 19.4% and 21.7%, respectively. By the present study, a new species, Stellantchasmus dermogenysi n. sp. (Digenea: Heterophyidae), is proposed in Thailand based on molecular evidences, in addition to minor morphological differences between S. falcatus and the new species.

First report of Euryhelmis parasites (Trematoda, Heterophyidae) in Africa: conservation implications for endemic amphibians.

In this study, we report, through molecular identification, the first African records of a digenean trematode parasite of the genus Euryhelmis. We recovered metacercariae encysted in an anuran, the endemic Moroccan painted frog (Discoglossus scovazzi), and a vulnerable caudate, the North African fire salamander (Salamandra algira), from four localities in North Africa (Morocco). Our records go back to the past century and have been confirmed in successive fieldwork seasons thereafter. Metacercarial stages of these parasites require amphibians as the last intermediate host, but the exact identity of the primary hosts and predators of the infected animals in Africa remain unknown. Our searches with basic local alignment search tool (BLAST) from Genbank revealed that hosts were infected by parasites of Euryhelmis costaricensis, which showed almost the same genetic identity (with only one substitution) to previous reports from Costa Rica and Japan, suggesting a recent introduction in Morocco. We proceed to discuss the likely role of introduced mustelids as the potential definitive hosts of trematode adults. Under this assumption, we conclude that the infestation of Discoglossus scovazzi and Salamandra algira might pose a risk to these threatened species.

Digeneans of northern fur seals Callorhinus ursinus (Pinnipedia: Otariidae) from five subpopulations on St. Paul Island, Alaska.

A parasitological survey of 651 northern fur seals Callorhinus ursinus L. from five subpopulations was conducted on St. Paul Island, Alaska, during July-August 2012-2014. Digenean trematodes were found in 210 of 651 fur seals with a total prevalence of 32.3%. Intensity of infection varied from 1 to 1540 parasites with mean intensity 18.4 ± 111.1 SD and median intensity of 2 specimens per host. Significant differences in prevalence and intensity of infection in northern fur seals between separate rookeries was not observed (Mann-Whitney test; p > 0.05). Four species of digeneans belonging to the families Heterophyidae (Apophallus zalophi Price, 1932, Phocitrema fusiforme Goto and Ozaki, 1930, and Galactosomum ubelakeri (Dailey, 1969)) and Troglotrematidae (Nanophyetus salmincola (Chapin, 1926)) were found. Nanophyetus salmincola is reported from C. ursinus for the first time. We obtained partial 28S rDNA sequences for all digenean species and conducted molecular phylogenetic analysis to demonstrate their phylogenetic relationships.

Two new species of Haplorchoides Chen, 1949 (Digenea: Heterophyidae) infecting an Australian siluriform fish, Neoarius graeffei Kner & Steindachner.

Combined morphological and molecular analyses are used to characterise two new species of Haplorchoides Chen, 1949 (Digenea: Heterophyidae) from an Australian siluriform fish. Haplorchoides maiwariensis n. sp. and H. daguilarensis n. sp. are described from the intestine of the Blue salmon catfish, Neoarius graeffei (Kner & Steindachner) (Siluriformes: Ariidae) from tidal reaches of the Brisbane River and from a freshwater creek off Lake Wivenhoe, Kipper Creek, Australia. The two new species most obviously differ from all previously described species of Haplorchoides in possessing clearly and reliably tripartite seminal vesicles. The two new species differ in the size and distribution of vitelline follicles, the size of the pigment granules and the form of the ventral sucker. Previously described species of Haplorchoides have been reported from Africa and Asia, principally from bagrid, schilbeid, silurid and sisorid catfishes; these are the first species reported from an ariid catfish. Complete ITS2 and partial 28S ribosomal DNA data were generated for both new species. The two species differ from each other by eight base pairs in the ITS2 region, and by 13 bp for the 28S region. The 28S rDNA sequence of H. daguilarensis agrees with a previously reported sequence from an unidentified species of Haplorchoides collected from N. graeffei in Lake Wivenhoe, Australia; we identify this previous report as relating to H. daguilarensis.

Description of Metagonimus pusillus sp. nov. (Trematoda: Heterophyidae): phylogenetic relationships within the genus.

As a result of experimental studies conducted in the Russian southern Far East, adult worms from the genus Metagonimus were obtained. A comparative analysis of the morphometry of these worms with other Metagonimus representatives showed that they are most similar to M. katsuradai Izumi, 1935 and M. otsurui Shimazu & Urabe, 2002 found in Japan, due to the ratio of suckers and the positions of the testicle, uterus and vitellaria. However, Russian worms differ from species in Japan by other metric characters: they differ from M. otsurui by the maximum size of most organs and from M. katsuradai by body width, pharynx length, and maximum size of testes and ovary. At the same time, they are identical to a trematode from the Russian southern Far East, which was previously identified as M. katsuradai. The validity of this species was also confirmed by genetic data. According to the 28S gene and the internal transcribed spacer 2 (ITS2) region of rDNA, as well as the cytochrome c oxidase I (cox1) gene of mtDNA, the Metagonimus specimens found in Russia differ from published genetic data for other members of this genus. However, both morphological similarity and molecular data showed that M. pusillus sp. nov., M. katsuradai and M. otsurui are most likely cryptic species. Furthermore, additional data based on a mitochondrial marker were provided for M. suifunensis Shumenko, Tatonova & Besprozvannykh, 2017 from Russia.