Hidden in the frog: morphological and molecular characterization of Halipegus ovocaudatus (Platyhelminthes: Trematoda), type species of the genus Halipegus Looss, 1899.

The type species of the genus Halipegus Looss, 1899, Halipegus ovocaudatus (Vulpian, 1859) is a generalist parasite of different species of amphibians, mainly anurans. To date, the phylogenetic position of this species has not been established. Specimens of H. ovocaudatus, were collected from three species of water frogs, Pelophylax ridibundus (Pallas), P. esculentus (Linnaeus), and P. lessonae (Camerano) inhabiting the Middle Volga region (European Russia). In our study, we provide detailed morphological redescription of these digeneans and combine it with molecular phylogenetic analyses based on partial sequences of the 28S rDNA and coI mtDNA genes. The phylogenetic position of H. ovocaudatus among the derogenids has been established as a sister clade to other representatives of the Halipeginae. We compared pairwise distances based on partial sequences of coI mtDNA genes of H. ovocaudatus and other representatives of Derogenidae. It was shown, that adults of H. ovocaudatus from different hosts are representatives of the same species. Specimens parasitizing three frog hosts from several localities differ slightly in body shape and size, which probably reflects host-induced intraspecific variability.

DESCRIPTIONS AND PHYLOGENETIC AFFINITIES OF TWO NEW SPECIES OF RHABDIAS STILES AND HASSALL, 1905 (NEMATODA: RHABDIASIDAE) FROM NORTH AMERICAN FROGS: ARE WE STILL ONLY SCRATCHING THE SURFACE?

Two new species of lung-dwelling nematodes are described from North American frogs: Rhabdias aurorae n. sp. from Rana aurora and Rhabdias conni n. sp. from Rana clamitans and Rana catesbeiana from Arkansas; the latter species was also found in Oklahoma and Georgia. Rhabdias aurorae n. sp. differs from other Nearctic congeners in the combination of the following characteristics: buccal capsule 22-25 µm wide, elongated tail covered with inflated cuticle, esophagus with prominent dilatation in anterior part and 6 small circumoral lips. Rhabdias conni n. sp. is morphologically closest to Rhabdias ranae Walton, 1929 and Rhabdias joaquinensisIngles, 1936; it differs from them in the shape of lateral pseudolabia, the dimensions of the body, and the egg size. Both new species were found to be significantly different from the Nearctic congeners in the nucleotide sequences of nuclear ribosomal DNA (18S-ITS-28S region), 12S, and CO1 mitochondrial genes. The 2 new species differ from other currently sequenced Nearctic congeners by 1.1-2.7% of nucleotide positions in the nuclear rDNA region, 1.3-3.4% in the 12S gene, and 3.4-9.4% in CO1 gene. Molecular phylogenetic analysis based on nuclear ribosomal DNA sequences placed both new species into the clade consisting of Nearctic and Neotropical Rhabdias spp. The position of Rh. aurorae n. sp. within the clade is uncertain because of a polytomy, but Rh. conni n. sp. is nested within the "Rh. joaquinensis complex" related to Rh. ranae and Rhabdias tarichaeKuzmin, Tkach, and Snyder, 2003. The phylogenetic analysis based on nuclear ribosomal DNA sequences has revealed 3 evolutionary host-switching events from anuran to caudatan hosts among Rhabdias spp. that occurred in the Nearctic and Palearctic. The molecular phylogeny also suggests that Rhabdias may have originally evolved in what is now Africa.

Lernaea cf. cyprinacea infection in agile frog (Rana dalmatina) tadpoles in Hungary.

The ectoparasitic copepod Lernaea cyprinacea (anchor worm) has more than 100 host species among teleost fishes and affects cyprinids both in fish farms and natural waters. In addition, while L. cyprinacea infection in amphibians has been recorded in Asia, North and South America, there is no data available in the literature on their presence in Europe. In this study, we first reported L. cf. cyprinacea parasitising an anuran tadpole in Europe. Specimens of L. cf. cyprinacea were observed attached to a tadpole of the agile frog (Rana dalmatina), which was caught during fishing of crucian carp fingerlings (Carassius carassius) from a small fish pond in Hungary during the summer of 2012. The infected tadpole was collected from a rearing pond, where juvenile crucian carps were kept. The tadpole was inspected in the laboratory, and digital photos were taken. The parasites were found attached to the body-tail junction and to the leg of the tadpole (at Gosner stage 41). The parasite species was identified as L. cf. cyprinacea based on morphological traits.

Blood parasites of water frogs (Pelophylax esculentus complex) from the Danube Delta, Romania.

Water frogs of the genus Pelophylax host a variety of parasites, from protozoa to helminths. Among the blood parasites, representatives of Apicomplexa, Trypanosoma and Nematoda show the highest prevalence. In this study, we focused on blood parasites of water frogs living in the Danube Delta, Romania. In total, 74 individuals of P. ridibundus and eight individuals of P. esculentus from six localities were examined. Blood parasites were detected microscopically and using a molecular marker (18S rDNA). 89.77% of frogs from all investigated localities were found to be infected with at least one parasitic group, specifically with haemogregarines (84.09%), nematodes (1.14%), and trypanosomes (63.64%). The parasitemia of haemogregarines and trypanosomes differed significantly among the studied locations. There was no statistically significant difference in parasitemia between male and female hosts. However, adults were found to have a significantly higher parasitemia in comparison with subadults infected with haemogregarines. Correlation between parasitemia and the body length of frogs infected with haemogregarines was also significant (r = 0.226). By comparing the 18S rDNA sequences with the corresponding GenBank sequences, Hepatozoon species identified in water frogs showed a close similarity (98.1-99.8%) to Hepatozoon magna. Trypanosomes showed the highest sequence similarity to Trypanosoma sp. isolate R10 clone L2-3, Trypanosoma ranarum, and Trypanosoma cobitis.

New species of Foleyellides (Nematoda: Onchocercidae: Waltonellinae), parasite of Lithobates brownorum (Amphibia: Ranidae) from South-eastern Mexico and genetic barcodes of the Mexican species of the genus.

Specimens of Foleyellides were collected from the body cavity of frogs in different regions of Mexico; Lithobates brownorum from Yucatán, Quintana Roo and Campeche; L. megapoda from Jalisco and Rhinella marina, from Guerrero. Foleyellides calakmulesis n. sp. is described based on specimens found parasitizing L. brownorum. The new species is distinguished from the other members of the genus by the combination of the following male characters: four pairs of caudal papillae different in size and the presence of a preanal plaque. Partial DNA sequences of the mitochondrial Cytochrome Oxidase C, subunit I of the four known Mexican species of Foleyellides and two potentially new species collected in this study were generated and compared, validating the erection of the new species.

Specific Diversity of Helminth Parasites of the Edible Frog Hoplobatrachus occipitalis (Günther, 1858) in Agricultural Landscapes in the South-East of Ivory Coast, Africa.

OBJECTIVE: This study describes the specific diversity of Helminth parasites of the edible frog Hoplobatrachus occipitalis (Günther, 1858) to assess the rate of infestation in three types of plantations (coconut, palm and banana plantations) in the south-east of Africa. METHODS AND RESULTS: A total of 156 frog specimens were collected in November 2019 from all the plantations and ten parasitic Helminth taxa were recorded. The overall prevalence (93.6%) showed a high infestation of the frog in these anthropized environments. The banana plantations that use the most fertilizers and pesticides had the highest prevalence (95.2%) suggesting pollution-related parasitic load. The number of parasites was higher in female frogs than in males, suggesting a sex-specific immune resistance. This study also highlights the parasite specificity and the sites of Helminth infestations. Trematodes of the genus Haematoelochus and Diplodiscus showed strict specificity in the lungs and large intestine/rectum of the host. The other parasites colonized the digestive tract with a more or less marked specificity. CONCLUSION: Our study provides several elements of response on the population of Helminth parasites of the edible frog Hoplobatrachus occipitalis, with a view to better knowledge, management, conservation and protection.

Gathering morphological and molecular data for identifying two species of trematodes from the neotropical Vaillant's frog, Lithobates vaillanti (Amphibia: Ranidae) in the highlands of Chiapas, south-eastern Mexico.

Mexico possesses a large diversity of amphibians partly due to its complex topography and transitional position between the Nearctic and Neotropical biogeographical regions. However, its helminth parasite fauna has been relatively poorly studied. Specimens of the Vaillant's frog, Lithobates vaillanti (Brocchi) were sampled in the tropical rain forest of Nahá, in the Chiapas Highlands, and examined for parasites. Two trematode species were collected from their hosts; morphologically, specimens were allocated to the genera Langeronia Caballero and Bravo-Hollis, 1949 and Haematoloechus Looss, 1899, respectively. Individuals were sequenced for two molecular markers (the mitochondrial cytochrome c oxidase gene, and the ribosomal gene 28S), and processed for morphological analyses, including scanning electron microscopy. The new evidence was not enough to accomplish the identification at species level of Langeronia sp. due to the lack of sequence data from the type localities of Langeronia parva Christian, 1970 and Langeronia macrocirra Caballero and Bravo-Hollis, 1949. Likewise, the newly generated data were useful to properly identify the adult specimens of lung flukes as Haematoloechus complexus Seely, 1906.

Effects of minimum Strigea robusta (Digenea: Strigeidae) cercariae doses and localization of cysts on the anomaly P manifestation in Pelophylax lessonae (Anura: Ranidae) tadpoles.

The anomaly P is a mass morphological anomaly found in some populations of anuran amphibians (water frogs of the genus Pelophylax and toads of the genera Bufo and Bufotes) caused by the parasitic flatworm Strigea robusta. Minimum dose of cercariae for the appearance of the anomaly P remains unknown. However, it is important information for understanding of host population dynamics after invasion and the effects of the parasite on the second intermediate hosts. Herein, the invasion properties of S. robusta in Pelophylax lessonae tadpoles (Anura: Ranidae) and minimum dose for appearance of mild and severe forms of the anomaly P syndrome were described after direct experiments with certain numbers of cercariae exposure. Experimental groups of tadpoles have been exposed to eight doses of cercariae (2, 4, 6, 8, 10, 12, 14 and 16). A total of 63.8% tadpoles survived to the end of this experiment. It was revealed that a mild form of the anomaly P (polydactyly) can appear after infection by two cercariae, while the severe form traits appear after infection by four cercariae. The mean number of detected encysted metacercariae was reached to 53.5%. Differences in infection rates can be explained by the presence of an individual immune response in tadpoles or by the presence of different genetic lineages of the parasite infecting the same snail, which have different infectious potential. Low doses of infection leading to the induction of anomalies characterize S. robusta as a highly pathogenic species for amphibian species that are susceptible to infection and show an abnormal phenotype.

A new species of Trichodina lishuiensis n. sp. (Ciliophora: Trichodinidae) in urinary bladder of Odorrana schmackeri (Amphibia: Ranidae) from Zhejiang, China.

Endoparasitic trichodinids are rather rare ciliates. In this study we describe a new species named Trichodina lishuiensis from the bladder of Odorrana schmackeri collected in Zhejiang, China, with the prevalence of 20% (9/45). We identified T. lishuiensis as a new species by morphological comparison and molecular analysis. The ciliates were observed using the dry-silver and protargol staining methods, as well as SEM (scanning electron microscopy). Trichodina lishuiensis is a small species (cell diameter 31.8-43.9 µm), with incompact denticles connection, medium-wide blades and thick rays. We also sequenced a 1712 bp-long fragment of the small subunit ribosomal RNA gene (SSU rRNA). Phylogenetic analyses showed that the new species clustered with Trichodina unionis. The route of transmission of Trichodina species in the urinary bladder remains a mystery. We hypothesize that the transmisison takes place during the amplexus, with eggs and sperm discharged from the cloaca, and that trichodinids 'accompany' the amphibian through its whole life cycle, but further studies are needed to test this hypothesis.

The phylogeographic puzzle of Pseudoacanthocephalus toshimai, an amphibian acanthocephalan in northern Japan.

The amphibian acanthocephalan, Pseudoacanthocephalus toshimai, was considered to be an island-endemic species in Hokkaido, Japan. However, the parasite was found from Rana ornativentris, Rana tagoi, Zhangixalus arboreus, and Bufo japonicus formosus in northern Honshu (Aomori and Iwate Prefectures), which is separated from Hokkaido by the Tsugaru Strait. The mitochondrial DNA-based phylogenetic and population genetic analyses of P. toshimai showed that the northern Honshu isolates are far distantly related to the Hokkaido isolates, and that a demographic population expansion occurred in Hokkaido during the recent geological past. The rich genetic diversity of P. toshimai in northern Honshu suggests a scenario that anuran hosts invaded Hokkaido together with P. toshimai via the land bridge of the Tsugaru Strait. However, the evolutionary history of Rana pirica, a main definitive host for P. toshimai in Hokkaido, is contradictory to the introduction scenario inferred from the parasite. The finding of several geographically mismatched isolates of P. toshimai from both northern Honshu and Hokkaido suggests a possibility that the migration of the parasite infrequently occurred between the two areas even after the land bridge disappeared. More detailed information on the evolutionary history of anurans is needed to resolve the biogeographical enigma of P. toshimai.

Multilocus Genotyping of Sympatric Hepatozoon Species Infecting the Blood of Ontario Ranid Frogs Reinforces Species Differentiation and Identifies an Unnamed Hepatozoon Species.

Intraerythrocytic gamonts of at least 2 named Hepatozoon species have been reported to infect the erythrocytes of ranid frogs in Ontario, Canada. Although gamonts of both species are morphometrically similar, the cytopathological changes that 1 of these species, Hepatozoon clamatae, causes to host erythrocytes, manifested by nuclear fragmentation, was used historically to distinguish this parasite from Hepatozoon catesbianae. Molecular characterization of these 2 Hepatozoon species has been equivocal in correlating genotype with gamont morphotype. Amplification and sequencing of multiple potential genotyping loci within the nuclear (18S ribosomal deoxyribonucleic acid [rDNA]; internal transcribed spacer 1), apicoplast (23S rDNA), and mitochondrial genomes (complete genomes, cytochrome c oxidase subunits I and III [COI and COIII], and cytochrome b) were conducted on Hepatozoon species that infect ranid frogs in Ontario. Sequence data were then used to evaluate the diversity of parasites present in these amphibian hosts and to assign genotypes to gamont morphotypes, if possible. Three distinct genotypes were identified at all loci; the data permitted the discovery of a third, formerly unrecognized Hepatozoon species in ranid frogs from Ontario. Although all genetic loci demonstrated differences between Hepatozoon species, mitochondrial COIII sequences were most suitable for genotypic differentiation of these parasites of frogs. Linking genotypes to gamont morphotypes proved impossible; genotypes identified as H. catesbianae and H. clamatae were found in infections with or without nuclear fragmentation of their host erythrocytes. This suggests that differentiating these species must rely on suitable genotyping methods for identification in the blood of their amphibian intermediate hosts.

Polystoma luohetong n. sp. (Monogenea: Polystomatidae) from Rana chaochiaoensis Liu (Amphibia: Ranidae) in China.

Polystoma chaochiaoensis from the urinary bladder of the chaochiao frog Rana chaochiaoensis Liu was briefly described in a symposium abstract and presented at the Third Symposium on Parasitology of China in 1990. Types were not assigned and the original specimens collected are no longer available. The morphological description was incomplete and no illustrations were provided. We consider Polystoma chaochiaoensis a nomen nudum and provide a full description for the species and assign types. Based on morphological characteristics and molecular data of partial 18S rDNA sequences, we describe this species as Polystoma luohetong n. sp. Out of 578 frogs examined, 16 male and 38 female frogs were infected (prevalence 9.3%; mean intensity 1.02). Polystoma luohetong n. sp. is distinguished from all other Polystoma species by the presense of a prominent crest on the hamulus as well as by the shape and size of marginal hooklets and the intestinal arrangement. Furthermore, the phylogentic analysis based on the 18S rRNA gene shows Polystoma luohetong n. sp. well nested within the Ploystoma clade and as a sister taxon to Polystoma integerrimum.

Molecular and Morphological Elucidation of the Life Cycle of the Frog Trematode Langeronia macrocirra (Digenea: Pleurogenidae) in Los Tuxtlas, Veracruz, Mexico.

The genus LangeroniaCaballero and Bravo-Hollis, 1949, currently contains 6 species of amphibian trematodes distributed in North and Middle America. The type species of the genus, Langeronia macrocirraCaballero and Bravo-Hollis, 1949, occurs in Mexico and is relatively commonly found as a parasite of leopard frogs. However, information regarding its life cycle is lacking. In this paper, we study the life cycle of L. macrocirra in Laguna Escondida, Los Tuxtlas, Veracruz. Definitive hosts (Rana spp.) as well as potential intermediate hosts (gastropods, bivalves, crustaceans, tadpoles, hemipterans, and odonate naiads) were sampled in the locality and studied to search for the presence of adults and larval stages of the trematode. Specimens were morphologically characterized, and some individuals were sequenced for 1 ribosomal gene (28S rRNA) and 1 mitochondrial gene (COI). DNA sequences of the adults obtained from leopard frogs were matched with those of the larval forms in their intermediate hosts (metacercariae, cercariae, and sporocysts) to demonstrate conspecificity. Further, we conducted a detailed study of the tegument of the body surface with scanning electron microscopy to characterize each of the developmental stages of the life cycle of L. macrocirra.

Meta-transcriptomic identification of Trypanosoma spp. in native wildlife species from Australia.

BACKGROUND: Wildlife species carry a remarkable diversity of trypanosomes. The detection of trypanosome infection in native Australian fauna is central to understanding their diversity and host-parasite associations. The implementation of total RNA sequencing (meta-transcriptomics) in trypanosome surveillance and diagnosis provides a powerful methodological approach to better understand the host species distribution of this important group of parasites. METHODS: We implemented a meta-transcriptomic approach to detect trypanosomes in a variety of tissues (brain, liver, lung, skin, gonads) sampled from native Australian wildlife, comprising four marsupials (koala, Phascolarctos cinereus; southern brown bandicoot, Isoodon obesulus; swamp wallaby, Wallabia bicolor; bare-nosed wombat, Vombatus ursinus), one bird (regent honeyeater, Anthochaera phrygia) and one amphibian (eastern dwarf tree frog, Litoria fallax). Samples corresponded to both clinically healthy and diseased individuals. Sequencing reads were de novo assembled into contigs and annotated. The evolutionary relationships among the trypanosomatid sequences identified were determined through phylogenetic analysis of 18S rRNA sequences. RESULTS: We detected trypanosome sequences in all six species of vertebrates sampled, with positive samples in multiple organs and tissues confirmed by PCR. Phylogenetic analysis indicated that the trypanosomes infecting marsupials were related to those previously detected in placental and marsupial mammals, while the trypanosome in the regent honeyeater grouped with avian trypanosomes. In contrast, we provide the first evidence for a trypanosome in the eastern dwarf tree frog that was phylogenetically distinct from those described in other amphibians. CONCLUSIONS: To our knowledge, this is the first meta-transcriptomic analysis of trypanosomes in native Australian wildlife, expanding the known genetic diversity of these important parasites. We demonstrated that RNA sequencing is sufficiently sensitive to detect low numbers of Trypanosoma transcripts and from diverse hosts and tissues types, thereby representing an effective means to detect trypanosomes that are divergent in genome sequence.

Strigea robusta causes polydactyly and severe forms of Rostand's anomaly P in water frogs.

BACKGROUND: Cases of polydactyly in natural populations of amphibians have attracted great interest from biologists. At the end of the 1940s, the French biologist Jean Rostand discovered a polymorphic syndrome in some water frog (Anura: Pelophylax) populations that included polydactyly and some severe morphological anomalies (he called it 'anomaly P'). The cause of this anomaly remains unknown for 70 years. In a previous study, we obtained anomaly P in the laboratory in tadpoles of water frogs that developed together with molluscs Planorbarius corneus (Mollusca: Gastropoda) collected in the field. We thus proposed the 'trematode hypothesis', according to which the infectious agent responsible for anomaly P is a trematode species. METHODS: Metacercariae from tadpoles with anomaly P were identified using ITS2 gene sequencing as Strigea robusta (Trematoda: Strigeidae). To verify teratogenic features of the species, cercariae of S. robusta were tested for the possibility to cause anomalies. Identification of cercariae species was made using morphological and molecular methods (sequencing of ITS2 and 28S rRNA). The tadpoles were exposed to parasites at four doses of cercariae (control, low, medium and high) and divided into two groups: "early" (at 25-27 Gosner stages) and "late" (at 29-34 Gosner stages) exposure. RESULTS: A total of 58 (72.5%) tadpoles survived until metamorphosis under the dose-dependent experiment with the trematode S. robusta. Differences in the survival rates were observed between the exposed and unexposed tadpoles both in the group of "early" tadpoles and "late" tadpoles. The exposure of tadpoles to the cercariae of S. robusta induced anomaly P in 82% of surviving tadpoles. The severe forms developed only in "early" stages under all doses of cercariae exposure. Polydactyly predominantly developed in the "late" stages; under a light exposure dose, polydactyly also developed in "early" tadpoles. Laboratory-hatched tadpoles reared together with infected snails had different rates of survival and complexity of deformations associated with the period of coexistence. CONCLUSIONS: The experiments with direct cercariae exposure provide compelling evidence that S. robusta leads to anomaly P in tadpoles of water frogs. The manifestation of anomaly P turned out to be dependent on the stage of development, cercariae dose, and the location of the cysts.

Low prevalence of spargana infection in farmed frogs in the Yangtze River Delta of China.

Frogs are the main source of infection for human sparganosis. In this study, the prevalence and pathogenicity of plerocercoid larvae (sparganum) in frogs collected from the Yangtze River Delta in East China were investigated. A total of 386 frogs belonging to five species were purchased from farmers' markets across all three provincial level areas in the Yangtze River Delta region. The overall prevalence was 4.9% (19/386), and 39 spargana were detected visually, with the intensity ranging from 1 to 11. The spargana infection rate was 7.7% (11/143) in Jiangsu Province and 4.4% (8/181) in Shanghai City, while no spargana infection was detected in Zhejiang Province. In five tested frog species, only Rana nigromaculata and R. limnocharis were found to harbor spargana infection, with a prevalence of 7.7% (13/168) and 6.3% (6/95), respectively. There was no significant difference among the months of the experimental period, July to September. The spargana mostly parasitized the muscle tissues of frogs, especially in the hind legs. All the spargana were identified by molecular analysis based on cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes, and all plerocercoids were Spirometra erinaceieuropaei. Nine mice were infected orally with 1 to 3 scoleces, and 77.8% (14/18) of plerocercoids were found in mice at the 30th day post infection. No obvious clinical symptoms were observed in the mice; however, histopathological analysis showed an inflammatory cellular response in all tissues except intestinal tissue. Hematologic analysis showed an increased number of white blood cells (WBCs) at the 18th day post infection. These results indicated that R. nigromaculata and R. limnocharis are a potential source of zoonotic sparganosis in the Yangtze River Delta of China, and farmed frogs may substantially reduce zoonotic risk as compared to eating wild frogs. Our findings will provide data for frog food safety and prevention and control of sparganosis in the region.

Developmental stages of Amphibiocystidium sp., a parasite from the Italian stream frog (Rana italica).

Amphibian parasites of the genus Amphibiocystidium are members of the class Ichthyosporea (=Mesomycetozoea), within the order Dermocystida. Most of the species in the Dermocystida fail to grow in ordinary culture media, so their life cycle has only been partially constructed by studies in host tissues. However, to date, there have been few reports on the life cycle of Amphibiocystidium parasites with respect to the developmental life stages of both Dermocystidium and Rhinosporidium parasites. In this study, we provide light and electron microscopic findings of developmental phenotypes of Amphibiocystidium sp., a parasite previously characterized in the Italian stream frog (Rana italica), which has caused an ongoing infection in a natural population of Central Italy. These phenotypes exhibited distinct morphological characteristics that were similar to A. ranae from the skin of R. temporaria, but showed histochemical properties particularly comparable with those of maturing phenotypes of Rhinosporidium seeberi, and compatible with fungal-like parasites. Therefore, for Amphibiocystidium sp. phenotypes, we suggest adopting the terminology used for maturing stages of R. seeberi, such as juvenile sporangia, early mature sporangia and mature sporangia. The characterization of these developmental stages will be useful to increase the understanding of the life cycle of parasites of the genus Amphibiocystidium and of the interactions with their amphibian hosts.

Morphological and molecular characteristics of hemoparasites in vaillant's frogs (Lithobates vaillanti).

Amphibian populations are declining around the world, and the main reasons are the environmental changes and pathogens. However, there are few studies addressing the interaction and impact of the different pathogens that affect amphibians, such as hemoparasites. These parasites had been described as common in some amphibian species, but unfortunately, their description and characterization are unclear and scarcely spread. The objective of the present study was to evaluate the morphological and molecular characterization of hemoparasites present in vaillant's frogs. Seven frogs of Lithobates vaillanti were captured at the biological station La Florida in Tabasco, Mexico. Blood smears were performed, and results show that 100% of the animals have hemoparasites. Three types of hemoparasites were found. Eighty-five percent of the frogs were positive to Hepatozoon sp., 57% to Lankesterella sp., and 28% to Trypanosoma sp. According to the molecular analysis of the obtained sequences of Trypanosoma sp. and Hepatozoon sp., both protozoans were positioned in between the clusters of parasites of different geographical regions. Nevertheless, no species names were assigned to any of these parasites because more sequences and analysis are needed.

Large-scale survey of a neglected agent of sparganosis Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidae) in wild frogs in China.

BACKGROUND: In China, frogs play an understudied role in the spread of human sparganosis (caused by the larval form of Spirometra). However, our knowledge about the prevalence of sparganum infection in frogs remains fragmented, and the taxonomic identification of the parasite is still controversial. METHODOLOGY/PRINCIPAL FINDINGS: The prevalence of sparganum infection in wild frogs was surveyed at 145 geographical locations from 28 of the 34 provinces/autonomous regions/municipalities in China for six years. The collected sparganum isolates from the different locations were subjected to molecular identification by a multiplex PCR assay and then were analysed with clustering analysis. In the survey, sparganum infection was found in 8 out of 13 of the collected frog species, and the most frequently infected species was Pelophylax nigromaculatus (the infection rate was up to 14.07%). Infected frogs were found in 80 of the 145 surveyed locations. The sparganum infection rates in the wild frogs in several regions of China were still high (above 10%), especially in South and Southwest China. A total of 72 spargana were selected for molecular identification, and the clustering analysis showed that sequences from the Chinese isolates were very similar to those identified as from Spirometra erinaceieuropaei. However, the taxonomy of the genus remains confused and further analysis is required. CONCLUSIONS: Eating wild frogs is associated with considerable health risks in China. Several traditional Chinese folk remedies may increase the risk of infection. The sparganum isolates in China are most likely from S. erinaceieuropaei, but new studies, especially comprehensive morphological analyses, are needed in the future.

Helminth Species and Infracommunities in Frogs Pelophylax ridibundus and P. esculentus (Amphibia: Ranidae) in Northern Ukraine.

PURPOSE: The study aims to characterise and compare the helminth assemblages and helminth infracommunities in the marsh frog, Pelophylax ridibundus and the edible frog, P. esculentus collected in the northern part of Ukraine. METHODS: Occurrence and abundance of the helminths were analysed by calculating the prevalence, intensity, and mean abundance of infection; similarities between the infracommunities were estimated by the Bray-Curtis index and visualised using nMDS plots. Dissimilarities were estimated using the ANOSIM and SIMPER routines. RESULTS: In total, 27 helminth species were found in 143 frogs. Pelophylax ridibundus (n = 86) harboured 20 species of helminths, 24 species were found in P. esculentus (n = 57), and 17 species were shared by the two hosts. Oswaldocruzia bialata and larval Strigea sp. were absent in P. ridibundus, while they reached the prevalence of 30% and 10%, respectively, in P. esculentus. Cosmocerca ornata, Diplodiscus subclavatus, Opisthioglyphe ranae, and Codonocephalus urniger had significantly larger prevalence in P. ridibundus, whereas Haematoloechus asper was found to be more prevalent in P. esculentus. Acanthocephalus ranae, Icosiella neglecta, Haematoloechus variegatus, Pleurogenes claviger, Pleurogenoides medians, and Prosotocus confusus were equally common in both hosts. Helminth infracommunities in the two hosts had identical species richness (1-10 species, 4 on average); abundance was significantly higher in P. ridibundus. CONCLUSIONS: Helminth assemblages of the two hosts in northern Ukraine are rather similar; however, small but significant differences were found in their species composition, parameters of infection in some species, and structure of helminth infracommunities.