A new species of Gorgoderina (Digenea: Gorgoderidae) from Rana berlandieri in Los Tuxtlas tropical rainforest, Mexico, with the elucidation of its life cycle.

Species in the genus Gorgoderina Looss, 1902 are parasites of the urinary bladder of amphibians and include around 50 species described globally. Molecular data on species of the genus are scarce, as is the information of their life-cycle patterns. During a survey on the genetic characterization of the frog trematodes in the tropical rain forest of Los Tuxtlas, in the Gulf of Mexico slope of Mexico, specimens of two morphotypes of Gorgoderina were sampled from the Rio Grande leopard frog, Rana berlandieri. One of them represented an undescribed species which is described herein as Gorgoderina rosamondae n. sp., whereas the other one was morphologically very similar to an apparently widely distributed North American species, G. attenuata, which has been previously reported in the same geographical area. Specimens of both morphotypes were sequenced for two nuclear and one mitochondrial genes. Phylogenetic trees corroborated the distinction of the new species, and data on the internal transcribed spacer 2 revealed genetic differences between G. attenuata sequenced from frogs in USA and specimens of Gorgoderina sp. from Los Tuxtlas, indicating the possibility that they also represent an undescribed species. COI sequences showed high genetic divergence values between the new species and Gorgoderina sp. from Los Tuxtlas (8.63-9.99%). Additionally, COI sequences of the larval forms (sporocyst, cercariae and metacercariae) sampled in the same locality from their first and second intermediate hosts (Pisidium sp. and Agriogomphus tumens, respectively) showed conspecificity, and the 3 host life-cycle of the new species was elucidated.

Echinostoma mekongi: Discovery of Its Metacercarial Stage in Snails, Filopaludina martensi cambodjensis, in Pursat Province, Cambodia.

Echinostoma mekongi was reported as a new species in 2020 based on specimens collected from humans in Kratie and Takeo Province, Cambodia. In the present study, its metacercarial stage has been discovered in Filopaludina martensi cambodjensis snails purchased from a local market nearby the Tonle Sap Lake, Pursat Province, Cambodia. The metacercariae were fed orally to an experimental hamster, and adult flukes were recovered at day 20 post-infection. They were morphologically examined using light and scanning electron microscopes and molecularly analyzed by sequencing of their mitochondrial cox1 and nad1 genes. A total of 115 metacercariae (1-8 per snail) were detected in 60 (60.0%) out of 100 Filopaludina snails examined. The metacercariae were round, 174 µm in average diameter (163-190 µm in range), having a thin cyst wall, a head collar armed with 37 collar spines, and characteristic excretory granules. The adult flukes were elongated, ventrally curved, 7.3 (6.4-8.2)×1.4 (1.1-1.7) mm in size, and equipped with 37 collar spines on the head collar (dorsal spines in 2 alternating rows), being consistent with E. mekongi. In phylogenetic analyses, the adult flukes showed 99.0-100% homology based on cox1 sequences and 98.9-99.7% homology based on nad1 sequences with E. mekongi. The results evidenced that F. martensi cambodjensis snails act as the second intermediate host of E. mekongi, and hamsters can be used as a suitable experimental definitive host. As local people favor to eat undercooked snails, these snails seem to be an important source of human infection with E. mekongi in Cambodia.

Morphological and molecular characterization of larval digenean trematodes (Parvatrema: Gymnophallidae) and their pathological effects on the clam Leukoma thaca (=Protothaca thaca) (Bivalvia:Veneridae) (Molina, 1782) from northern Chile.

Trematodes are one of the largest taxa of mollusk parasites. The clam Leukoma thaca is an economically exploited bivalve found along the south-eastern Pacific coast of Peru and Chile. This bivalve is parasitized by various unidentified larval stages of digeneans in the mantle, gonads and digestive gland. The aims of this study were to determine and describe the different larval stages of the digeneans based on morphological characteristics, to identify them at the species level by performing molecular analyses, and to evaluate pathologies associated with the parasites of this clam. Individuals of L. thaca were collected in San Jorge Bay (23°S), Chile, between November 2018 and February 2019. Morphological description was carried out using in vivo and fixed specimens, and analyses including histological and scanning electron microscopy were performed. Individuals were also isolated for molecular analysis using nuclear ribosomal internal transcribed spacer 1 (ITS1), including partial subunit 18S rDNA (18S) and small subunit 5.8S gene (5.8S). Morphological characteristics indicated that the metacercaria larval stage belongs to the family Gymnophallidae, genus Parvatrema, which was supported by molecular analysis. Molecular results revealed that metacercaria, sporocysts and cercaria stages found in this clam belong to the same species of Parvatrema (genetic distance 0%), evidencing that this species uses L. thaca as the first and second intermediate host. Pathologies examined in the host were similar in nature to those reported in other gymnophallids in bivalves, but high prevalence of cercariae (20%) in gonads suggested an important castrator effect on the host.

Morphological and Molecular Characteristics of Clinostomid Metacercariae from Korea and Myanmar.

Morphological and molecular characterization of clinostomid metacercariae (CMc) was performed with the specimens collected in fish from Korea and Myanmar. Total 6 batches of clinostomid specimens by the fish species and geographical localities, 5 Korean and 1 Myanmar isolates, were analyzed with morphological (light microscopy and SEM) and molecular methods (the cytochrome c oxidase 1 gene and internal transcribed spacer 1/5.8S rRNA sequence). There were some morphological variations among CMc specimens from Korea. However, some morphometrics, i.e., the size of worm body and each organ, ratio of body length to body width, and morphology of cecal lumens, were considerably different between the specimens from Korea and Myanmar. The surface ultrastructures were somewhat different between the specimens from Korea and Myanmar. The CO1 sequences of 5 Korean specimens ranging 728-736 bp showed 99.6-100% identity with Clinostomum complanatum (GenBank no. KM923964). They also showed 99.9-100% identity with C. complanatum (FJ609420) in the ITS1 sequences ranging 692-698 bp. Meanwhile, the ITS1 sequences of Myanmar specimen showed 99.9% identity with Euclinostomum heterostomum (KY312847). Five sequences from Korean specimens clustered with the C. complanatum genes, but not clustered with Myanmar specimens. Conclusively, it was confirmed that CMc from Korea were morphologically and molecularly identical with C. complanatum and those from Myanmar were E. heterostomum.

First report of Diplostomidae metacercariae (Trematoda: Digenea) in African catfish Clarias gariepinus (Siluriformes: Clariidae) in Brazil / Primeiro relato de metacercárias de Diplostomidae (Trematoda: Digenea) em bagre africano Clarias gariepinus (Siluriformes, Clariidae) no Brasil

Abstract Clarias gariepinus is a fish from North of South Africa and was later introduced in several countries, including Brazil. The present study aimed to describe the first report of Diplostomidae metacercariae in C. gariepinus in Brazil. For this, 30 C. gariepinus were captured in a lake in the city of Campos dos Goytacazes, RJ, Brazil. Fishes were euthanized using freezing and necropsied for collection of parasites. The organs were dissected and analyzed for the presence of parasites that were processed for light and scanning electron microscopy. Trematodes were stained with Semichon's Carmine and Gomori's Trichrome, observed and schematized under a light microscope with image analysis software. A total of 190 trematodes were collected from the gills, suprabranchial organs, heart, stomach, intestinal mesentery, liver and body cavity of the fish. The parasites had a foliaceous body divided by a discrete constriction, without genital primordia, and a holdfast organ present at the posterior region, typical of metacercariae of the family Diplostomidae. It was classified as the 'Diplostomulum' morphotype, based on the morphology of the reserve bladder structure. This is the first report of the metacercariae of Diplostomidae parasitizing C. gariepinus in the Americas. This fish acts as an intermediate or paratenic host of this digenean in Brazil.

Resumo Clarias gariepinus é um peixe do Norte da África do Sul e posteriormente introduzido em diversos países, incluindo o Brasil. O presente estudo teve como objetivo descrever o primeiro relato de metacercárias em C. gariepinus no Brasil. Para tanto, 30 C. gariepinus foram capturados em um lago na cidade de Campos dos Goytacazes, RJ, Brasil. Os peixes foram eutanasiados por congelamento e necropsiados para a coleta de parasitos. Os órgãos foram dissecados e analisados para a presença de parasitos que foram processados para microscopia ótica e electronica de varredura. Os Trematodeos foram corados com carmim de Semichon e tricrômico de Gomori, observados e esquematizados sob microscópio ótico equipado com software de análise de imagens. Um total de 190 trematodeos foram coletados das brânquias, órgãos suprabrânquiais, coração, estômago, mesentério intestinal, fígado e cavidade corporal dos peixes. Os parasitos tinham corpo foliáceo, dividido por uma discrete contrição, sem primórdio genital, e órgão tribocítico presente na região posterior, típico das metacercárias da fámilia Diplostomidae. Foram classificados como do morfotipo 'Diplostomulum', baseado na morfologia da estrutura da bexiga de reserva. Este é o primeiro relato de metacercárias de Diplostomidae parasitando C. gariepinus nas Américas. Este peixe atua como hospedeiro intermediário ou paratênico deste Digenea no Brasil.

First report of Diplostomidae metacercariae (Trematoda: Digenea) in African catfish Clarias gariepinus (Siluriformes: Clariidae) in Brazil.

Clarias gariepinus is a fish from North of South Africa and was later introduced in several countries, including Brazil. The present study aimed to describe the first report of Diplostomidae metacercariae in C. gariepinus in Brazil. For this, 30 C. gariepinus were captured in a lake in the city of Campos dos Goytacazes, RJ, Brazil. Fishes were euthanized using freezing and necropsied for collection of parasites. The organs were dissected and analyzed for the presence of parasites that were processed for light and scanning electron microscopy. Trematodes were stained with Semichon's Carmine and Gomori's Trichrome, observed and schematized under a light microscope with image analysis software. A total of 190 trematodes were collected from the gills, suprabranchial organs, heart, stomach, intestinal mesentery, liver and body cavity of the fish. The parasites had a foliaceous body divided by a discrete constriction, without genital primordia, and a holdfast organ present at the posterior region, typical of metacercariae of the family Diplostomidae. It was classified as the 'Diplostomulum' morphotype, based on the morphology of the reserve bladder structure. This is the first report of the metacercariae of Diplostomidae parasitizing C. gariepinus in the Americas. This fish acts as an intermediate or paratenic host of this digenean in Brazil.

Fasciola gigantica: Ultrastructural cytochemistry of the tegumental surface in newly- excysted metacercariae and in vitro-penetrated juvenile flukes informs a concept of parasite defence at the interface with the host.

Cytochemical staining techniques were carried out en bloc with in vitro excysted and gut-penetrated Fasciola gigantica larvae in order to visualise the glycocalyx of the tegument, a structure which comprises the parasite component of the host-parasite interface, yet is incompletely preserved by conventional fixation and preparation techniques for electron microscopy. Positive reactivity with ruthenium red and periodic acid-thiocarbohydrazine-osmium (PATCO) techniques revealed that the glycocalyx is polyanionic and carbohydrate-rich throughout its depth. It comprises a trilaminate arrangement, with a thin dense zone and fibrillar layer closely apposed to the outer aspect of the apical plasma membrane, invested by an irregular thick mucopolysaccharide capsule. The latter, not recorded in adult flukes, may represent a specific adaptation to facilitate invasion in the face of host immunity, and may also protect the parasite surface from the action of host- and parasite-derived proteases. Early in the invasion of a naïve host, the glycocalyx may be partly responsible for triggering the responses of innate immunity, while later in infection, or when an anamnestic response is initiated in an immunocompetent host, the antibodies and activated lymphocytes of specific acquired immunity are invoked to interact with the parasite surface. The cytochemical properties of the glycocalyx, together with its potential for dynamic turnover due to exocytosis of the T0 tegumental secretory bodies, are likely to aid neutralisation of potentially damaging immune effectors and ensure their removal from the vicinity of the parasite by sloughing in complex with glycocalyx components.

Characterization of Clinostomum metacercariae using microscopic and molecular approaches

One of the fundamental aspects in understanding the biology, diversity and epidemiology of a parasite lies in its proper identification. In the present study, morphological and molecular characterization of Clinostomum metacercariae recovered from an ornamental fish, Trichogaster fasciata, was carried out in order to ascertain its identity. To serve the purpose, scanning electron micrographs and gene sequences for two commonly used molecular markers, i.e., nuclear ribosomal internal transcribed spacer 2 (rDNA-ITS2) and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) were obtained. The sequences were further used for generating similarity index matrix as well as inferring phylogenies. Light and electron microscopic observations on metacercariae of the parasite revealed that it belongs to the genus Clinostomum. Identification of the same up to the level of species was made possible through sequence and phylogenetic analyses. The ITS2 sequence analyses of our species (KX758630) showed similarity to unidentified Clinostomum sp. reported from Nigeria (KY865625) and China (KP110579), and C. tilapiae recorded from South Africa (KX034048) and Nigeria (KY649353). However, the CO1 gene analyses suggested it to be highly identical to C. philippinense and the same was also corroborated in the phylogenetic analysis. Thus, morphological and molecular characterization revealed that the recovered metacercariae belong to the species C. philippinense. Additionally, a brief description of secondary structures of ITS2 of various species of Clinostomum has also been discussed.

Fasciola gigantica: Comparison of the tegumental ultrastructure in newly excysted metacercariae and in vitro penetrated juvenile flukes indicates intracellular sources of molecules with vaccinal and immunomodulatory potential.

Using in vitro procedures to prepare newly excysted metacercariae and gut-penetrated juvenile Fasciola gigantica, the ultrastructural features of the tegumental syncytium and perikarya of these ephemeral stages in the host-invasion process were compared. The T0-type tegumental cells in newly excysted metacercariae are packed with stored T0 granules which, following transport to the surface membrane of the syncytium, discharge by exocytosis to maintain the glycocalyx. The T0 cells become depleted of T0 granules during the penetration process, shrink in size, and initiate autophagy in the cytoplasm to facilitate metamorphosis from a storage function to active biosynthesis. The novel products appear to include lysosomes which contribute to the autophagosomes, and T1 granules, necessary for maintenance of the glycocalyx and immunoprotection, as the invasion process continues into the host liver. Residual bodies, the end-products of autophagy, are eliminated from the apical membrane of the tegumental syncytium into the host-parasite interface. There they may present a transient source of parasite-derived molecules, including lysosomal cathepsin-type proteases, with potential for interaction with the host's immune system, and so might be exploited as targets for vaccinal and immunomodulatory studies.

New Record of Thapariella anastomusa (Trematoda: Thapariellidae) Metacercariae in Northern Thailand.

The family Thapariellidae has been reported in only 3 countries since 1990. The objective of this study was to identify Thapariella anastomusa metacercariae in snails in Thailand based on morphological traits using a light (LM) and a scanning electron microscope (SEM). A total of 94 Filopaludina snails were collected and identified as 50 F. martensi martensi and 44 F. doliaris. Metacercariae of T. anastomusa were recovered from the snails by the crushing method. The overall prevalence was 22.3% (21/94), and the mean intensity was 17.0 per snail. The prevalence in F. martensi martensi was 24.0% (12/50) and F. doliaris 20.5% (9/44) with the mean intensity of 18.8 and 14.8 per snail, respectively. SEM revealed traits such as a concave ventral body and well-developed oral and ventral suckers. This study represents the first report of T. anastomusa in South East Asia. While LM and SEM observations provide novel insights into T. anastomusa metacercarial morphology and life history, the trematode's life cycle remains unclear. To date, there has been no report of T. anastomusa causing infections in humans. However, the snails F. martensi martensi and F. doliaris carrying the infective stages of T. anastomosa are frequently consumed by Thai people. This consumption, particularly uncooked snails, may present a risk of Thapariella infections in humans.

Echinostoma macrorchis (Digenea: Echinostomatidae): Metacercariae in Cipangopaludina chinensis malleata Snails and Adults from Experimental Rats in Korea.

A survey was performed to know the infection status of echinostome metacercariae in Cipangopaludina chinensis malleata snails from Korea. Total 75 snails collected in 5 localities, i.e., Imsil-gun, Jeollabuk-do, Hwasun-gun and Shinan-gun (Aphae and Jido), Jeollanam-do, and Jinju-si, Gyeongsangnam-do, were examined for metacercariae by the artificial digestion method. Infection rates of metacercariae were 80.0%, 66.7%, 100%, 60.0%, and 73.3%, and their densities were 39, 32, 183, 19, and 30 per snail infected, respectively. The metacercariae were round, 105-118×105-118 µm in size, with a thin cyst wall, collar spines on the head collar, and excretory granules in 2 canals of excretory tube. Adult flukes were elongated, ventrally curved, and 5,167×939 µm in average size. Head collar distinct, bearing 45 collar spines with 5 end groups on each side. Oral sucker subterminal, pharynx well developed, and esophagus somewhat short. Cirrus sac well developed, with a saccular seminal vesicle, and ventral sucker very large. Ovary elliptical and on the median line of the body. Testes tandem and slightly lobed. Eggs operculated, elliptical, and 90-103×55-60 µm in size. By scanning electron microscopy, the head collar was prominent with 45 collar spines resembling horns of younger stags. Scale-like tegumental spines were densely distributed on the body surface between the head collar and ventral sucker. Conclusively, it has been first confirmed that the life cycle of E. macrorchis is indigenously maintained in Korea, and C. chinensis malleata snails are popularly infected with the metacercariae of this echinostome.

Artyfechinostomum malayanum: Metacercariae Encysted in Pila sp. Snails Purchased from Phnom Penh, Cambodia.

The metacercariae of Artyfechinostomum malayanum (Leiper, 1911) Mendheim, 1943 were discovered in Pila sp. snails purchased from a market in Phnom Penh, Cambodia. They were isolated from the snails using the artificial digestion technique and were orally fed to 2 hamsters, 1 rat, and 2 mice to obtain the adult flukes. The metacercariae were round, 145-165 µm in diameter, having a cyst wall of 6-10 µm in thickness, a head collar and collar spines, and characteristic features of excretory granules. Adult flukes were recovered in the small intestines of the animals at days 14 and 32 post infection and were morphologically observed using a light microscope and a scanning electron microscope. They were plump or elongated, ventrally curved, 6.0-8.1×1.6-2.0 mm in size, and characterized by the head collar bearing 43 collar spines, including 5 end group ones on each side, a long cirrus sac extending beyond the posterior margin of the ventral sucker, a submedian ovary, and 2 deeply lobed testes. Eggs in uteri were operculate, ovoid to ellipsoid, and 120-135×68-75 µm in size. In scanning electron microscopy, the head collar was prominent with collar spines looking like horns. Scale-like tegumental spines were densely distributed on the ventral surface between the head collar and ventral sucker. Sensory papillae were distributed mainly on the tegument around suckers. By this study, it has been first confirmed that the life cycle of A. malayanum exists in Cambodia.

Morphological and molecular characterisation of three Indian Neascus-type metacercariae (Digenea: Diplostomidae).

Diplostomid digeneans are important parasites whose larval stages infect freshwater snails and fish as first and second intermediate hosts respectively. Diplostomid digeneans as adults are parasitic in many fish-eating birds and mammals. Our understanding of the species diversity of diplostomid digeneans remains incomplete, especially in the Indian sub-continent. Here, we describe three Neascus-type metacercariae (N. hanumanthai, N. gussevi, and N. xenentodoni) from freshwater fish specimens of Channa punctata (Bloch 1793), Trichogaster fasciata (Bloch and Schneider 1801) and Xenentodon cancila (Hamilton, 1822) respectively, collected in India. Next, we characterised these metacecariae using nuclear (28S and ITS1) and mitochondrial DNA (cox1) to determine their systematic and phylogenetic position. Molecular identification using interspecific variation for all three molecular markers revealed a closer relationship between N. hanumanthai and N. gussevi (1.9%-2.4%) than either of N. hanumanthai and N. gussevi to N. xenentodoni (3.1%-3.7% and 4.4%-4.0% respectively). In phylogenetic analyses, estimated by neighbour-joining (NJ) and maximum likelihood (ML) methods, N. gussevi and N. hanumanthai nested as sister groups of Posthodiplostomum Dubois, 1936 for all three markers used; N. xenentodoni, however, nested as a sister taxa of all other diplostomes when using 28S and ITS1 and nested as a sister taxa of Bolbophorus, Alaria and Neodiplostomum using cox1. These findings suggest that N. hanumanthai and N. gussevi are members of Posthodiplostomum, but that N. xenentodoni belongs to a separate and unknown genus. Similarly, by proteomics, we found that the cox1 protein sequences and structures were similar between N. hanumanthai and N. gussevi but distinct for N. xenentodoni.

Morphological and molecular characterization of the metacercaria of Paragonimus caliensis, as a separate species from P. mexicanus in Costa Rica.

The trematode Paragonimus mexicanus is the etiological agent of paragonimiasis, a food-borne zoonotic disease in Latin America. This species, as well as Paragonimus caliensis, have been reported from Costa Rica, but it is not known if the two are synonymous. Two types of Paragonimus metacercariae from freshwater pseudothelphusid crabs from several localities in Costa Rica were recognized by light microscopy. Morphologically, these corresponded to descriptions of P. mexicanus and P. caliensis. Metacercariae of the former species lacked a membrane or cyst and their bodies were yellow in color. Those of P. caliensis were contained in a transparent thin cyst and were pink in color. Morphotypes of metacercariae were determined using scanning electron microscopy (SEM). Based on the number and distribution of papillae in the ventral sucker, three morphotypes were found for P. mexicanus and two for P. caliensis. Analysis of DNA sequences (nuclear ribosomal 28S and ITS2 genes, and partial mitochondrial cox1 gene) confirmed the presence of P. mexicanus and provided the first molecular data for P. caliensis. The two species are phylogenetically distinct from each other and distant from the Asian species. The confirmation of P. caliensis as a separate species from P. mexicanus raises several questions about the ecology, biological diversity, and epidemiology of the genus Paragonimus in Costa Rica.

Redescription of Clinostomum phalacrocoracis metacercariae (Digenea: Clinostomidae) in cichlids from Lake Kinneret, Israel.

Clinostomidae are digeneans characterized by a complex taxonomic history, continuously under revision based on both morphological and molecular analysis. Among the 14 species considered valid so far Clinostomum phalacrocoracis has been well described only at the adult stage, whereas the morphology of the metacercarial stage has been reported only once. During a parasitological survey carried out on 262 wild cichlids sampled from Lake Kinneret (Israel) metacercariae referable to C. phalacrocoracis were found in 18 fingerlings. In this study, we report this clinostomid species for the first time in wild fish from Israel describing the metacercarial stage of Clinostomum phalacrocoracis, coupling its morphological description with molecular analysis carried out on ITS rDNA and COI mtDNA sequences.

Maritrema orensense and Maritrema bonaerense (Digenea: Microphallidae): descriptions, life cycles, and comparative morphometric analyses.

We elucidate the life cycle of Maritrema orensense for the first time and experimentally confirm that of the sympatric Maritrema bonaerense. In Argentinean estuaries, both species parasitize the cochliopid snail Heleobia australis as first intermediate host, the grapsid crabs Neohelice granulata and Cyrtograpsus angulatus as second intermediate hosts, and gulls as definitive hosts. Here, we describe the daughter sporocysts and cercariae of M. orensense and redescribe these stages for M. bonaerense. Sporocysts of M. orensense are shorter, with fewer developed cercariae than M. bonaerense. The cercariae of M. orensense have longer, larger, and more undulating cephalic glands than M. bonaerense. We redescribe metacercariae and adults of both species and compare them with the previous descriptions. Intestinal ceca length, vitellaria shape and extension, and egg size are the most relevant characteristics in metacercariae and adults for differentiating the species. Hence, the detailed morphological description and comparative analyses of morphometrics obtained from natural and experimental infections permit clear differentiation of M. orensense and M. bonaerense at each life stage.

Morphology, ultrastructure, genetics, and morphometrics of Diplostomum sp. (Digenea: Diplostomidae) metacercariae infecting the European flounder, Platichthys flesus (L.) (Teleostei: Pleuronectidae), off the northwest coast of Portugal.

The morphology, ultrastructure, genetics, and morphometrics of a species of Diplostomum von Nordmann 1832 (Digenea: Diplostomidae), isolated from the European flounder (Platichthys flesus (L.)) caught off the northwest coast of Portugal, are characterized. The metacercarial stage was found unencysted in the lens capsule of the eye. Light microscopical observations revealed the existence of some variability in specimen shape and size, with two morphotypes, referred to as "round" and "long", being apparent. Scanning electron microscopy revealed a smooth, unarmed tegument, with the lappet region being the most irregular and porose. Both the oral and ventral suckers were provided with a series of papillae, which presented very distinctive ultrastructural features and were particularly conspicuous in the case of the ventral sucker. The two morphotypes detected were found to have 100% genetic correspondence in the 18S + ITS1 + 5.8S region of the rDNA. Since the genetic data for this metacercaria differed from those of the species of Diplostomum available in GenBank, a description of a new genotype (accession number GQ370809) is provided. The molecular phylogenetic analyses, in conjunction with principal components and cluster analyses based on morphometric data, revealed the existence of consistent differences between the Diplostomum sp. metacercariae from flounder compared with Diplostomum spathaceum, Diplostomum mergi, Diplostomum pseudospathaceum, and Diplostomum paracaudum. The latter of these species was found to be the most similar to the present material. Our results do not support an evolutionary separation of the European and North American species of Diplostomum.

Hybridization experiments indicate incomplete reproductive isolating mechanism between Fasciola hepatica and Fasciola gigantica.

Experiments on hybridization between Fasciola hepatica and Fasciola gigantica were carried out to clarify whether a reproductive isolating mechanism appears between the two Fasciola species. Molecular evidence for hybridization was based on the DNA sequence of the internal transcribed spacer 1 (ITS1) region in nuclear ribosomal DNA, which differs between the species. The results suggested that there were not pre-mating but post-mating isolating mechanisms between the two species. However, viable adults of the hybrids F1 and F2 were produced from both parental F. hepatica and F. gigantica. The hybrids inherited phenotypic characteristics such as ratio of body length and width and infectivity to rats from parental Fasciola hepatica and F. gigantica. These findings suggest that reproductive isolation is incomplete between Fasciola hepatica and F. gigantica. Adults of the hybrids F1 and F2 were completely different in mode of reproduction from aspermic Fasciola forms that occur in Asia and seem to be offspring originated from hybridization between F. hepatica and F. gigantica and to reproduce parthenogenetically.

Morphological and molecular study of Microphallus primas (Digenea: Microphallidae) metacercaria, infecting the shore crab Carcinus maenas from northern Portugal.

The present study describes the anatomy and surface topography of the metacercaria of Microphallusprimas (Jägerskiöld, 1909) infecting the shore crab Carcinus maenas (L.) in Aveiro estuary, northern Portugal. The metacercaria species identification resulted from the combined use of morphological and molecular data, particularly the 28S rDNA gene. The metacercariae encysted preferentially in the host's hepatopancreas and also in the gonads. Isolated cysts were present in two distinct forms, spherical and oval, and were shown to be the identical species by the internal transcribed spacer 1 (ITS1) sequence. Chemically excysted metacercariae were studied by light (LM) and scanning electron microscopy (SEM). Their specific characteristics observed include the particular aspect of the vesiculo-prostatic pouch surrounded by a very thin membrane, the presence of a prominent muscular papilla, and an obvious metraterm. The dorsal and ventral tegumental surfaces of the metacercaria were densely packed with similar squamous spines, which decreased in number and size towards the hindbody. The edges of the posterior and ventral face of the body were coated with numerous microvilli, whose function remains unknown. In order to identify the species of metacercariae, we compared a 28S partial rDNA sequence of the two forms of cysts with the same 28S partial region of M. primas available in GenBank. With this comparison, we determined that the sequences had a 100% similarity and therefore belonged to the same species, i.e., M. primas.

[The influence of Echinostoma caproni metacercariae (Trematoda) on the survival of biomphalaria molluscs (Pulmonata)].

The infrapopulation of the Echinostoma caproni partenites has a development of prolong character (Ataev et al, 2005). However, in laboratory conditions, Biomphalaria molluscs infested with this parasite die within 1--3 weeks after the beginning of cercariae emission. It has been suggested that autoinvasion of the mollusc host with the cercariae, which use it as second intermediate host, is the cause of this phenomenon. Studying the dynamics of metacercariae accumulation in the host (both infected and non-infected with the Echinostoma caproni rediae) and experiments where quantity of cercariae around molluscs reduced by different ways, confirmed this hypothesis. Evidently, pathogenicity of metacercariae for molluscs is lesser in nature, because the concentration of cercariae reduces to the values, which do not result in lethal effect: some part of cercariae dies, but another part uses other animals as a host (Haas, 2000).