Interrelationships of Anenterotrema (Digenea: Dicrocoeliidae) from Neotropical bats (Mammalia: Chiroptera) with description of a new species from Molossus molossus in Brazil.

Anenterotrema is a small genus of dicrocoeliids (Digenea: Dicrocoeliidae) containing 6 species found in Neotropical bats. Members of this genus are characterized by the lack of digestive system organs and, unlike the majority of dicrocoeliids, parasitize the intestine of their definitive hosts. In this study, we have morphologically examined newly collected specimens belonging to four species of Anenterotrema from Brazil, Ecuador, and Panama. Based on the data in original descriptions and our analysis of quality new specimens, we resurrected Anenterotrema freitasi, previously synonymized with A. eduardocaballeroi. We also described a new species of Anenterotrema from Molossus molossus in the Brazilian Amazon. The new species differs from congeners in several morphological features, most prominently in the size and structure of its highly muscular, protuberant ventral sucker. It is also characterized by the lack of the semi-circular thickening of the tegument around the oral sucker typical for some members of the genus. We used partial DNA sequences of the large ribosomal subunit gene (28S) and mitochondrial cytochrome c oxidase subunit 1 gene (cox1) to test the monophyly of Anenterotrema, and study the interrelationships and determine the inter- and intraspecific variation of three Anenterotrema spp. collected from different bat species in Brazil, Ecuador and Panama.

Morphological and molecular evidence reveals a new species of Lyperosomum Looss, 1899 (Digenea: Dicrocoeliidae) from Melanerpes aurifrons (Wagler, 1829) from northern Mexico.

A new species of the genus Lyperosomum Looss, 1899, from the intestine of the golden-fronted woodpecker (Melanerpes aurifrons) from northern Mexico is described. Lyperosomum cuauhxinqui sp. n. is morphologically distinguished from other congeneric species from the Americas by a higher oral/ventral sucker ratio and its body length and width. The sequences of domains D1-D3 of the large subunit (LSU) of nuclear ribosomal DNA and cytochrome c oxidase subunit 1 (cox 1) from the mitochondrial DNA of the new species were obtained and compared with available sequences from GenBank. The genetic divergence estimated between the new species and other congeneric species ranged from 2 to 6% and 13.4 to 17.3% for LSU and cox 1, respectively. Phylogenetic analyses based on the two (LSU and cox 1) molecular markers consistently showed that L. cuauhxinqui sp. n. was nested within the genus Lyperosomum, with strong bootstrap support (100%) and Bayesian posterior probabilities (1.0). In particular, the LSU tree indicated that the sequence of the new species is closely related to sequences from Zonorchis alveyi, Zonorchis delectans and Zonorchis sp. from Central America, suggesting that these sequences should be transferred to the genus Lyperosomum. The new species represents the first record from Mexico and the fifth species identified in the Americas. Our study also revealed that the taxonomy of the genus Lyperosomum should be re-examined by combining molecular, morphological and ecological characteristics.

Molecular phylogenetics and mitogenomics of three avian dicrocoeliids (Digenea: Dicrocoeliidae) and comparison with mammalian dicrocoeliids.

BACKGROUND: The Dicrocoeliidae are digenetic trematodes mostly parasitic in the bile ducts and gall bladder of various avian and mammalian hosts. Until recently their systematics was based on morphological data only. Due to the high morphological uniformity across multiple dicrocoeliid taxa and insufficient knowledge of relative systematic value of traditionally used morphological characters, their taxonomy has always been unstable. Therefore, DNA sequence data provide a critical independent source of characters for phylogenetic inference and improvement of the system. METHODS: We examined the phylogenetic affinities of three avian dicrocoeliids representing the genera Brachylecithum, Brachydistomum and Lyperosomum, using partial sequences of the nuclear large ribosomal subunit (28S) RNA gene. We also sequenced the complete or nearly complete mitogenomes of these three isolates and conducted a comparative mitogenomic analysis with the previously available mitogenomes from three mammalian dicrocoeliids (from 2 different genera) and examined the phylogenetic position of the family Dicrocoeliidae within the order Plagiorchiida based on concatenated nucleotide sequences of all mitochondrial genes (except trnG and trnE). RESULTS: Combined nucleotide diversity, Kimura-2-parameter distance, non-synonymous/synonymous substitutions ratio and average sequence identity analyses consistently demonstrated that cox1, cytb, nad1 and two rRNAs were the most conserved and atp6, nad5, nad3 and nad2 were the most variable genes across dicrocoeliid mitogenomes. Phylogenetic analyses based on mtDNA sequences did not support the close relatedness of the Paragonimidae and Dicrocoeliidae and suggested non-monophyly of the Gorgoderoidea as currently recognized. CONCLUSIONS: Our results show that fast-evolving mitochondrial genes atp6, nad5 and nad3 would be better markers than slow-evolving genes cox1 and nad1 for species discrimination and population level studies in the Dicrocoeliidae. Furthermore, the Dicrocoeliidae being outside of the clade containing other xiphidiatan trematodes suggests a need for the re-evaluation of the taxonomic content of the Xiphidiata.

Cepaea spp. as a source of Brachylaima mesostoma (Digenea: Brachylaimidae) and Brachylecithum sp. (Digenea: Dicrocoeliidae) larvae in Poland.

Parasite diagnostics were carried out on 11 Polish populations of Cepaea spp. In three of them, coming from the roadside ditches of a village (Rytel, northern Poland), very high (up to 60%) prevalence of Brachylaima mesostoma was observed. This study provides the first molecular evidence of the presence of B. mesostoma inside Cepaea spp. in Europe. In a few snails from a population found in a private garden in a small town (Chelmza, northern Poland), larvae of Brachylecithum sp. were present. Cercariae and/or metacercariae of B. mesostoma were observed in both species of Cepaea: C. hortensis and C. nemoralis, whereas larvae of Brachylecithum sp. were found only in C. nemoralis. Both species of parasites inhabited snail hepatopancreas whose structure was significantly damaged by larvae. There was no significant connection between parasite invasion and snail host morphotype. The research did not allow the reasons for the high prevalence of B. mesostoma in Cepaea spp. to be explained, and also did not explicitly indicate how the parasite invaded Cepaea spp. individuals making them, at the same time a second intermediate host. However, it poses important questions about the life cycle of the parasite that may threaten extensively kept small-size farms of poultry.

Dicrocoeliidae Family: Major Species Causing Veterinary Diseases.

This chapter analyses the taxonomic position of Dicrocoeliidae family and several of its genus and species. The biology of the major species causing veterinary diseases such Dicrocoelium dendriticum, Dicrocoelium hospes, Dicrocoelium chinensis, Eurytrema pancreaticum and Platynosomum fastosum, has been reviewed. All these species have an indirect life cycle, involving two intermediate hosts (molluscs as first and ants, grasshoppers and lizards as second). Dicrocoelium dendriticum is a very widespread hepatic trematode in the ruminants of many countries in Europe, Asia, North Africa and North America, even affecting humans. Dicrocoelium hospes is widely distributed in the savanna areas of Africa south of the Sahara, whilst D. chinensis has mainly been found in ruminants in East Asia and some European countries (probably imported from Asia). Eurytrema pancreaticum is a common parasite whose adults live in ruminant bile ducts, gall bladder, pancreatic ducts and intestines in Europe, Madagascar, Asia and South America. Adult P. fastosum live in the liver, gall bladder and pancreas of birds and mammals in Europe, Africa, Asia, North, Central and South America. Information about the epidemiology, pathology, clinical aspect, diagnosis, treatment, control, prevention and economic impact mainly of Dicrocoeliosis produced by D. dendriticum, as well as of Eurytrematodosis and Platynosomiosis, has been included.

A new species of Metadelphis Travassos, 1944 (Digenea: Dicrocoeliidae) from the gall-bladder of Molossus molossus (Pallas) (Chiroptera: Molossidae) in the Brazilian eastern Amazon.

Metadelphis tkachi n. sp. is described based on material from the gall-bladder of Molossus molossus (Pallas) (Chiroptera: Molossidae) in the Brazilian eastern Amazon. The main differentiating characters of the new species include lobed, tandem testes and elongated clusters of vitelline follicles commencing at the level of the seminal receptacle. The new species can be distinguished from Metadelphis lenti (Santos & Gibson, 1998) and Metadelphis apharyngotrema (Marshall & Miller, 1979) by the shape and position of the testes; from Metadelphis evandroi Travassos, 1944 by having tandem, lobed testes and shorter clusters of vitelline follicles; from Metadelphis compactus Travassos, 1955 by having tandem testes and more elongated clusters of vitelline follicles, and from Metadelphis alverangai Travassos, 1955 by having smaller testes and body, and vitellarium with large follicles. New morphological data are reported for M. lenti based on light and scanning electron microscopy; intraspecific variation was observed.

Description and Phylogenetic Relationships of Pojmanskatrema balcanica n. gen., n. sp. (Digenea: Dicrocoeliidae) from the Eurasian Water Shrew Neomys fodiens (Mammalia: Soricidae) in Bulgaria.

BACKGROUND: We describe a new dicrocoeliid collected from the Eurasian water shrew Neomys fodiens in Bulgaria based on the combination of morphological characteristics and molecular data. RESULTS: Pojmanskatrema balcanica n. sp., n. gen. is distinguished from all other species of family Dicrocoeliidae Looss, 1900 in having a postovarian vitellarium situated in the posterior half of the body, postacetabular, nearly tandem, lobed testes, and relatively short body. CONCLUSIONS: Molecular phylogenetic analysis based on 28S rDNA sequences of the new species and previously sequenced dicrocoeliid taxa has demonstrated close relationship of Pojmanskatrema n. gen. with the genus Eurytrema. Considering the non-monophyletic nature of previously recognized dicrocoeliid sub-families we support the recently proposed dissolution of the sub-families within this family.

Phylogenetic relationships within Dicrocoeliidae (Platyhelminthes: Digenea) from birds from the Czech Republic using partial 28S rDNA sequences.

Partial (D1-D3) 28S rRNA gene sequences from 16 isolates of digenean parasites of the family Dicrocoeliidae recovered from 16 bird species from the Czech Republic were used for phylogenetic reconstruction. Comparison with sequences available from GenBank suggests that the genus Brachylecithum is paraphyletic, requiring further validation and possible systematic revision. Although partial 28S rDNA is relatively conserved, analyses suggest that the following taxa are synonymous: Lutztrema attenuatum = L. monenteron = L. microstomum, Brachylecithum lobatum = B. glareoli. Zonorchis petiolatus is reassigned back to the genus Lyperosomum with L. collurionis as a junior synonym. The study revealed how complicated the systematics of the family Dicrocoeliidae is currently. The morphology of the group is variable, and the current distinguishing characters at species and even generic level are not sufficiently distinctive; it is difficult to identify the specimens correctly and identification of GenBank isolates is not reliable. Extensive sampling of isolates for both molecular and morphological studies is necessary to resolve the relationships within the family.

DNA sequences confirm low specificity to definitive host and wide distribution of the cat pathogen Platynosomum illiciens (= P. fastosum) (Trematoda: Dicrocoeliidae).

Although feline platynosomosis has been commonly reported in several parts of the world, the taxonomy and epidemiological chain related to cat liver flukes remain controversial. In this study, nuclear ribosomal ITS, 28S, and mitochondrial cox1 sequences obtained for Platynosomum illiciens from cat, marmoset, lizard, and snail found naturally infected in Brazil reveal no significant molecular differences between these isolates. Moreover, sequence data confirm that Brazilian P. illiciens from different hosts is conspecific with parasites obtained from cats in Vietnam, supporting wide distribution of the species. The lack of pronounced specificity of P. illiciens to definitive hosts is confirmed here for the first time using molecular approach. The results are discussed in context of the epizootology of platynosomosis.

Characterization of the complete nuclear ribosomal DNA sequences of Eurytrema pancreaticum.

Eurytrema pancreaticum is one of the most common trematodes of cattle and sheep, and also infects humans occasionally, causing great economic losses and medical costs. In this study, the sequences of the complete nuclear ribosomal DNA (rDNA) repeat units of five E. pancreaticum individuals were determined for the first time. They were 8306-8310 bp in length, including the small subunit (18S) rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), large subunit (28S) rDNA and intergenic spacer (IGS). There were no length variations in any of the investigated 18S (1996 bp), ITS1 (1103 bp), 5.8S (160 bp), ITS2 (231 bp) or 28S (3669 bp) rDNA sequences, whereas the IGS rDNA sequences of E. pancreaticum had a 4-bp length variation, ranging from 1147 to 1151 bp. The intraspecific variations within E. pancreaticum were 0-0.2% for 18S rDNA, 0-0.5% for ITS1, 0% for 5.8S rDNA and ITS2, 0-0.2% for 28S rDNA and 2.9-20.2% for IGS. There were nine types of repeat sequences in ITS1, two types in 28S rDNA, but none in IGS. A phylogenetic analysis based on the 18S rDNA sequences classified E. pancreaticum in the family Dicrocoeliidae of Plagiorchiata, closely related to the suborder Opisthorchiata. These results provide useful information for the further study of Dicrocoeliidae trematodes.

Platynosomum fastosum (Trematoda: Dicrocoeliidae) from Cats in Vietnam: Morphological Redescription and Molecular Phylogenetics.

The present study was performed to reveal the morphological characteristics and molecular phylogenetic position of Platynosomum fastosum Kossack, 1910. A total 167 specimens of P. fastosum were collected in 8 (4.9%) out of 163 sets of gall-bladders and bile ducts of cats. The number of worms was 1-105 per infected cat. This species was characterized by having a long and slender body, slightly larger ventral sucker than the oral sucker, indistinct prepharynx, small pharynx, short esophagus, bifurcation midway between 2 suckers, and ceca extending to the posterior end of the body. The length of the partial sequences of ITS1 and 5.8S rDNA of P. fastosum were 990 bp, GC-rich. AT/GC ratio was 0.9, there were 9 polymorphic sites, and intraspecific variations ranged from 0.1% to 0.9%. Phylogenetic analyses by neighbor-joining phylogram inferred from ITS1 rDNA sequences revealed that the genetic distance between P. fastosum specimens ranged from 0.3 to 1.5% while the smallest interspecific distance among dicrocoeliid species was 20.9 %. The redescription and genetic characters of P. fastosum are taxonomically important to recognize future different species of the genus Platynosomum showing high intraspecific and morphological variability.

Redescription and phylogenetic relationships of the rare Lyperosomum sarothrurae Baer, 1959 (Digenea: Dicrocoeliidae).

Lyperosomum sarothrurae Baer, 1959 is a rare dicrocoeliid described based on a single specimen from Belgian Congo, now Democratic Republic of the Congo, and never reported again. The original description lacked several important details of the species morphology. This work provides morphological redescription of Lyp. sarothrurae based on specimens from Kibale National Park, Uganda which is about 200 kilometres from the type locality. Results of re-examination of the holotype are also provided. Morphology of Lyp. sarothrurae shows some characteristics intermediate between those of the species rich genus Lyperosomum Looss, 1899 and Megacetabulum Oshmarin, 1964 that includes only 3 species. Due to the lack of clear morphological differences between the two genera, Megacetabulum is considered here as a junior synonym of Lyperosomum. Molecular phylogenetic analysis including Lyp. sarothrurae and all currently available partial sequences of the nuclear ribosomal 28S RNA gene of dicrocoeliid species is also included and its results are discussed.

Morphological and molecular characterization of Eurytrema cladorchis parasitizing cattle (Bos indicus) in Bangladesh.

There is always controversy regarding identification of different species in the genus Eurytrema. Identification has been based mainly on morphology, which can be misleading and subject to differing interpretation among the scientists. Therefore, the aim of this study was to identify Eurytrema flukes both by morphology and molecular properties on the basis of 18-subunit ribosomal RNA (18S rRNA) gene as well as internal transcribed spacer 2 (ITS2) to clarify their phylogenetic status. Among six different agroecological areas of Bangladesh, 22 Eurytrema flukes were recovered from the bile ducts of 22 cattle in Bandarban, a hill district. The flukes were identified as Eurytrema cladorchis through morphometric and morphological studies. Phylogenetic analyses were conducted by neighbor-joining phylogram inferred from both 18S rRNA (1784 bp) gene and ITS2 (229 bp) sequences. A monophyletic clade was constructed by the E. cladorchis from Bangladesh; however, the clade was distinct from those formed by Eurytrema pancreaticum and Eurytrema coelomaticum. This study first described the existence of E. cladorchis from Bangladesh and may provide useful information for both morphological and molecular properties that may further help to clarify phylogenetic relationships within the genus Eurytrema and also for other digeneans.

Keys to the species of Athesmia Loss, 1899 (Digenea: Dicrocoeliidae: Dicrocoeliinae), with the description of a new species from the clapper rail, Rallus longirostris Boddaert (Gruiformes: Rallidae), from Galveston, Texas, U.S.A.

Eleven specimens of a previously undescribed species of Athesmia Looss, 1899 were recovered from six clapper rails, Rallus longirostris Boddaert (Rallidae), collected from November, 1984 through October, 1995 from the Galveston, Texas area of the Gulf of Mexico. Species of Athesmia can be divided into two body types based on the posterior extent of the ceca relative to the vitelline field (vitellarium): the attilae type where the ceca may be uneven, but they both extend at least to near the level of the posterior margin of the vitelline field, or more commonly surpass the vitelline field posteriorly and the heterolechithodes type where the cecum on the side of the vitelline field terminates well above the posterior margin of the vitelline field, usually near its anterior extremity, while the cecum opposite the vitelline field extends at least to the level of the posterior margin of the vitelline field, or more commonly surpasses the vitelline field posteriorly. Athesmia ralli n. sp. is assigned to the attilae type. The new species is most similar to Athesmia butensis Petri, 1942, but differs from this species by having a longer space from posterior testis to ovary (600-790 compared to less than 200), which represent a larger percentage of the total body length (7-8% compared to 2%) and a larger maximum egg size (47 by 29 compared to 42 by 25). The new species further differs from A. butensis by having a somewhat larger body, a longer forebody and the vitelline field reaches posteriorly to the posterior third of the body in the new species but is confined to the middle third of the body in A. butensis. A key to species also is provided.

Phylogenetic relationships of the genus Eurytrema from domestic and wild animal based on 18S rRNA sequences.

Since Loss (1907) established the genus Eurytrema, there were more than eleven species found worldwide from America, Europe to Asia. Adult worms are generally found in pancreatic and bile ducts of wild and domestic ruminants. Some species from wild animal and domestic animal have already differentiated. In this study, we amplified and sequenced the partial 18S rRNA sequences of some Eurytrema species found in wild and domestic animals. The phylogenetic analysis was conducted to show the genetic relationship of these Eurytrema species. The results demonstrated that same species of Eurytrema from domestic animal and wild animal or from separated geological region have a considerable degree of genetic differentiation. Analysis of the 18S rRNA sequences indicated that Eurytrema fukienensis is an independent species and suggested that it may represent the intermediate species between wild and domestic animal.

Occurrence of infection by Platynosomum illiciens (Braun, 1901) in captive neotropical primates.

Platynosomum illiciens (Trematoda, Plagiorchida) is a trematode parasite reported in felids and falconiforms. It was identified in the gall bladder of eight captive neotropical necropsied primates from the National Primate Center (CENP), Ananindeua, State of Pará, Brazil. This is the first description of Platynosomum illiciens as a parasite of primates.

Gastrointestinal parasites of Howler monkeys (Alouatta palliata) inhabiting the fragmented landscape of the Santa Marta mountain range, Veracruz, Mexico.

In recent years populations of howler monkeys (Alouatta palliata) in southeastern Mexico have decreased substantially due to the transformation and loss of natural habitats. This is especially evident in the Santa Marta mountain range, Veracruz, Mexico where several studies have evaluated the impact of fragmentation on howler monkey populations in order to propose management programs for their conservation. The conditions generated by fragmentation likely change the rates of parasitic infection and could decrease howler survival. In this study, gastrointestinal parasite species richness, prevalence, and egg density of infection were determined in howler groups inhabiting five forest fragments at the Santa Marta mountain range. Two hundred and seventy-eight fresh fecal samples were collected between October 2002 and April 2003. Three parasite species were found during the dry and the wet season in all forest fragments sampled: one unidentified species of Eimeriidae; Trypanoxyuris minutus (Oxyuridae); and Controrchis biliophilus (Dicrocoeliidae). Both the prevalence of T. minutus and infection density for all parasites differed between seasons and fragments (the largest fragment consistently differed from other fragments). Host density, distance to the nearest town, fragment size, fragment shape, and total basal area of food trees explained parasite prevalence, but each species had a different pattern. Although parasite richness was lower, prevalence and density were higher than values reported for howlers in conserved forests. These results suggest that the establishment of biological corridors and animal translocation programs must take into account the parasite ecology of each fragment to avoid higher infection rates and preclude potential consequent mortality.

[On the fauna of trematodes from the family Dicrocoeliidae (Plagiorchidida) parasitizing animals in Vietnam].

A review of the fauna of trematodes from the family Dicrocoeliidae parasitizing animals in Vietnam has been conducted. According to original and literature data, 57 trematode species from 15 genera were recorded. Data on hosts, localization, and distribution of all these species in the territory of Vietnam are given.

A new dicrocoeliid from the bank vole Clethrionomys glareolus (Rodentia: Microtidae) from Poland.

A new dicrocoeliid trematode, Brachylecithum glareoli n. sp., is described from the biliary ducts of the bank vole, Clethrionomys glareolus, in southwest Poland. This is the first dicrocoeliid species described in rodents from Poland. It is characterized mainly by the maximum body width at the level of the vitellaria; large, longitudinally oval testes; round, or transversely oval, ovary that is smaller than the testes; vitellaria located in the midbody; cirrus sac dorsally overlapping ventral sucker, but never reaching beyond half of its length; and large, distinctly elongated eggs.