Echinostoma chankensis nom. nov., other Echinostoma spp. and Isthmiophora hortensis in East Asia: morphology, molecular data and phylogeny within Echinostomatidae.

Life cycles, and morphological and molecular data were obtained for Echinostoma chankensis nom. nov., Echinostoma cinetorchis, Echinostoma miyagawai and Isthmiophora hortensis from East Asia. It was established that, based on both life cycle and morphology data, one of the trematodes is identical to the worms designated as Euparyphium amurensis. Genetic data showed that this trematode belongs to Echinostoma. The complex data on biological, morphological and genetic characterizations establish that the distribution of the morphologically similar species, I. hortensis and Isthmiophora melis, in the Old World are limited by the East Asian and European regions, respectively. Data on mature worms of East Asian E. miyagawai revealed morphological and genetic identity with E. miyagawai from Europe. However, E. miyagawai from Europe differs from E. miyagawai from the type locality (East Asia) in terms of reaching maturity and the morphology of cercariae. These data indicate that the European worm, designated E. miyagawai, does not belong to this species. An analysis of the phylogenetic relationships of Echinostomatidae was conducted based on the 28S, ITS2 and nad1 markers. Analysis using the nad1 gene for the known representatives of Echinostomatidae is carried out for the first time, showing that nuclear markers are ineffective separate from mitochondrial ones.

Cercarial trematodes in freshwater snails from Bangkok, Thailand: prevalence, morphological and molecular studies and human parasite perspective.

We investigated the prevalence, morphological characters and molecular classifications of trematode cercariae in freshwater snails randomly collected from 59 sampling localities in Bangkok from May 2018 to March 2019. We used a crushing technique to observe the cercarial stage inside each snail body and amplified the internal transcribed spacer 2 regions of cercarial DNA using polymerase chain reaction methodology. The associated phylogenetic tree was reconstructed using Bayesian inference analyses. A total of 517 of 15 621 examined snails were infected with trematode cercariae, and the infected snails were classified into 11 species of seven families with a 3.31% overall prevalence of the infection. The Bithynia siamensis siamensis snail displayed the highest prevalence of infection (16.16%), whereas the Physella acuta snail exhibited the lowest prevalence (0.08%) of infection. Eight morphological types of cercariae were observed. The highest prevalence of infection was observed in mutabile cercaria (1.86%). Based on molecular investigations, the phylogram revealed eight cercaria types assigned to at least nine digenean trematode families, of which five belong to groups of human intestinal flukes. Although, with the exception of schistosome cercaria, trematode cercariae are not known to directly damage humans, understanding the general biology of trematode cercariae (including diversity, distribution, infection rates and host range) is important and necessary for the prevention and control of parasitic transmission that impacts aquatic cultivations, livestock farming and human health.

Parvatrema duboisi (Digenea: Gymnophallidae) Life Cycle Stages in Manila Clams, Ruditapes philippinarum, from Aphae-do (Island), Shinan-gun, Korea.

Life cycle stages, including daughter sporocysts, cercariae, and metacercariae, of Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974 (Digenea: Gymnophallidae) have been found in the Manila clam Ruditapes philippinarum from Aphaedo (Island), Shinan-gun, Jeollanam-do, Korea. The daughter sporocysts were elongated sac-like and 307-570 (av. 395) µm long and 101-213 (av. 157) µm wide. Most of the daughter sporocysts contained 15-20 furcocercous cercariae each. The cercariae measured 112-146 (av. 134) µm in total length and 35-46 (av. 40) µm in width, with 69-92 (av. 85) µm long body and 39-54 (av. 49) µm long tail. The metacercariae were 210-250 (av. 231) µm in length and 170-195 (av. 185) µm in width, and characterized by having a large oral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, strongly suggesting P. duboisi. The metacercariae were experimentally infected to ICR mice, and adults were recovered at day 7 post-infection. The adult flukes were morphologically similar to the metacercariae except in the presence of up to 20 eggs in the uterus. The daughter sporocysts and metacercariae were molecularly (ITS1-5.8S rDNA-ITS2) analyzed to confirm the species, and the results showed 99.8-99.9% identity with P. duboisi reported from Kyushu, Japan and Gochang, Korea. These results confirmed the presence of various life cycle stages of P. duboisi in the Manila clam, R. philippinarum, playing the role of the first as well as the second intermediate host, on Aphae-do (Island), Shinan-gun, Korea.

New data on the nervous system of Cercaria parvicaudata Stunkard & Shaw, 1931 (Trematoda: Renicolidae): revisiting old hypotheses.

Data on the interposition of the immunoreactive nerve cords in Cercaria parvicaudata Stunkard & Shaw, 1931 (Trematoda: Renicolidae) and its chaetotaxy were obtained. The nervous system of C. parvicaudata was described using immunostaining of 5-hydroxytryptamine and FMRFamide immunoreactive nerve elements. The morphology and distribution of sensory receptors were analysed using scanning electron microscopy and the silver nitrate impregnation technique. Our integrated approach to the study of the nervous system revealed a clear colocalization of surface papillae with nerve cords and commissures in C. parvicaudata. The structure of the nervous system in C. parvicaudata differs partly from the classical model that defines the entire nomenclature of chaetotaxy.

Pronocephaloid cercariae (Platyhelminthes: Trematoda) from gastropods of the Queensland coast, Australia.

The superfamily Pronocephaloidea Looss, 1899 comprises digeneans occurring in the gut and respiratory organs of fishes, turtles, marine iguanas, birds and mammals. Although many life cycles are known for species of the Notocotylidae Lühe, 1909 maturing in birds and mammals, relatively few are known for the remaining pronocephaloid lineages. We report the cercariae of five pronocephaloid species from marine gastropods of the Queensland coast, Australia. From Lizard Island, northern Great Barrier Reef, we report three cercariae, two from Rhinoclavis vertagus (Cerithiidae) and one from Nassarius coronatus (Nassariidae). From Moreton Bay, southern Queensland, an additional two cercariae are reported from two genotypes of the gastropod worm shell Thylacodes sp. (Vermetidae). Phylogenetic analysis using 28S rRNA gene sequences shows all five species are nested within the Pronocephaloidea, but not matching or particularly close to any previously sequenced taxon. In combination, phylogenetic and ecological evidence suggests that most of these species will prove to be pronocephalids parasitic in marine turtles. The Vermetidae is a new host family for the Pronocephaloidea.

Molecular and morphological characterization of the cercariae of Lecithodendrium linstowi (Dollfus, 1931), a trematode of bats, and incrimination of the first intermediate snail host, Radix balthica.

Lecithodendrium linstowi is one of the most prevalent and abundant trematodes of bats, but the larval stages and intermediate hosts have not been identified. We present the first molecular and morphological characterization of the cercariae of L. linstowi based on a phylogenetic analysis of partial fragments of LSU and ITS2 rDNA. The first intermediate host was incriminated as Radix balthica by DNA barcoding using cox1 and ITS2 sequences, although the snail morphologically resembled Radix peregra, emphasizing the requirement for molecular identification of lymnaeids as important intermediate hosts of medical and veterinary impact. The application of molecular data in this study has enabled linkage of life cycle stages and accurate incrimination of the first intermediate host.

Life cycle and biology of Tristriata anatis (Digenea: Notocotylidae): morphological and molecular approaches.

Notocotylids are common digeneans parasitising birds and mammals. They have a two-host life cycle with cercariae encysting in the open. Particular life cycles remain unknown for majority of notocotylid species, including a common parasite of sea ducks Tristriata anatis. Here we resolve the life cycle of T. anatis by means of D2 LSU, ITS1 and CO1 DNA sequence analysis, showing that the first intermediate hosts for this species are periwinkles Littorina spp. Morphological descriptions of rediae and cercariae are provided for the first time, and we also supplement the existing morphological data on adults. Apart from differential diagnosis, we discuss some features of cercariae and rediae biology, geographical distribution and host range. Our molecular data confirm that genus Tristriata is monotypic and that T. anatis has circumpolar distribution. CO1 sequence analysis has shown that isolation exists between the Atlantic and Pacific populations of T. anatis, suggesting that there are geographical races. We suppose that their formation may be linked to the Last Ice Age events, when trans-Arctic bird migrations ceased and periwinkle ranges shrunk. These made transfer of parasites across the Arctic impossible, and it still has not resumed. We discuss the possible influence of host vagility and adults' lifespan on digeneans' potential for geographical colonisation.

Trematode infections of the freshwater snail Biomphalaria pfeifferi from a south-east Nigerian community with emphasis on cercariae of Schistosoma.

Knowledge of trematode infections in Biomphalaria pfeifferi from south-eastern Nigeria is scarce, due to the absence of Schistosoma mansoni infection in the region. Therefore, the present study sought to describe trematode infections in B. pfeifferi from the River Uzuru in the Nigeria Cement Factory area, Nkalagu, south-eastern Nigeria. Four hundred and sixty snails were checked for trematode infections, and mice were exposed to the Schistosoma cercariae shed from the snails. Adult worms were harvested from the mice 13 weeks post-infection, while sections of the liver and spleen were examined. Primary school children living in the area were screened for S. mansoni infection and assessed for activities involving water contact. The edges of the river were also searched for burrows and rodents. The five cercaria morphotypes found were armatae xiphidiocercariae, echinocercariae, Schistosoma cercariae, cystophorous cercariae and cercariaeum cercariae. The overall prevalence and mean intensity of trematode infections were 39.78% and 195.46, with the prevalence and mean intensity of most cercaria morphotypes higher in the hot-dry than in the cool-dry season. The infected mice showed S. mansoni-like characteristics but the stool samples of the schoolchildren were negative for S. mansoni eggs. Water-contact activities in the River Uzuru were minimal. Burrows were seen at the river edges but no Schistosoma eggs were recovered from captured rats. This is the first report of Schistosoma cercariae and other cercaria morphotypes in B. pfeifferi from south-eastern Nigeria. Additional molecular investigations are needed to identify correctly these Schistosoma cercariae, due to their public health implication.

[Description of virgulate Cercaria etgesh larva Nov. (Xiphidiocercariae): A new type of virgule organ].

A new species of virgulate cercaria is described. The morphology and chaetotaxy of cercariae are studied. The new type of virgule organ is described. Every description is illustrated by drawings.

The first record of Centrocestus formosanus (Nishigori, 1924) (Digenea: Heterophyidae) in Egypt.

The life cycle of Centrocestus formosanus (Digenea: Heterophyidae) was to be successfully completed in the laboratory in the present study. Hundreds of the thiarid snail, Melanoides tuberculata, were collected from the main water course Mansouriya Canal, Giza Governorate, Egypt. The snails were individually exposed to artificial light to determine possible infection with trematode larvae. Fifteen snails were found infected with opthalmopleurolophocercous cercariae (infection index of 1.97). These opthalmopleurolophocercous cercariae shedded from snails were collected and placed in an aquarium with fish intermediate host, Gambusia affinis, to obtain metacercariae encysted in the gills. The gills with metacercariae were fed to albino rats, Rattus norvegicus, to obtain the adult worms. Adult worms were recovered in the small intestine of rats at 7 days after infection and they were identified as Centrocestus formosanus based on the morphological characteristics and the comparison with the previous descriptions in the literature. They were small, 518 × 324 µm in average size and had characteristic 32 circumoral spines around the oral sucker. The morphological characteristics of the developmental stages, from cercariae to adults, of this heterophyid fluke were given here. Therefore, the presence of this fluke is to be confirmed for the first time in Egypt by the present study.

Biomphalaria straminea (Mollusca: Planorbidae) as an intermediate host of Drepanocephalus spp. (Trematoda: Echinostomatidae) in Brazil: a morphological and molecular study.

Species of trematodes belonging to the genus Drepanocephalus are intestinal parasites of piscivorous birds, primarily cormorants (Phalachrocorax spp.), and are widely reported in the Americas. During a 4-year malacological study conducted on an urban lake in Brazil, 27-collar-spined echinostome cercariae were found in 1665/15,459 (10.7 %) specimens of Biomphalaria straminea collected. The cercariae were identified as Drepanocephalus spp. by sequencing the 18S (SSU) rDNA, ITS1/5.8S rDNA/ITS2 (ITS), 28S (LSU) rDNA region, cytochrome oxidase subunit 1 (CO1), and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) markers. In experimental life cycle studies, metacercariae developed in laboratory-reared guppies (Poecilia reticulata); however, attempts to infect birds and rodents were unsuccessful. Two closely related morphotypes of cercariae were characterized. One species, identified by molecular markers as a genetic variant of Drepanocephalus auritus (99.9 % similarity at SSU, ITS, LSU; 97.2 % at CO1; 95.8 % at ND1), differs slightly from an archived North American isolate of this species also sequenced as part of this study. A second species, putatively identified as Drepanocephalus sp., has smaller cercariae and demonstrates significant differences from D. auritus at the CO1 (11.0 %) and ND1 (13.6 %) markers. Aspects related to the morphological taxonomic identification of 27-collar-spined echinostome metacercariae are briefly discussed. This is the first report of the involvement of molluscs of the genus Biomphalaria in the transmission of Drepanocephalus and the first report of D. auritus in South America.

MORPHOLOGY OF TWO NEW SPECIES OF CERCARIAE VIRGULAE (LÜHE, 1909) GROUP.

Two new species of virgulous cercariae are described. Morphological descriptions and differential diagnoses are given. Each description is supplemented with a detailed picture.

Muscle system of Diplodiscus subclavatus (Trematoda: Paramphistomida) cercariae, pre-ovigerous, and ovigerous adults.

The musculature of cercariae, pre-ovigerous, and ovigerous adults of Diplodiscus subclavatus was studied by means of TRITC-conjugated phalloidin staining of filamentous actin and confocal scanning laser microscopy. The body wall appears to include four muscle layers as follows: circular, outer longitudinal, diagonal, and inner longitudinal. Two layers of longitudinal muscle fibers are arranged in different modes due to the secondary transformed paramphistomid body construction. The organization of the acetabulum turned out to be more complex than ever described, with a radial layer, two layers of circular, two layers of meridional, an additional starry layer of muscle fibers, as well as a few separate muscle layers of the accessory sucker. Within the pharynx, I found a group of alar muscle fibers, never described before for any paramphistomids, and some morphological features which were not considered to be characteristic for D. subclavatus (namely--the middle semicircular layer and the transverse muscle fibers in the pre-sphincteric space). No significant reorganizations of the somatic musculature occur throughout the development from the cercaria to the ovigerous adult worm, so the metamorphosis goes in the manner of completion. The cercarial tail includes a layer of circular muscle fibers and a longitudinal muscle layer beneath. The latter consists of two medial longitudinal bundles of smooth muscle fibers and two lateral longitudinal bands of obliquely striated muscle fibers which are partially divided in halves.

Gigantobilharzia melanoidis n.sp. (Trematoda: Schistosomatidae) from Melanoides tuberculata (Gastropoda: Thiaridae) in the United Arab Emirates.

Delicate filamentous schistosomatids detected in the intestinal veins of experimentally infected chickens are here described as a new parasite species, Gigantobilharzia melanoidis, and details of its life cycle are given. It is the first complete description of a schistosome species that uses Melanoides tuberculata as an intermediate host. Apharyngeate ocellate brevifurcate cercariae found in 65 out of 950 M. tuberculata collected in a pond in Al Aweer, United Arab Emirates were used as infection material. The new species can be distinguished from the other species of the genus by the following combination of characters: caecal reunion in males situated anterior to seminal vesicle, a very short gynecophoric canal (gynecophoric canal length/body length ratio lower than 0.05) supported by 12-14 thickened bands. Cercariae of G. melanoidis can be distinguished from other Gigantobilharzia cercariae described in the literature based on the combination of these characters: flame cell formula 2[3 + 3 + (1)] = 14 and relatively longer tail stem in relation to body (tail stem length/body length ratio = 2). Under laboratory conditions at a temperature between 24 and 26 °C, M. tuberculata started to shed cercariae 7 weeks after exposure to miracidia. The prepatent period of G. melanoidis in experimentally infected chicken lasted between 43 and 49 days. The parasite inhabits the blood vessels mainly of the small intestine. Sections of adult worms and eggs were also found in histocuts of parenchymatous organs. Results of phylogenetic analysis corroborated that G. melanoidis is a distinct species; however, they also confirmed that the genus Gigantobilharzia is in need of revision and in future might be split into several genera.

Two new species of Maritrema Nicoll, 1907 (Digenea: Microphallidae) from New Zealand: morphological and molecular characterisation.

Two new species of the microphallid genus Maritrema (Maritrema) Nicoll, 1907 are described from freshwaters in the South Island of New Zealand. Maritrema deblocki n. sp. occurs as an adult in the mallard Anas platyrhynchos (L.); Maritrema poulini n. sp. is found as sporocysts/cercariae in Potamopyrgus antipodarum (Gray) and as metacercariae in two species of amphipod and two species of isopod. We use morphological and molecular characterisation to distinguish between the two species, and compare them to their four morphologically closest congeners. M. deblocki n. sp. and M. poulini n. sp. are distinguished from each other by the relative sucker size, the positions of the genital pore and ovary, the convergence of the vitelline ribbons, and overall size. With the aid of molecular data, we matched life cycle stages of M. poulini n. sp. and assessed its use of multiple second intermediate hosts. Phylogenetic analyses included sequences for the two new species and the available microphallid sequences for the large ribosomal subunit and the internal transcribed spacer 1 of the ribosomal RNA gene. Closer to each other than to any other species, the sister species together with Maritrema novaezealandense Martorelli, Fredensborg, Mouritsen & Poulin, 2004, Maritrema heardi (Kinsella & Deblock, 1994), Maritrema eroliae Yamaguti, 1939 and Maritrema oocysta (Lebour, 1907) formed a well-supported clade. In addition, we clarify the taxonomic identity of several unidentified Microphallus spp. in the recent ecological literature from New Zealand and propose corrected spellings for a number of Maritrema species epithets.

A review of the currently recognised zoogonid cercariae, including the identification and emergence ecology of Cercaria capricornia XI (Digenea: Zoogonidae) from Nassarius olivaceus (Gastropoda: Nassariidae) in Central Queensland, Australia.

In the present study, we review the known zoogonid cercariae, summarise their life-cycles and first intermediate host distributions, and present a new cercaria, Cercaria capricornia XI (Digenea: Zoogonidae), which was found in one of three nassariid gastropods, Nassarius olivaceus (Bruguière), surveyed in the intertidal zone in the Capricornia region of Central Queensland, Australia. Morphological data and molecular analysis of the ITS2 rDNA region support placement of this cercaria in the family Zoogonidae but do not allow any further resolution of its identity. There are now fifteen cercariae described as belonging to the Zoogonidae; thirteen of these, including the present species, infect neogastropods as first intermediate hosts and two use vetigastropods. This study reinforces the pattern that the Nassariidae is by far the most commonly reported family for the Zoogonidae. Given its richness we predict that the Nassariidae will prove to harbour many more zoogonid species.

Diplostomum von Nordmann, 1832 (Digenea: Diplostomidae) in the sub-Arctic: descriptions of the larval stages of six species discovered recently in Iceland.

Frequent infections with Diplostomum spp. (Digenea: Diplostomidae) were found in the freshwater snail Radix peregra (Müller) and three fish species, the salmonids Salmo trutta fario L., Salvelinus alpinus (L.) and the gasterosteid Gasterosteus aculeatus L., collected in four lakes in south-western Iceland in 2012. Detailed analysis of the isolates integrating molecular, morphological and ecological data revealed that these belong to Diplostomum spathaceum (Rudolphi, 1819) and five putative new species (three infecting both snails and fish). This paper provides detailed descriptions of the metacercariae of the six species-level lineages of Diplostomum spp. and of the cercariae of three of the lineages discovered in Iceland with comments on the application of ITS1 rDNA for species distinction within Diplostomum von Nordmann, 1832 in the light of the novel data. We strongly suggest the use of molecular evidence based on cox1 gene sequences (in addition to ITS1-5.8S-ITS2 sequences) in association with detailed assessment of the morphology of the larval stages in future studies of Diplostomum spp. in fish and snails.

The sexual adult of Cercaria praecox Walker, 1971 (Digenea: Fellodistomidae), with the proposal of Oceroma n. g.

A sexual adult trematode that is considered to be conspecific with the distinctive larval trematode Cercaria praecox Walker, 1971 is reported from the kyphosid fish Scorpis lineolata Kner in Moreton Bay, Queensland, Australia. The sexual adult is consistent with the cercarial body of Cercaria praecox in having a single caecum with an asymmetrical appendix, symmetrical testes immediately posterior to the ventral sucker, and the ovary and vitellarium both well posterior to the testes. This combination of characters is distinct within the Fellodistomidae Nicoll, 1909 and requires the proposal of a new genus, Oceroma n. g. Analysis of 28S rDNA sequences demonstrates that this species forms a clade with Coomera Dove & Cribb, 1995 within the Fellodistomidae. The life-cycle of the species is predicted to require two hosts and to involve the direct ingestion of the cercaria.

[On the validity of the genus Otidilepis Yamaguti, 1959 (Cestoda: Hymenolepididae) and the classification of the rostellar hooks of its type species, O. tetraciS (Cholodkowsky, 1906)].

The type material on Otidilepis tetracis (Cholodkowsky, 1906), the type species of the genus Otidilepis Yamaguti, 1959 is reinvestigated. The validity of the genus Otidilepis is confirmed. It is clearly distinguished from the genus Hispaniolepis Lopez Neyra, 1942 by the form and dimensions of the rostellar hooks as well as by the proportions of the attachment organs of the scolex (diameter of suckers: width of the rostellum). "Orthodiorchoid" type based on the hooks of D. acuminata (Clerc, 1902), type species of the genus Diorchis Clerc, 1903, is introduced. The former "diorchoid" type, based on the hooks of D. americana Ransom, 1909, is renamed in "americanoid". Rostellar hooks of O. tetracis belong to the americanoid type.

Idiopyrgus souleyetianus (Gastropoda: Tomichiidae) as an intermediate host of the liver fluke Amphimerus sp. (Trematoda: Opisthorchiidae) in Brazil.

Despite the importance of fish-borne trematodes of the family Opisthorchiidae as causative agents of human liver fluke disease, studies on these parasites outside Asia are relativally scarce. In South America, human focus of amphimerosis is known in Ecuador since the mid-20th century, and Amphimerus spp. have also been reported in wild and domestic mammals. Nevertheless, the knowledge on the snails that act as the first intermediate host of these potentially zoonotic parasites are scarce. Herein, a new cercaria of the pleurolophocercous morphotype found in the freshwater snail Idiopyrgus souleyetianus from Brazil was subjected to morphological and molecular studies. Multigene phylogenetic analyses based on 28S, 5.8S-ITS-2 and Cox-1 sequences enabled the identification of Amphimerus sp., a species distinct from that reported in humans from Ecuador. This cercariae was morphologically compared with other opisthorchiid cercariae known. The possible occurrence of human amphimerosis in Brazil is discussed.