Two avian schistosome cercariae from Nepal,including a Macrobilharzia-like species from Indoplanorbis exustus

As part of a global survey of schistosomes, a total of 16,109 freshwater snails representing 14 species were collected from lakes, ponds, rivers, rice fields and swamps mostly in the Terai region of southern Nepal. Only two snails were found to harbor avian schistosome cercariae even though Nepal is well known for its rich avian diversity. One schistosome infection was from an individual of Radix luteola and on the basis of phylogenetic analyses using 28S rDNA and cox1 sequences, grouped as a distinctive and previously unknown lineage within Trichobilharzia. This genus is the most speciose within the family Schistosomatidae. It includes 40 described species worldwide, and its members mostly infect anseriform birds (ducks) and two families of freshwater snails (Lymnaeidae and Physidae). The second schistosome cercaria was recovered from an individual of Indoplanorbis exustus that was also actively emerging a Petasiger-like echinostome cercaria. Although I. exustus is commonly infected with mammalian schistosomes of the Schistosoma indicum species group on the Indian subcontinent, this is the first specifically documented avian schistosome reported in this snail. Both cercariae reported here are among the largest of all schistosome cercariae recovered to date. The I. exustus-derived schistosome clustered most closely with Macrobilharzia macrobilharzia, although it seems to represent a distinct lineage. Specimens of Macrobilharzia have thus far not been recovered from snails, being known only as adult worms from anhingas and cormorants. This study is the first to characterize by sequence data avian schistosomes recovered from Asian freshwater habitats. This approach can help unravel the complex of cryptic species causing cercarial dermatitis here and elsewhere in the world.     

Comparative life histories of two diplostomid trematodes, Ornithodiplostomum ptychocheilus and Posthodiplostomum minimum

A lack of comparative studies limits our understanding of interspecific variation in parasite life histories, especially for species that incorporate asexual and resting stages into their life cycles. Ornithodiplostomum ptychocheilus and Posthodiplostomum minimum are sympatric diplostomid trematodes that share the same first (pond snail, Physa gyrina) and second (fathead minnow, Pimephales promelas) intermediate hosts within lakes in Alberta, Canada. Interspecific differences in the body sizes of adults, cercariae, and metacercariae were evaluated from parasites recovered from laboratory-raised chickens, snails, and minnows, respectively. Differences in adult worm fecundity and cercariae production were estimated from chickens and snails exposed to known numbers of parasite larvae. Posthodiplostomum minimum adults, metacercariae, and eggs were 97, 86, and 5% larger than those of 0. ptychocheilus, respectively. The average numbers of eggs produced per day by P. minimum was 3 times higher than for O. ptychocheilus, and P. minimum produced approximately twice as many cercariae per day. Although the larger of the 2 species had higher egg and cercariae production, conclusions regarding covariation between adult worm size and the reproductive rates of other life cycle stages require further studies on related species.     

A putative new genus of avian schistosome transmitted by Biomphalaria straminea (Gastropoda: Planorbidae) in Brazil,with a discussion on the potential involvement in human cercarial dermatitis

Human cercarial dermatitis (HCD) caused by avian schistosomes is an emerging health issue in different parts of the world. Nevertheless, parasite diversity, life cycle, and involvement in HCD remain poorly known or neglected in South America. Herein, we reported data obtained during a long-term malacological survey carried out in Pampulha Reservoir, an urban eutrophic waterbody from Brazil between 2009 and 2012. An ocellate brevifurcate cercaria emerged from 55 of 16,235 (0.34%) specimens of Biomphalaria straminea. Samples of the cercariae were subjected to morphological, experimental, and molecular study (analysis of partial sequences of nuclear 28S and mitochondrial cox1 genes). The molecular analysis revealed that the larva corresponds to an avian schistosome; however, it does not correspond to any named genus. A close related isolate was previously reported in Biomphalaria sudanica from Kenya (molecular divergences of 0.54% and 9.62% for 28S and cox1, respectively). The morphology of this cercaria was compared with other avian schistosome larvae from Biomphalaria spp. Attempts to infect experimentally ducks (Cairina moschata) and mice revealed cutaneous manifestations after exposure to cercariae, but adult parasites were not obtained in these hosts. Phylogenetic analysis suggests this parasite is a putative new genus and species of avian schistosome. The potential involvement of the larvae herein described in cases of HCD in Brazil cannot be ruled out. Surprisingly, HCD was not reported in the country so far, which can be related to difficulties in its diagnosis in areas of overlap with human schistosomes.     

Developmental regulation of protein kinase A expression and activity in Schistosoma mansoni

cAMP-dependent protein kinases (PKAs) are the main transducers of cAMP signalling in eukaryotic cells. Recently we reported the identification and characterisation of a PKA catalytic subunit (SmPKA-C) in Schistosoma mansoni that is required for adult schistosome viability in vitro. To gain further insights into the role of SmPKA-C in biological processes during the schistosome life cycle, we undertook a quantitative analysis of SmPKA-C mRNA expression in different life cycle stages. Our data shows that SmPKA-C mRNA expression is developmentally regulated, with the highest levels of expression in cercariae and adult female worms. To evaluate the biological role of SmPKA-C in these developmental stages, cercariae and adult worms were treated with various concentrations of PKA inhibitors. Treatment of cercariae with H-89 or PKI 14-22 amide resulted in loss of viability suggesting that, as in adults, PKA is an essential enzyme activity in this infectious larval stage. In adult worms, in vitro exposure to sub-lethal concentrations of H-89 or PKI 14-22 amide resulted in inhibition of egg production in a dose-dependent manner. Furthermore, using a murine model of schistosome infection where S. mansoni fecundity is impaired, we show that reduced rates of egg production in vivo correlate with significant reductions in SmPKA-C mRNA expression and PKA activity. Finally, restoration of parasite egg production in vivo also resulted in normalisation of SmPKA-C mRNA expression and PKA activity. Taken together, our data suggest that PKA signalling is required for cercarial viability and may play a specific role in the reproductive activity of adult worms.     

Molecular taxonomic position of the elephant schistosome, Bivitellobilharzia nairi, newly discovered in Sri Lanka

Bivitellobilharzia nairi (Mudaliar and Ramanujachar, 1945) Dutt and Srivastava, 1955 was first recorded in India. A number of adult worm specimens of this schistosome species were recovered from a domestic elephant, which died in 1999 in Sri Lanka. This is the first report of this schistosome from Sri Lanka. In the present study, in order to clarify the phylogenetic relationship with other species of schistosomes, sequences from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene from B. nairi were analyzed. Two intraspecific variations were seen within 13 individuals in the ITS2 region. In the CO1 region of the mitochondrial DNA, there were four haplotypes in the nucleotide sequences and two haplotypes in the amino acid sequences. Phylogenetic analysis using the nuclear DNA showed that B. nairi was basal to all of species of the genus Schistosoma. The 28S tree also showed that the mammalian lineage was monophyletic. However, phylogenetic analysis using the mitochondrial DNA showed that B. nairi was nested within the genus Schistosoma. The taxonomical position for this species as well as the contradiction between the results from the nuclear and mitochondrial genes were discussed.     

Schistosomes in the North: A unique finding from a prosobranch snail using molecular tools

Samples of schistosome cercariae from three different snail species (Lymnaea stagnalis, Radix auricularia and Valvata (Tropidina) macrostoma) collected from lakes in Central Finland were analyzed using molecular techniques. Based on sequences of ITS region of rDNA, the parasite isolates from L. stagnalis and R. auricularia belong to Trichobilharzia szidati and T. franki, respectively. This confirms a wide distribution of these two species in Europe. On the other hand, the isolates from V. macrostoma represent a unique finding — they belong to yet unknown schistosome species falling into the bird schistosome clade. Therefore, identification of natural final hosts and morphological characterization of particular developmental stages need to be performed in the future.     

Non-resident definitive host presence is sufficient to sustain avian schistosome populations

To control swimmer’s itch in northern Michigan inland lakes, USA, one species of bird, the common merganser (Mergus merganser), has been relocated from several lakes since 2015. Relocation efforts are driven by a desire to reduce the prevalence of the swimmer’s itch-causing parasite Trichobilharzia stagnicolae. The intention of this state-sponsored control effort was to interrupt the life cycle of T. stagnicolae and reduce parasite egg contribution into the environment from summer resident mergansers such that infections of the intermediate snail host Stagnicola emarginata declined. Reduced snail infection prevalence was expected to substantially reduce the abundance of the swimmer’s itch-causing cercarial stage of the parasite in water. With no official programme in place to assess the success of this relocation effort, we sought to study the effectiveness and impact of the removal of a single definitive host from a location with high definitive host and parasite diversity. This was assessed through a comprehensive, lake-wide monitoring study measuring longitudinal changes in the abundance of three species of avian schistosome cercariae in four inland Michigan lakes. Environmental measurements were also taken at these lakes to understand how they can affect swimmer’s itch incidence. This study demonstrates that the diversity of avian schistosomes at the study lakes would likely make targeting a single species of swimmer’s itch-causing parasite meaningless from a swimmer’s itch control perspective. Our data also suggest that removing the common merganser is not an effective control strategy for the T. stagnicolae parasite, likely due to contributions of the parasite made by non-resident birds, possibly migrants, in the autumn and spring. It appears likely that only minimal contact time between the definitive host and the lake ecosystem is required to contribute sufficient parasite numbers to maintain a thriving population of parasite species with high host specificity.     

The life cycle of Paraquimperia tenerrima: a parasite of the European eel Anguilla anguilla

Previous studies on the life history of the nematode eel specialist Paraquimperia tenerrima (Nematoda: Quimperiidae) have failed to determine whether an intermediate host is required in the life cycle. In the laboratory, eggs failed to hatch below 10 degrees C, hatching occurring only at temperatures between 11 and 30 degrees C. Survival of the free-living second stage larvae (L2) was also temperature dependent, with maximal survival between 10 and 20 degrees C. Total survival of the free-living stages (eggs and L2) is unlikely to exceed a month at normal summer water temperatures, confirming that parasite could not survive the 6 month gap between shedding of eggs in spring and infection of eels in early winter outside of a host. Eels could not be infected directly with L2, nor could a range of common freshwater invertebrate species. Third stage larvae (L3) resembling P. tenerrima were found frequently and abundantly in the swimbladder of minnows Phoxinus phoxinus from several localities throughout the year and were able to survive in this host in the laboratory for at least 6 months. Third stage larvae identical to these larvae were recovered from minnows experimentally fed L2 of P. tenerrima, and eels infected experimentally with naturally and experimentally infected minnows were found to harbour fourth stage larvae (L4) and juvenile P. tenerrima in their intestines. Finally, the whole life cycle from eggs to adult was completed in the laboratory, confirming that minnows are an obligate intermediate host for P. tenerrima.     

Testing parasite ‘intimacy’: the whipworm Trichuris muris in the European house mouse hybrid zone

Host‐parasite interaction studies across hybrid zones often focus on host genetic variation, treating parasites as homogeneous. ‘Intimately’ associated hosts and parasites might be expected to show similar patterns of genetic structure. In the literature, factors such as no intermediate host and no free‐living stage have been proposed as ‘intimacy’ factors likely constraining parasites to closely follow the evolutionary history of their hosts. To test whether the whipworm, Trichuris muris, is intimately associated with its house mouse host, we studied its population genetics across the European house mouse hybrid zone (HMHZ) which has a strong central barrier to gene flow between mouse taxa. T. muris has a direct life cycle and nonmobile free stage: if these traits constrain the parasite to an intimate association with its host we expect a geographic break in the parasite genetic structure across the HMHZ. We genotyped 205 worms from 56 localities across the HMHZ and additionally T. muris collected from sympatric woodmice (Apodemus spp.) and allopatric murine species, using mt‐COX1, ITS1‐5.8S‐ITS2 rDNA and 10 microsatellites. We show four haplogroups of mt‐COX1 and three clear ITS1‐5.8S‐ITS2 clades in the HMHZ suggesting a complex demographic/phylogeographic history. Microsatellites show strong structure between groups of localities. However, no marker type shows a break across the HMHZ. Whipworms from Apodemus in the HMHZ cluster, and share mitochondrial haplotypes, with those from house mice. We conclude Trichuris should not be regarded as an ‘intimate’ parasite of the house mouse: while its life history might suggest intimacy, passage through alternate hosts is sufficiently common to erase signal of genetic structure associated with any particular host taxon.     

New intermediate host records for the avian schistosomes Dendritobilharzia pulverulenta, Gigantobilharzia huronensis, and Trichobilharzia querquedulae from North America

Here we provide the first North American report of a naturally infected snail, Gyraulus parvus, harboring the larval stages of the cosmopolitan, arterial schistosome, Dendritobilharzia pulverulenta. The relatively small cercariae of this species are shed in the early morning, are sticky, and adhere to the water's surface film. We also provide a report of the snail host, Physa gyrina, of the widespread North American passerine schistosome, Gigantobilharzia huronensis. Finally, we provide unambiguous documentation that Physa gyrina is a natural snail host for Trichobilharzia querquedulae, a schistosome primarily of dabbling ducks.     

Identification and characterization of a UDP-GalNAc:GlcNAc{eta}-R {eta}->4-N-acetylgalactosaminyltransferase from cercariae of the schistosome Trichobilharzia ocellata. Catalysis of a key step in the synthesis of N,N'-diacetyllactosediamino (lacdiNAc)-type glycans

Three different stages of the avian schistosome Trichobilharziaocellata appeared to contain a novel N-acetylgalac-tosaminyltransferaseactivity. To investigate its function in the biosynthesis ofschistosome glycoconjugates, the enzyme was partially purifiedfrom cercariae, a free-living stage of the parasite, by affinitychromatography on UDP-Sepharose. Acceptor specificity studiesshowed that the enzyme catalyses the transfer of N-acetylgalactosamine(GalNAc) from UDP-GalNAc to oligosaccharides, glyco-peptidesand glycoproteins carrying a terminally     

A new acanthocolpid cercaria (Digenea) from Cantharus dorbignyi (Prosobranchia) in the Western Mediterranean

A new cercaria is recorded from the prosobranch snail Cantharus dorbignyi from the sublittoral region off the coast of Corsica. The redia, cercaria and metacercaria (the latter experimentally developed in the labrid fishes Symphodus ocellatus and S. rostratus) are described, along with the behaviour of naturally emitted cercariae. In view of the morphology of the larval stages, the parasite is thought likely to be an acanthocolpid. The morphology of the cercaria and metacercaria are compared with both known and suspected acanthocolpid cercariae and with adult acanthocolpids known to occur in the Mediterranean. Morphological and circumstantial evidence suggests that this cercaria may be a species of the genus Tormopsolus.     

Molecular identification of developmental stages in Opecoelidae (Digenea)

Nuclear ribosomal DNA sequences represent a useful tool for distinction of poorly differentiated developmental stages, such as trematode cercariae or metacercariae. Here, the complete internal transcribed spacer region of the ribosomal DNA (ITS 1 + 5.8S + ITS 2) was sequenced for 29 specimens of the digenean family Opecoelidae, including 16 adult specimens and 13 undescribed larval stages (nine cercariae and four metacercariae) occurring in various marine host organisms. Six cercariae and three metacercariae were found to match their corresponding adult form. This work also revealed that cercariae of the same species are able to infect more than one gastropod host species, suggesting that the specificity for the first intermediate host within the Digenea may be lower than previously thought.     

The life cycle of Cardicola forsteri (Trematoda: Aporocotylidae), a pathogen of ranched southern bluefin tuna, Thunnus maccoyi

Aporocotylids (fish blood flukes) are emerging as pathogens of fishes in both marine and freshwater aquaculture. Efforts to control these parasites are hampered by a lack of life cycle information. Here we report on the life cycle of Cardicola forsteri, which is considered a significant pathogen in southern bluefin tuna, Thunnus maccoyi, ranched in South Australia. We surveyed polychaetes, bivalves and gastropods from sites close to tuna pontoons. Infections consistent with the Aporocotylidae were found in terebellid polychaetes, a single Longicarpus modestus and five individuals of Reterebella aloba. All infections were comprised of hundreds of sporocysts in the body cavity of the host, each filled with developing and mature cercariae. Sequences of ITS-2 and lsrDNA from the infection from L. modestus were a perfect match with those of adult C. forsteri from T. maccoyi. This life cycle link is considered confirmed but it is possible that additional terebellid species are infected in South Australia; equally, other species of intermediate host are likely to be involved in other parts of the range of this cosmopolitan trematode. Sequences of the species from R. aloba did not match a known adult but phylogenetic analysis of lsrDNA suggests that it is also a species of Cardicola Short, 1953. These findings show that terebellid polychaetes are a major host group for marine aporocotylids, especially given that Cardicola is the largest marine aporocotylid genus. The two cercarial types are among the smallest known for the family and are unusual, but not unique, in having short, simple tails. We speculate that the form of the tail means that these cercariae are not active swimmers and are thus heavily dependent on currents for dispersal. Control of this parasite might be effected by moving the tuna pontoons appropriate distances to avoid encounter with current-dispersed cercariae, or by increasing the separation of the nets from the sea floor, either by raising the nets or moving to deeper water.     

Schistosoma mansoni tropomyosin: cDNA characterization, sequence, expression, and gene product localization

We have defined the polypeptide pattern of 3-hr Schistosoma mansoni schistosomula on nonequilibrium two-dimensional gels (NEPHGE). An acidic group of polypeptides with a molecular weight of about 40 kDa and a pI value of around 5.0 (numbered 48/59/53) were identified as antigens on Western blots probed with chronic human infection sera or vaccinated mouse sera. Polypeptides 48/49/53 from silver-stained NEPHGE gels produced antisera that were specific as demonstrated by Western blot analysis and immunoprecipitations of in vitro translation products. A cDNA clone (clone 1) from a S. mansoni adult worm pBR322 library was isolated by using cDNA probes made from size-fractionated mRNA and defined as encoding polypeptide 49 by hybridization selection of the mRNA which was in vitro translated and immunoprecipitated with specific mouse antiserum. A lambda gt 11 expression clone which contained an insert close to the full length mRNA was isolated from a S. mansoni cercariae library. The complete sequence of the mRNA was determined by sequencing the insert of this clone as well as primer extension of total RNA. The only open reading frame coding for 284 amino acids in the 1316 nucleotide sequence showed a 44.76 to 55.44% homology with the amino acid sequences of 18 different tropomyosins from various species. Computer-predicted secondary structure of schistosome tropomyosin was mainly alpha-helix which was very similar to other tropomyosins. Northern analysis showed the mRNA to be about 1.5 kb in size and detectable at much higher levels in the adult worm stage as compared to the cercariae and the egg stages. Western blot analysis likewise showed that greater amounts of tropomyosin were detected in extracts from adult worm stage as compared to extracts from cercariae and egg stages. Immunocytochemical analysis shows that tropomyosin is strongly associated with the tegument of adult worms. The restriction digestion pattern given by genomic Southern analysis suggests the existence of introns and/or multiple gene copies. Thus polypeptide 49, an immunodominant antigen, represents schistosome tropomyosin.