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Rhopalocercous Cercaria duplicata von Baer, 1827 develops in an intermediate host, the unionid bivalve Anodonta anatina (L.), but its adult form has been unknown. We examined eight fish species occurring in the presence of a highly infested population of A. anatina in the Zesławice reservoir (S Poland). Gravid Phyllodistomum specimens were obtained from the ureters of ide, Leuciscus idus (L.) and common rudd, Scardinius erythrophthalmus (L.). One of the rudd specimens was doubly infected, a trematode was also found in the urinary bladder. In addition, a gravid Phyllodistomum specimen was found in the ureter of a tench Tinca tinca (L.), caught in Lake Ilmėdas (Lithuania). In order to clarify the phylogenetic position of larval and adult gorgoderids and to establish their life cycle, ITS2 and 28S rDNA sequences were analysed. The analysis showed that adult Phyllodistomum specimens located in the ureters are conspecific with C. duplicata. The trematode found in the urinary bladder of S. erythrophthalmus was P. folium (Olfers, 1816). It is suggested that adult stages of C. duplicata should be referred to as Phyllodistomum duplicatum n. comb. The intercaecal position of the uterus and the deeply-lobed ovary are the main features distinguishing it from other Phyllodistomum species. Host specificity and ecology are discussed., (© 2024. The Author(s).)

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., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lymnaeid snails are some of the most widespread snails and are the first intermediate host of trematode parasites that affect human and livestock health. A full understanding of the genetic relationship of hosts and parasites is of paramount importance for effective parasite management. The present study assessed the prevalence of trematode larvae in lymnaeid snails and examined the genetic diversity of these snails collected across Thailand. We collected 672 lymnaeid snails from 39 locations in 22 provinces of six regions in Thailand. Subsequently, cercarial infection in the snails was observed by using the shedding method. Lymnaeid snails released 5 types of trematode cercariae, namely, xiphidiocercariae, echinostome cercariae I, echinostome cercariae II, furcocercous cercariae, and strigea cercariae. The phylogenetic analysis based on ITS2 and 28S rDNA sequences revealed 5 cercaria types assigned to four trematode families, of which two belong to the group of human intestinal flukes. Combination of shell morphology and sequence analysis of the mitochondrial COI and 16S rDNA genes, the lymnaeid snails were classified into two species, Radix rubiginosa and Orientogalba viridis. Moreover, the combined dataset of mtDNA genes (COI + 16S rDNA) from R. rubiginosa and O. viridis revealed 32 and 15 different haplotypes, respectively, of which only a few haplotypes were infected with cercariae. The genetic diversity and genetic structure revealed that R. rubiginosa and O. viridis experienced a bottleneck phenomenon, and showed limited gene flow between populations. Population demographic history analyses revealed that R. rubiginosa and O. viridis experienced population reductions followed by recent population expansion. These findings may improve our understanding of parasite-lymnaeid evolutionary relationships, as well as the underlying molecular genetic basis, which is information that can be used for further effective control of the spread of trematode disease., Competing Interests: Declaration of competing interest The authors declare there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This study aimed to describe the morphological and molecular characteristics of Paralecithodendrium longiforme (Digenea: Lecithodendriidae) adults and cercariae isolated in Thailand. Adult flukes were isolated from the Chinese pipistrelle bat (Hypsugo sp.), and cercariae were detected in the viviparid snail (Filopaludina martensi martensi) from Chiang Mai province. The morphological characteristics were observed and described using conventional methods, and the molecular characteristics with internal transcribed spacer 2 (ITS2) and 28S rDNA gene sequences. The adult flukes were fusiform, 0.84-0.98 mm in length, and 0.37-0.49 mm in width, and were distinguishable from other species by the presence of longitudinal uterine coils. The cercariae were nonvirgulate xiphidiocercariae, with the oral sucker bigger than the acetabulum, the tail without fin fold, a body size of 117.5-138.3 × 48.3-52.2 µm, and a tail size of 100.7-103.7 × 15.0-18.9 µm. Molecular studies revealed that the adults and cercariae shared 99.3% (ITS2) and 99.6% (28S rDNA) homology with each other. They were phylogenetically close to P. longiforme with an identity of 94.5% for ITS2 and 98.7% for 28S rDNA. This study provides new information on the natural definitive host and first intermediate host of P. longiforme in Thailand. The discovery of its cercarial stage in Filopaludina snails highlights the importance of monitoring the associated second intermediate host and prevention and control of this potentially zoonotic trematode.

Indoplanorbis exustus is a freshwater gastropod belonging to the family Planorbidae. This snail is widely distributed across the tropics and plays an important role as the intermediate host for trematodes. However, relatively little is understood regarding the genetic relationship between I. exustus and trematodes. The goals of this study were to investigate the current transmission status of trematode cercariae in I. exustus in Thailand and to examine the genetic diversity, genetic structure, and demographic history of I. exustus. We collected 575 I. exustus from 21 provinces across six regions of Thailand and investigated cercarial infections by using the shedding method. I. exustus from two provinces were infected with cercarial trematodes, and two types of cercarial stages were molecularly identified as furcocercous cercaria and xiphidiocercariae. Phylogenetic tree analysis based on 28S rDNA and ITS2 sequences demonstrated that furcocercous cercaria and xiphidiocercariae were closely clustered with a clade of Euclinostomum sp. and Xiphidiocercariae sp., respectively. Phylogenetic and network analyses of I. exustus haplotypes based on the COI, 16S rDNA, and ITS1 genes demonstrated four main clades. Only snails in clade A were distributed in all regions of Thailand and harbored trematode cercariae. The level of genetic diversity of I. exustus was relatively high, but most populations were not genetically different, thus suggesting the appearance of gene flow within the I. exustus populations. Overall, the haplotype network was star-shaped, thus suggesting the recent demographic expansion of populations. This result was also supported by the unimodal mode of the mismatch distribution graph and the large negative values of the neutrality tests. Therefore, the I. exustus snail was likely another freshwater snail of the invasive species in Thailand. This information will aid in monitoring the spread of the parasitic trematodes carried by I. exustus from different populations., Competing Interests: The authors declare there are no conflicts of interest., (Copyright: © 2024 Dumidae et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In South America, the knowledge of trematode diversity parasitizing freshwater fishes is still scarce, as less than 5% of the freshwater fish fauna has been examined for parasites. A similar situation applies to studies on digenean life cycles, which have become increasingly rare. Among the digenean families parasitizing freshwater fishes in the region, Haploporidae is considered the richest in species diversity. However, information about the developmental stages of haploporid life cycles remains fragmentary. Particularly, in Argentina, nine cercariae attributed to the family Haploporidae have been described using morphological analysis, and only two life cycles of this family have been completely elucidated. In this study a new type of cercaria, morphologically assigned to the family Haploporidae and collected from the snail Heleobia parchappii (Cochliopidae) in Los Padres shallow lake, Buenos Aires province, was identified using morphological and molecular techniques. The molecular analysis, based on 28S and ITS2 sequences, revealed that the cercariae were 100% identical to adult specimens of Saccocoelioides nanii (Haploporidae) parasitizing the fish Prochilodus lineatus (Prochilodontidae) from Los Talas, Buenos Aires province. Our results not only provide information about the life cycle of S. nanii but also show that a molecular and morphological approach can be extremely useful in identifying the developmental stages of digeneans and elucidating their life cycles.

Lymnaeids are aquatic snails playing an important role in the transmission of many parasitic trematode species of veterinary and medical importance. In this study, we assessed the presence of cercarial flukes in naturally infected lymnaeid snails from Phayao province, Thailand, and determined the species diversity of both the intermediate snail hosts and parasite larvae. A total of 3,185 lymnaeid snails were collected from paddy fields at 31 sites in eight districts of Phayao province between October 2021 and December 2022. Larval fluke infection was assessed using the cercarial shedding method. The collected snails as well as emerging cercariae were identified at the species level via morphological and molecular methods. The sequences of snail internal transcribed spacer region 2 (ITS2) and cercarial 28S ribosomal RNA gene (28S rDNA) and cytochrome C oxidase1 (Cox1) were determined by PCR amplification and sequencing. Three species of lymnaeid snails were detected in this study, including Radix (Lymnaea) rubiginosa (Michelin, 1831), Radix (Lymnaea) swinhoei (Adams, 1866) and Austropeplea viridis (Quoy & Gaimard, 1832), of which R. rubiginosa was the most abundant, followed by A. viridis and R. swinhoei. The overall rate of trematode cercarial infection in the lymnaeid snails was 2.8% (90/3,185); the cercarial infection rate in R. rubiginosa and A. viridis was 3.5% (60/1,735) and 3.1% (30/981), respectively. No larval fluke infection was observed in the studied R. swinhoei (0/469). Nine morphotypes of cercariae were detected at 15 sites from four districts. The emerging cercariae were molecularly identified as Clinostomum sp., Aporocotylidae sp., Apharyngostrigea sp., Trichobilharzia sp., Apatemon sp., Pegosomum sp., Petasiger sp., Echinostoma revolutum and Plagiorchis sp. These findings emphasize the occurrence and diversity of trematode cercariae among naturally infected lymnaeid snails in Phayao province and could contribute to broadening our understanding of the host-parasite relationships between trematodes and their first intermediate hosts as well as developing effective interventions to control trematode parasitic diseases., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Parasites have evolved a variety of astonishing strategies to survive within their hosts, yet the most challenging event in their personal chronicles is the passage from one host to another. It becomes even more complex when a parasite needs to pass through the external environment. Therefore, the free-living stages of parasites present a wide range of adaptations for transmission. Parasitic flatworms from the group Digenea (flukes) have free-living larvae, cercariae, which are remarkably diverse in structure and behavior. 1 , 2 One of the cercariae transmission strategies is to attain a prey-like appearance for the host. 3 This can be done through the formation of a swimming aggregate of several cercariae adjoined together by their tails. 4 Through the use of live observations and light, electron, and confocal microscopy, we described such a supposedly prey-mimetic colony comprising cercariae of two distinct morphotypes. They are functionally specialized: larger morphotype (sailors) enable motility, and smaller morphotype (passengers) presumably facilitate infection. The analysis of local read alignments between the two samples reveals that both cercaria types have identical 18S, 28S, and 5.8S rRNA genes. Further phylogenetic analysis of these ribosomal sequences indicates that our specimen belongs to the digenean family Acanthocolpidae, likely genus Pleorchis. This discovery provides a unique example and a novel insight into how morphologically and functionally heterogeneous individuals of the same species cooperate to build colonial organisms for the purpose of infection. This strategy bears resemblance to the cooperating castes of the same species found among insects. 5 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lymnaeid snails play a crucial role in the transmission of trematode cercariae as an intermediate host that can infect humans, ruminants like buffalo, and other animals, resulting in serious economic losses. The purpose of the study was to identify the morphological and molecular characteristics of snails and cercariae collected from water bodies near buffalo farms that were integrated with palm oil in Perak, Malaysia. The presence or absence of snails in 35 water bodies was examined via cross-sectional study. From three marsh wetlands, 836 lymnaeid snails were gathered in total. Each snail's shell was morphologically identified to determine its family and species. The cercarial stage inside each snail's body was observed using the crushing method and trematode cercariae types were determined. In addition, the target gene Cytochrome c oxidase subunit 1 (Cox1) and the ribosomal internal transcribed spacer 2 (ITS2) region were used to identify the snail species and cercarial types according to the species level. The findings indicated that the collected snails belong to the family lymnaeidae and Radix rubiginosa species. In snails, the cercarial emergence infection rate was 8.7%. Echinostome, xiphidiocercariae, gymnocephalous, brevifurcate-apharyngeate distome cercariae (BADC), and longifurcate-pharyngeal monostome cercariae (LPMC) are the five morphological cercarial types that were observed. The cercariae were identified using morphological and molecular techniques, and they are members of the four families which are Echinostomatidae, Plagiorchiidae, Fasciolidae, and Schistosomatidae. Interestingly, this is the first study on R. rubiginosa and several trematode cercariae in Perak water bodies near buffalo farms that are integrated with palm oil. In conclusion, our research shown that a variety of parasitic trematodes in Perak use R. rubiginosa as an intermediate host., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The identity of a larval bucephalid infecting the eastern oyster, Crassostrea virginica (Gmelin, 1791), from a tidal river in Virginia is investigated using ribosomal deoxyribonucleic acid (rDNA). The internal transcribed spacer (ITS1, 5.8S, ITS2) region and a portion of the 28S rDNA were isolated from genomic DNA from sporocysts containing cercariae and compared with sequences from GenBank and from our previous collections of possibly related bucephalids. The studied larval bucephalid was 100% identical at the ITS1, 5.8S, and partial 28S rDNA sequences with Prosorhynchoides paralichthydis (Corkum, 1961) Curran and Overstreet, 2009; however, it differed from P. paralichthydis by 6 bases plus 3 deletions in the ITS2 region. This level of variation at the ITS2 region has been demonstrated among some Indo-Pacific species of Prosorhynchoides Dollfus, 1929, suggesting that the larval bucephalid represents an unidentified or innominate species of Prosorhynchoides that is closely related to P. paralichthydis., (© American Society of Parasitologists 2023.)

Background: The trematode parasite Schistosoma mansoni uses an aquatic snail intermediate and a vertebrate definitive host to complete its life cycle. We previously showed that a key transmission trait-the number of cercariae larvae shed from infected Biomphalaria spp. snails-varies significantly within and between different parasite populations and is genetically controlled by five loci. We investigated the hypothesis that the success of parasite genotypes showing high propagative fitness in the intermediate snail host may be offset by lower reproductive fitness in the definitive vertebrate host., Methods: We investigated this trade-off hypothesis by selecting parasite progeny producing high or low number of larvae in the snail and then comparing fitness parameters and virulence in the rodent host. We infected inbred BALB/c mice using two Schistosoma mansoni parasite lines [high shedder (HS) and low shedder (LS) lines] isolated from F2 progeny generated by genetic crosses between SmLE (HS parent) and SmBRE (LS parent) parasites. We used the F3 progeny to infect two populations of inbred Biomphalaria glabrata snails. We then compared life history traits and virulence of these two selected parasite lines in the rodent host to understand pleiotropic effects of genes determining cercarial shedding in parasites infecting the definitive host., Results: HS parasites shed high numbers of cercariae, which had a detrimental impact on snail physiology (measured by laccase-like activity and hemoglobin rate), regardless of the snail genetic background. In contrast, selected LS parasites shed fewer cercariae and had a lower impact on snail physiology. Similarly, HS worms have a higher reproductive fitness and produced more viable F3 miracidia larvae than LS parasites. This increase in transmission is correlated with an increase in virulence toward the rodent host, characterized by stronger hepato-splenomegaly and hepatic fibrosis., Conclusions: These experiments revealed that schistosome parasite propagative and reproductive fitness was positively correlated in intermediate and definitive host (positive pleiotropy). Therefore, we rejected our trade-off hypothesis. We also showed that our selected schistosome lines exhibited low and high shedding phenotype regardless of the intermediate snail host genetic background. ​., (© 2023. The Author(s).)

Cercariae, the free-living larval stages of trematodes, have adopted an amazing variety of transmission strategies. One of them is prey-mimetism, i.e. cercariae mimicking prey to attract motile hosts to be eaten. In a period between 2002 and 2019, we examined small planorbid snails, Bathyomphalus contortus, Gyraulus parvus and Planorbis planorbis from lakes in Finland and Iceland and from the Curonian Lagoon in Lithuania. Cercariae with conspicuously enlarged tails and unusual swimming behaviour, likely mimicking invertebrate prey, were detected and studied by the use of morphological and molecular (cox1, ITS1-5.8S-ITS2 and 28S rDNA) methods. Cercariae of two species belonging to the genus Apatemon (Strigeidae) were recognised. We consider Apatemon sp. 5 ex P. planorbis from the Curonian Lagoon identical to Cercaria globocaudata U. Szidat, 1940. Cercariae ex G. parvus from Iceland and ex B. contortus from Finland were conspecific, and we named them Apatemon sp. 6; these cercariae could not be associated with any known species. For the first time, we verified that cercariae of the Bulbocauda group belong to the genus Apatemon. We provide a mini-review on records of furcocercariae of the family Strigeidae with enlarged tails reported in freshwaters of the northern hemisphere and reveal that it is not only Apatemon but also Australapatemon and most likely Strigea which belong to the Bulbocauda group, rendering it a purely ecological assemblage. Understanding which invertebrate swimming behaviour these cercariae are mimicking will enhance our knowledge of the processes behind trematode transmission and will help to assess evolutionary pathways of host-finding strategies in trematodes., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Schistosoma japonicum is one of the major infectious agents of human schistosomiasis, mainly endemic in China and the Philippines. We have previously reported the finding of two schistosome isolates, each with a different cercarial emergence pattern adapted to their different hosts. However, there are currently no whole-genome sequencing studies to investigate the underlining genetics of the adaptive traits. We sampled schistosomes in 2013 and 2020 from a hilly area Shitai (ST) and a marshland area Hexian (HX) of Anhui, China. Ten to 15 male or female adult worms from each site/year were sent for whole genome sequencing. Genetics were analyzed, and selection signals along genomes were detected. Gene enrichment analysis was performed for the genome regions under selection. The results revealed considerable genetic differentiation between the two isolates. The genome "windows" affected by natural selection were fewer in ST (64 windows containing 78 genes) than in HX (318 windows containing 276 genes). Twelve significantly enriched genes were identified in ST, but none in HX. These genes were mainly related to specific DNA binding and intercellular signaling transduction. Some functional region changes identified along the genome of the hilly schistosome may be related to its unique late afternoon cercarial emergence., (© H.-Y. Sun et al., published by EDP Sciences, 2023.)

The studies of opisthorchiids larval stages associated with Bithyniidae snails can provide important and the most reliable data for opisthorchiidoses foci characterization due to the low mobility of such opisthorchiid's host species as Bithyniidae snails. The foci of opisthorchiosis (caused by Opisthorchis felineus) and metorchiosis (caused by Metorchis bilis) are overlapping in the basins of the Ob and Irtysh Rivers. Thus, difficulties with determining the species of cercariae significantly reduce the accuracy of epidemiological conclusions regarding opisthorchiosis, which has a much higher medical significance. Moreover, M. bilis cercariae identification is complicated by the fact that the focus of metorchiosis caused by Metorchis xanthosomus (infecting fish-eating predatory birds) occurs on the same territory. In this study, we for the first time carried out the molecular genetic identification of West-Siberian opisthorchiid cercariae to verify morphological identification and confirmed O. felineus's main association with Bithynia troschelii snail and M. bilis'-with B. tentaculata snail. Thus, our study applied a two-stage approach: the morphological identification of any opisthorchiid-like cercariae in Bithynia snails and subsequent molecular genetic precise species determination of cercariae in the reduced samples., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A survey was conducted in the lowland areas of Binh Dinh and Phu Yen provinces in south central Vietnam to study the diversity of digenean cercariae within freshwater snails. These regions are known as highly endemic areas for the transmission of liver flukes, Fasciola spp. and Opisthorchis viverrini. In total, 13 snail species were found, and 3 of these were dominant, comprising 51.9% of all collected snails, i.e., Bithynia funiculata, Melanoides tuberculata, and Radix auriculata. Cercariae were found in 5 snail species, and among these, Indoplanorbis exustus had the highest overall prevalence of infection (38.5%). No cercarial infections were found in viviparid and ampullariid snails. Morphological identification resulted in 10 cercariae types that were illustrated and measured. Among them, xiphidiocercariae and echinostomata were the most common, causing 70.55% of all cercariae infections. Bithynia species had the most diverse digenean fauna. Molecular data revealed the presence of cercariae of 13 digenean species, including O. viverrini. The 18S rDNA sequences of echinostomata and xiphidiocercariae type 3, which were shed from different snail hosts, were similar to 2 and 3 digenean species, respectively. In conclusion, our findings highlight the diversity of digenean fauna in Binh Dinh and Phu Yen provinces., (© American Society of Parasitologists 2022.)

As part of surveillance of snail-borne trematodiasis in Knowsley Safari (KS), Prescot, United Kingdom, a collection was made in July 2021 of various planorbid ( n = 173) and lymnaeid ( n = 218) snails. These were taken from 15 purposely selected freshwater habitats. In the laboratory emergent trematode cercariae, often from single snails, were identified by morphology with a sub-set, of those most accessible, later characterized by cytochrome oxidase subunit 1 ( cox 1) DNA barcoding. Two schistosomatid cercariae were of special note in the context of human cercarial dermatitis (HCD), Bilharziella polonica emergent from Planorbarius corneus and Trichobilharzia spp. emergent from Ampullacaena balthica . The former schistosomatid was last reported in the United Kingdom over 50 years ago. From cox 1 analyses, the latter likely consisted of two taxa, Trichobilharzia anseri , a first report in the United Kingdom, and a hitherto unnamed genetic lineage having some affiliation with Trichobilharzia longicauda . The chronobiology of emergent cercariae from P. corneus was assessed, with the vertical swimming rate of B. polonica measured. We provide a brief risk appraisal of HCD for public activities typically undertaken within KS educational and recreational programmes.

The best way to study digenean diversity combines molecular genetic methods, life-cycle studies and elaborate morphological descriptions. This approach has been barely used for one of the most widespread digenean taxa parasitizing fish – the superfamily Hemiuroidea. Here, we applied the integrative approach to the hemiuroideans from the family Derogenidae parasitizing fish at the White and Barents Seas. Analysis of 28S, 18S, 5.8S rDNA, ITS2 and cox1 gene sequences from sexually adult worms (maritae) showed genetic heterogeneity for 2 derogenid species known from this area: Derogenes varicus and Progonus muelleri . Thus, 2 pairs of genetic lineages were found: DV1 and DV2, PM1 and PM2, respectively. Data from other regions indicate that 2 more lineages of D. varicus probably exist. Based on previous records from the White and Barents Seas, we hypothesized that the cercariae found in the moonsnails (family Naticidae) belong to the Derogenidae and may help to differentiate these lineages as species. According to our results, Cercaria appendiculata from Cryptonatica affinis matched DV1, similar nameless cercariae from Euspira pallida and Amauropsis islandica matched DV2, and Cercaria octocauda from C. affinis matched PM1. We provide new data on the structure of these cercariae and discuss the life-cycle pattern of the studied digeneans.

Larval didymozoids (Trematoda: Digenea) were discovered parasitizing the hemocoel of the heteropod Firoloida desmarestia (redia mean intensity = 13) and the chaetognaths Flaccisagitta enflata and Flaccisagitta hexaptera (metacercaria mean intensity = 1) during a 2014-2016 systematic study of parasites of zooplankton collected in the central and southern regions of the Gulf of California, Mexico. Didymozoid infection route during the early life cycle was inferred combining morphological (light microscopy) and molecular (mitochondrial cytochrome c oxidase subunit I gene, cox1) evidence. Didymozoid rediae parasitizing F. desmarestia were observed, just after field collection of the host, containing hundredths of completely developed cystophorous cercariae, releasing them though the birth pore at approximately one cercaria every 12 s. Cercariae lost their tails developing into a 'young metacercaria' in 1 d at 22 °C without need of an intermediate host. Molecular analysis of cox1 showed that rediae found in F. desmarestia belong to two distinct didymozoid species (Didymozoidae sp. 1 and sp. 2). Metacercariae parasitizing chaetognaths were morphologically identified as Didymozoidae type Monilicaecum and cox1 sequences showed that metacercariae of chaetognaths matched with these two Didymozoidae sp. 1, and sp. 2 species found parasitizing F. desmarestia, plus a third distinct Didymozoidae sp. 3. These are the first DNA sequences of cox1 gene from didymozoid larvae for any zooplankton taxonomic group in the world. We concluded that F. desmarestia is the first intermediate host of rediae and cercariae, and the chaetognaths are the second intermediate hosts where non-encysted metacercariae were found. The definitive host is still unknown because cox1 sequences of present study did not genetically match with any available cox1 sequence of adult didymozoid. Our results demonstrate a potential overlap in the distribution of two carnivorous zooplankton taxonomic groups that are intermediate hosts of didymozoids in the pelagic habitat. The didymozoid specimens were not identified to species level because any of the cox1 sequences generated here matched with the sequences of adult didymozoids currently available in GenBank and Bold System databases. This study provides baseline information for the future morphological and molecular understanding of the Didymozoidae larvae that has been previously based on the recognition of the 12 known morphotypes., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: The Zanzibar Archipelago (Pemba and Unguja islands) is targeted for the elimination of human urogenital schistosomiasis caused by infection with Schistosoma haematobium where the intermediate snail host is Bulinus globosus. Following multiple studies, it has remained unclear if B. nasutus (a snail species that occupies geographically distinct regions on the Archipelago) is involved in S. haematobium transmission on Zanzibar. Additionally, S. haematobium was thought to be the only Schistosoma species present on the Zanzibar Archipelago until the sympatric transmission of S. bovis, a parasite of ruminants, was recently identified. Here we re-assess the epidemiology of schistosomiasis on Pemba and Unguja together with the role and genetic diversity of the Bulinus spp. involved in transmission., Methodology/principal Findings: Malacological and parasitological surveys were conducted between 2016 and 2019. In total, 11,116 Bulinus spp. snails were collected from 65 of 112 freshwater bodies surveyed. Bulinus species identification were determined using mitochondrial cox1 sequences for a representative subset of collected Bulinus (n = 504) and together with archived museum specimens (n = 6), 433 B. globosus and 77 B. nasutus were identified. Phylogenetic analysis of cox1 haplotypes revealed three distinct populations of B. globosus, two with an overlapping distribution on Pemba and one on Unguja. For B. nasutus, only a single clade with matching haplotypes was observed across the islands and included reference sequences from Kenya. Schistosoma haematobium cercariae (n = 158) were identified from 12 infected B. globosus and one B. nasutus collected between 2016 and 2019 in Pemba, and cercariae originating from 69 Bulinus spp. archived in museum collections. Schistosoma bovis cercariae (n = 21) were identified from seven additional B. globosus collected between 2016 and 2019 in Pemba. By analysing a partial mitochondrial cox1 region and the nuclear ITS (1-5.8S-2) rDNA region of Schistosoma cercariae, we identified 18 S. haematobium and three S. bovis haplotypes representing populations associated with mainland Africa and the Indian Ocean Islands (Zanzibar, Madagascar, Mauritius and Mafia)., Conclusions/significance: The individual B. nasutus on Pemba infected with S. haematobium demonstrates that B. nasutus could also play a role in the local transmission of S. haematobium. We provide preliminary evidence that intraspecific variability of S. haematobium on Pemba may increase the transmission potential of S. haematobium locally due to the expanded intermediate host range, and that the presence of S. bovis complicates the environmental surveillance of schistosome infections., Competing Interests: The authors have declared that no competing interests exist.

Lymnaeid snails play an essential role in transmitting fasciolosis as intermediate hosts. Therefore, this study aims to use the molecular method to identify liver fluke in lymnaeid snails. A total of 320 lymnaeid snails were collected from a rice field. The samples were dissected to collect cercaria and identified using polymerase chain reaction. Furthermore, the internal transcribed spacer 2 (ITS2) was used as the target gene to identify the species of cercaria. The result showed that 3.75% (12/320) of the snails were infected by Fasciola gigantica, while the phylogenetic tree based on ITS2 showed that the cercaria in this study was monophyletic and similar to species from several countries in Southeast Asia, including China. Furthermore, the haplotype network showed that all four cercaria samples were similar with sequences from several countries. This study suggests that the F. gigantica cercaria isolated from lymnaeid snails in Kulon Progo, Yogyakarta, Indonesia, has a sequence similar to that of other species in Southeast Asian countries, although no hybrid type was detected in these sequences. This is the first report on the molecular identification of cercaria F. gigantica isolated from lymnaeid snails in Yogyakarta, Indonesia., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The adult fluke Stomylotrema vicarium (Stomylotrematidae, Microphalloidea) was described for the first time in Theristicus caerulescens in 1901, but the complete life cycle has remained unknown to date. Here, we found a stomylotrematid trematode in the digestive gland of the endemic apple snail Pomacea americanista. The digestive gland's tubuloacini were compressed by the trematode larvae placed on connective tissues and haemocoel spaces. Non-virgulate, stylet-bearing cercariae showed three pairs of penetration glands with a body, oral sucker and stylet morphometrically similar to those of stylet-bearing, unencysted young metacercariae of S. vicarium found in the aquatic coleopteran Megadytes glaucus, and at a lesser extent with cercariae of S. gratiosus found in the apple snail Pomacea maculata. The larvae molecular phylogeny was inferred using the markers rRNA 28S and ITS1, being these sequences grouped with the sequences of S. vicarium obtained from adult flukes. Together, these findings indicate that the life cycle of S. vicarium begins in P. americanista, thus supporting the hypothesis that the ampullariid snails act as a first intermediate host.

Extracellular Vesicles (EVs) are an integral component of cellular/organismal communication and have been found in the excreted/secreted (ES) products of both protozoan and metazoan parasites. Within the blood fluke schistosomes, EVs have been isolated from egg, schistosomula, and adult lifecycle stages. However, the role(s) that EVs have in shaping aspects of parasite biology and/or manipulating host interactions is poorly defined. Herein, we characterise the most abundant EV-enriched protein in Schistosoma mansoni tissue-migrating schistosomula (Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1)). Comparative sequence analysis demonstrates that lev1 orthologs are found in all published Schistosoma genomes, yet homologs are not found outside of the Schistosomatidae. Lifecycle expression analyses collectively reveal that smlev1 transcription peaks in cercariae, is male biased in adults, and is processed by alternative splicing in intra-mammalian lifecycle stages. Immunohistochemistry of cercariae using a polyclonal anti-recombinant SmLEV1 antiserum localises this protein to the pre-acetabular gland, with some disperse localisation to the surface of the parasite. S. mansoni-infected Ugandan fishermen exhibit a strong IgG1 response against SmLEV1 (dropping significantly after praziquantel treatment), with 11% of the cohort exhibiting an IgE response and minimal levels of detectable antigen-specific IgG4. Furthermore, mice vaccinated with rSmLEV1 show a slightly reduced parasite burden upon challenge infection and significantly reduced granuloma volumes, compared with control animals. Collectively, these results describe SmLEV1 as a Schistosomatidae-specific, EV-enriched immunogen. Further investigations are now necessary to uncover the full extent of SmLEV1's role in shaping schistosome EV function and definitive host relationships., Competing Interests: The authors have declared that no competing interests exist.

Schistosomes require both molluscan and mammalian hosts for development. The larval cercaria exits the snail host and swims to identify and invade the mammalian host. The cercaria has two macrostructures, the head and the tail. The head invades the host, where it matures into an adult worm. The tail is lost after host invasion. Translation in the cercaria differs in each macrostructure, with higher levels of translation in the cercarial tail and little to no translational activity in the cercarial head. We compared the transcriptome and proteome of the cercarial head and tail and observed stark differences between the two macrostructures. We identified unique and differentially expressed transcripts and proteins, including ribosomal components expressed in higher levels in tails than in heads, which may explain the differences in translation levels between heads and tails. We also characterized the weak correlation between transcription and translation in infectious cercarial heads and tails., (© 2021. The Author(s).)

Trematodes of the genus Himasthla are usual parasites of coastal birds in nearshore ecosystems of northern European seas and the Atlantic coast of North America. Their first intermediate hosts are marine and brackish-water gastropods, while second intermediate hosts are various invertebrates. We analysed sequences of partial 28S rRNA and nad1 genes and the morphology of intramolluscan stages, particularly cercariae of Himasthla spp. parasitizing intertidal molluscs Littorina spp. in the White Sea, the Barents Sea and coasts of North Norway and Iceland. We showed that only three Himasthla spp. are associated with periwinkles in these regions. Intramolluscan stages of H. elongata were found in Littorina littorea, of H. littorinae, in both L. saxatilis and L. obtusata, and of Cercaria littorinae obtusatae, predominantly, in L. obtusata. Other Himasthla spp. previously reported from Littorina spp. in North Atlantic are either synonymous with one of these species or described erroneously. Based on a comparison of newly generated 28S rDNA sequences with GenBank data, rediae and cercariae of C. littorinae obtusatae were identified as belonging to H. leptosoma. Some previously unknown morphological features of young and mature rediae and cercariae of the three Himasthla spp. are described. We provide a key to the rediae and highlight characters important for identification of cercariae. Genetic diversity within the studied species was only partially determined by their specificity to the molluscan host. The nad1 network constructed for H. leptosoma lacked geographical structure, which is explained by a high gene flow owing to highly vagile definitive hosts, shorebirds.

Cercarial emission of schistosomes is a determinant in the transmission to the definitive host and constitutes a good marker to identify which definitive host is responsible for transmission, mainly in introgressive hybridization situations. Our goal was to test the hypothesis that micro-mammals play a role in Schistosoma haematobium, S. bovis, and/or S. haematobium x S. bovis transmission. Small mammal sampling was conducted in seven semi-lacustrine villages of southern Benin. Among the 62 animals trapped, 50 individuals were investigated for Schistosoma adults and eggs: 37 Rattus rattus, 3 Rattus norvegicus, 9 Mastomys natalensis, and 1 Crocidura olivieri. Schistosoma adults were found in four R. rattus and two M. natalensis, with a local prevalence reaching 80% and 50%, respectively. Two cercarial chronotypes were found from Bulinus globosus experimentally infected with miracidia extracted from naturally infected M. natalensis: a late diurnal and nocturnal chronotype, and an early diurnal, late diurnal, and nocturnal chronotype. The cytochrome C oxidase subunit I mtDNA gene of the collected schistosomes (adults, miracidia, and cercariae) belonged to the S. bovis clade. Eleven internal transcribed spacer rDNA profiles were found; four belonged to S. bovis and seven to S. haematobium x S. bovis. These molecular results together with the observed multi-peak chronotypes add M. natalensis as a new host implicated in S. haematobium x S. bovis transmission. We discuss the origin of the new chronotypes which have become more complex with the appearance of several peaks in a 24-h day. We also discuss how the new populations of offspring may optimize intra-host ecological niche, host spectrum, and transmission time period.

Swimmer's itch or human cercarial dermatitis (HCD) appears as a skin rash caused by an allergic reaction to larval (cercariae) flatworm parasites of the family Schistosomatidae. In our study, two cases of HCD were analyzed; both of them were reported in people swimming in Lake Pluszne. In the summer of 2018, a sample of 397 snails was collected at swimming sites in that area. Five Lymnaea stagnalis (1.9%) were found to host cercariae of bird schistosomes. Positive samples were selected by amplification of the Internal Transcribed Spacers (ITS) gene region. Sequence analysis confirmed that they were homologous with European isolates of Trichobilharzia szidati . The cases reported in this article are the first confirmed cases of HCD in this lake. This study demonstrates that there is a rationale for conducting screening studies of regions with a high recreational potential.

Foodborne zoonotic trematode (FZT) infections are common neglected tropical diseases in Southeast Asia. Their complicated life cycles involve freshwater snails as intermediate hosts. A cross-sectional study was conducted in Yen Bai and Thanh Hoa provinces in North and Central Vietnam, to investigate the diversity of cercariae of potential FZT and to construct the phylogenetic relationship of trematode cercariae based on the Internal Transcribed Spacer 2 (ITS2) region. Among 17 snail species collected from various habitats, 13 were infected by 10 cercarial groups among which parapleurolophocercous, pleurolophocercous, and echinostome cercariae were of zoonotic importance. The monophyletic tree separated cercarial sequences into different groups following the description of the cercariae families in which Haplorchidae, Opisthorchiidae, Echinochasmidae, and Echinostomatidae are important families of FZT. The overall prevalence was different among snail species and habitats and showed a seasonal trend. Parapleurolophocercous and echinostome cercariae emerged as the most common cercariae in snails in Yen Bai, while monostome, echinostome, and megalura cercariae were most common in Thanh Hoa. Using a molecular approach, we identified Parafossarulus striatulus as the first intermediate snail host of Clonorchis sinensis in Thac Ba Lake. Melanoides tuberculata and Bithynia fuchsiana were we identified preferred intermediate snail hosts of a diverse range of trematode species including intestinal flukes (i.e., Haplorchis pumilio and Echinochasmus japonicus) in Yen Bai and Thanh Hoa, respectively.

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.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Digenean trematodes are parasitic platyhelminths that use several hosts in their life cycles and are thereby embedded in various ecosystems affected by local environmental conditions. Their presence in a habitat will reflect the presence of different host species and, as such, they can serve as ecological indicators. Only limited information on the occurrence of trematodes and their link to other trophic levels in the Danish freshwater ecosystems is currently available.Therefore, the main aim of the present study was to increase our knowledge in this field., Methods: Snails were sampled from 21 freshwater lakes in Denmark, following which shedding procedures were performed, cercariae were recoved and the released parasites were identified using molecular tools (PCR and sequencing)., Results: A total of 5657 snail hosts belonging to ten species were identified, revealing a highly diverse parasite fauna comprising 22 trematode species. The overall trematode prevalence was 12.6%, but large variations occurred between host species. The snail host Lymnaea stagnalis showed the highest prevalence and also exhibited the highest diversity, accounting for 47.6% of the species richness., Conclusions: This survey contributes updated information on parasite-host relations and compatibility and may assist in describing the ecological structure of the investigated Danish freshwater ecosystems.

Background: Leeches (Hirudinida) play a significant role as intermediate hosts in the circulation of trematodes in the aquatic environment. However, species richness and the molecular diversity and phylogeny of larval stages of strigeid trematodes (tetracotyle) occurring in this group of aquatic invertebrates remain poorly understood. Here, we report our use of recently obtained sequences of several molecular markers to analyse some aspects of the ecology, taxonomy and phylogeny of the genera Australapatemon and Cotylurus, which utilise leeches as intermediate hosts., Methods: From April 2017 to September 2018, 153 leeches were collected from several sampling stations in small rivers with slow-flowing waters and related drainage canals located in three regions of Poland. The distinctive forms of tetracotyle metacercariae collected from leeches supplemented with adult Strigeidae specimens sampled from a wide range of water birds were analysed using the 28S rDNA partial gene, the second internal transcribed spacer region (ITS2) region and the cytochrome c oxidase (COI) fragment., Results: Among investigated leeches, metacercariae of the tetracotyle type were detected in the parenchyma and musculature of 62 specimens (prevalence 40.5%) with a mean intensity reaching 19.9 individuals. The taxonomic generic affiliation of metacercariae derived from the leeches revealed the occurrence of two strigeid genera: Australapatemon Sudarikov, 1959 and Cotylurus Szidat, 1928. Phylogenetic reconstructions based on the partial 28S rRNA gene, ITS2 region and partial COI gene confirmed the separation of the Australapatemon and Cotylurus clades. Taking currently available molecular data and our results into consideration, recently sequenced tetracotyle of Australapatemon represents most probably Au. minor; however, unclear phylogenetic relationships between Au. burti and Au. minor reduce the reliability of this conclusion. On the other hand, on the basis of the obtained sequences, supplemented with previously published data, the metacercariae of Cotylurus detected in leeches were identified as two species: C. strigeoides Dubois, 1958 and C. syrius Dubois, 1934. This is the first record of C. syrius from the intermediate host., Conclusions: The results of this study suggest the separation of ecological niches and life cycles between C. cornutus (Rudolphi, 1808) and C. strigeoides/C. syrius, with potential serious evolutionary consequences for a wide range of host-parasite relationships. Moreover, phylogenetic analyses corroborated the polyphyletic character of C. syrius, the unclear status of C. cornutus and the separate position of Cotylurus raabei Bezubik, 1958 within Cotylurus. The data demonstrate the inconsistent taxonomic status of the sequenced tetracotyle of Australapatemon, resulting, in our opinion, from the limited availability of fully reliable, comparative sequences of related taxa in GenBank.

In this study, two potentially new species of turtle blood flukes (TBFs) (Digenea: Spirorchiidae) have been recorded from South Brazil. The spirorchiid parasites infect the vascular system of turtles, thereby compromising their health. The life cycle of these parasites is not well studied. The larval stage of cercaria is found in intermediate gastropod hosts, with some species presenting similar morphological characteristics, which can result in misinterpretations when using only morphological taxonomy for species identification. In this study, we recorded a single morphotype belonging to the family Spirorchiidae in Biomphalaria occidentalis in an urban aquatic ecosystem in Brazil. However, molecular data (28S rDNA and cytochrome oxidase subunit I) confirmed the presence of two species of Spirorchiidae in the sampled environment; both phylogenetically close to genera previously studied in freshwater turtles from the Peruvian Amazon. In this study, species characterization was possible because of molecular tools. We recommend using more than one molecular marker in future studies focusing on TBFs, which need attention about their evolutionary history and ecology to understand their distribution in South America.

We found unusual digenean intramolluscan stages, sporocysts and cercariae, in gastropods Sulcospira dautzenbergiana (Morelet) (Caenogastropoda: Pachychilidae) from Southern Vietnam and named them Cercaria cattieni 1. These cercariae have a stylet and thus belong to the Xiphidiata. However, such combination of characters as extremely large body size and I-shaped excretory bladder has not been found before in any other xiphidiocercariae. We obtained COI, ITS1, 5.8S + ITS2, and 28S rDNA sequences for C. cattieni 1. The latter allowed us to specify the phylogenetic position of the discovered cercariae: C. cattieni 1 falls within the superfamily Microphalloidea and is most closely grouped to Pachypsolus irroratus (Rudolphi, 1819) (Pachypsolidae), the sea turtle parasite. Information on the family Pachypsolidae is limited. Judging from the molecular phylogeny, C. cattieni 1 might be the larva of the Pachypsolidae, documented for the first time.

Background: Parasitic flatworms (Trematoda: Digenea) represent one of the most remarkable examples of drastic morphological diversity among the stages within a life cycle. Which genes are responsible for extreme differences in anatomy, physiology, behavior, and ecology among the stages? Here we report a comparative transcriptomic analysis of parthenogenetic and amphimictic generations in two evolutionary informative species of Digenea belonging to the family Psilostomatidae., Methods: In this study the transcriptomes of rediae, cercariae and adult worm stages of Psilotrema simillimum and Sphaeridiotrema pseudoglobulus, were sequenced and analyzed. High-quality transcriptomes were generated, and the reference sets of protein-coding genes were used for differential expression analysis in order to identify stage-specific genes. Comparative analysis of gene sets, their expression dynamics and Gene Ontology enrichment analysis were performed for three life stages within each species and between the two species., Results: Reference transcriptomes for P. simillimum and S. pseudoglobulus include 21,433 and 46,424 sequences, respectively. Among 14,051 orthologous groups (OGs), 1354 are common and specific for two analyzed psilostomatid species, whereas 13 and 43 OGs were unique for P. simillimum and S. pseudoglobulus, respectively. In contrast to P. simillimum, where more than 60% of analyzed genes were active in the redia, cercaria and adult worm stages, in S. pseudoglobulus less than 40% of genes had such a ubiquitous expression pattern. In general, 7805 (36.41%) and 30,622 (65.96%) of genes were preferentially expressed in one of the analyzed stages of P. simillimum and S. pseudoglobulus, respectively. In both species 12 clusters of co-expressed genes were identified, and more than a half of the genes belonging to the reference sets were included into these clusters. Functional specialization of the life cycle stages was clearly supported by Gene Ontology enrichment analysis., Conclusions: During the life cycles of the two species studied, most of the genes change their expression levels considerably, consequently the molecular signature of a stage is not only a unique set of expressed genes, but also the specific levels of their expression. Our results indicate unexpectedly high level of plasticity in gene regulation between closely related species. Transcriptomes of P. simillimum and S. pseudoglobulus provide high quality reference resource for future evolutionary studies and comparative analyses.

Cercariae invasion of the human skin is the first step in schistosome infection. Proteases play key roles in this process. However, little is known about the related hydrolytic enzymes in Schistosoma japonicum. Here, we investigated the biochemical features, tissue distribution and biological roles of a cathepsin B cysteine protease, SjCB2, in the invasion process of S. japonicum cercariae. Enzyme activity analysis revealed that recombinant SjCB2 is a typical cysteine protease with optimum temperature and pH for activity at 37°C and 4.0, respectively, and can be totally inhibited by the cysteine protease inhibitor E-64. Immunoblotting showed that both the zymogen (50 kDa) and mature enzyme (30.5 kDa) forms of SjCB2 are expressed in the cercariae. It was observed that SjCB2 localized predominantly in the acetabular glands and their ducts of cercariae, suggesting that the protease could be released during the invasion process. The protease degraded collagen, elastin, keratin, fibronectin, immunoglobulin (A, G and M) and complement C3, protein components of the dermis and immune system. In addition, proteomic analysis demonstrated that SjCB2 can degrade the human epidermis. Furthermore, it was showed that anti-rSjCB2 IgG significantly reduced (22.94%) the ability of the cercariae to invade the skin. The cysteine protease, SjCB2, located in the acetabular glands and their ducts of S. japonicum cercariae. We propose that SjCB2 facilitates skin invasion by degrading the major proteins of the epidermis and dermis. However, this cysteine protease may play additional roles in host-parasite interaction by degrading immunoglobins and complement protein., Competing Interests: The authors have declared that no competing interests exist.

As a unique feature among otherwise hermaphroditic trematodes, Schistosoma species are gonochoric parasites whose sex is genetically determined (ZZ for males and ZW for females). However, schistosome larvae are morphologically identical, and sex can only be discriminated by molecular methods. Here, we integrated published Schistosoma. japonicum transcriptome and genome data to identify W chromosome-specific genes as sex biomarkers. Three W chromosome-specific genes of S. japonicum were identified as sex biomarkers from a panel of 12 genes expressed only in females. An efficient duplex real-time PCR (qPCR) method for sexing cercariae was developed which could identify the sex of cercariae within 2 h without DNA extraction. Moreover, this method can be used to identify not only single-sex but also mixed-sex schistosome-infected snails. We observed a nearly equal proportion of single-male, single-female, and mixed-sex schistosome infections in artificially infected snails. Sex-known schistosome-infected snail models can be efficiently constructed with the aid of duplex qPCR. A field study revealed that single-sex schistosome infections were predominant among naturally infected snails. Finally, a schistosomiasis mouse model based on sex-known cercariae infection was shown to be more reliable than a model based on sex-unknown cercariae infection. The developed duplex qPCR method for sexing S. japonicum cercariae can be widely used for schistosomiasis modeling, genetic experiments, and field-based molecular epidemiological studies., Competing Interests: The authors have declared that no competing interests exist.

Background: Schistosoma japonicum is a waterborne parasite that causes schistosomiasis in humans and in more than 40 animal species. Schistosoma japonicum shows distinct genetic differentiation among geographical populations and multiple hosts, but the genetic diversity of different developmental stages of S. japonicum from is less studied. Such studies could elucidate ecological mechanisms in disease transmission by analysing feedbacks in individual physiology and population state., Methods: After infection using cercariae from a pool of snails shedding together (Method I) and infection using mixed equal numbers of cercariae from individually shed snails (Method II), different developmental stages of S. japonicum were genotyped with microsatellite loci, including 346 cercariae, 701 adult worms and 393 miracidia. Genetic diversity and molecular variation were calculated at different population levels. Kinships (I') among cercariae at intra-snail and inter-snail levels were evaluated. Genetic distance (Dsw) was compared between paired and unpaired worms, and partner changing was investigated through paternity identification for miracidia., Results: The cercaria clones in individual snails varied from 1 to 8 and the kinship of cercariae within individual snails was significant higher (P < 0.001) than that among different snails after deleting near-identical multi-locus genotypes (niMLGs). The allelic diversity of worms in Method I was lower (P < 0.001) than that in Method II, and allele frequency among mice in Method I was also less consistent. The parents of some miracidia were worms that were not paired when collected. The Dsw between each female of paired and unpaired males was much larger (P < 0.001) than that between the female and male in each pair., Conclusions: Most of the infected snails contained multiple miracidia clones. The aggregation of genetically similar S. japonicum miracidia in individual snails and the unbalanced distribution of miracidia among snails suggests a non-uniform genetic distribution of cercariae among snails in the field. This further influenced the genetic structure of adult worms from infections with different cercariae sampling methods. Schistosoma japonicum in mice can change paired partner, preferring to mate with genetically similar worms. These characteristics provide implications for understanding the balance in genetic diversity of S. japonicum related to the transmission of schistosomiasis.

Schistosomiasis remains a parasitic infection which poses serious public health consequences around the world, particularly on the African continent where cases of introgression/hybridization between human and cattle schistosomiasis are being discovered on a more frequent basis in humans, specifically between Schistosoma haematobium and S. bovis. The aim of this paper is to analyze the occurrence of S. bovis in cattle and its relationship with S. haematobium in an area where cattle and humans share the same site in Benin (West Africa). We used the chronobiology of cercarial emergence as an ecological parameter and both molecular biology (COI mtDNA and ITS rDNA) of the larvae and morphology of the eggs as taxonomic parameters. The results showed a chronobiological polymorphism in the cercarial emergence rhythm. They showed for the first time the presence of S. bovis in Benin, the presence of introgressive hybridization between S. bovis and S. haematobium in domestic cattle, and the presence of atypical chronobiological patterns in schistosomes from cattle, with typical S. haematobium shedding pattern, double-peak patterns, and nocturnal patterns. Our results showed that the chronobiological life-history trait is useful for the detection of new hosts and also may reveal the possible presence of introgressive hybridization in schistosomes. Our results, for the first time, place cattle as reservoir host for S. haematobium and S. bovis x S. haematobium. The consequences of these results on the epidemiology of the disease, the transmission to humans, and the control of the disease are very important.

Background: Urogenital schistosomiasis, caused by infection with Schistosoma haematobium, is endemic in Niger but complicated by the presence of Schistosoma bovis, Schistosoma curassoni and S. haematobium group hybrids along with various Bulinus snail intermediate host species. Establishing the schistosomes and snails involved in transmission aids disease surveillance whilst providing insights into snail-schistosome interactions/compatibilities and biology., Methods: Infected Bulinus spp. were collected from 16 villages north and south of the Niamey region, Niger, between 2011 and 2015. From each Bulinus spp., 20-52 cercariae shed were analysed using microsatellite markers and a subset identified using the mitochondrial (mt) cox1 and nuclear ITS1 + 2 and 18S DNA regions. Infected Bulinus spp. were identified using both morphological and molecular analysis (partial mt cox1 region)., Results: A total of 87 infected Bulinus from 24 sites were found, 29 were molecularly confirmed as B. truncatus, three as B. forskalii and four as B. globosus. The remaining samples were morphologically identified as B. truncatus (n = 49) and B. forskalii (n = 2). The microsatellite analysis of 1124 cercariae revealed 186 cercarial multilocus genotypes (MLGs). Identical cercarial genotypes were frequently (60%) identified from the same snail (clonal populations from a single miracidia); however, several (40%) of the snails had cercariae of different genotypes (2-10 MLG's) indicating multiple miracidial infections. Fifty-seven of the B. truncatus and all of the B. forskalii and B. globosus were shedding the Bovid schistosome S. bovis. The other B. truncatus were shedding the human schistosomes, S. haematobium (n = 6) and the S. haematobium group hybrids (n = 13). Two B. truncatus had co-infections with S. haematobium and S. haematobium group hybrids whilst no co-infections with S. bovis were observed., Conclusions: This study has advanced our understanding of human and bovid schistosomiasis transmission in the Niger River Valley region. Human Schistosoma species/forms (S. haematobium and S. haematobium hybrids) were found transmitted only in five villages whereas those causing veterinary schistosomiasis (S. bovis), were found in most villages. Bulinus truncatus was most abundant, transmitting all Schistosoma species, while the less abundant B. forskalii and B. globosus, only transmitted S. bovis. Our data suggest that species-specific biological traits may exist in relation to co-infections, snail-schistosome compatibility and intramolluscan schistosome development.

Objective: To characterize a species of the genus Tricula and parasitized trematodes in schistosomiasis-endemic areas of Yunnan Province using a molecular analysis, so as to understand their taxonomic positions., Methods: Tricula spp. and Oncomelania snails were collected from Xiangyun County, Yunnan Province, and cercaria parasitizing snails were observed using crushing followed by microscopy. Cercaria parasitizing Tricula snails at various morphologies were sampled using a shedding method. Genomic DNA was extracted from snail soft tissues and cercariae, and the 16S rRNA , COI , 28S rDNA genes in snails and the ND1 and 28S rDNA genes in cercariae were amplified using a PCR assay and sequenced. The species of Tricula snails and their parasitized trematodes was characterized using sequence alignment and phylogenetic analysis., Results: Among 382 Tricula snails detected, there were three types of trematode cercariae found, including the non-forked (20.94%, 80/382), double-forked (3.40%, 13/382) and swallow shapes (7.07%, 27/382). Sequence and phylogenetic analyses showed that the 16S rRNA , COI and 28S rDNA gene sequences of this species of Tricula had high homology to those in Delavaya dianchiensis , and were clustered in a branch. Sequencing analysis of the ND1 and 28S rDNA genes revealed that the non-forked cercariae belonged to the family Pleu- rogenidae, the swallow-shaped cercariae belonged to the family Opecoelidae, and the double-forked cercariae belonged to another species of the genus Schistosoma that was different from S. sinensium and S. ovuncatum ., Conclusions: The species and taxonomy of Triculla spp. and their parasitized trematodes are preliminarily determined in schistosomiasis-endemic areas of Yunnan Province; however, further studies are required to investigate the more definite taxonomy and pathogenicity.

Microcercous cercariae possess a very short tail and are produced by digenean species of several families including medically important species, such as members of the genera Paragonimus Braun, 1899, Nanophyetus Chapin, 1927 and Troglotrema Odhner, 1914. During our survey of cercariae of Paragonimus spp. in Vietnam, we found microcercous cercariae from ten (0.29%) out of 3,400 snails of Triculinae gen. sp. 2. They were morphologically and molecularly analysed for species identification. The molecular analysis, based on ITS2 sequences, revealed two distinct species: four specimens were identical to Paragonimus proliferus Hsia et Chen, 1964 (Paragonimidae Dollfus, 1939), and the other six specimens were closest to members of the family Troglotrematidae Odhner, 1914 and were temporarily named Troglotrematidae gen. sp. Morphologically, cercariae of the two species found in this study are similar to each other in their gross characteristics but can be distinguished from one another by subtle morphological details. The cercaria of P. proliferus has an I-shaped excretory bladder and does not have mucous gland cells. In contrast, that of Troglotrematidae gen. sp. has a Y-shaped excretory bladder and mucous gland cells. Besides, the redia of P. proliferus is elongate with a short intestine and contains 5-6 cercariae whereas that of Troglotrematidae gen. sp. is more round with a longer intestine and harbours 3-4 cercariae. Our results have shown the importance of the shape of the excretory bladder and the presence/absence of mucous gland cells of the cercaria as well as the shape and size of the redia, and its intestinal length as valuable taxonomic characters of intramolluscan trematode larvae. In addition, the finding of similar microcercous cercariae of different species in the same snail species suggests that careful attention to morphological details is required in the differentiation of Paragonimus cercariae and those of closely related species.

Background: The East Route Project (ERP) of the South-to-North Water Diversion Project (SNWDP) stretches across schistosomiasis endemic and non-endemic areas in China, which may lead to the dispersal of Oncomelania hupensis, the intermediate host of Schistosoma japonicum, from permissive areas along the Yangtze River Basin to non-permissive areas in northern China. A previous survey demonstrated that O. hupensis could survive and breed for 13 years (12 generations) after being transferred to a non-permissive area, and could be infected by S. japonicum. However, it is not clear if the migrated snails will change their ability to transmit S. japonicum., Methods: We infected mice with the cercariae released from the infected transferred snails bred in Jining city of Shandong Province (non-permissive areas) for 13 years. The mice in the control group were infected with cercariae derived from the snails collected in their original habitat (Jiangdu county of Jiangsu Province, permissive areas). Then, we explored the pathogenicity to mice including worm burden, liver egg count and pathology. Additionally, the gene expression profiles of the adult male and female worms recovered from the infected mice were analyzed by RNA sequencing., Results: The worm burden, liver egg count and pathology of the mice infected with cercariae released from transferred snails bred in non-permissive areas for 13 years showed no significant differences, when compared with the control cercariae. Slight changes occurred at the transcription level between adult male and female worms recovered from mice infected with cercariae derived from snails bred in permissive and non-permissive areas. Only fourteen genes were significantly differentially expressed in the comparison of adult female worms, and no significantly differentially expressed gene was found in the comparison of adult male worms., Conclusions: Our findings strongly suggest that transferred snails did not change their schistosomiasis transmission ability and the worms derived from them retained the original pathogenicity, even after migrating from permissive to non-permissive areas for 13 years. Therefore, a long-term surveillance system of snails along the SNWDP is urgently needed to prevent the diffusion of O. hupensis and reduce the risk of transmission of schistosomiasis.

Digenean larvae of hermaphroditic generation - cercariae - are known to be polymorphic at genetic and behavioural levels. Cercariae arise as a result of parthenogenetic reproduction of intramolluscan stages, and represent a clone if a snail was infected with a single miracidium. Here we investigated cercarial clones of Himasthla elongata - namely, the infectivity of cercariae with normal (negative) and deviant (positive) photoreaction. In our study, most H. elongata clones showed intraclonal variance in their response to light. The proportion of photopositive cercariae ranged between 0.2% and 60% in different H. elongata clones. Photopositive larvae demonstrated significantly reduced rates of encystment in Mytilus edulis haemolymph in vitro and in young mussels. We discuss the possible mechanisms behind intraclonal variations, such as non-specific genomic rearrangements.

Diphterostomum brusinae (Zoogonidae) is a digenean species that has been recorded worldwide parasitizing marine fishes. Several species have been synonymized with D. brusinae because they lack conspicuous morphological differences. However, due to the breadth of its geographic distribution and the variety of hosts involved in the life cycles, it is likely to be an assemblage of cryptic species. Diphterostomum flavum n. sp. is described here as a morphologically cryptic relative of D. brusinae, in the fish Pinguipes brasilianus (Pinguipedidae) off the Patagonian coast, Southwestern Atlantic Ocean, and its life cycle is elucidated through morphology and molecular analysis. This species uses the gastropod Buccinanops deformis (Nassariidae) as first and second intermediate host with metacercariae encysting within sporocysts. They also, however, use the polychaete Kinbergonuphis dorsalis (Onuphidae) as second intermediate host. No morphological differences were found between adults of D. flavum n. sp. and D. brusinae; however, the number of penetration glands of the cercariae, a diagnostic feature, differed (9 vs. 3 pairs), as well as the ITS2 sequences for the two species. This work provides morphological and molecular evidence of cryptic diversification among species described as D. brusinae, in which the only clear differences are in larval morphology and host spectrum. The strict specificity to the snail acting as the first intermediate host and the variety of fishes with different feeding habits acting as definitive hosts support the likely existence of multiple cryptic species around the world., (© C. Gilardoni et al., published by EDP Sciences, 2020.)

Schistosomiasis is one of the major parasitic diseases with more than  200 million people infected worldwide every year. Praziquantel is the drug of choice against the schistosomiasis although the use of a single drug to treat such a large amount of infected people appears particularly worrisome. For this reason, the search of new schistosomicidal compounds is viewed as an urgent goal and a number of screening campaigns have been carried out in the past years. The larval stage of Schistosoma (schistosomula) has been widely used in order to identify new compounds against the parasite. Here we describe detailed practical procedures for a luminescence-based assay proven to be highly effective for the selection of schistosomicidal compounds on small and medium-high scale. The assay is based on the quantitation of the parasite ATP, a good indicator of metabolically active cells, as measure of schistosomula viability. This assay is fast and reproducible, and it is suitable either for manual or for semiautomated screenings.

In recent years, the prevalence and infection intensity of Schistosoma japonicum in endemic areas of the Philippines have significantly decreased due to yearly population-based treatment strategies, yet transmission rates remain high and uninterrupted. An important indicator of active disease transmission is the presence of Schistosoma japonicum and its snail intermediate host Oncomelania hupensis quadrasi in freshwater habitats. In this study, we sought to apply a species-specific real-time PCR (qPCR) assay for the detection of S. japonicum and O. hupensis quadrasi in freshwater samples using environmental DNA approach that can complement the commonly utilized malacological survey in determining potential transmission foci in order to have a more effective snail surveillance strategy for schistosomiasis japonica in endemic areas. The newly developed assay was specific to S. japonicum and O. hupensis quadrasi with no amplification detected against non-target trematode Fasciola spp. and snails such as Lymnaea spp., Pomacea canaliculata, and Melanoides spp. that typically co-exist in the same environment. The assay effectiveness was determined using 19 environmental water samples collected from Northern Samar (N = 5 sites), Leyte (N = 11 sites) and Compostela Valley (N = 3 sites) and compared to malacological survey for determining O. hupensis quadrasi snail colonies and snail crushing to visualize S. japonicum cercariae. TaqMan qPCR targeting a short fragment of the cytochrome c oxidase subunit 1 (cox1) gene was positive for S. japonicum in 9 sites, for O. hupensis quadrasi in 9 sites, and for both S. japonicum and O. hupensis quadrasi in 5 sampling sites. Moreover, it was able to detect O. hupensis quadrasi in 3 out of 12 sites found negative and 6 out of 7 sites found positive through malacological survey, and in 4 of the 5 snail sites positive for snails with cercariae. Overall, this method can complement malacological surveys for monitoring of schistosomes in endemic areas of the Philippines, especially those with high risk of human infection., Competing Interests: The authors have declared that no competing interests exist.

Schistosomes are obligate helminths responsible for over 218 million cases of human schistosomiasis in 78 countries around the world. Infection occurs when free-swimming cercariae penetrate human skin and initiate developmental progression into parasitic obligate worms that consume red blood cells. Transcriptomic studies of infectious cercariae reveal abundant mRNAs associated with energy metabolism and host invasion. However, the cercaria is mostly transcriptionally quiescent, suggesting that most mRNAs are primed prior to cercarial escape from the snail host. The use of transcriptomics to understand protein expression presumes that transcription and translation are functionally coupled and the cercarial stage has categorically been treated as a single unit for -omic analysis. Per contra, the relationship between transcription and translation in infectious cercariae has not been described. To understand the correlation between transcription and translation in cercariae, we separately measured nascent translation levels in cercarial heads, cercarial tails and in the developing schistosomula, the next stage of its life cycle. The loss of the cercarial tail is essential for the transformation from a cercaria to a schistosomulum. We observed that translation was initially limited and the translation rate accelerated during the first 72-hours after tail loss. When we tested nascent translation in cercarial heads, cercarial tails, whole cercariae, and 4-hour schistosomula, we found that translation is significantly upregulated in the cercarial tail when compared to the cercarial head and that translation was undetectable in heads using immunofluorescent image quantification (p = .0005). These data represent a major shift in how we understand the cercarial stage. The cercarial head is mostly transcriptionally and translationally quiescent while being sufficient for progression into a schistosomulum. In addition, transcription and translation are not linked in Schistosoma mansoni cercaria. Thus, our current conceptual approach of treating the cercaria as a single functional unit for -omic studies may be insufficient to understand cercarial development., Competing Interests: The authors have declared that no competing interests exist.

Accurate discrimination of the Schistosoma japonicum cercariae gender is very important for establishing monosexual infection animal models and for standardizing the real intensity of infection. In this study, a multiplex PCR technique consisting of two pairs of primers, of which one amplifies a 185-bp band specific for the W chromosome and the other amplifies a 420-bp band for the Z chromosome, was established to sex the S. japonicum cercariae. For male cercariae (ZZ), a single 420-bp band is expected, and for female cercariea (ZW), two distinct 185-bp and 420-bp bands can be observed. There was no cross-reaction with S. mansoni, S. haematobium, Clonorchis sinensis, Paragonimus westermani, and Trichinella spiralis. After sexing the cercariae escaped from a single snail, mice in group A were infected with 60 male cercariae and mice of group B were infected with 40 female cercariae. Meanwhile, mice in group C were infected with 10 male and 10 female cercariae that were sexed by multiplex PCR. At 45 days postinfection, male and female adult worms were recovered to verify the accuracy of multiplex PCR for sexing S. japonicum cercariae and to calculate the male and female survival rate and paired worm ratio. Our results showed that the multiplex PCR technique could distinguish male cercariae with 100% accuracy. However, sometimes the discrimination results of multiplex PCR mis-scored mixed sexual cercariae as female cercariae. The mean male adult worm burden in mice of group C was 10.7 ± 2.4, and the mean female adult worm burden was 7.7 ± 2.5. There was a significant difference between the male worm burden and female worm burden in group C. The P value was 0.013. The real paired worm ratio of group C was 74.2% (95%CI 56.6~91.8%). These results demonstrated a male-biased sex ratio in the mice model with equilibrated sex ratio cercariae infection, as predicted by our multiplex PCR technique. In conclusion, our multiplex PCR technique is an effective tool for sexing S. japonicum cercariae, especially for distinguishing male cercariae, which is of great value for establishing monosexual cercariae infection mice models to harvest male adult worms for anti-schistosomal drug screening.

Background: The full scope of the genes expressed by schistosomes during intramolluscan development has yet to be characterized. Understanding the gene products deployed by larval schistosomes in their snail hosts will provide insights into their establishment, maintenance, asexual reproduction, ability to castrate their hosts, and their prolific production of human-infective cercariae. Using the Illumina platform, the intramolluscan transcriptome of Schistosoma mansoni was investigated in field-derived specimens of the prominent vector species Biomphalaria pfeifferi at 1 and 3 days post infection (d) and from snails shedding cercariae. These S. mansoni samples were derived from the same snails used in our complementary B. pfeifferi transcriptomic study. We supplemented this view with microarray analyses of S. mansoni from B. glabrata at 2d, 4d, 8d, 16d, and 32d to highlight robust features of S. mansoni transcription, even when a different technique and vector species was used., Principal Findings: Transcripts representing at least 7,740 (66%) of known S. mansoni genes were expressed during intramolluscan development, with the greatest number expressed in snails shedding cercariae. Many transcripts were constitutively expressed throughout development featuring membrane transporters, and metabolic enzymes involved in protein and nucleic acid synthesis and cell division. Several proteases and protease inhibitors were expressed at all stages, including some proteases usually associated with cercariae. Transcripts associated with G-protein coupled receptors, germ cell perpetuation, and stress responses and defense were well represented. We noted transcripts homologous to planarian anti-bacterial factors, several neural development or neuropeptide transcripts including neuropeptide Y, and receptors that may be associated with schistosome germinal cell maintenance that could also impact host reproduction. In at least one snail the presence of larvae of another digenean species (an amphistome) was associated with repressed S. mansoni transcriptional activity., Conclusions/significance: This in vivo study, emphasizing field-derived snails and schistosomes, but supplemented with observations from a lab model, provides a distinct view from previous studies of development of cultured intramolluscan stages from lab-maintained organisms. We found many highly represented transcripts with suspected or unknown functions, with connection to intramolluscan development yet to be elucidated., Competing Interests: The authors have declared that no competing interests exist.

The phylogenetic position of most xiphidiocercariae from subgroups Cercariae virgulae and Cercariae microcotylae remains unknown or unclear, even at the family level. In this paper, we studied the morphology and molecular phylogeny of 15 microcotylous and virgulate cercariae (11 new and four previously described ones). Based on morphological and molecular data, we suggested five distinct morphological types of xiphidiocercariae, which are a practical alternative to Cercariae virgulae and Cercariae microcotylae subgroups. Four of these types correspond to actual digenean taxa (Microphallidae, Lecithodendriidae, Pleurogenidae and Prosthogonimidae), while the fifth is represented by Cercaria nigrospora Wergun, 1957, which we classified on the basis of molecular data for the first time. We reassessed the relative importance of morphological characters used for the classification of virgulate and microcotylous cercariae, and discussed the main evolutionary trends within xiphidiocercariae. Now stylet cercariae can be reliably placed into several sub-taxa of Microphalloidea on the basis of their morphological features.