An inside out journey: biogenesis, ultrastructure and proteomic characterisation of the ectoparasitic flatworm Sparicotyle chrysophrii extracellular vesicles.

BACKGROUND: Helminth extracellular vesicles (EVs) are known to have a three-way communication function among parasitic helminths, their host and the host-associated microbiota. They are considered biological containers that may carry virulence factors, being therefore appealing as therapeutic and prophylactic target candidates. This study aims to describe and characterise EVs secreted by Sparicotyle chrysophrii (Polyopisthocotyla: Microcotylidae), a blood-feeding gill parasite of gilthead seabream (Sparus aurata), causing significant economic losses in Mediterranean aquaculture. METHODS: To identify proteins involved in extracellular vesicle biogenesis, genomic datasets from S. chrysophrii were mined in silico using known protein sequences from Clonorchis spp., Echinococcus spp., Fasciola spp., Fasciolopsis spp., Opisthorchis spp., Paragonimus spp. and Schistosoma spp. The location and ultrastructure of EVs were visualised by transmission electron microscopy after fixing adult S. chrysophrii specimens by high-pressure freezing and freeze substitution. EVs were isolated and purified from adult S. chrysophrii (n = 200) using a newly developed ultracentrifugation-size-exclusion chromatography protocol for Polyopisthocotyla, and EVs were characterised via nanoparticle tracking analysis and tandem mass spectrometry. RESULTS: Fifty-nine proteins involved in EV biogenesis were identified in S. chrysophrii, and EVs compatible with ectosomes were observed in the syncytial layer of the haptoral region lining the clamps. The isolated and purified nanoparticles had a mean size of 251.8 nm and yielded 1.71 × 108 particles · mL-1. The protein composition analysis identified proteins related to peptide hydrolases, GTPases, EF-hand domain proteins, aerobic energy metabolism, anticoagulant/lipid-binding, haem detoxification, iron transport, EV biogenesis-related, vesicle-trafficking and other cytoskeletal-related proteins. Several identified proteins, such as leucyl and alanyl aminopeptidases, calpain, ferritin, dynein light chain, 14-3-3, heat shock protein 70, annexin, tubulin, glutathione S-transferase, superoxide dismutase, enolase and fructose-bisphosphate aldolase, have already been proposed as target candidates for therapeutic or prophylactic purposes. CONCLUSIONS: We have unambiguously demonstrated for the first time to our knowledge the secretion of EVs by an ectoparasitic flatworm, inferring their biogenesis machinery at a genomic and transcriptomic level, and by identifying their location and protein composition. The identification of multiple therapeutic targets among EVs' protein repertoire provides opportunities for target-based drug discovery and vaccine development for the first time in Polyopisthocotyla (sensu Monogenea), and in a fish-ectoparasite model.

Small intestinal flukes of the genus Metagonimus (Digenea: Heterophyidae) in Europe and the Middle East: A review of parasites with zoonotic potential.

The heterophyid trematode Metagonimus romanicus (Ciurea, 1915) (Digenea) is redescribed on the basis of type material from domestic dogs (Canis familiaris) in Romania, vouchers from experimentally infected cats (Felis catus) and adults recovered from golden hamsters (Mesocricetus auratus) infected with metacercariae from scales of chub (Squalius cephalus) and common nase (Chondrostoma nasus) (Cypriniformes: Leuciscidae) in Hungary. This trematode, endemic to Europe and neighbouring regions (northwestern Türkiye), was previously misidentified as M. yokogawai (Katsurada, 1912), a zoonotic parasite of humans in East Asia. However, the two species differ considerably both genetically and morphologically, e.g., in the position of the ventral sucker, the presence of the prepharynx, the anterior extent of the vitelline follicles and the posterior extent of the uterus. Metagonimus ciureanus (Witenberg, 1929) (syn. Dexiogonimus ciureanus Witenberg, 1929), described from domestic cats and dogs in Israel, is a valid species distributed in the Middle East and Transcaucasia, which is also confirmed by molecular data. It differs from all Metagonimus species, including M. romanicus, in having symmetrical testes instead of the oblique testes of the other congeners. The zoonotic significance of M. romanicus and M. ciureanus is unclear, but appears to be low in Europe, mainly because raw or undercooked, whole fish with scales are generally not consumed. Accidental infection of fishermen by metacercariae in the scales when cleaning fish is more likely, but has never been reported. Remains of cyprinoids with scales infected with metacercariae of Metagonimus spp. can be an important natural source of infection for dogs, cats, and other carnivores, which can serve as a reservoir for these parasites.

Title: Petits trématodes intestinaux du genre Metagonimus (Digenea : Heterophyidae) en Europe et au Moyen-Orient : revue de parasites à potentiel zoonotique. Abstract: Le trématode Heterophyidae Metagonimus romanicus (Ciurea, 1915) (Digenea) est redécrit sur la base de matériel type provenant de chiens domestiques (Canis familiaris) en Roumanie, de vouchers issus de chats (Felis catus) infectés expérimentalement et d'adultes collectés chez des hamsters dorés (Mesocricetus auratus) infectés par des métacercaires provenant d'écailles de chevesne commun (Squalius cephalus) et de nase commun (Chondrostoma nasus) (Cypriniformes : Leuciscidae) de Hongrie. Ce trématode, endémique d'Europe et des régions voisines (nord-ouest de la Turquie), avait été précédemment identifié à tort comme étant M. yokogawai (Katsurada, 1912), un parasite zoonotique des humains en Asie de l'Est. Cependant, les deux espèces diffèrent considérablement sur le plan génétique et morphologique, par exemple par la position de la ventouse ventrale, la présence du prépharynx, l'étendue antérieure des follicules vitellins et l'étendue postérieure de l'utérus. Metagonimus ciureanus (Witenberg, 1929) (syn. Dexiogonimus ciureanus Witenberg, 1929), décrite chez des chats et des chiens domestiques en Israël, est une espèce valide répartie au Moyen-Orient et en Transcaucasie, ce qui est également confirmé par des données moléculaires. Cette espèce diffère de toutes les espèces de Metagonimus, y compris M. romanicus, par ses testicules symétriques au lieu des testicules obliques des autres congénères. L'importance zoonotique de M. romanicus et M. ciureanus n'est pas claire, mais semble faible en Europe, principalement parce que les poissons n'y sont généralement pas consommés crus ou insuffisamment cuits et entiers avec les écailles. L'infection accidentelle des pêcheurs par des métacercaires présents dans les écailles lors du nettoyage du poisson est plus probable mais n'a jamais été signalée. Les restes de poissons cyprinoïdes avec les écailles, infectés par des métacercaires de Metagonimus spp. peuvent être une source naturelle importante d'infection pour les chiens, les chats et autres carnivores, qui peuvent servir de réservoir à ces parasites.

New species of Echinobothrium van Beneden, 1849 (Cestoda: Diphyllidea) from Indo-Pacific maskrays (Neotrygon Castelnau) off Australia and Borneo.

Four new diphyllidean species of the genus Echinobothrium van Beneden, 1849 are described from Indo-Pacific maskrays (Neotrygon Castelnau, Dasyatidae). Echinobothrium giraffaeous sp. n. from Neotrygon leylandi (Last) off northern Australia, Echinobothrium ivanovae sp. n. from Neotrygon orientalis Last, White et Serét off Borneo, and Echinobothrium bethae sp. n. from Neotrygon varidens (Garman) off Borneo are distinguished from all but one of the 33 valid species of the genus by the possession of the outermost A hooks with an extended base into which the bases of the three outermost B hooks are inserted. Echinobothrium rhynchobati (Khalil et Abdul-Salam, 1989) is the only known species with this unique feature, but its rostellum has a system of interlocking knobs and sockets that articulate bases of the A and B type hooks with one another, which is not present in any of the newly described species. Echinobothrium tyleri sp. n. from Neotrygon australiae Last, White et Serét off northern Australia is distinguished from all known species of Echinobothrium by its unique rostellar hook formula {2-3 18/17 2-3}. With the present addition of four new species, the central Indo-Pacific realm becomes the major hotspot for Echinobothrium, from which 13 species have been reported.

Human diphyllobothriosis in Taiwan: A review of cases and molecular evidence of Dibothriocephalus nihonkaiensis.

Diphyllobothriosis is an infectious disease caused by the consumption of raw freshwater or marine fish containing larvae of broad tapeworms (Diphyllobothriidae). In the present study, we critically reviewed all cases of human diphyllobothriosis reported from Taiwan, including unpublished reports from hospitals in Taipei. Genotyping based on mitochondrial DNA marker (cox1) confirmed that two of the recent cases were caused by Dibothriocephalus nihonkaiensis, which is not native to Taiwan and was probably imported with Pacific salmon infected with larvae of D. nihonkaiensis. The causative species previously reported in Taiwan could not be definitively confirmed. However, considering the distribution of Dibothriocephalus latus, which is not endemic in Taiwan, past cases diagnosed as D. latus are questionable.

TAPEWORMS (CESTODA: OPHIOTAENIA) FROM THE NORTHERN COTTONMOUTH (AGKISTRODON PISCIVORUS).

The northern cottonmouth, Agkistrodon piscivorus (Lacépède, 1789) (Viperidae: Crotalinae), occurs in the southeastern United States and is one of the few semiaquatic vipers in the world. Three proteocephalid tapeworms of the genus OphiotaeniaLa Rue, 1911 (Cestoda), have been described from this venomous snake. A critical evaluation of type specimens and tapeworms recently collected from A. piscivorus has revealed that only 2 species, Ophiotaenia marenzelleri (Barrois, 1898) and Ophiotaenia grandisLa Rue, 1911 (new synonym Ophiotaenia agkistrodontis [Harwood, 1933]), are specific parasites of this crotaline viper; both species are redescribed here. Ophiotaenia grandis was originally described from a mixture of 2 species: 'true' O. grandis, which is indistinguishable from O. agkistrodontis described 22 yr later and thus considered to be a junior synonym of O. grandis, and O. marenzelleri. Ophiotaenia marenzelleri, also reported from the pygmy rattlesnake, Sistrurus miliarius (Linnaeus, 1766), is a larger tapeworm with a massive scolex, a large cirrus sac, a very large, annular vaginal sphincter near the equatorial gonopore, and an oval, 3-layered embryophore surrounded by a nearly square hyaline outer membrane. Ophiotaenia grandis is much smaller and more slender and has a narrower scolex, a smaller cirrus sac and vaginal sphincter, a 2-layered embryophore, and a distinctly pre-equatorial gonopore. In addition to O. marenzelleri and O. grandis, other Ophiotaenia species typical of semiaquatic snakes (Colubridae: Natricinae) may be present in the northern cottonmouth, which serves only as a postcyclic or occasional host. There is also a tabular summary of 18 species of Ophiotaenia from semiaquatic snakes worldwide, with information on their hosts, distribution, and taxonomically important characters, including key measurements.

Ups and downs of infections with the broad fish tapeworm Dibothriocephalus latus in Europe (Part II) and Asia from 1900 to 2020.

The broad fish tapeworm, Dibothriocephalus latus (Diphyllobothriidea), is the most important causative agent of diphyllobothriosis, a fish-borne zoonosis, in Europe. Part I of this review focused on the occurrence of D. latus in northwestern and central Europe, particularly in Fennoscandia, the Baltic, the Alpine lakes and Danube River regions during 1900-2020. Part II summarises data on D. latus from the European and Asian parts of Russia and from Asian countries. The tapeworm has occurred throughout Russia, with the most important foci in (i) the Republic of Karelia in the northwest of European Russia, (ii) the Volga River basin in the central and southern parts of European Russia, (iii) the Ob-Irtysh rivers region in the Ural region, (iv) the Yenisei-Lena rivers region in Siberia, and (v) the Lake Baikal basin in Siberia. The incidence of diphyllobothriosis has declined in recent decades, especially in European Russia, but zoonosis is still prevalent in some regions of Siberia. Cases reported from Arctic regions, the region around Lake Baikal, and the Pacific coast, including the Amur basin, however, were probably misidentifications with D. dendriticus and/or D. nihonkaiensis. No other Asian country where D. latus findings represented either imported cases or misidentifications had natural focus of diphyllobothriosis. Patterns of distribution of D. latus occurrence were similar in all Eurasian foci between 1900 and 2020. The numbers of records were associated with historical and epidemiological milestones of particular time periods.

The evolution of endoparasitism and complex life cycles in parasitic platyhelminths.

Within flatworms, the vast majority of parasitism is innate to Neodermata, the most derived and diversified group of the phylum Platyhelminthes.1,2 The four major lineages of Neodermata maintain various combinations of life strategies.3 They include both externally (ecto-) and internally feeding (endo-) parasites. Some lineages complete their life cycles directly by infecting a single host, whereas others succeed only through serial infections of multiple hosts of various vertebrate and invertebrate groups. Food sources and modes of digestion add further combinatorial layers to the often incompletely understood mosaic of neodermatan life histories. Their evolutionary trajectories have remained molecularly unresolved because of conflicting evolutionary inferences and a lack of genomic data.4 Here, we generated transcriptomes for nine early branching neodermatan representatives and performed detailed phylogenomic analyses to address these critical gaps. Polyopisthocotylea, mostly hematophagous ectoparasites, form a group with the mostly hematophagous but endoparasitic trematodes (Trematoda), rather than sharing a common ancestor with Monopisthocotylea, ectoparasitic epithelial feeders. Phylogenetic placement of the highly specialized endoparasitic Cestoda alters depending on the model. Regardless of this uncertainty, this study brings an unconventional perspective on the evolution of platyhelminth parasitism, rejecting a common origin for the endoparasitic lifestyle intrinsic to cestodes and trematodes. Instead, our data indicate that complex life cycles and invasion of vertebrates' gut lumen, the hallmark features of these parasites, evolved independently within Neodermata. We propose the demise of the traditionally recognized class Monogenea and the promotion of its two subclasses to the class level as Monopisthocotyla new class and Polyopisthocotyla new class.

Secretion of extracellular vesicles during ontogeny of the tapeworm Schistocephalus solidus.

We provide the first ultrastructural evidence of the secretion of extracellular vesicles (EVs) across all parasitic stages of the tapeworm Schistocephalus solidus (Müller, 1776) (Cestoda: Diphyllobothriidea) using a laboratory life cycle model. We confirmed the presence of EV-like bodies in all stages examined, including the hexacanth, procercoids in the copepod, Macrocyclops albidus (Jurine, 1820), plerocercoids from the body cavity of the three-spined stickleback, Gasterosteus aculeatus Linnaeus, and adults cultivated in artificial medium. In addition, we provide description of novel tegumental structures potentially involved in EV biogenesis and the presence of unique elongated EVs similar to those previously described only in Fasciola hepatica Linnaeus, 1758 (Trematoda), Hymenolepis diminuta (Rudolphi, 1819) (Cestoda), and Trypanosoma brucei Plimmer et Bradford, 1899 (Kinetoplastida).

Fish tapeworms (Cestoda) in the molecular era: achievements, gaps and prospects.

The tapeworms of fishes (Chondrichthyes and Actinopterygii) account one-third (1670 from around 5000) of the total tapeworm (Platyhelminthes: Cestoda) species diversity. In total 1186 species from 9 orders occur as adults in elasmobranchs (sharks, rays and chimaeras), and 484 species from 8 orders mature in ray-finned fishes (referred to here as teleosts). Teleost tapeworms are dominated by freshwater species (78%), but only 3% of elasmobranch tapeworms are known from freshwater rays of South America and Asia (Borneo). In the last 2 decades, vast progress has been made in understanding species diversity, host associations and interrelationships among fish tapeworms. In total, 172 new species have been described since 2017 (149 from elasmobranchs and 23 from teleosts; invalidly described taxa are not included, especially those from the Oriental region). Molecular data, however, largely limited to a few molecular markers (mainly 28S rDNA, but also 18S and cox1), are available for about 40% of fish tapeworm species. They allowed us to significantly improve our understanding of their interrelationships, including proposals of a new, more natural classification at the higher-taxonomy level (orders and families) as well as at the lower-taxonomy level (genera). In this review, we summarize the main advances and provide perspectives for future research.

Archigetes Leuckart, 1878 (Cestoda, Caryophyllidea): diversity of enigmatic fish tapeworms with monoxenic life cycles.

The caryophyllidean genus Archigetes Leuckart, 1878 is unique among all tapeworms in that its species can mature in invertebrate hosts (Oligochaeta), i.e., have a monoxenic (direct) life cycle. All five species were described as progenetic plerocercoids in oligochaetes and two of them also as adults from cypriniform fishes. Two species, A. sieboldi Leuckart, 1878 and A. iowensis Calentine, 1962, were found in North America in non-native common carp (Cyprinus carpio). A molecular study of caryophyllideans from the southern United States has revealed the occurrence of three new species in native freshwater fishes (Catostomidae, Ictiobinae): Archigetes loculotruncatus n. sp. from Ictiobus bubalus, I. niger and Carpiodes cyprinus is the largest representative of the genus and differs by a loculotruncate scolex. Archigetes megacephalus n. sp. from Ictiobus niger, I. bubalus and I. cyprinellus is characterised by a prominent, bothrioloculodiscate scolex. Archigetes vadosus n. sp. from I. bubalus is typified by a globular scolex with very shallow loculi; it differs from the closely related A. sieboldi in the shape of the body, with a distinct neck region and a scolex wider than the remaining body. Archigetes iowensis Calentine, 1962 becomes a junior synonym of Paraglaridacris limnodrili (Yamaguti, 1934). The generic diagnosis of Archigetes is amended and a key to identification of North American taxa is provided. Species of Archigetes and Paraglaridacris differ from each other most conspicuously in the structure of the ovary, which is follicular in Archigetes versus compact in Paraglaridacris.

TITLE: Archigetes Leuckart, 1878 (Cestoda, Caryophyllidea) : diversité de cestodes de poissons énigmatiques à cycle de vie monoxène. ABSTRACT: Le genre Archigetes Leuckart, 1878 (Caryophyllidea) est unique parmi les cestodes en ce sens que ses espèces peuvent atteindre la maturité chez des hôtes invertébrés (Oligochaeta), c'est-à-dire avoir un cycle de vie monoxénique (direct). Les cinq espèces ont été décrites à partir de plérocercoïdes progénétiques chez des oligochètes et deux d'entre elles aussi à partir d'adultes chez des poissons cypriniformes. Deux espèces, A. sieboldi Leuckart, 1878 et A. iowensis Calentine, 1962, ont été trouvées en Amérique du Nord chez des carpes communes (Cyprinus carpio), non indigènes. Une étude moléculaire des caryophyllidés du sud des États-Unis a révélé la présence de trois nouvelles espèces chez les poissons d'eau douce indigènes (Catostomidae, Ictiobinae). Archigetes loculotruncatus n. sp. d'Ictiobus bubalus, I. niger et Carpiodes cyprinus est le plus grand représentant du genre et est caractérisé par un scolex loculotronqué. Archigetes megacephalus n. sp. d'Ictiobus niger, I. bubalus et I. cyprinellus est caractérisé par un scolex proéminent et bothrioloculodisqué. Archigetes vadosus n. sp. d'I. bubalus est caractérisé par un scolex globulaire avec des loges très peu profondes, et diffère d'A. sieboldi, étroitement apparenté, par la forme du corps, avec une région du cou distincte et un scolex plus large que le reste du corps. Archigetes iowensis Calentine, 1962 devient un synonyme plus récent de Paraglaridacris limnodrili (Yamaguti, 1934). Le diagnostic générique d'Archigetes est modifié et une clé d'identification des taxons nord-américains est fournie. Les espèces d'Archigetes et de Paraglaridacris diffèrent de la manière la plus visible les unes des autres par la structure de l'ovaire, qui est folliculaire chez Archigetes par opposition à compact chez Paraglaridacris.

Genetic interrelationships of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidea), the causative agent of sparganosis in Europe.

The geographic distribution of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidea), the causative agent of food/water-borne sparganosis, is restricted to Europe, where infected canids, felids, mustelids, suids, and reptiles have been documented from Poland, Ukraine, Belarus, Russia, Serbia, Estonia, Latvia, and Finland. The main objective of the current study was to map the molecular divergence of S. erinaceieuropaei from Finland using the complete sequences of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1 mtDNA). Seven cox1 haplotypes were determined in 15 tapeworms from Eurasian lynx (Lynx lynx) from three localities in southern Finland. In addition, the first inter-population study of S. erinaceieuropaei based on currently obtained data on cox1 from Finland and previously published data from Finland, Latvia, Ukraine, and Poland, was performed. The haplotype network showed a star-like pattern without specific subdivision of lineages according to the locality. Samples from Finland, Latvia, and Poland shared several haplotypes and formed the common Baltic lineage. The haplotype of S. erinaceieuropaei from Ukraine was unique and placed on a separate mutational pathway, suggesting a different lineage of the parasite.

TITLE: Interrelations génétiques de Spirometra erinaceieuropaei (Cestoda, Diphyllobothriidea), l'agent causal de la sparganose en Europe. ABSTRACT: La distribution géographique de Spirometra erinaceieuropaei (Cestoda : Diphyllobothriidea), l'agent causal de la sparganose d'origine alimentaire/hydrique, est limitée à l'Europe, où des canidés, félidés, mustélidés, suidés et reptiles infectés ont été documentés en Pologne, Ukraine, Biélorussie, Russie, Serbie, Estonie, Lettonie et Finlande. L'objectif principal de la présente étude était de cartographier la divergence moléculaire de S. erinaceieuropaei de Finlande à l'aide des séquences complètes du gène mitochondrial de la sous-unité 1 de la cytochrome c oxydase (ADNmt cox1). Sept haplotypes cox1 ont été déterminés chez quinze cestodes du Lynx d'Eurasie (Lynx lynx) de trois localités du sud de la Finlande. En outre, la première étude inter-populationnelle de S. erinaceieuropaei basée sur les données actuellement obtenues sur cox1 de Finlande et sur des données précédemment publiées de Finlande, Lettonie, Ukraine et Pologne, a été réalisée. Le réseau d'haplotypes a montré un motif en étoile sans subdivision spécifique des lignées selon la localité. Des échantillons de Finlande, Lettonie et Pologne partagent plusieurs haplotypes et forment la lignée commune de la Baltique. L'haplotype de S. erinaceieuropaei d'Ukraine est unique et placé sur une voie de mutation distincte suggérant une lignée différente du parasite.

The Acute Immune Responses of the Common Carp Cyprinus carpio to PLGA Microparticles-The Interactions of a Teleost Fish with a Foreign Material.

Poly lactic-co-glycolic acid (PLGA) particles safely and effectively deliver pharmaceutical ingredients, with many applications approved for clinical use in humans. In fishes, PLGA particles are being considered as carriers of therapeutic drugs and vaccine antigens. However, existing studies focus mainly on vaccine antigens, the endpoint immune responses to these (e.g., improved antibody titres), without deeper understanding of whether fishes react to the carrier. To test whether or not PLGA are recognized by or interact at all with the immune system of a teleost fish, we prepared, characterized and injected PLGA microparticles intraperitoneally into common carp. The influx, phenotype of inflammatory leukocytes, and their capacity to produce reactive oxygen species and phagocytose PLGA microparticles were tested by flow cytometry, qPCR, and microscopy. PLGA microparticles were indeed recognized. However, they induced only transient recruitment of inflammatory leukocytes that was resolved 4 days later whereas only the smallest µm-sized particles were phagocytosed. The overall response resembled that described in mammals against foreign materials. Given the similarities between our findings and those described in mammals, PLGA particles can be adapted to play a dual role as both antigen and drug carriers in fishes, depending on the administered dose and their design.

Ups and downs of infections with the broad fish tapeworm Dibothriocephalus latus in Europe from 1900 to 2020: Part I.

The broad fish tapeworm, Dibothriocephalus latus (Diphyllobothriidea), is the most frequent causative agent of diphyllobothriosis, a fish-borne zoonosis, in Europe. Diphyllobothriosis is characterized by the transmission of D. latus larvae to humans via the consumption of raw, marinated, smoked or inadequately cooked fish products. The most important European foci of diphyllobothriosis have been Fennoscandia, the Baltic region, the Alpine lakes region, the Danube River region, and several endemic regions in Russia. This review provides basic data on the biology, life cycle, host specificity, methods of identification of D. latus, and a detailed summary of its occurrence in intermediate and definitive hosts in Fennoscandia and the Baltic, Alpine, and Danube regions during the last 120 years (1900-2020). Deeper insight into the unique pattern of distribution of D. latus in endemic regions is provided. The numbers of records are associated with several milestones of particular time periods. The first milestone (historical), which influenced studies on D. latus in Europe, was the period during and after World War II (1941-1950). The second milestone (epidemiological) was the decade 1981-1990, when previous massive health campaigns led to a marked decline of diphyllobothriosis in Europe and less published data on D. latus. Based on recent data, the broad fish tapeworm is either absent or present at very low prevalences in Fennoscandia and the Baltic and Danube regions, but the Alpine lakes region represents a continuous ongoing circulation of the parasite in the natural environment and humans.

Tapeworms as pathogens of fish: A review.

Tapeworms (Cestoda) represents a species rich (about 5000 species) group of flatworms (Neodermata) parasitizing all groups of vertebrates including humans, with about 1000 species parasitizing elasmobranchs and almost 500 occurring in teleosts as adults. They are common parasites of cultured fish, both as adults and larvae (metacestodes), but only few adult tapeworms are actually pathogenic for their fish hosts. In contrast, cestode larvae can be harmful for fish, especially plerocercoids migrating throughout their tissue and internal organs. Current knowledge of host-parasite relationships, including immune response of fish infected with tapeworms, is still insufficient to enable adequate control of cestodoses, and most data available were obtained several decades ago. Treatment of fish infected with adult tapeworms is effective, especially with praziquantel, whereas the treatment of metacestodes is problematic. Control measures include interruption of the complex life cycle and prevention of transport of uninspected fish to new region.

Evolutionary transitions in broad tapeworms (Cestoda: Diphyllobothriidea) revealed by mitogenome and nuclear ribosomal operon phylogenetics.

Broad tapeworms (Diphyllobothriidea) are parasites whose adults are capable of infecting a wide range of freshwater, marine and terrestrial tetrapods including humans. Previous works examining the evolution of habitat and host use in this group have been hampered by the lack of a well-resolved phylogeny. In order to produce a robust phylogenetic framework for diphyllobothriideans, we sequenced the complete mitochondrial genome of 13 representatives, carefully chosen to cover the major clades, and two outgroup species representing the Spathebothriidea and Haplobothriidea. In addition, complementary data from the nuclear ribosomal operon was sequenced for 10 representative taxa. Mitogenomes and ssrDNA and lsrDNA were used towards elucidating the phylogenetic framework for the Diphyllobothriidea. The Cephalochlamydidae is confirmed as the earliest diverging diphyllobothriidean lineage, and Solenophoridae and Diphyllobothriidae are sister groups. We infer a probable freshwater origin of the diphyllobothriideans. The ancestral condition for life cycle complexity could not be unambiguously resolved. However, we infer exclusive use of a three-host life cycle following the origin of the Solenophoridae + Diphyllobothriidae. Regarding definitive host use, although we infer reptiles as the most likely ancestral condition, this result should be revisited with a more densely sampled phylogeny in future studies. Freshwater habitat is used by the early diverging lineages within the Solenophoridae + Diphyllobothriidae clade. For the latter, habitat use shifts between freshwater and marine environments, and definitive host use includes marine and terrestrial mammals and birds. We use mitochondrial genomes to distinguish Schistocephalus species occurring in different species of sticklebacks and demonstrate conspecificity of Ligula cf. intestinalis specimens collected from two Fennoscandian ringed seal subspecies.

Marine fish imported from Argentina as source of human diphyllobothriosis in Europe? Ecological evidence from dolphins.

Diphyllobothriosis caused by the Pacific broad tapeworm Adenocephalus pacificus (syn. Diphyllobothrium pacificum) is an emerging parasitic disease reported also from non-endemic areas, including Europe (Spain). The origin of these human cases is unknown but should be related to fresh marine fish imported from endemic areas. In this study, we molecularly confirmed common dolphins Delphinus delphis off Argentina as euparatenic transit hosts of A. pacificus. Preliminary analysis of their stomach content, together with data from previous studies from the Southwest Atlantic, showed that common dolphins feed almost exclusively on schooling Argentine hake Merluccius hubbsi and Argentine anchovy Engraulis anchoita. Therefore, we suggest that Argentine hake and Argentine anchovy may represent the intermediate hosts of A. pacificus in the Southwest Atlantic, but also in Europe to where M. hubbsi is imported on ice (unfrozen).

Diphyllobothrium sprakeri n. sp. (Cestoda: Diphyllobothriidae): a hidden broad tapeworm from sea lions off North and South America.

BACKGROUND: The systematic of several marine diphyllobothriid tapeworms of pinnipeds has been revised in recent years. However, 20 species of Diphyllobothrium from phocids and otariids are still recognized as incertae sedis. We describe a new species of Diphyllobothrium from the intestine of California sea lions Zalophus californianus (Lesson) (type-host) and South American sea lions Otaria flavescens (Shaw). METHODS: Zalophus californianus from the Pacific coast of the USA and O. flavescens from Peru and Argentina were screened for parasites. Partial fragments of the large ribosomal subunit gene (lsrDNA) and the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene were amplified for 22 isolates. Properly fixed material from California sea lions was examined using light and scanning electron microscopy. RESULTS: A total of four lsrDNA and 21 cox1 sequences were generated and aligned with published sequences of other diphyllobothriid taxa. Based on cox1 sequences, four diphyllobothriid tapeworms from O. flavescens in Peru were found to be conspecific with Adenocephalus pacificus Nybelin, 1931. The other newly generated sequences fall into a well-supported clade with sequences of a putative new species previously identified as Diphyllobothrium sp. 1. from Z. californianus and O. flavescens. A new species, Diphyllobothrium sprakeri n. sp., is proposed for tapeworms of this clade. CONCLUSIONS: Diphyllobothrium sprakeri n. sp. is the first diphyllobothriid species described from Z. californianus from the Pacific coast of North America, but O. flavescens from Argentina, Chile and Peru was confirmed as an additional host. The present study molecularly confirmed the first coinfection of two diphyllobothriid species in sea lions from the Southern Hemisphere.

Hidden diversity of the most basal tapeworms (Cestoda, Gyrocotylidea), the enigmatic parasites of holocephalans (Chimaeriformes).

Gyrocotylideans are evolutionary ancient parasitic flatworms, and like their hosts-a relict group of holocephalan fishes (Chimaeriformes)-they are considered to be "living fossils" of a vanished past. However, the species diversity, host associations and biogeography of these most basal tapeworms are poorly known. Herein, we provide evidence of a conspicuous contrast between the genetic and morphological data based on an examination of newly collected and properly processed Gyrocotyle specimens (hologenophores) isolated from holocephalans off Taiwan and Argentina. Our molecular data, inferred from three genes (COI, 28S rRNA, 18S rRNA), showed unexpected genetic interrelationships among isolates of the genus Gyrocotyle, because each of the four genotypes from Taiwan clustered with isolates of distinct gyrocotylideans from the North Atlantic. Three genotypes of Gyrocotyle from Taiwan were morphologically almost indistinguishable from each other but represented distinct genetic lineages; a single specimen of Gyrocotyle sp. genotype 4 exhibited a clear genetic and morphological distinctness, though its formal description as a new species would be premature. Additionally, specimens of Gyrocotyle rugosa Diesing, 1850, from the type host Callorhinchus callorynchus from Argentina, provided the first genetic data on the type species of the genus and enabled us to characterise it, which is necessary for future taxonomic studies. The finding of some specimens of Gyrocotyle sp. genotype 3 in Chimaera phantasma, and another one in C. cf. argiloba, together with the putative conspecificity of an unidentified gyrocotylidean from Callorhinchus milii off Australia and G. rugosa from C. callorynchus off Argentina, represent evidence that one gyrocotylidean species may parasitise more than one holocephalan host species. Existing taxonomic problems and conflicts between morphological and molecular data on species of Gyrocotyle can only be resolved if hologenophores from type hosts and localities of nominal taxa are properly characterised genetically and morphologically.

Sparganosis (Spirometra) in Europe in the Molecular Era.

Sparganosis is a relatively neglected foodborne and waterborne disease caused by species of the tapeworm genus Spirometra, the global distribution of which has not been sufficiently recognized. Known mainly as a zoonosis of East Asia, its species are native to all inhabited continents including Europe. Spirometra has been reported from numerous wildlife species from 17 European countries, and a critical review confirmed 17 autochthonous and 8 imported human clinical cases. We present the first molecular evidence of the coincident presence of 2 species in Europe and review the current distribution to raise awareness of the parasite in this region. Spirometra erinaceieuropaei is restricted to Europe and Spirometra mansoni represents a lineage distributed mainly across Asia and Oceania that reaches Europe. The parasite is common in Eastern Europe and its distribution has potential to expand along with its invasive or migrating mammal hosts, spreading the risks of human infection.

Extracellular vesicles secreted by model tapeworm Hymenolepis diminuta: biogenesis, ultrastructure and protein composition.

We provided the first known evidence of the presence and release of extracellular vesicles in adults of important model tapeworm Hymenolepis diminuta. Two different subtypes have been observed on the surface of the worm and among the secretory products confirmed by several microscopical methods. Proteomic analysis revealed the presence of parasite-specific proteins as well as those of the host in purified extracellular vesicles. Among the protein cargo, we identified potential drug targets, vaccine candidates and H. diminuta antigens. Finally, the protein composition further revealed proteins participating in the endosomal complex required for transport-dependent biogenesis pathway.