Variation in Paramarteilia canceri infections in velvet crab Necora puber.

Sustainable management of crustacean populations requires an understanding of the range of factors affecting different crustacean species. Recently, a high prevalence of a paramyxid parasite, Paramarteilia canceri, was reported in velvet crabs Necora puber in Ireland. Similar parasites have been known to cause mass mortalities in bivalves and, as velvet crabs are an important commercial species, these parasite infections are cause for concern. The main objective of this study was to examine variation in P. canceri infections in relation to host biology and season over a 2 yr period. In addition, we tested a range of host tissues and organs to gain more information on the host-parasite interaction. The parasite was present in all tissues and organs investigated, including the gonad and eggs of a berried female. Parasite prevalence was highest in the cuticular epithelium and hepatopancreas. Both annual and seasonal variation was found in parasite prevalence and parasite load. No difference was found in parasite prevalence or parasite load with either crab size or crab sex. Granulomas as a response to infection were significantly more abundant in infected velvet crab individuals. The results of this study provide important information on the host-parasite interaction between P. canceri and the velvet crab and highlight the importance of including parasite monitoring in the management of crustacean fisheries.

Schistosomes in the Persian Gulf: novel molecular data, host associations, and life-cycle elucidations.

Avian schistosomes, comprise a diverse and widespread group of trematodes known for their surprising ability to switch into new hosts and habitats. Despite the considerable research attention on avian schistosomes as causatives of the human cercarial dermatitis, less it is known about the diversity, geographical range and host associations of the marine representatives. Our molecular analyses inferred from cox1 and 28S DNA sequence data revealed presence of two schistosome species, Ornithobilharzia canaliculata (Rudolphi, 1819) Odhner, 1912 and a putative new species of Austrobilharzia Johnston, 1917. Molecular elucidation of the life-cycle of O. canaliculata was achieved for the first time via matching novel and published sequence data from adult and larval stages. This is the first record of Ornithobilharzia from the Persian Gulf and globally the first record of this genus in a potamidid snail host. Our study provides: (i) new host and distribution records for major etiological agents of cercarial dermatitis and contributes important information on host-parasite relationships; (ii) highlights the importance of the molecular systematics in the assessment of schistosome diversity; and (iii) calls for further surveys to reach a better understanding of the schistosome diversity and patterns of relationships among them, host associations, transmission strategies and distribution coverage.

Novel molecular data for monogenean parasites of sparid fishes in the Mediterranean and a molecular phylogeny of the Microcotylidae Taschenberg, 1879.

During a study of the monogeneans of four sparid fishes (Diplodus vulgaris, Pagellus bogaraveo, Pagrus pagrus and Sparus aurata) from the Western Mediterranean off Algeria, a large collection of parasites was characterised molecularly (28S rRNA and cox1 genes). A total of 46 partial sequences (23 for each gene) were generated from 38 isolates of monogeneans which included four species (Atrispinum acarne, Microcotyle erythrini (sensu stricto), Sparicotyle chrysophrii and Prostatomicrocotylinae gen. sp.) of the family Microcotylidae, two putative species of the family Capsalidae (Encotyllabe spp.), and one species (Choricotyle chrysophryi) of the family Diclidophoridae. Our study provides (i) the first molecular data for the Capsalidae in the Mediterranean; (ii) the first record of a member of the Prostatomicrocotylinae in the Mediterranean and in a sparid fish (D. vulgaris); (iii) the first cox1 sequences for A. acarne; (iv) the second record of M. erythrini (s.s.) from P. pagrus; and (v) the second confirmed by molecular data record of S. chrysophrii in wild populations of S. aurata. The first phylogenetic hypotheses for the family Microcotylidae developed here, revealed the monophyly of the subfamily Prostatomicrocotylinae and the genus Microcotyle but the relationships among the subfamilies were still largely unresolved with the best represented subfamily Microcotylinae being polyphyletic. Our results highlight the importance of molecular methods in the assessment of monogenean diversity and the need for a thorough taxon-sampling approach to increase the accuracy of phylogenetic reconstruction of the relationships of the large and taxonomically complex polyopisthocotylean family Microcotylidae.

Molecular and morphological characterisation of Diplostomum phoxini (Faust, 1918) with a revised classification and an updated nomenclature of the species-level lineages of Diplostomum (Digenea: Diplostomidae) sequenced worldwide.

We characterised morphologically and molecularly Diplostomum phoxini (Faust, 1918) based on cercarial isolates from the snail Ampullaceana balthica (L.) (Gastropoda: Lymnaeidae) and metacercariae from the Eurasian minnow, Phoxinus phoxinus (L.) (Cypriniformes: Leuciscidae), and provided molecular evidence for the identification of the snail intermediate host. Phylogenetic analyses based on the cytochrome c oxidase subunit 1 (cox1) gene depicted 44 molecularly characterised species and genetically distinct lineages of Diplostomum, and resulted in: (i) a re-identification/re-classification of 98 isolates plus D. baeri sampled in North America; (ii) re-definition of the composition of the D. baeri species complex which now includes nine molecularly characterised species/lineages; (iii) re-definition of the composition of the D. mergi species complex which now includes seven molecularly characterised species/lineages; and (iv) an updated nomenclature for the molecularly characterised species-level lineages of Diplostomum.

Uncharted digenean diversity in Lake Tanganyika: cryptogonimids (Digenea: Cryptogonimidae) infecting endemic lates perches (Actinopterygii: Latidae).

BACKGROUND: Lake Tanganyika is considered a biodiversity hotspot with exceptional species richness and level of endemism. Given the global importance of the lake in the field of evolutionary biology, the understudied status of its parasite fauna is surprising with a single digenean species reported to date. Although the most famous group within the lake's fish fauna are cichlids, the pelagic zone is occupied mainly by endemic species of clupeids (Actinopterygii: Clupeidae) and lates perches (Actinopterygii: Latidae, Lates Cuvier), which are an important commercial source for local fisheries. In this study, we focused on the lake's four lates perches and targeted their thus far unexplored endoparasitic digenean fauna. METHODS: A total of 85 lates perches from four localities in Lake Tanganyika were examined. Cryptogonimid digeneans were studied by means of morphological and molecular characterisation. Partial sequences of the nuclear 28S rRNA gene and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were sequenced for a representative subset of the specimens recovered. Phylogenetic analyses were conducted at the family level under Bayesian inference. RESULTS: Our integrative approach revealed the presence of six species within the family Cryptogonimidae Ward, 1917. Three out of the four species of Lates were found to be infected with at least one cryptogonimid species. Two out of the three reported genera are new to science. Low interspecific but high intraspecific phenotypic and genetic diversity was found among Neocladocystis spp. Phylogenetic inference based on partial 28S rDNA sequences revealed a sister group relationship for two of the newly erected genera and their close relatedness to the widely distributed genus Acanthostomum Looss, 1899. CONCLUSIONS: The present study provides the first comprehensive characterisation of the digenean diversity in a fish family from Lake Tanganyika which will serve as a baseline for future explorations of the lake's digenean fauna. Our study highlights the importance of employing an integrative approach for revealing the diversity in this unique host-parasite system.

Considerations on the taxonomy and morphology of Microcotyle spp.: redescription of M. erythrini van Beneden & Hesse, 1863 (sensu stricto) (Monogenea: Microcotylidae) and the description of a new species from Dentex dentex (L.) (Teleostei: Sparidae).

BACKGROUND: Microcotyle erythrini van Beneden & Hesse, 1863 (Platyhelminthes: Monogenea) and other closely related species of the genus are often considered as cryptic. Records in hosts other than the type-host with no species confirmation by molecular analyses have contributed to this situation. METHODS: Gill parasites of five sparid fishes, Boops boops (L.), Pagellus erythrinus (L.), P. acarne (Risso), Dentex dentex (L.) and Pagrus pagrus (L.), from the Western Mediterranean off Spain were collected. Specimens of Microcotyle spp. were characterised both molecularly and morphologically. Partial fragments (domains D1-D3) of the 28S rRNA gene and the cytochrome c oxidase subunit 1 (cox1) gene were amplified and used for molecular identification and phylogenetic reconstruction. Principal components analysis was used to look for patterns of morphological separation. RESULTS: Molecular analyses confirmed the identity of three species: M. erythrini ex P. erythrinus and Pa. pagrus; M. isyebi Bouguerche, Gey, Justine & Tazerouti, 2019 ex B. boops; and a species new to science described herein, M. whittingtoni n. sp. ex D. dentex. The specific morphological traits and confirmed hosts (P. erythrinus and Pa. pagrus) are delimited here in order to avoid misidentifications of M. erythrini (sensu stricto). Microcotyle erythrini (s.s.) is mostly differentiated by the shape of its haptor, which is also longer than in the other congeners. New morphological and molecular data are provided for M. isyebi from the Spanish Mediterranean enlarging the data on its geographical range. Microcotyle whittingtoni n. sp. is described from D. dentex and distinguished from the remaining currently recognised species of the genus by the number and robustness of the clamps. CONCLUSIONS: New diagnostic morphological traits useful to differentiate Microcotyle spp. are suggested: (i) haptor dimensions including lobes; (ii) the thickness of the clamps; (iii) the size and shape of spines of the genital atrium; (iv) the extension of the posterior extremities of vitelline fields; and (v) the shape of egg filaments. The use of new morphological approaches may allow considering these species of Microcotyle as being pseudocryptic. The use of representative undamaged specimens that have been genetically confirmed as conspecific is considered crucial to avoid abnormally wide morphological ranges that prevent species differentiation.

Advantages and disadvantages of 3D printing for pre-operative planning of revision hip surgery.

Total hip arthroplasty (THA) is one of the most successful and cost-effective interventions in medicine nowadays, providing reliable pain relief and functional improvement to those with osteoarthritis or inflammatory arthritis of the hip (P Kinov, B Antonov,'Possibilities for surgical treatment of acetabular osteolysis subsequent to revision arthroplasty of hip joint', Orthop Trauma. 52, 2015). Revision hip arthroplasty or severe reconstructive procedures in cases with significant anatomical defects require skilled surgical staff and accurate preoperative planning, including bone insufficiency, deficiency, discontinuity (anatomical assessment) as well as augmentation, cage or other requirements. (implant planning). Some authors recommend preoperative 3D model planning for precise anatomical assessment and preoperative training. This method has some limitations but could be successfully used in addition to conventional surgery.

Heterophyid trematodes (Digenea) from penguins: A new species of Ascocotyle Looss, 1899, first description of metacercaria of Ascocotyle (A.) patagoniensis Hernández-Orts et al. (2012), and first molecular data.

Two species of heterophyid trematodes were found in the Magellanic penguin, Spheniscus magellanicus (Forster), from Patagonia, Argentina. Ascocotyle (Ascocotyle) patagoniensis Hernández-Orts et al. (2012) is re-described based on new, properly fixed specimens (original material from South American sea lion, Otaria flavescens Shaw, was from frozen hosts). Metacercariae of this species are reported and described for the first time from the heart of the silversides, Odontesthes argentinensis (Valenciennes) and O. smitti (Lahille), from Patagonia. Ascocotyle (Phagicola) cameliae n. sp. is described from the intestine of S. magellanicus. The new species is placed into the subgenus Phagicola Faust, 1920 because of the presence of a single row of circumoral spines and uterine loops and vitelline follicles being confined posterior to the ventral sucker. However, it differs distinctly from other members of this subgenus by the number (19-24) and length (23-31 µm) of massive circumoral spines and by the morphology of the ventrogenital sac with a large, simple gonotyl devoid of refractile bodies. Molecular data (partial 28S rDNA sequences) for both species are also provided. Matching sequences from metacercarial and adult stages helped elucidate partially the life-cycle of A. (A.) patagoniensis. The interspecific relationships and phylogenetic position of Ascocotyle were further assessed on a broad phylogeny on the Opisthorchioidea Looss, 1899. Ascocotyle (P.) ornamentata Shalaby et al. (1993) described from decomposed worms (all circumoral spines were detached) found in a dog in Egypt, with no type-specimens of this species deposited in a repository collection, is considered to be species inquirenda.

Myogenic fibrosis of the flexor tendons after amphetamine drug abuse.

Myogenic fibrosis of the flexor tendons in a 23-year-old patient was caused by intense drug abuse. He was presented in the clinic with spastic flexor tendon contracture of his right hand. The patient was treated by tendon elongation with a satisfactory result. The treatment of flexor tendon contracture in those cases is very difficult and needs to be done in a complex way.

Species of Lepidapedon Stafford, 1904 (Digenea: Lepidapedidae) from deep-sea fishes in the Western Mediterranean: molecular and morphological evidence.

The species diversity of Lepidapedon Stafford, 1904 (Lepidapediae) in the Western Mediterranean was assessed based on samples from five deep-sea gadiform fishes collected between the Balearic Islands and the Catalonian coast of Spain: Lepidion lepidion (Risso) and Mora moro (Risso) (both Moridae); Coelorinchus mediterraneus Iwamoto & Ungaro and Trachyrincus scabrus (Rafinesque) (both Macrouridae); and Phycis blennoides (Brünnich) (Phycidae). Integration of morphological and molecular evidence (28S rRNA gene and the mitochondrial nad1 gene) revealed that the deep-sea fishes in the Western Mediterranean share two species of Lepidapedon. Lepidapedon desclersae Bray & Gibson, 1995 was recovered in all five fish species of three families [Moridae (L. lepidion and M. moro); Macrouridae (C. mediterraneus and T. scabrus); and Phycidae (P. blennoides)] and L. guevarai Lopez-Roman & Maillard, 1973 was recovered in three fish species of three families [Phycidae (P. blennoides); Macrouridae (T. scabrus); and Moridae (L. lepidion)]. Therefore, both species are considered to be euryxenic but restricted to gadiforms. The voucher material for the two species from different fish hosts is described in detail and the host-related variability based on the morphometric data is assessed.

Exploring the diversity of Diplostomum (Digenea: Diplostomidae) in fishes from the River Danube using mitochondrial DNA barcodes.

BACKGROUND: Metacercariae of Diplostomum are important fish pathogens, but reliable data on their diversity in natural fish populations are virtually lacking. This study was conducted to explore the species diversity and host-parasite association patterns of Diplostomum spp. in a large riverine system in Europe, using molecular and morphological data. METHODS: Twenty-eight species of fish of nine families were sampled in the River Danube at Nyergesújfalu in Hungary in 2012 and Stúrovo in Slovakia in 2015. Isolates of Diplostomum spp. were characterised morphologically and molecularly. Partial sequences of the 'barcode' region of the cytochrome c oxidase subunit 1 (cox1) and complete sequences of the nicotinamide adenine dinucleotide dehydrogenase subunit 3 (nad3) mitochondrial genes were amplified for 76 and 30 isolates, respectively. The partial cox1 sequences were used for molecular identification of the isolates and an assessment of haplotype diversity and possible host-associated structuring of the most prevalent parasite species. New primers were designed for amplification of the mitochondrial nad3 gene. RESULTS: Only lens-infecting Diplostomum spp. were recovered in 16 fish species of five families. Barcoding of representative isolates provided molecular identification for three species/species-level genetic lineages, D. spathaceum, D. pseudospathaceum and 'D. mergi Lineage 2', and three single isolates potentially representing distinct species. Molecular data helped to elucidate partially the life-cycle of 'D. mergi Lineage 2'. Many of the haplotypes of D. spathaceum (16 in total), D. pseudospathaceum (15 in total) and 'D. mergi Lineage 2' (7 in total) were shared by a number of fish hosts and there was no indication of genetic structuring associated with the second intermediate host. The most frequent Diplostomum spp. exhibited a low host-specificity, predominantly infecting a wide range of cyprinid fishes, but also species of distant fish families such as the Acipenseridae, Lotidae, Percidae and Siluridae. The nad3 gene exhibited distinctly higher levels of interspecific divergence in comparison with the cox1 gene. CONCLUSIONS: This first exploration of the species diversity and host ranges of Diplostomum spp., in natural fish populations in the River Danube, provided novel molecular, morphological and host-use data which will advance further ecological studies on the distribution and host ranges of these important fish parasites in Europe. Our results also indicate that the nad3 gene is a good candidate marker for multi-gene approaches to systematic estimates within the genus.

From mammals back to birds: Host-switch of the acanthocephalan Corynosoma australe from pinnipeds to the Magellanic penguin Spheniscus magellanicus.

Trophically-transmitted parasites are regularly exposed to potential new hosts through food web interactions. Successful colonization, or switching, to novel hosts, occur readily when 'donor' and 'target' hosts are phylogenetically related, whereas switching between distantly related hosts is rare and may result from stochastic factors (i.e. rare favourable mutations). This study investigates a host-switching event between a marine acanthocephalan specific to pinnipeds that is apparently able to reproduce in Magellanic penguins Spheniscus magellanicus from Brazil. Detailed analysis of morphological and morphometrical data from acanthocephalans from penguins indicates that they belong to Corynosoma australe Johnston, 1937. Partial fragments of the 28S rRNA and mitochondrial cox1 genes were amplified from isolates from penguins and two pinniped species (i.e. South American sea lion Otaria flavescens and South American fur seal Arctocephalus australis) to confirm this identification. Infection parameters clearly differ between penguins and the two pinniped species, which were significantly lower in S. magellanicus. The sex ratio of C. australe also differed between penguins and pinnipeds; in S. magellanicus was strongly biased against males, while in pinnipeds it was close to 1:1. Females of C. australe from O. flavescens were smaller than those from S. magellanicus and A. australis. However, fecundity (i.e. the proportion of fully developed eggs) was lower and more variable in females collected from S. magellanicus. At first glance, the occurrence of reproductive individuals of C. australe in Magellanic penguins could be interpreted as an adaptive colonization of a novel avian host through favourable mutations. However, it could also be considered, perhaps more likely, as an example of ecological fitting through the use of a plesimorphic (host) resource, since the ancestors of Corynosoma infected aquatic birds.

New molecular and morphological data for opecoelid digeneans in two Mediterranean sparid fishes with descriptions of Macvicaria gibsoni n. sp. and M. crassigula (Linton, 1910) (sensu stricto).

Molecular and morphological data were gathered for specimens of species of Macvicaria Gibson & Bray, 1982 and Pseudopycnadena Saad-Fares & Maillard, 1986 (Digenea: Opecoelidae) collected from two sparid fishes, Diplodus vulgaris (Geoffroy Saint-Hilaire) and Sparus aurata L., off the Algerian coast of the Western Mediterranean. Phylogenetic analyses based on ITS1-5.8S-ITS2 and partial 28S rDNA sequences provided evidence for the distinct species status of eight Mediterranean species of Macvicaria. Novel molecular data are provided for four species, M. gibsoni n. sp. and M. crassigula (Linton, 1910) (sensu stricto) based on specimens from D. vulgaris, and M. mormyri (Stossich, 1885) and M. maamouriae Antar, Georgieva, Gargouri & Kostadinova, 2015 based on specimens from S. aurata; descriptions of the molecular voucher material of the former three species are provided. Two species were delineated within the "crassigula" species complex of Macvicaria, M. gibsoni n. sp. and M. crassigula (s. str.), the distinctive differentiating features being the distribution of the dorsal vitelline fields in the forebody, confluent in M. gibsoni n. sp. and non-confluent in M. crassigula (s. str.), and the molecular data for both markers. Sequences associated with morphological description are also provided for the type-species of Pseudopycnadena, P. fischthali Saad-Fares & Maillard, 1986, based on material from D. vulgaris.

Skoulekia erythrini n. sp. (Digenea: Aporocotylidae): a parasite of Pagellus erythrinus (L.) (Perciformes: Sparidae) from the western Mediterranean with an amendment of the generic diagnosis.

A new aporocotylid, Skoulekia erythrini n. sp., is described from the heart, cephalic kidney and gill blood vessels of the common pandora Pagellus erythrinus (L.) collected from several localities of the western Mediterranean off Spain. The new species differs from the type- and only species of the genus, S. meningialis Alama-Bermejo, Montero, Raga & Holzer, 2011 in possessing a symmetrical body (vs laterally curved), short tegumental spines (3-4 vs 7-10 µm) without hooked ends, diffuse (vs conspicuous) oesophageal gland-cells, a relatively longer oesophagus, a testis that is shorter in relation to body length and a much smaller seminal vesicle (17-34 × 10-26 vs 33-101 × 27-97 µm). Elongated ellipsoidal eggs of S. erythrini n. sp. were found trapped in gill vessels in histological sections. Phylogenetic analyses based on partial 28S rDNA and ITS2 sequences supported the placement of the new species within Skoulekia and the close relationships of this genus with Psettarium Goto & Ozaki, 1929 and Pearsonellum Overstreet & Køie, 1986. Skoulekia meningialis is described from a new sparid host, Diplodus puntazzo (Walbaum), collected off Santa Pola, Spain. The new morphological data for the two Skoulekia spp. and a re-examination of three paratypes of S. meningialis pinpointed features amending both the description of S. meningialis and the generic diagnosis of Skoulekia.

Molecular characterisation of four echinostomes (Digenea: Echinostomatidae) from birds in New Zealand, with descriptions of Echinostoma novaezealandense n. sp. and Echinoparyphium poulini n. sp.

Morphological and molecular characterisation of echinostome specimens (Digenea: Echinostomatidae) recovered in one Anas platyrhynchos L. and one Cygnus atratus (Latham) (Anseriformes: Anatidae) from New Zealand revealed the presence of two known species, Echinostoma miyagawai Ishii, 1932 and Echinoparyphium ellisi (Johnston & Simpson, 1944) and two species new to science. Comparative morphological and phylogenetic analyses supported the distinct species status of Echinostoma novaezealandense n. sp. ex Branta canadensis (L.), A. platyrhynchos and C. atratus, and Echinoparyphium poulini n. sp. ex C. atratus. Echinostoma novaezealandense n. sp., a species of the "revolutum" species complex characterised by the possession of a head collar armed with 37 spines, keyed down to E. revolutum but was distinguished from the latter in having a much narrower body with almost parallel margins, longer oesophagus, wider cirrus-sac, larger seminal vesicle, much smaller ventral sucker, ovary, Mehlis' gland and testes, more anteriorly located ovary and testes, and distinctly smaller eggs (81-87 × 42-53 vs 106-136 × 55-70 µm). This new species appears similar to Echinostoma acuticauda Nicoll, 1914 described in Australia but differs in having a longer forebody, more posteriorly located ovary and testes, and much smaller eggs (81-87 × 42-53 vs 112-126 × 63-75 µm). Echinoparyphium poulini n. sp. is differentiated from the four species of Echinoparyphium possessing 37 collar spines considered valid as follows: from E. chinensis Ku, Li & Chu, 1964 in having a much smaller body, four (vs five) angle spines and simple seminal vesicle (vs bipartite); from E. schulzi Matevosyan, 1951 in having a less robust body at a comparable body length, much smaller ventral sucker, ovary and testes, and longer but narrower eggs (87-109 × 50-59 vs 70-85 × 60-84 µm); and from the two smaller forms, E. serratum Howell, 1968 and E. aconiatum Dietz, 1909, in a number of additional metrical features correlated with body size and especially in the possession of much larger collar spines. Partial fragments of the mitochondrial nad1 and 28S rRNA genes were amplified for representative isolates of the four species and analysed together with sequences for Echinostoma spp. and Echinoparyphium spp. available on GenBank. Phylogenetic analyses based on the mitochondrial nad1 gene revealed congruence between the molecular data and species identification/delineation based on morphology; this was corroborated by the 28S rDNA sequence data.

Molecular analyses reveal high species diversity of trematodes in a sub-Arctic lake.

To identify trematode diversity and life-cycles in the sub-Arctic Lake Takvatn, Norway, we characterised 120 trematode isolates from mollusc first intermediate hosts, metacercariae from second intermediate host fishes and invertebrates, and adults from fish and invertebrate definitive hosts, using molecular techniques. Phylogenies based on nuclear and/or mtDNA revealed high species richness (24 species or species-level genetic lineages) and uncovered trematode diversity (16 putative new species) from five families typical in lake ecosystems (Allocreadiidae, Diplostomidae, Plagiorchiidae, Schistosomatidae and Strigeidae). Sampling potential invertebrate hosts allowed matching of sequence data for different stages, thus achieving molecular elucidation of trematode life-cycles and exploration of host-parasite interactions. Phylogenetic analyses also helped identify three major mollusc intermediate hosts (Radix balthica, Pisidium casertanum and Sphaerium sp.) in the lake. Our findings increase the known trematode diversity at the sub-Arctic Lake Takvatn, showing that digenean diversity is high in this otherwise depauperate sub-Arctic freshwater ecosystem and indicating that sub-Arctic and Arctic ecosystems may be characterised by unique trematode assemblages.

Morphology and molecules reveal the alien Posthodiplostomum centrarchi Hoffman, 1958 as the third species of Posthodiplostomum Dubois, 1936 (Digenea: Diplostomidae) in Europe.

Metacercariae of two species of Posthodiplostomum Dubois, 1936 (Digenea: Diplostomidae) were subjected to morphological and molecular studies: P. brevicaudatum (von Nordmann, 1832) from Gasterosteus aculeatus (L.) (Gasterosteiformes: Gasterosteidae), Bulgaria (morphology, cox1 and ITS1-5.8S-ITS2) and Perca fluviatilis L. (Perciformes: Percidae), Czech Republic (morphology, cox1, ITS1-5.8S-ITS2 and 28S); and P. centrarchi Hoffman, 1958 from Lepomis gibbosus (L.) (Perciformes: Centrarchidae), Bulgaria (morphology, cox1 and ITS1-5.8S-ITS2) and Slovakia (cox1 and ITS1-5.8S-ITS2). In addition, cercariae of P. cuticola (von Nordmann, 1832) from Planorbis planorbis (L.) (Mollusca: Planorbidae), Lithuania (morphology and cox1) and metacercariae of Ornithodiplostomum scardinii (Schulman in Dubinin, 1952) from Scardinius erythrophthalmus (L.) (Cypriniformes: Cyprinidae), Czech Republic, were examined (morphology, cox1, ITS1-5.8S-ITS2 and 28S). These represent the first molecular data for species of Posthodiplostomum and Ornithodiplostomum Dubois, 1936 from the Palaearctic. Phylogenetic analyses based on cox1 and ITS1-5.8S-ITS2, using O. scardinii as the outgroup and including the three newly-sequenced Posthodiplostomum spp. from Europe and eight published unidentified (presumably species-level) lineages of Posthodiplostomum from Canada confirmed the distinct status of the three European species (contrary to the generally accepted opinion that only P. brevicaudatum and P. cuticola occur in the Palaearctic). The subspecies Posthodiplostomum minimum centrarchi Hoffmann, 1958, originally described from North America, is elevated to the species level as Posthodiplostomum centrarchi Hoffman, 1958. The undescribed "Posthodiplostomum sp. 3" of Locke et al. (2010) from centrarchid fishes in Canada has identical sequences with the European isolates of P. centrarchi and is recognised as belonging to the same species. The latter parasite, occurring in the alien pumpkinseed sunfish Lepomis gibbosus in Europe, is also supposed to be alien for this continent. It is speculated that it colonised Europe long ago and is currently widespread (recorded in Bulgaria, Slovakia and Spain); based on the cox1 sequence of an adult digenean isolate from the Ebro Delta, Spain, only the grey heron (Ardea cinerea L.) (Ciconiiformes: Ardeidae) is known to be its definitive host in Europe.

Molecular evidence for the existence of species complexes within Macvicaria Gibson & Bray, 1982 (Digenea: Opecoelidae) in the western Mediterranean, with descriptions of two new species.

Morphological and molecular characterisation of specimens of Macvicaria spp. (Digenea: Opecoelidae) from five species of Mediterranean sparids (Teleostei: Sparidae) sampled in the Bizerte Lagoon and the Bay of Bizerte (Tunisia) revealed the presence of two species complexes designated as "obovata" and "crassigula" groups. Detailed comparative morphological and phylogenetic analyses revealed that two of the specimen sets, one from each complex, represent species new to science. M. maamouriae n. sp. from Sparus aurata L. and Lithognathus mormyrus (L.) appeared genetically similar to M. obovata (Molin, 1859) but differs in having a much larger ventral sucker relative to body width, a cirrus-sac extending dorsally to the posterior margin of the ventral sucker or more posterior, vitelline fields comprising distinctly more abundant vitelline follicles, reaching the level of the pharynx both ventrally and dorsally and confluent dorsally in the forebody, and an ovary contiguous with the anterior testis. Macvicaria bartolii n. sp. from Diplodus annularis (L.) and Spondyliosoma cantharus (L.) belongs to the "crassigula" group and is characterised by having almost exclusively dorsal vitelline follicles forming two non-confluent fields in the forebody. Additional morphological data are provided for Macvicaria dubia (Stossich, 1905), a poorly known parasite of Oblada melanura (L.). Morphological descriptions are associated with ITS1-5.8S-ITS2 and 28S rDNA sequences for the three Mediterranean species of Macvicaria Gibson & Bray, 1982. The results of our study suggest that further exploration of the species diversity within this genus should be based on both morphological and molecular data.

Integrative taxonomic approach to the cryptic diversity of Diplostomum spp. in lymnaeid snails from Europe with a focus on the 'Diplostomum mergi' species complex.

BACKGROUND: Recent molecular studies have discovered substantial unrecognised diversity within the genus Diplostomum in fish populations in Europe and North America including three species complexes. However, data from the first intermediate host populations are virtually lacking. This study addresses the application of an integrative taxonomic approach to the cryptic species diversity of Diplostomum spp. in natural lymnaeid snail populations in Europe with a focus on the 'D. mergi' species complex. METHODS: Totals of 1,909 Radix auricularia, 349 Radix peregra, 668 Stagnicola palustris and 245 Lymnaea stagnalis were sampled at five reservoirs of the Ruhr river system in Germany and screened for infections with Diplostomum spp. Cercariae were examined and identified alive, fixed and under scanning electron microscopy. Sequences from the barcode region of the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene and from the internal transcribed spacer cluster (ITS1-5.8S-ITS2) of the rRNA gene were amplified for 51 and 13 isolates, respectively. RESULTS: Detailed morphological and molecular analyses provided evidence for three named species (Diplostomum spathaceum, D. pseudospathaceum and D. parviventosum), and a further four species-level lineages ('D. mergi Lineages 2-4' and 'Diplostomum sp. Clade Q' in the lymnaeid snail populations from the Ruhr river basin. The paper provides the first descriptions of molecularly identified cercariae of D. spathaceum and of the cercariae of D. parviventosum, three lineages of the 'D. mergi' species complex and of 'Diplostomum sp. Clade Q'. CONCLUSION: The integration of molecular and morphological evidence for Diplostomum spp. achieved in this study will serve as a baseline for species identification of these important parasites of snail and fish populations and thus advance further studies on the distribution of Diplostomum spp. in Europe.

An integrative taxonomic investigation of the diversity of digenean parasites infecting the intertidal snail Austrolittorina unifasciata Gray, 1826 (Gastropoda: Littorinidae) in Australia.

We investigated for the first time the digenean parasites of Austrolittorina unifasciata Gray (Gastropoda: Littorinidae), a periwinkle snail inhabiting the rocky shores of Australia. Here we present detailed morphological descriptions and molecular data (sequences for the mitochondrial cox1 and the nuclear 28S rRNA gene) for the cercariae and intramolluscan stages of the digenean parasites found. Five species, one each of the families Notocotylidae Lühe, 1909, Gorgocephalidae Manter, 1966 and Philophthalmidae Looss, 1899, and two of the family Renicolidae Dollfus, 1939, were recorded and characterised molecularly. Phylogenetic analyses at the superfamily level provided evidence for the familial and generic affiliation of the species and their relationships with congeners. This study is the first to provide data on the life cycle of a species of the family Gorgocephalidae, a parasite of kyphosid fish for which only adult stages had, thus far, been described. The relatively high prevalence of this species allowed mapping of the cox1 haplotype distribution of Gorgocephalus sp. Aus along the southern coast of New South Wales.