Genomic analysis of Paenibacillus larvae isolates from the Czech Republic and the neighbouring regions of Slovakia.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybee larvae. In the Czech Republic, two large infested regions were recognised. This study aimed to analyse P. larvae strains occurring in the Czech Republic in the years 2016-2017 and to characterise the genetic structure of their population with the use of Enterobacterial Repetitive Intergenic Consensus genotyping (ERIC), multilocus sequence typing (MLST) and whole genome sequence (WGS) analysis. The results were complemented by the analysis of isolates collected in the year 2018 in areas of Slovakia located near the Czechia-Slovakia border. ERIC genotyping revealed that 78.9% of tested isolates belonged to the ERIC II genotype and 21.1% to ERIC I genotype. MLST showed six sequence types with ST10 and ST11 being the most frequent among isolates. Within six isolates we found discrepancies in correlations between MLST and ERIC genotypes. The use of MLST and WGS analysis of isolates revealed that each of the large infested geographic regions had its own dominating P. larvae strains. We assume that these strains represented primary sources of infection in the affected areas. In addition, the sporadic presence of strains identified by core genome analysis as genetically related was unveiled in geographically distant regions suggesting possible human-mediated transmission of AFB.

Central European Strigeidae Railliet, 1919 (Trematoda: Strigeidida): Molecular and comparative morphological analysis suggests the reclassification of Parastrigea robusta Szidat, 1928 into Strigea Abildgaard, 1790.

Strigeidae Railliet, 1919 are digenean parasites of birds and mammals that are characteristic by their cup-shaped forebody and bilobed holdfast organ. Despite that the family is taxonomically unsettled, particularly due to a very limited number of visible autapomorphic identification features, molecular phylogenetics have never been applied to analyze the relationships among European members of Strigeidae except for the genus Ichthyocotylurus. Here, we analyze the Strigeidae found during the examination of Czech birds performed from 1962 to 2017, and we provide comparative measurements and host spectra, including prevalence and intensity; we also provide and analyze sequences of four DNA loci of 12 of the Strigeidae species. We suggest the reclassification of Parastrigea robusta Szidat, 1928 as Strigea robusta (Szidat, 1928) Heneberg and Sitko, 2018 comb. n. The genera Strigea Abildgaard, 1790 and Parastrigea Szidat, 1928 appear paraphyletic, and morphological diagnostic features of genera within Strigeini Dubois, 1936 are invalid. The mute swan Cygnus olor hosts two Cotylurus spp., Cotylurus syrius Dubois, 1934 and a second species with molecular identification features shared in part with Cotylurus cornutus (Rudolphi, 1808) and Cotylurus gallinulae Lutz, 1928. New host records are provided for seven species. Analyses of non-European genera of the Strigeidae are needed to provide an updated key to Strigeini.

Central European parasitic flatworms of the Cyclocoelidae Stossich, 1902 (Trematoda: Plagiorchiida): molecular and comparative morphological analysis suggests the reclassification of Cyclocoelum obscurum (Leidy, 1887) into the Harrahium Witenberg, 1926.

Cyclocoelidae Stossich, 1902 are medium-sized to large digenean bird parasites. Although these parasites bear few visible autapomorphic signs, and their diagnostic characters are unstable in response to the pressure applied during preparation, the numerous hitherto suggested re-classifications within the family have not been supported by any molecular analysis. We analyse here cyclocoelids found during the extensive examination of central European birds performed from 1962 to 2016, provide comparative measurements, host spectra, prevalence and intensity, and provide and analyse sequences of four DNA loci of five of the cyclocoelid species. Cyclocoleum Brandes, 1892 appears paraphyletic; thus we suggest the re-classification of Cyclocoleum obscurum (Leidy, 1887) as Harrahium obscurum (Leidy, 1887) Sitko and Heneberg comb. n. Molecular phylogenetics questioned also the validity of Cyclocoelinae Stossich, 1902 and Hyptiasminae Dollfus, 1948, which formed a single clade, whereas Allopyge Johnston, 1913, Prohyptiasmus Witenberg, 1923 and Morishitium Witenberg, 1928 formed another clade. Haematotrephinae Dollfus, 1948 are newly characterized as having a pretesticular or intertesticular ovary that forms a triangle with the testes. Analyses of non-European genera of the Cyclocoelidae and an examination of the position of families within Echinostomata La Rue, 1926 are needed.

Conservation of the Red Kite Milvus milvus (Aves: Accipitriformes) Is Not Affected by the Establishment of a Broad Hybrid Zone with the Black Kite Milvus migrans migrans in Central Europe.

Among Accipitriformes sensu stricto, only a few species have been reported to form hybrid zones; these include the red kite Milvus milvus and black kite Milvus migrans migrans. M. milvus is endemic to the western Palearctic and has an estimated total population of 20-24,000 breeding pairs. The species was in decline until the 1970s due to persecution and has declined again since the 1990s due to ingestion of rodenticide-treated baits, illegal poisoning and changes in agricultural practices, particularly in its core range. Whereas F1 M. milvus × M. migr. migrans hybrid offspring have been found, F2 and F3 hybrids have only rarely been reported, with low nesting success rates of F1 hybrids and partial hybrid sterility likely playing a role. Here, we analyzed the mitochondrial (CO1 and CytB) and nuclear (Myc) DNA loci of 184 M. milvus, 124 M. migr. migrans and 3 F1 hybrid individuals collected across central Europe. In agreement with previous studies, we found low heterozygosity in M. milvus regardless of locus. We found that populations of both examined species were characterized by a high gene flow within populations, with all of the major haplotypes distributed across the entire examined area. Few haplotypes displayed statistically significant aggregation in one region over another. We did not find mitochondrial DNA of one species in individuals with the plumage of the other species, except in F1 hybrids, which agrees with Haldane´s Rule. It remains to be investigated by genomic methods whether occasional gene flow occurs through the paternal line, as the examined Myc gene displayed only marginal divergence between M. milvus and M. migr. migrans. The central European population of M. milvus is clearly subject to free intraspecific gene flow, which has direct implications when considering the origin of individuals in M. milvus re-introduction programs.

Central European parasitic flatworms of the family Renicolidae Dollfus, 1939 (Trematoda: Plagiorchiida): molecular and comparative morphological analysis rejects the synonymization of Renicola pinguis complex suggested by Odening.

The Renicolidae are digenean parasites of piscivorous and molluscivorous birds. Although they exhibit few morphological autapomorphies and are highly variable, the numerous suggested re-classifications within the family have never been supported by any molecular analyses. We address the possible synonymization of species within the Renicola pinguis complex suggested previously by Odening. We provide and analyse sequences of two nuclear (ITS2, 28S rDNA) and two mitochondrial (CO1, ND1) DNA loci of central European species of the Renicolidae, namely Renicola lari, Renicola pinguis and Renicola sternae sp. n., and we also provide first sequences of Renicola sloanei. The combined molecular and comparative morphological analysis confirms the previously questioned validity of the three Renicola spp. of highly similar morphology, which display strict niche separation in terms of host specificity and selectivity. We identify two previously unreported clades within the genus Renicola; however, only one of them is supported by the analysis of adult worms. We also provide comparative measurements of the three examined closely related central European renicolids, and describe the newly proposed tern-specialized species Renicola sternae sp. n., which was previously repeatedly misidentified as Renicola paraquinta. Based on the extensive dataset collected in 1962-2015, we update the host spectrum of Renicolidae parasitizing central European birds (Renicola bretensis, R. lari, Renicola mediovitellata, R. pinguis, Renicola secunda and R. sternae sp. n.) and discuss their host-specific prevalence and intensity of infections.

Molecular and comparative morphological analysis of central European parasitic flatworms of the superfamily Brachylaimoidea Allison, 1943 (Trematoda: Plagiorchiida).

The Brachylaimoidea are digenean parasites of vertebrates, including humans, domestic animals, poultry and wild game. Numerous Brachylaimoidea, particularly adults of Brachylaima and Leucochloridium, are difficult to identify to species. We provide and analyse sequences of two nuclear (18S rDNA, ITS2) and two mitochondrial (CO1, ND1) DNA loci of central European species of the Brachylaimoidea, namely Leucochloridium holostomum, Leucochloridium paradoxum, Leucochloridium perturbatum, Leucochloridium subtilis, Leucochloridium vogtianum, Urotocus rossitensis, Urogonimus macrostomus, Michajlovia migrata, Leucochloridiomorpha lutea, Brachylaima arcuatus, Brachylaima fuscata and Brachylaima mesostoma. We identified three clades in the genus Leucochloridium, which do not correspond to the previously suggested subgenera Neoleucochloridium, Papilloleucochloridium and Leucochloridium. We reject classification of Urotocus and Urogonimus in Leucochloridiinae, and, instead, re-establish the subfamilies Urotocinae and Urogoniminae. We synonymize the genus Renylaima with the genus Brachylaima. We reject M. migrata as a member of Leucochloridiinae sensu stricto or Brachylaimidae suggested by some previous authors. We found that the previously sequenced Glaphyrostomum sp. does not cluster with any hitherto sequenced Brachylaimidae. We also provide comparative measurements of the examined central European Brachylaimoidea, address the the specificity of their localization in the host and discuss their host-specific prevalence and intensity of infections based on the extensive dataset of birds examined in 1962-2015.

Integrative taxonomy of European parasitic flatworms of the genus Metorchis Looss, 1899 (Trematoda: Opisthorchiidae).

Metorchis spp. are flukes (Platyhelminthes: Digenea) that infect vertebrates, including humans, dogs, cats, poultry and wild game, with cyprinid freshwater fish serving as typical second intermediate hosts. In their definitive hosts, the Metorchis spp. are difficult to identify to species. We provide and analyze sequences of two nuclear (18S rDNA and ITS2) and two mitochondrial (CO1 and ND1) DNA loci of four morphologically identified European species of the Metorchis, namely Metorchis albidus, Metorchis bilis, Metorchis crassiusculus and Metorchis xanthosomus, and of another opisthorchiid, Euamphimerus pancreaticus. DNA analysis suggests that the Metorchis specimens identified morphologically as M. albidus (from Lutra lutra), M. bilis (from Phalacrocorax carbo) and M. crassiusculus (from Aquila heliaca and Buteo rufinus) represent a single species. Thus, M. albidus (Braun, 1893) Loos, 1899 and M. crassiusculus (Rudolphi, 1809) Looss, 1899 are recognized as junior subjective synonyms of M. bilis (Braun, 1790) Odening, 1962. We also provide comparative measurements of the Central European Metorchis spp., and address their tissue specificity and prevalence based on the examination of extensive bird cohort from 1962 to 2015. M. bilis and M. xanthosomus can be morphologically diagnosed by measuring the extent of genitalia relative to body length and by the size ratio of their suckers. They also differ in their core definitive hosts, with ducks (Anas, Aythya) and coots (Fulica) hosting M. xanthosomus, and cormorants (Phalacrocorax), the birds of prey (Buteo, Aquila, etc.), piscivorous mammals (Lutra, Vulpes, Ursus, etc.) and humans hosting M. bilis. Previous reports on the Metorchis spp. contain numerous suspected misidentifications.

Assemblage of filamentous fungi associated with aculeate hymenopteran brood in reed galls.

Monotypic stands of common reed and the reed-gall-associated insect assemblages are distributed worldwide. However, fungi associated with these assemblages have not been characterized in detail. Here we examined 5200 individuals (12 species) of immature aculeate hymenopterans or their parasitoids collected at 34 sampling sites in Central Europe. We noticed fungal outgrowth on exoskeletons of 83 (1.60%) larvae and pupae. The most common host was eudominant Pemphredon fabricii. However, the less abundant aculeate hymenopteran reed gall inquilines were infected at higher prevalence, these included Trypoxylon deceptorium, Trypoxylon minus, Hoplitis leucomelana and Hylaeus moricei (all considered new host records). We identified three fungal species, Penicillium buchwaldii (72% of cases), Aspergillus pseudoglaucus (22%) and Penicillium quebecense (6%). When multibrooded nests were affected, only a part of individuals was infected in 62% of cases. The sampling site-specific infection rate reached up to 13%, thus fungal infections should be considered an important variable driving the abundance of gall inquilines. Infections of generalist host species were more frequent than those of reed gall specialists, suggesting that suboptimal conditions decreased the immunocompetence of non-specialized species, which only occasionally nest in reed galls and feed in reed beds.

Intermediate hosts of the trematode Collyriclum faba (Plagiochiida: Collyriclidae) identified by an integrated morphological and genetic approach.

BACKGROUND: The cutaneous monostome trematode Collyriclum faba (Bremser in Schmalz, 1831) is a bird parasite with a hitherto unknown life cycle and highly focal occurrence across the Holarctic and Neotropic ecozones. METHODS: Representative specimens of benthic organisms were sampled at multiple sites and dates within the known foci of C. faba occurrence in Slovakia. A combined approach involving detailed morphological examination and sequencing of two independent DNA loci was used for their analysis. RESULTS: We elucidated the complete life cycle of C. faba, which we determined to include the aquatic gastropod mollusk Bythinella austriaca (Frauenfeld, 1857) as the first intermediate host, the mayflies of the family Heptageniidae, Ecdyonurus venosus (Fabricius, 1775) and Rhithrogena picteti Sowa, 1971 x iridina (Kolenati, 1839), as the second intermediate hosts, and birds (primarily but not exclusively passeriform birds) as the definitive hosts. Bythinella austriaca occurs focally in the springs of tributaries of the Danube in the Alpine-Carpathian region. The restricted distribution of B. austriaca explains the highly focal distribution of C. faba noticed previously in spite of the broad distribution of its second intermediate and definitive host species. Utilization of both larval and adult Ephemeroptera spp. as the second intermediate hosts explains the known spectrum of the definitive host species, with the highest prevalence in species feeding on larvae of Ephemeroptera, such as Cinclus cinclus (Linnaeus, 1758) and Motacilla cinerea Tunstall, 1771, or adults of Ephemeroptera, such as Sylvia atricapilla (Linnaeus, 1758) and Regulus regulus (Linnaeus, 1758). In this study, we also determine the prevalence and DNA sequences of other immature trematode specimens found in the examined benthic organisms (particularly the families Microphallidae, Troglotrematidae and Nanophyetidae and Euryhelmis zelleri Grabda-Kazubska, 1980, Heterophyidae), and describe cercariae of C. faba. CONCLUSIONS: We determined the full life cycle of the Central European populations of C. faba. We speculate that other species of Bythinella and the closely related genus Amnicola may serve as first intermediate hosts in other parts of the distribution range of C. faba. Similarly, other Ephemeroptera of the family Heptageniidae may serve as the second intermediate hosts of C. faba in the Americas.

Integrative taxonomy of central European parasitic flatworms of the family Prosthogonimidae Lühe, 1909 (Trematoda: Plagiorchiida).

Species of the family Prosthogonimidae are considered the most pathogenic poultry trematodes worldwide, affecting particularly low intensity farming in rural areas. Adults of Prosthogonimus occur mainly in the bursa of Fabricius, oviduct and cloaca of ducks, geese, fowl and other birds feeding at least occasionally on dragonflies or damselflies (Odonata). We analyzed the central European species of the Prosthogonimidae, namely Prosthogonimus cuneatus, Prosthogonimus ovatus, Prosthogonimus pellucidus and Prosthogonimus rarus. We sequenced three nuclear (ITS2) and mitochondrial (CO1, ND1) DNA loci of four species isolated from Anas clypeata, Anas strepera, Anas platyrhynchos, Aythya ferina, Passer domesticus and Turdus merula. Intra- and inter-specific sequence variability revealed that all four species represent distinct well-defined entities. Our data, combined with previously published studies, suggest the return of the name Prosthogonimus rarus Braun, 1901 for Schistogonimus rarus (Braun, 1901). The genus name Schistogonimus Lühe, 1909 is considered a junior synonym of Prosthogonimus Lühe, 1899. We identified the existence of two clades, one represented by P. cuneatus and P. pellucidus, and another one formed by P. ovatus and P. rarus. We also provide comparative measurements of these four central European prosthogonimids, and address their tissue specificity, host-specific prevalence (based on the extensive bird cohort examined in years 1962-2014), and for some bird hosts we address also differences in the prevalence of Prosthogonimus spp. in natural and near-natural wetlands in comparison with fishponds utilized for intense carp production. We provide an updated key to European Prosthogonimus spp. based on their morphological characters.

Focal Philophthalmus gralli infection possibly persists in Melanoides tuberculata over two years following the definitive hosts' removal.

Philophthalmosis is a zoonotic disease associated largely with the spread of the invasive freshwater snail Melanoides tuberculata, serving as an intermediate host. Here we examined Philophthalmus gralli focal fenced infection site reported recently as being associated with Tinamus major and M. tuberculata in Alajuela, Costa Rica. Removal of the definitive hosts allowed us to address also the long-term survival strategy of the parasite. Initially, the snail intermediate hosts displayed high prevalence of P. gralli infection across all its age cohorts. Two years following the removal of definitive hosts, the infection rate decreased by one order of magnitude, while the snails aging less than one year displayed zero infection prevalence. Additionally, phylogenetic analysis of mitochondrial (ND1) and nuclear (ITS1, ITS2) DNA loci revealed negligible intrasite DNA variability of the specimens obtained at the study site in Costa Rica (but not of those obtained earlier in Peru or New Zealand), supporting strongly the hypothesis on focal origin of the infection. The observed dynamics of infection suggests the explanation for the high variability in P. gralli prevalence in intermediate hosts experienced worldwide. We noticed that the reports claiming >20% prevalence of M. tuberculata infection by P. gralli originated exclusively from foci with known eye infection of the definitive hosts, while the P. gralli infection penetrance <2% is typically associated with sites, where the infection of definitive hosts was not observed, suggesting that the infected definitive hosts were present onsite only in the past, or were present only at a site upstream or downstream of the respective sampling site. Thus, this is the first evidence on the possible persistence of eye-trematode infection site for over two years following the last confirmed outbreak in its adult hosts.

Active chi-like sequences are present in the ITS1 region of polyembryonic adult Collyriclum faba trematodes encysted in pairs.

Collyriclum faba (Plagiochiida: Collyriclidae) adults occur in pairs within subcutaneous cysts. Here, we tested the extensive C. faba infrapopulation for five DNA loci known to display variability among Central European C. faba individuals. The infrapopulation tested shared 100% similarity in four of the five mitochondrial and nuclear DNA loci tested. Contrariwise, the internal transcribed spacer 1 (ITS1) loci in all but one individual differed from each other. We found only 0.0-1.5 base substitutions per 1,000 sites within the cysts, while we found 0.7-9.0 substitutions between the cysts of the single host and 3.0-9.0 substitutions when comparing C. faba individuals isolated from different host individuals. We observed the most of the ITS1 variability within 48 bp repetitive sequences featured by the chi-like sequence 5'-GCTTGTCTGCC-3' at their beginning. Similarly to the extensive C. faba infrapopulation examined, we determined the presence of highly variable number of repetitive sequences within the ITS1 locus of C. faba isolated from multiple host species and from various geographic locations. While similar variability was observed earlier in mutually unrelated specimens of several Schistosomatidae and Microphallidae species, here, we for the first time document it among multiple individuals of a single infracommunity possessing single mitochondrial haplotype. Lower ITS1 evolutionary divergence rates observed between individuals within the cysts when compared to those between the cysts suggest that the recombination occurs at multiple stages of the life cycle. We propose DNA recombination involving chi-like sequences to serve as a general feature shared by multiple families of digenetic trematodes to increase genetic diversity of their polyembryonic populations infecting their definitive hosts.