The genetic structure of populations of Isthmiophora melis (Schrank, 1788) (Digenea: Echinostomatidae). Does the host's diet matter?

BACKGROUND: Here we provide a comparative analysis of the genetic structure of populations (based on nad1 mtDNA) of Isthmiophora melis isolated from the American mink (Neogale vison), an introduced invasive species, commonly occurring in the territory of Poland, and from the striped field mouse (Apodemus agrarius). METHODS: A total of 133 specimens of I. melis were obtained from naturally infected N. vison collected from six localities in Poland (108 samples) and 25 individuals of I. melis from A. agrarius. All sequences of the nad1 gene obtained during the present study were assembled and aligned. The standard statistics for haplotype composition, i.e., the number of haplotypes, haplotype diversity, nucleotide diversity, and average number of nucleotide differences, were calculated. Haplotype analysis and visualization of haplotype frequency among populations were performed using a median-joining network. RESULTS: Based on the samples collected from different localities in Poland, our study revealed that the overall genetic diversity of I. melis isolated from the American mink and of the striped field mouse do not differ significantly. The median-joining network showed that the three main haplotypes are in the centre of a star-like structure, with the remaining haplotypes as the satellites, reflecting the recent expansion of the populations. CONCLUSIONS: The overall genetic diversity of I. melis isolated from the American mink and striped field mouse reveals a high level of homogeneity. Moreover, regional differences in the food composition of the definitive hosts play an important role in shaping the genetic structure of the trematode populations.

Life history strategies of Cotylurus spp. Szidat, 1928 (Trematoda, Strigeidae) in the molecular era - Evolutionary consequences and implications for taxonomy.

Species of Cotylurus Szidat, 1928 (Diplostomoidea: Strigeidae) are highly specialized digeneans that parasitize the gastrointestinal tract and bursa of Fabricius of water and wading birds. They have a three-host life cycle; the role of first intermediate host is played by pulmonate snails, while a wide range of water snails (both pulmonate and prosobranch) and leeches are reported as second intermediate hosts. Unfortunately, species richness, molecular diversity and phylogeny of metacercariae of Cotylurus spp. (tetracotyle) occurring in snails remain poorly understood. Thus, we have performed the parasitological and taxonomical examination of tetracotyles form freshwater snails from Poland, supplemented with adult Strigeidae specimens sampled from water birds. In this study we report our use of recently obtained sequences of two molecular markers (28S nuclear large ribosomal subunit gene (28S rDNA) and the cytochrome c oxidase subunit 1 (CO1) fragment), supplemented by results of a method of species delimitation (GMYC) and haplotype analysis to analyse some aspects of the ecology, taxonomy, and phylogeny of members of the genus Cotylurus. The provided phylogenetic reconstructions discovered unexpectedly high molecular diversity within Cotylurus occurring in snails, with clearly expressed evidence of cryptic diversity and the existence of several novel-species lineages. The obtained results revealed the polyphyletic character of C. syrius Dubois, 1934 (with three separate molecular species-level lineages) and C. cornutus (Rudolphi, 1809) Szidat, 1928 (with four separate molecular species-level lineages). Moreover, we demonstrated the existence of two divergent phylogenetical and ecological lineages within Cotylurus (one using leeches and other snails as second intermediate hosts), differing significantly in their life history strategies.

Leeches as the intermediate host for strigeid trematodes: genetic diversity and taxonomy of the genera Australapatemon Sudarikov, 1959 and Cotylurus Szidat, 1928.

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

Causes of Fatal Cyathostomiasis in Brown Booby (Sula leucogaster) from Brazil: Identification of Pathogen and Implications for Management.

Fatal infection by Cyathostoma (Cyathostoma) phenisci (Nematoda: Syngamidae), was identified in 2 of 52 brown boobies (Sula leucogaster) collected on beaches in the state of Rio de Janeiro, Brazil, and admitted to the veterinary clinic for rehabilitation. Both infected birds were in poor physical condition, with atrophied pectoral muscles, and died soon after starting treatment. The parasitological and pathological examination of the carcasses revealed the presence of C. (C.) phenisci in the trachea, resulting in tracheitis, as well as severe parasitic granulomatous bronchopneumonia caused by eggs deposited in the lungs. In our opinion, these serious pathological changes were the primary cause of chronic respiratory illness. This is the first description of fatal cyathostomiasis in a fish-eating avian host caused by infection by a member of the subgenus Cyathostoma (Cyathostoma). Therefore, it is reasonable to consider C. (C.) phenisci to be a real threat to a wide range of their definitive hosts, and cyathostomiasis should be considered in the differential diagnosis for fish-eating marine birds, even in cases without respiratory signs. This is also the first record of the genus Cyathostoma in Brazil.

The systematic position and structure of the genus Leyogonimus Ginetsinskaya, 1948 (Platyhelminthes: Digenea) with comments on the taxonomy of the superfamily Microphalloidea Ward, 1901.

The systematic position, phylogenetic relationships and composition of the genus Leyogonimus Ginetsinskaya, 1948 have always been uncertain. In the present study, we investigate the taxonomic position and phylogenetic relationships between the type-species L. polyoon (Linstow, 1887) and L. postgonoporus (Neiland, 1951) (previously classified as Macyella), based on newly obtained partial sequences of the nuclear large ribosomal subunit DNA. To test some of the previously proposed systematic arrangements, we have also sequenced specimens of Stomylotrema vicarium Braun, 1901 and Phaneropsolus sp. Our results clearly demonstrate that both L. polyoon and L. postgonoporus belong to the family Pleurogenidae Looss, 1899 within the superfamily Microphalloidea. Thus, the Leyogonimidae Dollfus, 1951 should be recognized as a synonym of the Pleurogenidae. Leyogonimus polyoon clearly constitutes a separate, sister branch to the clade consisting of Collyricloides massanae Vaucher, 1969 and L. postgonoporus. Based on these results, we resurrect the genus Macyella Neiland, 1951 with type-species M. postgonoporus. Besides, Collyricloides Vaucher, 1968 is synonymized with Macyella resulting in new combination Macyella massanae (Vaucher, 1968) comb. nov. Molecular phylogenetic analysis has demonstrated the lack of a close phylogenetic relationships between Stomylotema vicarium and Leyogonimus previously placed by several authors into the family Stomylotrematidae Poche, 1925. The status of the Phaneropsolidae Mehra, 1935 as independent family was confirmed with the addition of the newly sequenced Phaneropsolus sp. from China.

The genetic structure of hypoderatid mites (Actinotrichida: Astigmata) parasitizing great cormorant (Phalacrocorax carbo) during host post-breeding dispersal in Milicz, SW Poland.

The astigmatid family Hypoderatidae includes over 80 mite species exhibiting peculiar life cycle. Deutonymphs are parasitic instars inhabiting subcutaneous or visceral tissues of birds and rodents, while all other instars are nidicolous forms. In this study we investigated genetic diversity of deutonymphs of two hypoderatid species, Neottialges evansi and Phalacrodectes gaudi, collected from 16 individuals of the great cormorant Phalacrocorax carbo in SW Poland during host post-breeding dispersal. The initial alternative hypotheses were: (1) populations of deutonymphs in both mite species found in tissues of particular bird individuals form genetically distinct populations, (2) mites are panmictic among hosts. The topologies of NJ phylogenetic trees and median-joining haplotype networks reconstructed for COI haplotypes revealed lack of hostdependent genetic structure in populations of N. evansi and P. gaudi. Furthermore, high haplotype diversity (Hd) and low nucleotide diversity (Pi) prove high genetic differentiation of both mite species. We concluded, that unlimited dispersal of mites among cormorant specimens could be explained by host specific breeding behavior: free mating between cormorants originating from different European populations and low contribution of reunited pairs in breeding colonies in subsequent breeding seasons, reuse of the same nest material by different members of the colony, and host behavior called prospecting.

Unusual way of feeding by the deutonymph of Neottialges evansi (Actinotrichida, Astigmata, Hypoderatidae), a subcutaneous parasite of cormorants, revealed by fine structural analyses.

The parasitic deutonymphs of hypoderatid mites live within the subcutaneous layer of their avian hosts, where they become greatly engorged despite not having functional mouthparts. The method by which they take up nutrients has been mysterious up to now. Here, we report on the morphology of hypoderatid deutonymphs using scanning and transmission electron microscopy and describe structures that may resolve the mystery. The deutonymph of Neottialges evansi (Hypoderatidae) from the cormorant Phalacrocorax carbo is a simply organized stage lacking both mouthparts and a functional foregut. The structure of midgut and hindgut indicate that they are not capable of processing food. The midgut consists of highly branching flat cells and rarely shows a lumen. Almost the entire space between integument, gut remnants and other organs (synganglion, developing gonads) is filled by huge cells containing protein and glycogen granules and numerous lipid inclusions. The anal opening is minute. The structure of the cuticle and epidermis suggests that nutrients are not absorbed through the general integument. Thus the two main existing hypotheses about feeding modes in hypoderatid deutonymphs, anal vs. integumentary food absorbtion, are not supported. We suggest instead that two pairs of genital papillae showing peculiar microanatomical features are actively involved in movement of liquid materials between host and mite and most probably are the nutrient-intake organs. J. Morphol. 277:1368-1389, 2016. © 2016 Wiley Periodicals, Inc.

Taxonomic status of Syngamus nematodes parasitizing passeriform hosts from Central Europe: Morphological, morphometric and molecular identification.

The systematic position and validity of species within genus Syngamus have always been controversial. In this present work, we evaluated the taxonomic status and phylogenetic relationships between three species of Syngamus nematodes (Syngamus trachea, Syngamus taiga and Syngamus merulae) and one taxa, determined only to the generic level, collected from respiratory tracts of passeriform hosts from Central Europe using newly obtained sequences of 2 nuclear markers (internal transcribed spacers 1 and 2 [ITS1, ITS2]) and a fragment of mitochondrial cytochrome oxidase I [COI] gene. Our results clearly showed that S. trachea, S. taiga and S. merulae are valid, molecularly and morphologically distinct species. Moreover, molecular analysis of adult female of Syngamus sp. collected from tracheae of the European robin Erithacus rubecula clearly indicate that these comprise separate species. In the derived phylogeny, the Syngamus clade is divided into two sub-clades: one comprised Syngamus species with a characteristic, well-developed cuticular collar around the oral opening (S. trachea and S. taiga) and a second that groups taxa without or with rudimentary collar (S. merulae and currently sequenced Syngamus sp. from Erithacus rubecula). These results clearly suggest that the degree of collar development (well-developed vs. rudimentary/absent) may be an important phylogenetic feature for determining the structure of the genus Syngamus on subgeneric level. Additionally, our results support historical division of the genus Syngamus into two subgenera S. (Syngamus) and S. (Ornithogamus).

On the systematic position of Collyricloides massanae Vaucher, 1969 (Platyhelminthes: Digenea) with notes on distribution of this trematode species.

The systematic position of the Collyricloides massanae, a rare cyst-dwelling parasite, located on intestinal wall of European birds and rodents, have always been controversial. Based on newly obtained sequences of the 28 sDNA of C. massanae from avian and rodent host from Central Europe, and on the previously published sequences of several genera and families among Microphalloidea, we evaluate its taxonomic position and the phylogenetic relationships within the genera Collyriclum Kossack, 1911 and Collyricloides Vaucher, 1969 which form the family Collyriclidae Ward, 1917. In the cladogram, C. massanae appears among the Pleurogenidae, forming a clade with Gyrabascus amphoraeformis (Modlinger, 1930) and Cortrema magnicaudata (Bykhovskaya-Pavlovskaya, 1950). We reject the commonly accepted placement of Collyricloides as the sister genus to Collyriclum within the Collyriclidae. Besides, we present and discuss the unusual records of C. massanae in the bank vole Myodes glareolus from northeastern Poland.

Phylogenetic relationships and systematic position of the families Cortrematidae and Phaneropsolidae (Platyhelminthes: Digenea).

The systematic position and phylogenetic relationships of the family Cortrematidae Yamaguti, 1958 have always been controversial. In the present study, the phylogenetic relationships of this family and its constituent genera and families within the superfamily Microphalloidea were evaluated using previously published and newly obtained sequences of 28S rDNA of Cortrema magnicaudata (Bykhovskaya-Pavlovskaya, 1950) (Cortrematidae), Phaneropsolus praomydis Baer, 1971 and Microtrema barusi Sitko, 2013 (Phaneropsolidae). Results clearly demonstrate that the genus Cortrema Tang, 1951 is closest to Gyrabascus Macy 1935, both genera forming one of the clades within the family Pleurogenidae in the superfamily Microphalloidea and sharing several important morphological features. Thus, the family Cortrematidae should be considered among synonyms of the Pleurogenidae. Based on the analysis of morphology, C. corti Tang, 1951, C. testilobata (Bykhovskaya-Pavlovskaya, 1953) and C. niloticus Ashour, Ahmed et Lewis, 1994 are considered junior synonyms of C. magnicaudata. The phylogenetic position of P. praomydis as a family-level branch not showing close relationships with other families of the Microphalloidea, supports the status of the Phaneropsolidae as an independent family. The genus Parabascus Looss, 1907 previously considered within the Phaneropsolidae clearly belongs to the Pleurogenidae. In addition, the molecular phylogeny has demonstrated that the recently described phaneropsolid Microtrema barusi belongs to the microphallid genus Microphallus Ward, 1901. Therefore, Microtrema Sitko, 2013 is considered a junior synonym of Microphallus. Our analysis has also confirmed the status of Collyriclidae as a family within the Microphalloidea. Not yet sequenced representatives of other families within the Microphalloidea (e.g. Anenterotrematidae, Eumegacetidae, Renschtrematidae, Stomylotrematidae, etc.) need to be included in future molecular phylogenetic studies to better unravel the taxonomic structure and content of this diverse digenean superfamily.

Extrinsic- and intrinsic-dependent variation in component communities and patterns of aggregations in helminth parasites of great cormorant (Phalacrocorax carbo) from N.E. Poland.

In the present study, we investigate the effect extrinsic (habitat and season) and intrinsic (host's age and sex) factors on the richness, diversity, and structure of parasite component communities and aggregation patterns in the helminth fauna of the great cormorant Phalacrocorax carbo from northeastern Poland. The helminth fauna of cormorants from the brackish water habitat was far richer (30 species) than in those from freshwater lakes (18 species) and strongly depend on season and age of the host. The values of diversity index strongly varied in relation to habitat and host age with clear seasonal differences in the value of diversity index, i.e., its value increased over time in adults from the brackish water habitat and decreased in those from the freshwater lakes. The number of helminths in adult and immature birds varied, depending on the season and habitat: in the brackish water habitat, the overall percentage of helminths was higher in spring than in summer, while in the freshwater habitat a higher proportion of helminths was recorded in summer. During spring, in the brackish water habitat, we observed a higher level of aggregation (for all groups of helminths) than in autumn. The opposite pattern was found in the freshwater habitat. However, this regularity was typical of adult birds only. In immature hosts, the level of aggregation was not predictable and varied among the higher taxa. Our study clearly showed that processes determinate diversity, structure, richness, and patterns of aggregation in helminth assemblages of avian hosts are multi-origin and highly complex.

Cyathostoma (Cyathostoma) phenisci Baudet, 1937 (Nematoda: Syngamidae), a parasite of respiratory tract of African penguin Spheniscus demersus: morphological and molecular characterisation with some ecological and veterinary notes.

Here we provide a morphological and molecular analysis of the taxonomic status of Cyathostoma (Cyathostoma) phenisci Baudet, 1937, a rare nematode parasite of African penguin Spheniscus demersus. Taxonomical evaluation is supplemented wi th ecological and epidemiological analysis of the nematode's occurrence in the African penguin's population. Tracheae and air sacs of 13 among the 94 necropsied birds (overall prevalence 13.8%) contained a total of 33 nematode specimens (20 females, 13 males). The highest prevalence was observed in juveniles (6 infected, 25%) and "blues" (6 infected, 14.3%), followed by nestlings (1 infected, 7.7%); no nematodes were found in adults. Our morphological and morphometric analysis shows that C. phenisci is closely related to another species, Cyathostoma (Cyathostoma) verrucosum (Hovorka & Macko, 1959). The doubtful status of the latter species was confirmed by molecular data: comparison of ITS2 sequence of C. phenisci with previously deposited sequences of C. verrucosum showed 96.3% similarity in this region. On this basis, we recognized Cyathostoma (Cyathostoma) verrucosum (Hovorka & Macko, 1959) as a synonym of Cyathostoma (Cyathostoma) phenisci Baudet, 1937.

Population biology of Contracaecum rudolphii sensu lato (Nematoda) in the great cormorant (Phalacrocorax carbo) from northeastern Poland.

Parameters related to the occurrence, aggregation, and population structure of the nematode Contracaecum rudolphii Hartwich, 1964 , in the great cormorant ( Phalacrocorax carbo ) from northeastern Poland were analyzed. A total of 491 birds of different ages (adults, immatures, and nestlings) was examined; the cormorants studied were taken from both fresh- and brackish water habitats. Contracaecum rudolphii were found in stomachs of 454 birds (92.5%); the 46,244 nematodes included third- (L3) and fourth-stage larvae (L4), and sub-adult and adult females and males. The distribution of nematodes in the host population were highly aggregated. The occurrence of C. rudolphii was significantly dependent on the host's age and habitat, as well as on season; the proportion of development stages differed significantly depending on birds' age and season (the latter only in adult birds). The infrapopulations of C. rudolphii in the adult cormorants showed distinct density-dependent correlations: that is, as the infrapopulation size increased, the number of adult females C. rudolphii decreased, and the proportion of larvae and sub-adult females increased. A higher proportion of larvae and sub-adult females characterized the component population structure of the nematodes in the cormorant nestlings, compared with adult birds, probably because of immune system deficiency in the immature birds, coupled with the development of the nematode population. Seasonal changes in the C. rudolphii population, observed in the adult cormorants, were not related to seasonality of the L3 occurrence in food; instead, the changes are believed to have resulted from independent processes of elimination of the oldest nematodes and their replacement by larval stages that subsequently matured.

Larval Contracaecum sp. (Nematoda: Anisakidae) in the Great Cormorant [Phalacrocorax carbo (L., 1758)] from north-eastern Poland: a morphological and morphometric analysis.

Stomachs of the Great Cormorant (Phalacrocorax carbo) from north-eastern Poland were found to contain adult nematode Contracaecum rudolphii [Hartwich, G., 1964. Revision der Vogelparasitischen Nematoden Mitteleuropas II. Die Gattung Contracaecum Railliet & Henry, 1912. Mitt. Zool. Mus. Berlin 40, 15-53.] sensu lato and nematode larvae which, based on their morphology, were identified as the third (L3) and fourth (L4) stage larvae of Contracaecum sp. Morphology and biometry of the L3 isolated from the Great Cormorant were very similar to those of the L3 of C. rudolphii described by Bartlett [Bartlett, C.M., 1996. Morphogenesis of Contracaecum rudolphii (Nematoda: Ascaridoidea), a parasite of fish-eating birds, in its copepod precursor and fish intermediate hosts. Parasite 4, 367-376.]. In our opinion, L3 and L4 to be larval stages of C. rudolphii. The paper contains detailed descriptions of the L3 and L4 stages of C. rudolphii; the L4 morphology is described in detail for the first time ever. The descriptions are supplemented by drawings and SEM images. Morphology and biometry of larvae were compared with the literature data and discussed.

Digenean fauna of the great cormorant Phalacrocorax carbo sinensis (Blumenbach, 1798) in the brackish waters of the Vistula Lagoon and the Gulf of Gdansk (Poland).

The great cormorant Phalacrocorax carbo sinensis (Blumenbach, 1798) is one of the most important birds in the Vistula Lagoon and the Gulf of Gdansk area. As a typical piscivore, the bird plays a significant part in life cycles of parasites in aquatic ecosystems. Within January 2000-June 2001, a complete helminthological examination was performed on great cormorants collected in the nesting area at Katy Rybackie on the Vistula Spit (80 specimens) and in the Vistula Lagoon (10 specimens). The infection prevalence, mean intensity, and intensity range were 92.2%, 376.5, and 1-4524, respectively. The presence of 9 digenean species (Paryphostomum radiatum (Dujardin, 1845), Petasiger exaeretus Dietz, 1909, P. phalacrocoracis (Yamaguti, 1939), Mesorchis pseudoechinatus (Olsson, 1876), Metorchis xanthosomus (Creplin, 1846), Cryptocotyle concavum (Creplin, 1825), Hysteromorpha triloba (Rudolphi, 1819), Tylodelphys clavata (Nordmann, 1832), and Holostephanus dubinini Vojtek et Vojtkova, 1968) was recorded, P. phalacrocoracis being the most common parasite (prevalence 92.2%, mean intensity 323.8). Tylodelplhys clavata proved a parasite new for the great cormorant moreover the records of P. phalacrocoracis, H. triloba, and H. dubinini are the first in Poland, while C. concavum and M. pseudoechinatus were for the first time recorded in the great cormorant in Poland.