Aneta Kostadinova

Phylogenetic relationships of Echinostoma Rudolphi, 1809 (Digenea: Echinostomatidae) and related genera re-assessed via DNA and morphological analyses

In order to investigate the relationships within the Echinostomatidae two data sets of gene sequences were analysed. The first consisted of all previously published ND1 sequences (20) together with 17 new sequences. The latter represented six species from the cosmopolitan genera Echinostoma, Echinoparyphium, Hypoderaeum and Isthmiophora. The second data-set of ITS sequences again included all previously published sequences (12) and three new sequences from species of Echinostoma, Echinoparyphium and Isthmiophora. All new isolates, as well as voucher material from five previously sequenced isolates, were identified on the basis of morphological characters. The phylogenetic trees inferred from the ND1 data set helped to clarify the generic affiliation of all isolates and confirmed the morphological identifications. The only exception was Echinoparyphium aconiatum, whose current position in the genus Echinoparyphium was not supported by the sequence data. Although the ITS data provided insufficient resolution for an unequivocal solution to the relationships within the genus Echinostoma, it supported the identification of Echinoparyphium ellisi and the distinct species status of three isolates of Echinostoma revolutum as predicted from the ND1 data.

New cryptic species of the ‘revolutum’ group of Echinostoma (Digenea: Echinostomatidae) revealed by molecular and morphological data

BackgroundThe digenean species of Echinostoma (Echinostomatidae) with 37 collar spines that comprise the so-called ‘revolutum’ species complex, qualify as cryptic due to the interspecific homogeneity of characters used to differentiate species. Only five species were considered valid in the most recent revision of the group but recent molecular studies have demonstrated a higher diversity within the group. In a study of the digeneans parasitising molluscs in central and northern Europe we found that Radix auricularia, R. peregra and Stagnicola palustris were infected with larval stages of two cryptic species of the ‘revolutum’ complex, one resembling E. revolutum and one undescribed species, Echinostoma sp. IG. This paper provides morphological and molecular evidence for their delimitation.MethodsTotals of 2,030 R. auricularia, 357 R. peregra and 577 S. palustris were collected in seven reservoirs of the River Ruhr catchment area in Germany and a total of 573 R. peregra was collected in five lakes in Iceland. Cercariae were examined and identified live and fixed in molecular grade ethanol for DNA isolation and in hot/cold 4% formaldehyde solution for obtaining measurements from fixed materials. Partial fragments of the mitochondrial gene nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad 1) were amplified for 14 isolates.ResultsDetailed examination of cercarial morphology allowed us to differentiate the cercariae of the two Echinostoma spp. of the ‘revolutum’ species complex. A total of 14 partial nad 1 sequences was generated and aligned with selected published sequences for eight species of the ‘revolutum’ species complex. Both NJ and BI analyses resulted in consensus trees with similar topologies in which the isolates from Europe formed strongly supported reciprocally monophyletic lineages. The analyses also provided evidence that North American isolates identified as E. revolutum represent another cryptic species of the ‘revolutum’ species complex.ConclusionOur findings highlight the need for further analyses of patterns of interspecific variation based on molecular and morphological evidence to enhance the re-evaluation of the species and advance our understanding of the relationships within the ‘revolutum’ group of Echinostoma.

Composition and structure of the parasite faunas of cod, Gadus morhua L. (Teleostei: Gadidae), in the North East Atlantic

BackgroundAlthough numerous studies on parasites of the Atlantic cod, Gadus morhua L. have been conducted in the North Atlantic, comparative analyses on local cod parasite faunas are virtually lacking. The present study is based on examination of large samples of cod from six geographical areas of the North East Atlantic which yielded abundant baseline data on parasite distribution and abundance.Materials and MethodsA total of 826 fish was sampled in the Baltic, Celtic, Irish and North seas, Icelandic waters and Trondheimsfjord (Norway) in 2002 (spring and autumn) and 2003 (spring). The gills and internal organs (oesophagus, stomach, intestine, pyloric caeca, liver, heart, spleen, gall bladder and gonads) were examined for macroparasites following a standardised protocol. The taxonomic consistency of the identification was ensured thorough the entire study.ResultsWe discuss some problems in parasite identification, outline the composition of the parasite faunas in cod in the six North East Atlantic regions, provide novel data on parasite prevalence and abundance and a comparative assessment of the structure of the regional parasite faunas with respect to the higher-level taxonomic groupings, host specificity and zoogeographical distribution of the parasites. Altogether 57 different parasite forms were found including seven new host records (Diclidophora merlangi, Rhipidocotyle sp., Fellodistomum sp., Steringotrema sp., Cucullanus sp., Spinitectus sp., and Chondracanthus ornatus). The predominant groups of cod parasites were trematodes (19 species) and nematodes (13 species) including larval anisakids which comprised 58.2% of the total number of individuals.ConclusionOur study reveals relatively rich regional parasite faunas in cod from the North East Atlantic which are dominated by generalist parasites with Arcto-Boreal distribution. Further, it provides more detailed data on the distribution in the North East Atlantic of the majority of cod parasites which may serve as baselines for future studies on the effect of climate change. Based on the faunal comparisons, predictions can be made in relation to the structure and diversity of the parasite communities in the North East Atlantic regions studied.

Interrelationships of the Haploporinae (Digenea: Haploporidae): A molecular test of the taxonomic framework based on morphology

The taxonomic framework of the Haploporidae is evaluated and the relationships within the Haploporinae are assessed for the first time at the generic level using molecular data. Partial 28S and complete ITS2 rDNA sequences from representatives of six of the nine recognised genera within the Haploporinae were analysed together with published sequences representing members of two haploporid subfamilies and of the closely related family Atractotrematidae. Molecular analyses revealed: (i) a close relationship between the Atractotrematidae and the Haploporidae; (ii) strong support for the monophyly of the Haploporinae, Dicrogaster and Saccocoelium, and the position of Ragaia within the Haploporinae; (iii) evidence for rejection of the synonymy of Saccocoelioides and Lecithobotrys and the validity of the Dicrogasterinae; and (iv) support for the distinct status of Saccocoelium in relation to Haploporus. The wider sampling within the genera Dicrogaster and Saccocoelium confirmed the distinct status of the included species, thus rejecting previously suggested synonymies. Saccocoelioides, recently transferred to the Chalcinotrematinae, was nested within the Haploporinae and this was largely associated with the position of Forticulcita, resolved as the most basal haploporine genus. Forticulcita also possesses a well-delimited eversible intromittent copulatory organ, a feature unique in the Haploporidae which has not been previously considered an important apomorphy. This, in association with the present hypothesis of the Haploporinae based on molecular data, led us to erect Forticulcitinae subf. n. for Forticulcita; this resolved Saccocoelioides and, by extension the Chalcinotrematinae, as sister groups to the Haploporinae.

The Systematics of the Echinostomes

The family Echinostomatidae Looss, 1899 is a rather heterogeneous group of cosmopolitan, hermaphroditic digeneans that parasitize, as adults, numerous vertebrate hosts of all classes. This group exhibits a substantial taxonomic diversity (91 nominal genera are described), which is associated with the broad range of final hosts and a wide geographical distribution. Adult echinostomes are predominantly found in birds, but also parasitize mammals and occasionally reptiles and fishes. Their typical site of location is the intestine, but there are examples from the bile duct, gall bladder, ureters and urinary tubules of the kidneys, cloaca and bursa Fabricii of birds. The main distinguishing feature of the family Echinostomatidae is the presence of a circumoral head collar armed with one or two ventrally interrupted crowns of large spines. The pattern of the crown armature is an essential diagnostic character at both suprageneric and generic level, and in most cases the size of the spines is used at the species level. Consequently, illustrations of the collar spines, after cutting the entire collar at its base and mounting in glycerine, is desirable in order to study these characters accurately and consistently.

Molecular and morphological identification of larval opecoelids (Digenea: Opecoelidae) parasitising prosobranch snails in a Western Mediterranean lagoon

In a study of the digeneans parasitising molluscs in the Els Alfacs lagoon (Ebro Delta, Western Mediterranean) we found heavy infections with sporocysts emitting two types of cotylocercous cercariae in the prosobranch trochid gastropod Gibbula adansonii and with metacercariae in the prosobranch nassariid gastropod Cyclope neritea. A comparative analysis using ITS ribosomal DNA sequences from these larval stages and published sequences of 17 larval and adult opecoelid stages allowed us to elucidate the life-cycle of Macvicaria obovata and to confirm the identification of Cainocreadium labracis based on cercarial morphology. We provide molecular evidence for the identification and the first detailed morphological descriptions of the intramolluscan larval stages of the two opecoelid species as well as partial 28S rDNA sequences to aid future studies on systematic relationships within the Opecoelidae.

Descriptions of some unusual digeneans from Boops boops L. (Sparidae) and a complete checklist of its metazoan parasites

Six species of digeneans, including three new host records, are described from the bogue Boops boops off the Spanish NE Atlantic and the Mediterranean coasts. The species involved are: Robphildollfusium martinezgomezi López-Román, Gijón-Botella, Kim & Vilca-Choque, 1992, Magnibursatus caudofilamentosa (Reimer, 1971) Gibson & Køie, 1991, Lepocreadium album Stossich, 1890, Steringotrema pagelli (van Beneden, 1871) Odhner, 1911, Tetrochetus coryphaenae Yamaguti, 1934 and Stephanostomum euzeti Bartoli & Bray, 2004 (metacercaria). B.␣boops is a new host for 11 metazoan parasites (six digeneans, three acanthocephalans, one copepod and one isopod) recovered in this study. These are reported and incorporated into a complete checklist of the metazoan parasites of B. boops throughout its distributional range. It comprises summarised information for 67 species in 260 host-parasite records and includes the name of the parasite species, the locality of the host, and the author and date of the published record. The taxonomy is updated and annotations are made on the validity of the records and synonymies.

Morphological and molecular characterization of tetraphyllidean merocercoids (Platyhelminthes: Cestoda) of striped dolphins ( Stenella coeruleoalba ) from the Western Mediterranean

Two types of tetraphyllidean merocercoids, Phyllobothrium delphini and Monorygma grimaldii, are well known from most cetaceans world-wide. The role of cetaceans in the life-cycle of these merocercoids is unclear because their specific identity is as yet unknown. The problem is compounded by poor descriptions of both merocercoids. We used light and scanning electron microscopy, and histological techniques to provide a thorough description of merocercoids collected from 11 striped dolphins, Stenella coeruleoalba, from the Spanish Mediterranean. We also described, for the first time, specimens of P. delphini with immature proglottides. Our merocercoids were morphologically similar to those described previously, except in the structure of the apical organ. Intra- and inter-sample variability in the morphology of the apical organ suggested that it degenerates during larval development. A subsample of 16 specimens of P. delphini and M. grimaldii was characterized for the D2 variable region of the large subunit ribosomal RNA gene (LSU) and compared with published tetraphyllidean cestode LSU sequences. P. delphini showed 2 unique signatures that differed from one another by a single base, whereas all sequences of M. grimaldii were identical. This suggests that each type may represent a single species, contrary to previous speculations based on morphological data. All merocercoid specimens formed a clade together with Clistobothrium montaukensis. Based on the low degree of divergence, all specimens of this clade are predicted to be congeneric.

A first insight into the barcodes for African diplostomids (Digenea: Diplostomidae): Brain parasites in Clarias gariepinus (Siluriformes: Clariidae)

Diplostomid trematodes comprise a large and diverse group of widespread digeneans whose larval stages are important parasitic pathogens that may exert serious impacts in wild and cultured freshwater fish. However, our understanding of their diversity remains incomplete especially in the tropics. Our study is the first application of a DNA-based approach to diplostomid diversity in the African continent by generating a database linking sequences for the mitochondrial cytochrome c oxidase subunit 1 (cox1) barcode region and ITS1-5.8S-ITS2 rRNA gene cluster for brain-infecting diplostomid metacercariae from the catfish Clarias gariepinus. Analyses of newly-generated partial cox1 sequences for 34 larval isolates of Tylodelphys spp. from Tanzania and Diplostomum spp. from Tanzania and Nigeria revealed three strongly supported reciprocally monophyletic lineages of Tylodelphys spp. and one of an unknown species of Diplostomum. The average intraspecific divergence for the cox1 sequences for each recognised novel lineage was distinctly lower compared with interspecific divergence (0.46-0.75% vs 11.7-14.8%). The phylogenetic hypotheses estimated from Bayesian inference and maximum likelihood analyses of ITS1-5.8S-ITS2 data exhibited congruent strong support for the cox1-derived lineages. Our study thus provides molecular-based evidence for the existence of three distinct brain-infecting species co-occurring in natural populations of C. gariepinus. Based on phylogenetic analyses, we re-allocated Diplostomum mashonenseBeverley-Burton (1963) to the genus Tylodelphys as a new combination. We also generated cox1 and ITS1-5.8S-ITS2 sequences for an unknown species of Diplostomum from another African fish host, Synodontis nigrita.

Robinia aurata n. g., n. sp. (Digenea: Hemiuridae) from the mugilid Liza aurata with a molecular confirmation of its position within the Hemiuroidea

Robinia aurata n. g., n. sp. is described from Liza aurata (Mugilidae), the golden grey mullet, from the Ebro Delta, Spanish Mediterranean. The new genus differs from all other hemiurid genera in the combined possession of muscular flanges and a vestigial ecsoma. Within the Bunocotylinae, which currently accommodates 2 genera, Bunocotyle and Saturnius, the new genus exhibits a unique combination of blind caeca, Juels organ, post-ovarian bulk of the uterus in the hind-body, and tegumental papillae surrounding the oral and ventral sucker apertures. Furthermore, Robinia n. g. differs from both Bunocotyle and Saturnius in the nature of the muscular extensions around the oral sucker, with the shape of a muscular belt in the latter and numerous muscular papillae in the former. The phylogenetic hypothesis for the Bunocotylinae developed from sequence data analyses based on partial lsrDNA and complete ssrDNA combined (22 species) and V4 domain of the ssrRNA gene (37 species) supports the erection of the new genus and confirms its position within the Hemiuroidea. Both molecular analyses confirmed the monophyly of the Hemiuroidea, its division into 2 major clades and the polyphyly of the Derogenidae, as in previous studies, and suggest that the Gonocercinae (with 2 genera, Gonocerca and Hemipera), may require a distinct familial status. Finally, there was poor support for the distinct status of the Lecithasteridae and Hemiuridae, following previous suggestions based on different sequence data sets. A key to genera of the Bunocotylinae is presented.