Evaluation of genome skimming to detect and characterise human and livestock helminths.

The identification of gastrointestinal helminth infections of humans and livestock almost exclusively relies on the detection of eggs or larvae in faeces, followed by manual counting and morphological characterisation to differentiate species using microscopy-based techniques. However, molecular approaches based on the detection and quantification of parasite DNA are becoming more prevalent, increasing the sensitivity, specificity and throughput of diagnostic assays. High-throughput sequencing, from single PCR targets through to the analysis of whole genomes, offers significant promise towards providing information-rich data that may add value beyond traditional and conventional molecular approaches; however, thus far, its utility has not been fully explored to detect helminths in faecal samples. In this study, low-depth whole genome sequencing, i.e. genome skimming, has been applied to detect and characterise helminth diversity in a set of helminth-infected human and livestock faecal material. The strengths and limitations of this approach are evaluated using three methods to characterise and differentiate metagenomic sequencing data based on (i) mapping to whole mitochondrial genomes, (ii) whole genome assemblies, and (iii) a comprehensive internal transcribed spacer 2 (ITS2) database, together with validation using quantitative PCR (qPCR). Our analyses suggest that genome skimming can successfully identify most single and multi-species infections reported by qPCR and can provide sufficient coverage within some samples to resolve consensus mitochondrial genomes, thus facilitating phylogenetic analyses of selected genera, e.g. Ascaris spp. Key to this approach is both the availability and integrity of helminth reference genomes, some of which are currently contaminated with bacterial and host sequences. The success of genome skimming of faecal DNA is dependent on the availability of vouchered sequences of helminths spanning both taxonomic and geographic diversity, together with methods to detect or amplify minute quantities of parasite nucleic acids in mixed samples.

Phylogeny of hymenolepidids (Cestoda: Cyclophyllidea) from mammals: sequences of 18S rRNA and COI genes confirm major clades revealed by the 28S rRNA analyses.

The aim of the study is to test a hypothesis for the phylogenetic relationships among mammalian hymenolepidid tapeworms, based on partial (D1-D3) nuclear 28S ribosomal RNA (rRNA) genes, by estimating new molecular phylogenies for the group based on partial mitochondrial cytochrome c oxidase I (COI) and nuclear 18S rRNA genes, as well as a combined analysis using all three genes. New sequences of COI and 18S rRNA genes were obtained for Coronacanthus integrus, C. magnihamatus, C. omissus, C. vassilevi, Ditestolepis diaphana, Lineolepis scutigera, Spasskylepis ovaluteri, Staphylocystis tiara, S. furcata, S. uncinata, Vaucherilepis trichophorus and Neoskrjabinolepis sp. The phylogenetic analyses confirmed the major clades identified by Haukisalmi et al. (Zoologica Scripta 39: 631-641, 2010): Ditestolepis clade, Hymenolepis clade, Rodentolepis clade and Arostrilepis clade. While the Ditestolepis clade is associated with soricids, the structure of the other three clades suggests multiple evolutionary events of host switching between shrews and rodents. Two of the present analyses (18S rRNA and COI genes) show that the basal relationships of the four mammalian clades are branching at the same polytomy with several hymenolepidids from birds (both terrestrial and aquatic). This may indicate a rapid radiation of the group, with multiple events of colonizations of mammalian hosts by avian parasites.

The increased sensitivity of qPCR in comparison to Kato-Katz is required for the accurate assessment of the prevalence of soil-transmitted helminth infection in settings that have received multiple rounds of mass drug administration.

BACKGROUND: The most commonly used diagnostic tool for soil-transmitted helminths (STH) is the Kato-Katz (KK) thick smear technique. However, numerous studies have suggested that the sensitivity of KK can be problematic, especially in low prevalence and low intensity settings. An emerging alternative is quantitative polymerase chain reaction (qPCR). METHODS: In this study, both KK and qPCR were conducted on stool samples from 648 participants in an STH epidemiology study conducted in the delta region of Myanmar in June 2016. RESULTS: Prevalence of any STH was 20.68% by KK and 45.06% by qPCR. Prevalence of each individual STH was also higher by qPCR than KK, the biggest difference was for hookworm with an approximately 4-fold increase between the two diagnostic techniques. Prevalence of Ancylostoma ceylanicum, a parasite predominately found in dogs, was 4.63%, indicating that there is the possibility of zoonotic transmission in the study setting. In individuals with moderate to high intensity infections there is evidence for a linear relationship between eggs per gram (EPG) of faeces, derived from KK, and DNA copy number, derived from qPCR which is particularly strong for Ascaris lumbricoides. CONCLUSIONS: The use of qPCR in low prevalence settings is important to accurately assess the epidemiological situation and plan control strategies for the 'end game'. However, more work is required to accurately assess STH intensity from qPCR results and to reduce the cost of qPCR so that is widely accessible in STH endemic countries.

Advancing the multi-disciplinarity of parasitology within the British Society for Parasitology: studies of host-parasite evolution in an ever-changing world.

The study of parasites typically crosses into other research disciplines and spans across diverse scales, from molecular- to populational-levels, notwithstanding promoting an understanding of parasites set within evolutionary time. Today, the 2030 Sustainable Development Goals (SDGs) help frame much of contemporary parasitological research, since parasites can be found in all ecosystems, blighting human, animal and plant health. In recognition of the multi-disciplinary nature of parasitological research, the 2017 Autumn Symposium of the British Society for Parasitology was held in London to provide a forum for novel exchange across medical, veterinary and wildlife fields of study. Whilst the meeting was devoted to the topic of parasitism, it sought to foster mutualism, the antithesis perhaps of parasitism, by forging new academic connections and social networks to exchange novel ideas. The meeting also celebrated the longstanding career of Professor David Rollinson, FLS in the award of the International Federation for Tropical Medicine Medal for his efforts spanning 40 years of parasitological research. Indeed, David has done so much to explore and promote the fascinating biology of parasitism, as exemplified by the 15 manuscripts contained within this Special Issue.

Phylogenetic relationships within Dicrocoeliidae (Platyhelminthes: Digenea) from birds from the Czech Republic using partial 28S rDNA sequences.

Partial (D1-D3) 28S rRNA gene sequences from 16 isolates of digenean parasites of the family Dicrocoeliidae recovered from 16 bird species from the Czech Republic were used for phylogenetic reconstruction. Comparison with sequences available from GenBank suggests that the genus Brachylecithum is paraphyletic, requiring further validation and possible systematic revision. Although partial 28S rDNA is relatively conserved, analyses suggest that the following taxa are synonymous: Lutztrema attenuatum = L. monenteron = L. microstomum, Brachylecithum lobatum = B. glareoli. Zonorchis petiolatus is reassigned back to the genus Lyperosomum with L. collurionis as a junior synonym. The study revealed how complicated the systematics of the family Dicrocoeliidae is currently. The morphology of the group is variable, and the current distinguishing characters at species and even generic level are not sufficiently distinctive; it is difficult to identify the specimens correctly and identification of GenBank isolates is not reliable. Extensive sampling of isolates for both molecular and morphological studies is necessary to resolve the relationships within the family.

Curious bivalves: Systematic utility and unusual properties of anomalodesmatan mitochondrial genomes.

Mitogenomic trees for Bivalvia have proved problematic in the past, but several highly divergent lineages were missing from these analyses and increased representation of these groups may yet improve resolution. Here, we add seven new sequences from the Anomalodesmata and one unidentified semelid species (Bryopa lata, Euciroa cf. queenslandica, Laternula elliptica, Laternula truncata, Lyonsia norwegica, Myadora brevis, Tropidomya abbreviata, "Abra" sp.). We show that relationships in a mitogenomic tree for the Class are improved by the addition of seven anomalodesmatans from this highly divergent clade, but are still not completely consistent with relationships recovered in studies of nuclear genes. We suggest that some anomalous relationships (for instance the non-monophyly of Bivalvia) may be partially explained by compositional heterogeneity in the mitogenome and suggest that the addition of more taxa may help resolve both this effect and possible instances of long branch attraction. We also identify several curious features about anomalodesmatan mitogenomes. For example, many protein-coding gene boundaries are poorly defined in marine bivalves, but particularly so in anomalodesmatans, primarily due to non-conserved boundary sequences. The use of transcriptomic and genomic data together enabled better definition of gene boundaries, the identification of possible pseudogenes and suggests that most genes are translated monocistronically, which contrasts with many other studies. We also identified a possible case of gene duplication of ND5 in Myadora brevis (Myochamidae). Mitogenome size in the Anomalodesmata ranges from very small compact molecules, with the smallest for Laternula elliptica (Laternulidae) only 14,622bp, to Bryopa lata (Clavagellidae) which is at least 31,969bp long and may be >40,000bp. Finally, sampled species show a high degree of sequence divergence and variable gene order, although intraspecific variation in Laternula elliptica is very low.

The mitochondrial genome and ribosomal operon of Brachycladium goliath (Digenea: Brachycladiidae) recovered from a stranded minke whale.

Members of the Brachycladiidae are known to cause pathologies implicated in cetacean strandings and it is important to develop accurate diagnostic markers to differentiate these and other helminths found in cetaceans. Brachycladium goliath (van Beneden, 1858) is a large trematode found, as adults, usually in the hepatic (bile) and pancreatic ducts of various cetaceans. Complete sequences were determined for the entire mitochondrial genome, and phylogenetically informative nuclear genes contained within the ribosomal operon, from a small piece of an individual worm taken from a common minke whale Balaenoptera acutorostrata Lacépède, 1804. Genomic DNA was sequenced using an Illumina MiSeq platform. The mtDNA is 15,229 bp in length consisting of 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 2 non-coding regions of which the larger is comprised of 4 tandemly repeated units (260 bp each). The ribosomal RNA operon is 9297 bp long. These data provide a rich resource of molecular markers for diagnostics, phylogenetics and population genetics in order to better understand the role, and associated pathology of helminth infections in cetaceans.

Diverse Applications of Environmental DNA Methods in Parasitology.

Nucleic acid extraction and sequencing of genes from organisms within environmental samples encompasses a variety of techniques collectively referred to as environmental DNA or 'eDNA'. The key advantages of eDNA analysis include the detection of cryptic or otherwise elusive organisms, large-scale sampling with fewer biases than specimen-based methods, and generation of data for molecular systematics. These are particularly relevant for parasitology because parasites can be difficult to locate and are morphologically intractable and genetically divergent. However, parasites have rarely been the focus of eDNA studies. Focusing on eukaryote parasites, we review the increasing diversity of the 'eDNA toolbox'. Combining eDNA methods with complementary tools offers much potential to understand parasite communities, disease risk, and parasite roles in broader ecosystem processes such as food web structuring and community assembly.

Evolution: a turn up for the worms.

Extensive sequencing of transcriptomes reveals the evolutionary relationships among the major flatworm lineages, suggesting new roots and conflicting routes to parasitism.

New digeneans (Opecoelidae) from hydrothermal vent fishes in the south eastern Pacific Ocean, including one new genus and five new species.

A new genus and five new species of digeneans are reported from fishes at hydrothermal vent sites in the South East Pacific Rise region. Biospeedotrema n. gen. (Opecoelidae: Stenakrinae) is distinguished from other stenakrines by the more or less symmetrical testicular configuration, with the uterus passing between the testes, sometimes distinctly into the post-testicular region. Biospeedotrema jolliveti n. gen., n. sp. from Ventichthys biospeedoi (Ophidiidae) is distinguished by the vitelline fields which extend only slightly into the post-testicular region, the intestinal bifurcation is dorsal to the ventral sucker, the genital pore is slightly dextrally submedian or median, the cirrus sac is short and the caeca are broad and overlap the testes, usually reaching into the post-testicular region. Biospeedotrema parajolliveti n. sp. from Thermichthys hollisi differs from Biospeedotrema jolliveti in being squat, always just wider than long, the tegument is wrinkled, the testes are lobate, and the caeca only just reach to the testes. Biospeedotrema biospeedoi n. sp. from T. hollisi differs from its congeners in its body-shape, uterine extent posterior to the testes and the small vitellarium. Caudotestis ventichthysi n. sp. (Opecoelidae: Stenakrinae) from V. biospeedoi is distinguished from its five congeners in various combinations of caecal length, cirrus sac length, internal seminal vesicle shape, vitelline extent and distribution, forebody length and egg-size. Buticulotrema thermichthysi n. sp. (Opecoelidae: Opecoelininae) from T. hollisi (Bythitidae) is distinguished from its only congener by its very long, very strongly muscular oesophagus, bifurcating dorsally to the posterior part of the ventral sucker, the long, narrow pars prostatica and distal male duct and the sinistral genital pore at the level of the pharynx. The phylogenetic position for three of these species, Buticulotrema thermichthysi, Biospeedotrema jolliveti and Biospeedotrema biospeedoi, is assessed based on ssrDNA and lsrDNA sequences, which verify the position of these species in the Opecoelidae. 

The complete mitochondrial genome of a turbinid vetigastropod from MiSeq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny.

A need to increase sampling of mitochondrial genomes for Vetigastropoda has been identified as an important step towards resolving relationships within the Gastropoda. We used shotgun sequencing of genomic DNA, using an Illumina MiSeq, to obtain the first mitochondrial genome for the vetigastropod family Turbinidae, doubling the number of genomes for the species-rich superfamily Trochoidea. This method avoids the necessity of finding suitable primers for long PCRs or primer-walking amplicons, resulting in a timely and cost-effective method for obtaining whole mitochondrial genomes from ethanol-preserved tissue samples. Bayesian analysis of amino acid variation for all available gastropod genomes including the new turbinid mtgenome produced a well resolved tree with high nodal support for most nodes. Major clades within Gastropoda were recovered with strong support, with the exception of Littorinimorpha, which was polyphyletic. We confirm here that mitogenomics is a useful tool for molluscan phylogenetics, especially when using powerful new models of amino acid evolution, but recognise that increased taxon sampling is still required to resolve existing differences between nuclear and mitochondrial gene trees.

Adding resolution to ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with large fragments of mtDNA.

The construction of a stable phylogeny for the Cestoda, indicating the interrelationships of recognised orders and other major lineages, has proceeded iteratively since the group first received attention from phylogenetic systematists. Molecular analyses using nuclear ribosomal RNA gene fragments from the small (ssrDNA) and large (lsrDNA) subunits have been used to test competing evolutionary scenarios based on morphological data but could not arbitrate between some key conflicting hypotheses. To the ribosomal data, we have added a contiguous fragment of mitochondrial (mt) genome data (mtDNA) of partial nad1-trnN-trnP-trnI-trnK-nad3-trnS-trnW-cox1-trnT-rrnL-trnC-partial rrnS, spanning 4034-4447 bp, where new data for this region were generated for 18 species. Bayesian analysis of mtDNA and rDNA as nucleotides, and where appropriate as amino acids, demonstrated that these two classes of genes provide complementary signal across the phylogeny. In all analyses, except when using mt amino acids only, the Gyrocotylidea is sister group to all other Cestoda (Nephroposticophora), and Amphilinidea forms the sister group to the Eucestoda. However, an earliest-diverging position of Amphilinidea is strongly supported in the mt amino acid analysis. Amphilinidea exhibit a unique tRNA arrangement (nad1-trnI-trnL2-trnP-trnK-trnV-trnA-trnN-nad3), whereas Gyrocotylidea shares that of the derived lineages, providing additional evidence of the uniqueness of amphilinid genes and genomes. The addition of mtDNA to the rDNA genes supported the Caryophyllidea as the sister group to (Spathebothriidea+remaining Eucestoda), a hypothesis consistently supported by morphology. This relationship suggests a history of step-wise evolutionary transitions from simple monozoic, unsegmented tapeworms to the more familiar polyzoic, externally segmented (strobilate) forms. All our data partitions recovered Haplobothriidea as the sister group to Diphyllobothriidae. The sister-group relationship between Diphyllidea and Trypanorhyncha, as previously established using rDNA, is not supported by the mt data, although it is supported by the combined mt and rDNA analysis. With regards to the more derived taxa, in all except the mt amino acid analysis, the following topology is supported: (Bothriocephalidea (Litobothriidea (Lecanicephalidea (Rhinebothriidea (Tetraphyllidea, (Acanthobothrium, Proteocephalidea), (Nippotaeniidea, Mesocestoididae, Tetrabothriidea, Cyclophyllidea)))))), where the Tetraphyllidea are paraphyletic. Evidence from the mt data provides strong (nucleotides) to moderate (amino acids) support for Tetraphyllidea forming a group to the inclusion of Proteocephalidea, with the latter consistently forming the sister group to Acanthobothrium. The interrelationships among Nippotaeniidea, Mesocestoididae, Tetrabothriidea and Cyclophyllidea remain ambiguous and require further systematic attention. Mitochondrial and nuclear rDNA data provide conflicting signal for certain parts of the cestode tree. In some cases mt data offer results in line with morphological evidence, such as the interrelationships of the early divergent lineages. Also, Tetraphyllidea, although remaining paraphyletic with the inclusion of the Proteocephalidea, does not include the most derived cestodes; a result which has consistently been obtained with rDNA.

A molecular phylogeny of bryozoans.

We present the most comprehensive molecular phylogeny of bryozoans to date. Our concatenated alignment of two nuclear ribosomal and five mitochondrial genes includes 95 taxa and 13,292 nucleotide sites, of which 8297 were included. The number of new sequences generated during this project are for each gene:ssrDNA (32), lsrDNA (22), rrnL (38), rrnS (35), cox1 (37), cox3 (34), and cytb (44). Our multi-gene analysis provides a largely stable topology across the phylum. The major groups were unambiguously resolved as (Phylactolaemata (Cyclostomata (Ctenostomata, Cheilostomata))), with Ctenostomata paraphyletic. Within Phylactolaemata, (Stephanellidae, Lophopodidae) form the earliest divergent clade. Fredericellidae is not resolved as a monophyletic family and forms a clade together with Plumatellidae, Cristatellidae and Pectinatellidae, with the latter two as sister taxa. Hyalinella and Gelatinella nest within the genus Plumatella. Cyclostome taxa fall into three major clades: i. (Favosipora (Plagioecia, Rectangulata)); ii. (Entalophoroecia ((Diplosolen, Cardioecia) (Frondipora, Cancellata))); and iii. (Articulata ((Annectocyma, Heteroporidae) (Tubulipora (Tennysonia, Idmidronea)))), with suborders Tubuliporina and Cerioporina, and family Plagioeciidae each being polyphyletic. Ctenostomata is composed of three paraphyletic clades to the inclusion of Cheilostomata: ((Alcyonidium, Flustrellidra) (Paludicella (Anguinella, Triticella)) (Hislopia (Bowerbankia, Amathia)) Cheilostomata); Flustrellidra nests within the genus Alcyonidium, and Amathia nests within the genus Bowerbankia. Suborders Carnosa and Stolonifera are not monophyletic. Within the cheilostomes, Malacostega is paraphyletic to the inclusion of all other cheilostomes. Conopeum is the most early divergent cheilostome, forming the sister group to ((Malacostega, Scrupariina, Inovicellina) ((Hippothoomorpha, Flustrina) (Lepraliomorpha, Umbonulomorpha))); Flustrina is paraphyletic to the inclusion of the hippothoomorphs; neither Lepraliomorpha nor Umbonulomorpha is monophyletic. Ascophorans are polyphyletic, with hippothoomorphs grouping separately from lepraliomorphs and umbonulomorphs; no cribrimorphs were included in the analysis. Results are discussed in the light of molecular and morphological evidence. Ancestral state reconstruction of larval strategy in Gymnolaemata revealed planktotrophy and lecithotrophy as equally parsimonious solutions for the ancestral condition. More comprehensive taxon sampling is expected to clarify this result. We discuss the extent of non-bryozoan contaminant sequences deposited in GenBank and their impact on the reconstruction of metazoan phylogenies and those of bryozoan interrelationships.

Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics.

Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags ('barcodes'). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three 'bait' sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species 'barcodes' that currently use the cox1 gene only.

First molecular estimate of cyclostome bryozoan phylogeny confirms extensive homoplasy among skeletal characters used in traditional taxonomy.

The Cyclostomata are the only extant representatives of the class Stenolaemata, an ancient group of exclusively marine bryozoans. Previous cladistic analyses of cyclostome bryozoans, based exclusively on skeletal characters, revealed extensive homoplasy amongst morphological traits. This study presents the first molecular phylogeny for Cyclostomata and confirms the previous findings of homoplasy. Almost complete lsr and ssrDNA fragments were sequenced for 22 taxa of cyclostome bryozoans, plus the outgroup (Pectinatella magnifica and Flustrellidra hispida). Three well-supported major clades were found, but their inter relationships are unclear. Suborder Tubuliporina was polyphyletic, with representatives found in all three major clades. The tubuliporine family Plagioeciidae was resolved as polyphyletic; Plagioecia grouped with Lichenoporidae and Densiporidae, whereas Entalophoroecia, Diplosolen and Cardioecia formed a paraphyletic subgroup that included Frondiporidae and Horneridae. The suborder Cerioporina was also polyphyletic; Densiporidae grouped with Plagioecia and Lichenoporidae, whereas Heteroporidae nested in a paraphyletic subgroup of tubuliporines, with the Crisiidae forming the sister-group to this clade. Cinctiporidae could not be placed unambiguously. Morphological character mapping was performed in order to find evidence favouring one of the three possible hypotheses of inter relationships of the major clades, but the results were ambiguous. This study questions the extent to which morphological characters can be used in phylogenetic studies of cyclostome bryozoans, both fossil and extant, and how far their morphology is the result of ecophenotypic plasticity and convergent evolution. The finding of numerous non-monophyletic taxa has implications for extinction rate assessment and for the use of fossil cyclostomes to calibrate molecular trees.

Platyhelminth systematics and the emergence of new characters.

Since the inclusion of molecular data in modern phylogenetic analyses, significant progress in resolving the origins and radiation of flatworms has been made, although some key problems remain. Here I review developments in the supply and use of systematic characters that provide the basis for diagnosis and phylogeny reconstruction, that in turn have driven systematic revisions and the interpretation of broader evolutionary patterns and processes; focus is placed on the parasitic taxa. Although useful tools have been refined to the point of becoming established systematic markers of broad utility, attention to the need for denser gene and taxon sampling is addressed in the light of unresolved questions and current trends in molecular systematics, from nucleotide to genome. Tradition and the nature of available comparative information tends to dictate the choice of systematic markers, but faced with incongruent phylogenies, the emergence of new technologies and the need for rapid species diagnosis, there is a pressing need to assess and standardize our choice of tools so they are fit for purpose, available to all and used widely. I present a brief review of existing and potential sources of phylogenetic characters and discuss their likely value in the context of the systematics and diagnostics of parasitic flatworms.

On the position of Archigetes and its bearing on the early evolution of the tapeworms.

The tapeworm Archigetes sieboldi Leuckart, 1878 (Platyhelminthes: Cestoda: Caryophyllidea) has been cited as a likely representative of the "protocestode" condition, owing to its lack of segmentation and ability to attain sexual maturity in the invertebrate host (aquatic oligochaetes). The idea has been variously amplified or rejected in the literature, although the actual phylogenetic position of the species has not been investigated until now. New collections of Archigetes sp. from both its vertebrate and invertebrate hosts provided the opportunity to estimate its phylogenetic position with the use of molecular systematics, while prompting new analyses aimed at assessing the early diversification of the Cestoda. Additional collections representing the Amphilinidea, Caryophyllidea, and Gyrocotylidea were combined with published gene sequences to construct data sets of complete 18S (110 taxa) and partial (D1-D3) 28S (107 taxa) rDNA sequences, including 8 neodermatan outgroup taxa. Estimates resulting from Bayesian inference, maximum likelihood, and maximum parsimony analyses of the separate and combined data sets supported a derived position of the genus within the Caryophyllidea, and thus reject the idea that Archigetes sp. may exemplify a "primitive" condition. Topological constraint analyses rejected the hypothesis that Archigetes represents the most basal lineage of the Eucestoda, but did not rule out that it could represent the earliest branching taxon of the Caryophyllidea. In all analyses, the Eucestoda were monophyletic and supported basal positions of the nonsegmented Caryophyllidea and Spathebothriidea relative to other major lineages of the Eucestoda, implying that segmentation is a derived feature of the common ancestor of the di- and tetrafossate eucestodes. However, constraint analyses could not provide unequivocal evidence as to the precise branching patterns of the cestodarian, spathebothriidean, and caryophyllidean lineages. Phylogenetic analyses favor the interpretation that sexual maturity of Archigetes sp. in the invertebrate host, and similar examples in members of the Spathebothriidea, are the result of progenesis and have little if any bearing on understanding the protocestode condition.

The mitochondrial genome of Gyrodactylus derjavinoides (Platyhelminthes: Monogenea)--a mitogenomic approach for Gyrodactylus species and strain identification.

Systematists and evolutionary biologists are constantly on the lookout for new sources of characters to discriminate amongst taxa and estimate interrelationships within and between taxa. Entire mitochondrial genomes provide a wealth of data, both at the nucleotide and amino acid level. Molecular markers are of particular utility when applied to small, morphologically conserved taxa, as is the case for many monogenean ectoparasites of fish. Gyrodactylus species display a considerable degree of anatomical conservatism, complicating diagnostics based solely on morphology, and some are significant pests of wild and cultured fish. Here we sequenced the complete mitochondrial genome of Gyrodactylus derjavinoides Malmberg, Collins, Cunningham & Behiar 2007, one of the most frequently found gyrodactylid species on salmonids in Scandinavia, and compared it with the recently published genomes of Gyrodactylus salaris Malmberg, 1957 and Gyrodactylus thymalli Zitnan 1960. Through comparative sliding window analysis we identified regions of high sequence variability and designed new primer sequences. In total, 6 new primer pairs have been developed, amplifying fragments of cox1, cox3, nad1, nad2, nad4, nad5 and atp6. Together, they amplify regions capturing almost half the nucleotide variability present in the complete mitochondrial genome. These degenerate primers should also work for other Gyrodactylus species parasitizing salmonids. In addition, we developed a multiplex assay that simultaneously amplifies four fragments in a single PCR reaction. Besides the diagnostic value, these fragments can be used for studying the transmission dynamics of Gyrodactylus, providing crucial information for an improved understanding of the spread and epidemiology of these important fish pathogens.

Bird schistosomes of wildfowl in the Czech Republic and Poland.

In 2005, we dissected 102 wildfowl from the Czech Republic and 73 wildfowl from Poland including representatives of Anseriformes, Gruiformes and Gaviiformes. Schistosome infection was found in a total of 21 (29%) and 23 (23%) birds from Poland and the Czech Republic, respectively. All infected birds belonged to the order Anseriformes. The prevalences of nasal and visceral species were, respectively, 22% and 16% in Poland and 6% and 19% in the Czech Republic. Four species of schistosomes were found: Bilharziella polonica Kowalewski, 1895, Trichobilharzia regenti Horák, Koláfová et Dvorák, 1998, T. szidati Neuhaus, 1952, and an undetermined schistosome from the intestinal wall of Anas penelope L. The finding of T. szidati represents the first record of the parasite from natural final host since the species description.