Evolutionary transitions in broad tapeworms (Cestoda: Diphyllobothriidea) revealed by mitogenome and nuclear ribosomal operon phylogenetics.

Broad tapeworms (Diphyllobothriidea) are parasites whose adults are capable of infecting a wide range of freshwater, marine and terrestrial tetrapods including humans. Previous works examining the evolution of habitat and host use in this group have been hampered by the lack of a well-resolved phylogeny. In order to produce a robust phylogenetic framework for diphyllobothriideans, we sequenced the complete mitochondrial genome of 13 representatives, carefully chosen to cover the major clades, and two outgroup species representing the Spathebothriidea and Haplobothriidea. In addition, complementary data from the nuclear ribosomal operon was sequenced for 10 representative taxa. Mitogenomes and ssrDNA and lsrDNA were used towards elucidating the phylogenetic framework for the Diphyllobothriidea. The Cephalochlamydidae is confirmed as the earliest diverging diphyllobothriidean lineage, and Solenophoridae and Diphyllobothriidae are sister groups. We infer a probable freshwater origin of the diphyllobothriideans. The ancestral condition for life cycle complexity could not be unambiguously resolved. However, we infer exclusive use of a three-host life cycle following the origin of the Solenophoridae + Diphyllobothriidae. Regarding definitive host use, although we infer reptiles as the most likely ancestral condition, this result should be revisited with a more densely sampled phylogeny in future studies. Freshwater habitat is used by the early diverging lineages within the Solenophoridae + Diphyllobothriidae clade. For the latter, habitat use shifts between freshwater and marine environments, and definitive host use includes marine and terrestrial mammals and birds. We use mitochondrial genomes to distinguish Schistocephalus species occurring in different species of sticklebacks and demonstrate conspecificity of Ligula cf. intestinalis specimens collected from two Fennoscandian ringed seal subspecies.

Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial Genomes.

Long non-coding, tandem-repetitive regions in mitochondrial (mt) genomes of many metazoans have been notoriously difficult to characterise accurately using conventional sequencing methods. Here, we show how the use of a third-generation (long-read) sequencing and informatic approach can overcome this problem. We employed Oxford Nanopore technology to sequence genomic DNAs from a pool of adult worms of the carcinogenic parasite, Schistosoma haematobium, and used an informatic workflow to define the complete mt non-coding region(s). Using long-read data of high coverage, we defined six dominant mt genomes of 33.4 kb to 22.6 kb. Although no variation was detected in the order or lengths of the protein-coding genes, there was marked length (18.5 kb to 7.6 kb) and structural variation in the non-coding region, raising questions about the evolution and function of what might be a control region that regulates mt transcription and/or replication. The discovery here of the largest tandem-repetitive, non-coding region (18.5 kb) in a metazoan organism also raises a question about the completeness of some of the mt genomes of animals reported to date, and stimulates further explorations using a Nanopore-informatic workflow.

Molecular circumscription of new species of Gyrocotyle Diesing, 1850 (Cestoda) from deep-sea chimaeriform holocephalans in the North Atlantic.

Chimaeras, or ratfishes, are the only extant group of holocephalan fishes and are the sole host group of gyrocotylidean cestodes, which represent a sister group of the true tapeworms (Eucestoda). These unique, non-segmented cestodes have been known since the 1850s and multiple species and genera have been erected despite a general agreement that the delineation of species on the basis of morphology is effectively impossible. Thus, in the absence of molecular studies, the validity of gyrocotylid taxa and their specific host associations has remained highly speculative. Here we report the presence of Gyrocotyle spp. from rarely-caught deep-sea chimaeras collected in the North-East Atlantic, and describe two new species: G. haffii n. sp. from the bent-nose chimaera, Harriota raleighana Goode & Bean, and G. discoveryi n. sp. from the large-eyed rabbit fish, Hydrolagus mirabilis (Collett). Nuclear ribosomal sequence data were generated for individual parasites taken from different host species collected on different dates and from different localities and were combined with previously published sequences. Phylogenetic analyses supported the recognition of independent lineages and clusters, indicative of species, but were indecisive in recovering the root of the tree in analyses that included non-gyrocotylid outgroup taxa. The molecular data reveal variation not reflected in morphology and point to a complex picture of genetic divergence shaped by both isolation and migration in the deep-sea environment.

Phylogeny of hymenolepidid cestodes (Cestoda: Cyclophyllidea) from mammalian hosts based on partial 28S rDNA, with focus on parasites from shrews.

The aims of the study are to enrich the partial 28S rDNA dataset for hymenolepidids by adding new sequences for species parasitic in the genera Sorex, Neomys and Crocidura (Soricidae) and to propose a new hypothesis for the relationships among mammalian hymenolepidids. New sequences 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 analysis (based on 56 taxa) confirmed the major clades identified by Haukisalmi et al. (Zool Scr 39:631-641, 2010) based on analysis of 31 species: Ditestolepis clade, Hymenolepis clade, Rodentolepis clade and Arostrilepis clade; however, the support was weak for the early divergent lineages of the tree and for the Arostrilepis clade. Novelties revealed include the molecular evidence for the monophyly of Coronacanthus, the non-monophyletic status of Staphylocystis and the polyphyly of Staphylocystoides. The analysis has confirmed the monophyly of Hymenolepis, the monophyly of hymenolepidids from glirids, the position of Pararodentolepis and Nomadolepis as sister taxa, the polyphyly of Rodentolepis, the position of Neoskrjabinolepis and Lineolepis as sister taxa, and the close relationship among the genera with the entire reduction of rostellar apparatus. Resolved monophyletic groups are supported by the structure of the rostellar apparatus. The diversification of the Ditestolepis clade is associated with soricids. The composition of the other major clades suggests multiple evolutionary events of host switching, including between different host orders. The life cycles of Coronacanthus and Vaucherilepis are recognised as secondarily aquatic as these taxa are nested in terrestrial groups.

Integrated Approach Reveals Role of Mitochondrial Germ-Line Mutation F18L in Respiratory Chain, Oxidative Alterations, Drug Sensitivity, and Patient Prognosis in Glioblastoma.

Glioblastoma is the most common and malignant primary brain tumour in adults, with a dismal prognosis. This is partly due to considerable inter- and intra-tumour heterogeneity. Changes in the cellular energy-producing mitochondrial respiratory chain complex (MRC) activities are a hallmark of glioblastoma relative to the normal brain, and associate with differential survival outcomes. Targeting MRC complexes with drugs can also facilitate anti-glioblastoma activity. Whether mutations in the mitochondrial DNA (mtDNA) that encode several components of the MRC contribute to these phenomena remains underexplored. We identified a germ-line mtDNA mutation (m. 14798T > C), enriched in glioblastoma relative to healthy controls, that causes an amino acid substitution F18L within the core mtDNA-encoded cytochrome b subunit of MRC complex III. F18L is predicted to alter corresponding complex III activity, and sensitivity to complex III-targeting drugs. This could in turn alter reactive oxygen species (ROS) production, cell behaviour and, consequently, patient outcomes. Here we show that, despite a heterogeneous mitochondrial background in adult glioblastoma patient biopsy-derived cell cultures, the F18L substitution associates with alterations in individual MRC complex activities, in particular a 75% increase in MRC complex II_III activity, and a 34% reduction in CoQ10, the natural substrate for MRC complex III, levels. Downstream characterisation of an F18L-carrier revealed an 87% increase in intra-cellular ROS, an altered cellular distribution of mitochondrial-specific ROS, and a 64% increased sensitivity to clomipramine, a repurposed MRC complex III-targeting drug. In patients, F18L-carriers that received the current standard of care treatment had a poorer prognosis than non-carriers (373 days vs. 415 days, respectively). Single germ-line mitochondrial mutations could predispose individuals to differential prognoses, and sensitivity to mitochondrial targeted drugs. Thus, F18L, which is present in blood could serve as a useful non-invasive biomarker for the stratification of patients into prognostically relevant groups, one of which requires a lower dose of clomipramine to achieve clinical effect, thus minimising side-effects.

Australian spiny mountain crayfish and their temnocephalan ectosymbionts: an ancient association on the edge of coextinction?

Australian spiny mountain crayfish (Euastacus, Parastacidae) and their ecotosymbiotic temnocephalan flatworms (Temnocephalida, Platyhelminthes) may have co-occurred and interacted through deep time, during a period of major environmental change. Therefore, reconstructing the history of their association is of evolutionary, ecological, and conservation significance. Here, time-calibrated Bayesian phylogenies of Euastacus species and their temnocephalans (Temnohaswellia and Temnosewellia) indicate near-synchronous diversifications from the Cretaceous. Statistically significant cophylogeny correlations between associated clades suggest linked evolutionary histories. However, there is a stronger signal of codivergence and greater host specificity in Temnosewellia, which co-occurs with Euastacus across its range. Phylogeography and analyses of evolutionary distinctiveness (ED) suggest that regional differences in the impact of climate warming and drying had major effects both on crayfish and associated temnocephalans. In particular, Euastacus and Temnosewellia show strong latitudinal gradients in ED and, conversely, in geographical range size, with the most distinctive, northern lineages facing the greatest risk of extinction. Therefore, environmental change has, in some cases, strengthened ecological and evolutionary associations, leaving host-specific temnocephalans vulnerable to coextinction with endangered hosts. Consequently, the extinction of all Euastacus species currently endangered (75%) predicts coextinction of approximately 60% of the studied temnocephalans, with greatest loss of the most evolutionarily distinctive lineages.

The molecular phylogeny of the digenean family Opecoelidae Ozaki, 1925 and the value of morphological characters, with the erection of a new subfamily.

Large and small rDNA sequences of 41 species of the family Opecoelidae are utilised to produce phylogenetic inference trees, using brachycladioids and lepocreadioids as outgroups. Sequences were newly generated for 13 species. The resulting Bayesian trees show a monophyletic Opecoelidae. The earliest divergent group is the Stenakrinae, based on two species which are not of the type-genus. The next well-supported clade to diverge is constituted of three species of Helicometra Odhner, 1902. Based on this tree and the characters of the egg and uterus, a new subfamily, the Helicometrinae, is erected and defined to include the genera Helicometra, Helicometrina Linton, 1910 and Neohelicometra Siddiqi et Cable, 1960. The subfamily Opecoelinae is found to be monophyletic, but the Plagioporinae is paraphyletic. The single representative of the Opecoelininae (not of the type genus) is nested within a group of deep-sea 'plagioporines'. The two representatives of the Opistholebetidae are embedded within a group of shallow-water 'plagioporine' species. The Opistholebetidae is reduced to subfamily status pro tem as its morphological and biological characteristics are distinctive. This implies that as opecoelid systematics develops with more molecular evidence, several further subfamilies will be recognised. Many of the morphological characters were found to be homoplasious, but the characters defining the Helicometrinae and Opecoelinae, such as filamented eggs, reduced cirrus-sac and uterine seminal receptacle, are closely correlated with the inferred phylogeny.

The first multi-gene phylogeny of the Macrostomorpha sheds light on the evolution of sexual and asexual reproduction in basal Platyhelminthes.

The Macrostomorpha-an early branching and species-rich clade of free-living flatworms-is attracting interest because it contains Macrostomum lignano, a versatile model organism increasingly used in evolutionary, developmental, and molecular biology. We elucidate the macrostomorphan molecular phylogeny inferred from both nuclear (18S and 28S rDNA) and mitochondrial (16S rDNA and COI) marker genes from 40 representatives. Although our phylogeny does not recover the Macrostomorpha as a statistically supported monophyletic grouping, it (i) confirms many taxa previously proposed based on morphological evidence, (ii) permits the first placement of many families and genera, and (iii) reveals a number of unexpected placements. Specifically, Myozona and Bradynectes are outside the three classic families (Macrostomidae, Microstomidae and Dolichomacrostomidae) and the asexually fissioning Myomacrostomum belongs to a new subfamily, the Myozonariinae nov. subfam. (Dolichomacrostomidae), rather than diverging early. While this represents the first evidence for asexuality among the Dolichomacrostomidae, we show that fissioning also occurs in another Myozonariinae, Myozonaria fissipara nov. sp. Together with the placement of the (also fissioning) Microstomidae, namely as the sister taxon of Dolichomacrostomidae, this suggests that fissioning is not basal within the Macrostomorpha, but rather restricted to the new taxon Dolichomicrostomida (Dolichomacrostomidae+Microstomidae). Furthermore, our phylogeny allows new insights into the evolution of the reproductive system, as ancestral state reconstructions reveal convergent evolution of gonads, and male and female genitalia. Finally, the convergent evolution of sperm storage organs in the female genitalia appears to be linked to the widespread occurrence of hypodermic insemination among the Macrostomorpha.

Mating behavior and the evolution of sperm design.

Sperm are the most diverse of all animal cell types, and much of the diversity in sperm design is thought to reflect adaptations to the highly variable conditions under which sperm function and compete to achieve fertilization. Recent work has shown that these conditions often evolve rapidly as a consequence of multiple mating, suggesting a role for sexual selection and sexual conflict in the evolution of sperm design. However, very little of the striking diversity in sperm design is understood functionally, particularly in internally fertilizing organisms. We use phylogenetic comparative analyses covering 16 species of the hermaphroditic flatworm genus Macrostomum to show that a complex sperm design is associated with reciprocal mating and that this complexity is lost secondarily when hypodermic insemination--sperm injection through the epidermis--evolves. Specifically, the complex sperm design, which includes stiff lateral bristles, is likely a male persistence trait associated with sexual conflicts over the fate of received ejaculates and linked to female resistance traits, namely an intriguing postcopulatory sucking behavior and a thickened epithelium of the sperm-receiving organ. Our results suggest that the interactions between sperm donor, sperm, and sperm recipient can change drastically when hypodermic insemination evolves, involving convergent evolution of a needle-like copulatory organ, a simpler sperm design, and a simpler female genital morphology. Our study documents that a shift in the mating behavior may alter fundamentally the conditions under which sperm compete and thereby lead to a drastic change in sperm design.

Molecular evidence that the genus Cadenatella Dollfus, 1946 (Digenea: Plagiorchiida) belongs in the superfamily Haploporoidea Nicoll, 1914.

A re-examination of published lsrDNA sequence data of haploporid digeneans has shown that the genus Cadenatella Dollfus, 1946, hitherto considered a lepocreadioid, is correctly placed within the superfamily Haploporoidea Nicoll, 1914, although its relationships within the superfamily are not resolved. The morphological similarities and differences between Cadenatella and other haploporoids are discussed, and the subfamily Cadenatellinae Gibson & Bray, 1982 is considered the best repository for Cadenatella spp. at present.

Fatal Hymenolepis nana-associated visceral larva migrans in captive juvenile white-tailed antsangies (Brachytarsomysalbicauda).

White-tailed antsangies (Brachytarsomys albicauda) are Madagascan rodents uncommonly kept in captivity. Hymenolepis nana is a cestode with an unusual life cycle, incorporating direct, indirect and autoinfective stages. This case series represents the first reported outbreak of H. nana cestodiasis in white-tailed antsangies, summarizing macroscopic and histological findings in four cases. On post-mortem examination (PME), numerous cysticerci were detected consistently throughout the intestinal serosa, liver, mesenteric lymphatic vasculature and mesenteric lymph nodes. Pancreatic cysticerci were observed in one case. Adult tapeworms, larvae and eggs were found only in the small intestine, and faecal egg shedding was a feature. Histopathological examination identified adult, larval and encysted cestodes within the respective gross lesions, with pulmonary, pancreatic and splenic involvement detected in a single case. The cestodes sampled on PME were identified by polymerase chain reaction and DNA sequencing, with H. nana confirmed in all cases. Visceral larva migrans was consistent throughout all specimens, in contrast with the natural infections of standard rodent hosts, and may be considered a likely pathological feature of H. nana infection in white-tailed antsangies.

Analyses of mitochondrial amino acid sequence datasets support the proposal that specimens of Hypodontus macropi from three species of macropodid hosts represent distinct species.

BACKGROUND: Hypodontus macropi is a common intestinal nematode of a range of kangaroos and wallabies (macropodid marsupials). Based on previous multilocus enzyme electrophoresis (MEE) and nuclear ribosomal DNA sequence data sets, H. macropi has been proposed to be complex of species. To test this proposal using independent molecular data, we sequenced the whole mitochondrial (mt) genomes of individuals of H. macropi from three different species of hosts (Macropus robustus robustus, Thylogale billardierii and Macropus [Wallabia] bicolor) as well as that of Macropicola ocydromi (a related nematode), and undertook a comparative analysis of the amino acid sequence datasets derived from these genomes. RESULTS: The mt genomes sequenced by next-generation (454) technology from H. macropi from the three host species varied from 13,634 bp to 13,699 bp in size. Pairwise comparisons of the amino acid sequences predicted from these three mt genomes revealed differences of 5.8% to 18%. Phylogenetic analysis of the amino acid sequence data sets using Bayesian Inference (BI) showed that H. macropi from the three different host species formed distinct, well-supported clades. In addition, sliding window analysis of the mt genomes defined variable regions for future population genetic studies of H. macropi in different macropodid hosts and geographical regions around Australia. CONCLUSIONS: The present analyses of inferred mt protein sequence datasets clearly supported the hypothesis that H. macropi from M. robustus robustus, M. bicolor and T. billardierii represent distinct species.

Macrobothriotaenia ficta (Cestoda: Proteocephalidea), a parasite of sunbeam snake (Xenopeltis unicolor): example of convergent evolution.

The poorly known proteocephalidean cestode Macrobothriotaenia ficta (Meggitt, 1931) from the sunbeam snake Xenopeltis unicolor (Ophidia: Xenopeltidae) is redescribed on the basis of re-examination of its type specimens from Burma (Myanmar), and vouchers from Thailand and Vietnam. The peculiar morphology of the scolex, which is formed by four pedunculate lobe-bearing pincer-shaped suckers, is described for the first time using scanning electron microscopy. In scolex morphology, M. ficta closely resembles phyllobothriidean cestodes, parasites of elasmobranchs. However, this similarity does not reflect phylogenetic relatedness of these cestodes but instead presents an example of convergent morphological evolution of attachment organs of unrelated groups of cestodes that parasitize different groups of vertebrates. Besides scolex morphology, the genus is characterised by the possession of a very large cirrus-sac, which may reach up to the midline of proglottides, few testes (less than 60), vitelline follicles limited to the dorsal side of proglottides, a large vaginal sphincter, and eggs with a three-layered embryophore covered with rounded projections. Numerous errors in the diagnosis of M. ficta, which appeared in the literature as a result of uncritical compilation of data without examination of original material, are corrected. Multilocus phylogenetic analysis of nuclear ribosomal RNA genes ssr- and lsr-DNA and mitochondrial genes rrnL and cox1 place this species among other snake-parasitizing proteocephalideans of the genus Ophiotaenia. The convergent evolution of scolex morphology across distantly related taxa is discussed.

Exploring gene sequences and phylogenetic relationships of four terrestrial planarian species (Platyhelminthes; Tricladida; Geoplanidae) in Europe.

Partial mitochondrial cox1 gene sequences from four recently recognised European species of terrestrial planarians, and ribosomal ITS1 sequences for two of them, are presented: Marionfyfea adventor, Artioposthia exulans (both introduced from New Zealand), Australopacifica atrata (from Australia) and specimens putatively identified as Microplana edwardsi, presumed to be native to the UK. The sequences are compared with those from other terrestrial planarian species and analysed phylogenetically. Results indicate that the sister group of M. adventor comprises a clade constituted by at least the genus Arthurdendyus. The phylogenetic position of Ar. exulans remains less certain, Australopacifica atrata might be closely related to the species Parakontikia ventrolineata and Endeavouria septemlineata. The specimens of M. cf. edwardsi are distinct from all other Microplana species for which sequences are available.

Evidence that a lineage of teleost-infecting blood flukes (Aporocotylidae) infects bivalves as intermediate hosts.

The family Aporocotylidae is recognized as having the widest intermediate host usage in the Digenea. Currently, intermediate host groups are clearly correlated with definitive host groups; all known life cycles of marine teleost-infecting aporocotylids involve polychaetes, those of freshwater teleost-infecting aporocotylids involve gastropods, and those of chondrichthyan-infecting aporocotylids involve bivalves. Here we report the life cycle for a marine elopomorph-infecting species, Elopicola bristowi Orélis-Ribeiro & Bullard in Orélis-Ribeiro, Halanych, Dang, Bakenhaster, Arias & Bullard, 2017, as infecting a bivalve, Anadara trapezia (Deshayes) (Arcidae), as the intermediate host in Moreton Bay, Queensland, Australia. The cercaria of E. bristowi has a prominent finfold, distinct anterior and posterior widenings of the oesophagus, a tail with symmetrical furcae with finfolds, and develops in elongate to oval sporocysts. We also report molecular data for an unmatched aporocotylid cercaria from another bivalve, Megapitaria squalida (G. B. Sowerby I) (Veneridae), from the Gulf of California, Mexico, and six unmatched cercariae from a gastropod, Posticobia brazieri (E. A. Smith) (Tateidae), from freshwater systems of south-east Queensland, Australia. Phylogenetic analyses demonstrate the presence of six strongly-supported lineages within the Aporocotylidae, including one of elopomorph-infecting genera, Elopicola Bullard, 2014 and Paracardicoloides Martin, 1974, now shown to use both gastropods and bivalves as intermediate hosts. Of a likely 14 aporocotylid species reported from bivalves, six are now genetically characterised. The cercarial morphology of these six species demonstrates a clear distinction between those that infect chondrichthyans and those that infect elopomorphs; chondrichthyan-infecting aporocotylids have cercariae with asymmetrical furcae that lack finfolds and develop in spherical sporocysts whereas those of elopomorph-infecting aporocotylids have symmetrical furcae with finfolds and develop in elongate sporocysts. This morphological correlation allows predictions of the host-based lineage to which the unsequenced species belong. The Aporocotylidae is proving exceptional in is propensity for major switches in intermediate host use, with the most parsimonious interpretation of intermediate host distribution implying a minimum of three host switches within the family.

Next generation sequencing and comparative analyses of Xenopus mitogenomes.

BACKGROUND: Mitochondrial genomes comprise a small but critical component of the total DNA in eukaryotic organisms. They encode several key proteins for the cell's major energy producing apparatus, the mitochondrial respiratory chain. Additionally, their nucleotide and amino acid sequences are of great utility as markers for systematics, molecular ecology and forensics. Their characterization through nucleotide sequencing is a fundamental starting point in mitogenomics. Methods to amplify complete mitochondrial genomes rapidly and efficiently from microgram quantities of tissue of single individuals are, however, not always available. Here we validate two approaches, which combine long-PCR with Roche 454 pyrosequencing technology, to obtain two complete mitochondrial genomes from individual amphibian species. RESULTS: We obtained two new xenopus frogs (Xenopus borealis and X. victorianus) complete mitochondrial genome sequences by means of long-PCR followed by 454 of individual genomes (approach 1) or of multiple pooled genomes (approach 2), the mean depth of coverage per nucleotide was 9823 and 186, respectively. We also characterised and compared the new mitogenomes against their sister taxa; X. laevis and Silurana tropicalis, two of the most intensely studied amphibians. Our results demonstrate how our approaches can be used to obtain complete amphibian mitogenomes with depths of coverage that far surpass traditional primer-walking strategies, at either the same cost or less. Our results also demonstrate: that the size, gene content and order are the same among xenopus mitogenomes and that S. tropicalis form a separate clade to the other xenopus, among which X. laevis and X. victorianus were most closely related. Nucleotide and amino acid diversity was found to vary across the xenopus mitogenomes, with the greatest diversity observed in the Complex 1 gene nad4l and the least diversity observed in Complex 4 genes (cox1-3). All protein-coding genes were shown to be under strong negative (purifying selection), with genes under the strongest pressure (Complex 4) also being the most highly expressed, highlighting their potentially crucial functions in the mitochondrial respiratory chain. CONCLUSIONS: Next generation sequencing of long-PCR amplicons using single taxon or multi-taxon approaches enabled two new species of Xenopus mtDNA to be fully characterized. We anticipate our complete mitochondrial genome amplification methods to be applicable to other amphibians, helpful for identifying the most appropriate markers for differentiating species, populations and resolving phylogenies, a pressing need since amphibians are undergoing drastic global decline. Our mtDNAs also provide templates for conserved primer design and the assembly of RNA and DNA reads following high throughput "omic" techniques such as RNA- and ChIP-seq. These could help us better understand how processes such mitochondrial replication and gene expression influence xenopus growth and development, as well as how they evolved and are regulated.

An integrated pipeline for next-generation sequencing and annotation of mitochondrial genomes.

Mitochondrial (mt) genomics represents an understudied but important field of molecular biology. Increasingly, mt dysfunction is being linked to a range of human diseases, including neurodegenerative disorders, diabetes and impairment of childhood development. In addition, mt genomes provide important markers for systematic, evolutionary and population genetic studies. Some technological limitations have prevented the expanded generation and utilization of mt genomic data for some groups of organisms. These obstacles most acutely impede, but are not limited to, studies requiring the determination of complete mt genomic data from minute amounts of material (e.g. biopsy samples or microscopic organisms). Furthermore, post-sequencing bioinformatic annotation and analyses of mt genomes are time consuming and inefficient. Herein, we describe a high-throughput sequencing and bioinformatic pipeline for mt genomics, which will have implications for the annotation and analysis of other organellar (e.g. plastid or apicoplast genomes) and virus genomes as well as long, contiguous regions in nuclear genomes. We utilize this pipeline to sequence and annotate the complete mt genomes of 12 species of parasitic nematode (order Strongylida) simultaneously, each from an individual organism. These mt genomic data provide a rich source of markers for studies of the systematics and population genetics of a group of socioeconomically important pathogens of humans and other animals.

Orientobilharzia Dutt & Srivastava, 1955 (Trematoda: Schistosomatidae), a junior synonym of Schistosoma Weinland, 1858.

In the last few decades, phylogenetic studies of the family Schistosomatidae based on molecular markers have revealed that members of the genus Orientobilharzia Dutt & Srivastava, 1955 belong within Schistosoma Weinland, 1858. In this study, the original descriptions and redescriptions of Orientobilharzia species and related revisions are reviewed, and it is confirmed that the morphological characters correspond with the results of the molecular studies. The two genera differ only in the number of testes; however, this character varies to a large extent within particular genera of the subfamily Schistosomatinae and cannot be used to justify the separation of Orientobilharzia from Schistosoma. Also, we have verified claims suggesting the synonymy of certain species of Orientobilharzia; the four valid species of this genus are transferred to Schistosoma and two new synonymies are formally presented. The following nomenclatural changes are made: Schistosoma Weinland, 1858 [syn. Orientobilharzia Dutt & Srivastava, 1955 (syn. nov.)]; Schistosoma bomfordi Montgomery, 1906 (comb. restit.); S. turkestanicum Skrjabin, 1913 (comb. restit.) [syns Ori. turkestanica var. tuberculata (Bhalerao, 1932) (syn. nov.) and Ori. cheni Hsü & Yang, 1957 (syn. nov.)]; S. dattai (Dutt & Srivastava, 1952) n. comb.; and S. harinasutai (Kruatrachue, Bhaibulaya & Harinasuta, 1965) n. comb. The generic diagnosis of Schistosoma is amended and a revised key to the subfamily Schistosomatinae Stiles & Hassall, 1898 is presented.

Worms and bugs of the gut: the search for diagnostic signatures using barcoding, and metagenomics-metabolomics.

Gastrointestinal (GI) helminth infections cause significant morbidity in both humans and animals worldwide. Specific and sensitive diagnosis is central to the surveillance of such infections and to determine the effectiveness of treatment strategies used to control them. In this article, we: (i) assess the strengths and limitations of existing methods applied to the diagnosis of GI helminth infections of humans and livestock; (ii) examine high-throughput sequencing approaches, such as targeted molecular barcoding and shotgun sequencing, as tools to define the taxonomic composition of helminth infections; and (iii) discuss the current understanding of the interactions between helminths and microbiota in the host gut. Stool-based diagnostics are likely to serve as an important tool well into the future; improved diagnostics of helminths and their environment in the gut may assist the identification of biomarkers with the potential to define the health/disease status of individuals and populations, and to identify existing or emerging anthelmintic resistance.

Expansion of Cyclophyllidea Biodiversity in Rodents of Qinghai-Tibet Plateau and the "Out of Qinghai-Tibet Plateau" Hypothesis of Cyclophyllideans.

The Cyclophyllidea comprises the most species-rich order of tapeworms (Platyhelminthes, Cestoda) and includes species with some of the most severe health impact on wildlife, livestock, and humans. We collected seven Cyclophyllidea specimens from rodents in Qinghai-Tibet Plateau (QTP) and its surrounding mountain systems, of which four specimens in QTP were unsequenced, representing "putative new species." Their complete mitochondrial (mt) genomes were sequenced and annotated. Phylogenetic reconstruction of partial 28S rDNA, cox1 and nad1 datasets provided high bootstrap frequency support for the categorization of three "putative new species," assigning each, respectively, to the genera Mesocestoides, Paranoplocephala, and Mosgovoyia, and revealing that some species and families in these three datasets, which contain 291 species from nine families, may require taxonomic revision. The partial 18S rDNA phylogeny of 29 species from Taeniidae provided high bootstrap frequency support for the categorization of the "putative new species" in the genus Hydatigera. Combined with the current investigation, the other three known Taeniidae species found in this study were Taenia caixuepengi, T. crassiceps, and Versteria mustelae and may be widely distributed in western China. Estimates of divergence time based on cox1 + nad1 fragment and mt protein-coding genes (PCGs) showed that the differentiation rate of Cyclophyllidea species was strongly associated with the rate of change in the biogeographic scenarios, likely caused by the uplift of the QTP; i.e., species differentiation of Cyclophyllidea might be driven by host-parasite co-evolution caused by the uplift of QTP. We propose an "out of QTP" hypothesis for the radiation of these cyclophyllidean tapeworms.