Using 454 technology for long-PCR based sequencing of the complete mitochondrial genome from single Haemonchus contortus (Nematoda)

<p>Abstract</p> <p>Background</p> <p>Mitochondrial (mt) genomes represent a rich source of molecular markers for a range of applications, including population genetics, systematics, epidemiology and ecology. In the present study, we used 454 technology (or the GS20, massively parallel picolitre reactor platform) to determine the complete mt genome of <it>Haemonchus contortus </it>(Nematoda: Trichostrongylidae), a parasite of substantial agricultural, veterinary and economic significance. We validate this approach by comparison with mt sequences from publicly available expressed sequence tag (EST) and genomic survey sequence (GSS) data sets.</p> <p>Results</p> <p>The complete mt genome of <it>Haemonchus contortus </it>was sequenced directly from long-PCR amplified template utilizing genomic DNA (~20–40 ng) from a single adult male using 454 technology. A single contig was assembled and compared against mt sequences mined from publicly available EST (NemBLAST) and GSS datasets. The comparison demonstrated that the 454 technology platform is reliable for the sequencing of AT-rich mt genomes from nematodes. The mt genome sequenced for <it>Haemonchus contortus </it>was 14,055 bp in length and was highly AT-rich (78.1%). In accordance with other chromadorean nematodes studied to date, the mt genome of <it>H. contortus </it>contained 36 genes (12 protein coding, 22 tRNAs, <it>rrnL </it>and <it>rrnS</it>) and was similar in structure, size and gene arrangement to those characterized previously for members of the Strongylida.</p> <p>Conclusion</p> <p>The present study demonstrates the utility of 454 technology for the rapid determination of mt genome sequences from tiny amounts of DNA and reveals a wealth of mt genomic data in current databases available for mining. This approach provides a novel platform for high-throughput sequencing of mt genomes from nematodes and other organisms.</p

Orders out of chaos – molecular phylogenetics reveals the complexity of shark and stingray tapeworm relationships

Novel molecular data are presented to resolve the long-standing issue of the non-monophyly of the elasmobranch-hosted tapeworm order Tetraphyllidea relative to the other acetabulate eucestode orders. Bayesian Inference analyses of various combinations of full ssrDNA, and full or partial lsrDNA (D1-D3), sequence data, which included 134 species representing 97 genera across the 15 eucestode orders, were conducted. New ssrDNA data were generated for 82 species, partial lsrDNA data for 53 species, and full lsrDNA data for 29 species. The monophyly of each of the elasmobranch-hosted orders Cathetocephalidea, Litobothriidea, Lecanicephalidea, and Rhinebothriidea was confirmed, as was the non-monophyly of the Tetraphyllidea. Two relatively stable groups of tetraphyllidean taxa emerged and are hereby designated as new orders. The Onchoproteocephalidea n. ord. is established to recognize the integrated nature of one undescribed and ten described genera of hook-bearing tetraphyllideans, previously of the family Onchobothriidae, with the members of the order Proteocephalidea. The Phyllobothriidea n. ord. is established for a subset of 12 non-hooked genera characterized by scoleces bearing four bothridia each with an anterior accessory sucker; most parasitise sharks and have been assigned to the Phyllobothriidae at one time or another. Tentative ordinal placements are suggested for 8 additional genera; placements for the remaining tetraphyllidean genera have not yet emerged. We propose these 17 genera remain in the “Tetraphyllidea”. Among these, particularly labile across analyses were Anthobothrium, Megalonchos, Carpobothrium, Calliobothrium, and Caulobothrium. The unique association of Chimaerocestus with holocephalans, rather than with elasmobranchs, appears to represent a host-switching event. Both of the non-elasmobranch hosted clades of acetabulate cestodes (i.e., Proteocephalidea and Cyclophyllidea and their kin) appear to have had their origins with elasmobranch cestodes. Across analyses, the sister group to the clade of “terrestrial” cestode orders was found to be an elasmobranch-hosted genus; as was the sister to the freshwater fish and tetrapod-hosted Proteocephalidea. Whilst further data are required to resolve outstanding nomenclatural and phylogenetic issues, the present analyses contribute significantly to an understanding of the evolutionary radiation of the entire Cestoda. Clearly, elasmobranch tapeworms comprise the backbone of cestode phylogeny

The mitochondrial genomes of Ancylostoma caninum and Bunostomum phlebotomum – two hookworms of animal health and zoonotic importance

<p>Abstract</p> <p>Background</p> <p>Hookworms are blood-feeding nematodes that parasitize the small intestines of many mammals, including humans and cattle. These nematodes are of major socioeconomic importance and cause disease, mainly as a consequence of anaemia (particularly in children or young animals), resulting in impaired development and sometimes deaths. Studying genetic variability within and among hookworm populations is central to addressing epidemiological and ecological questions, thus assisting in the control of hookworm disease. Mitochondrial (mt) genes are known to provide useful population markers for hookworms, but mt genome sequence data are scant.</p> <p>Results</p> <p>The present study characterizes the complete mt genomes of two species of hookworm, <it>Ancylostoma caninum </it>(from dogs) and <it>Bunostomum phlebotomum </it>(from cattle), each sequenced (by 454 technology or primer-walking), following long-PCR amplification from genomic DNA (~20–40 ng) isolated from individual adult worms. These mt genomes were 13717 bp and 13790 bp in size, respectively, and each contained 12 protein coding, 22 transfer RNA and 2 ribosomal RNA genes, typical for other secernentean nematodes. In addition, phylogenetic analysis (by Bayesian inference and maximum likelihood) of concatenated mt protein sequence data sets for 12 nematodes (including <it>Ancylostoma caninum </it>and <it>Bunostomum phlebotomum</it>), representing the Ascaridida, Spirurida and Strongylida, was conducted. The analysis yielded maximum statistical support for the formation of monophyletic clades for each recognized nematode order assessed, except for the Rhabditida.</p> <p>Conclusion</p> <p>The mt genomes characterized herein represent a rich source of population genetic markers for epidemiological and ecological studies. The strong statistical support for the construction of phylogenetic clades and consistency between the two different tree-building methods employed indicate the value of using whole mt genome data sets for systematic studies of nematodes. The grouping of the Spirurida and Ascaridida to the exclusion of the Strongylida was not supported in the present analysis, a finding which conflicts with the current evolutionary hypothesis for the Nematoda based on nuclear ribosomal gene data.</p

Bryozoan genera Fenestrulina and Microporella no longer confamilial; multi-gene phylogeny supports separation

Bryozoans are a moderately diverse, mostly marine phylum with a fossil record extending to the early Ordovician. Compared to other phyla, little is known about their phylogenetic relationships at both lower and higher taxonomic levels. Hence, an effort is being made to elucidate the phylogenetic relationships among bryozoans. Here, we present newly sequenced nuclear and mitochondrial genes for 21 cheilostome bryozoans and compile these with existing orthologous molecular data. Using these data, we focus on reconstructing the phylogenetic relationships of Fenestrulina and Microporella, two species-rich genera. They are currently placed in a globally distributed family, Microporellidae, defined by having a semicircular primary orifice and a proximal ascopore, although there are indirect inferences in the morphological literature that suggest they might not be confamilial. Our six-gene phylogenetic analysis reveals that the genera Fenestrulina and Microporella are each monophyletic, with the sister clade to Microporella comprising non-microporellids. These genera thus have a polyphyletic relationship and should not be placed in the same family. Our result supports the reinstatement of the family Fenestrulinidae Jullien, 1888 for Fenestrulina and genera with comparable frontal shield and ooecial morphologies. Our well-supported phylogeny based on independent molecular data lends credit to existing phylogenetic hypotheses based on morphological observations but does not conform to the current classification of these particular bryozoans. This illustrates the general need for a rethink of bryozoan higher-level systematics, ideally based on both morphological and molecular data

In search of phylogenetic congruence between molecular and morphological data in bryozoans with extreme adult skeletal heteromorphy

peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=tsab20© Crown Copyright 2015. This document is the author's final accepted/submitted version of the journal article. You are advised to consult the publisher's version if you wish to cite from it

Phylogenetically Widespread Polyembryony in Cyclostome Bryozoans and the Protracted Asynchronous Release of Clonal Brood-Mates

The file attached is the Published/publisher’s pdf version of the articl

Are well-studied marine biodiversity hotspots still blackspots for animal barcoding?

Marine biodiversity underpins ecosystem health and societal well-being. Preservation of biodiversity hotspots is a global challenge. Molecular tools, like DNA barcoding and metabarcoding, hold great potential for biodiversity monitoring, possibly outperforming more traditional taxonomic methods. However, metabarcoding-based biodiversity assessments are limited by the availability of sequences in barcoding reference databases; a lack thereof results in high percentages of unassigned sequences. In this study we (i) present the current status of known vs. barcoded marine species at a global scale based on online taxonomic and genetic databases; and (ii) compare the current status with data from ten years ago. Then we analyzed occurrence data of marine animal species from five Large Marine Ecosystems (LMEs) classified as biodiversity hotspots, to identify any consistent disparities in COI barcoding coverage between geographic regions and at phylum level. Barcoding coverage varied among LMEs (from 36.8% to 62.4% COI-barcoded species) and phyla (from 4.8% to 74.7% COI-barcoded species), with Porifera, Bryozoa and Platyhelminthes being highly underrepresented, compared to Chordata, Arthropoda and Mollusca. We demonstrate that although barcoded marine species increased from 9.5% to 14.2% since the last assessment in 2011, about 15,000 (corresponding to 7.8% increase) new species were described from 2011 to 2021. The next ten years will thus be crucial to enroll concrete collaborative measures and long term initiatives (e.g., Horizon 2030, Ocean Decade) to populate barcoding libraries for the marine realm.the Department of Biological, Geological and Environmental Sciences (BiGeA) of the University of Bologna (UniBo). The CoMBoMed initiative was supported by the European Marine Research Network (EUROMARINE Network), the Inter-Departmental Research Centre for Environmental Sciences (CIRSA – UniBo), the Cultural Heritage Department (DBC - UniBo, https://beniculturali.unibo.it/it), the Fondazione Flaminia and the ERANet Mar-Tera Project SEAMoBB (Solutions for sEmi-Automated Monitoring of Benthic Biodiversity).Peer reviewe

Next-generation mitogenomics: A comparison of approaches applied to caecilian amphibian phylogeny

Mitochondrial genome (mitogenome) sequences are being generated with increasing speed due to the advances of next-generation sequencing (NGS) technology and associated analytical tools. However, detailed comparisons to explore the utility of alternative NGS approaches applied to the same taxa have not been undertaken. We compared a 'traditional' Sanger sequencing method with two NGS approaches (shotgun sequencing and non-indexed, multiplex amplicon sequencing) on four different sequencing platforms (Illumina's HiSeq and MiSeq, Roche's 454 GS FLX, and Life Technologies' Ion Torrent) to produce seven (near-) complete mitogenomes from six species that form a small radiation of caecilian amphibians from the Seychelles. The fastest, most accurate method of obtaining mitogenome sequences that we tested was direct sequencing of genomic DNA (shotgun sequencing) using the MiSeq platform. Bayesian inference and maximum likelihood analyses using seven different partitioning strategies were unable to resolve compellingly all phylogenetic relationships among the Seychelles caecilian species, indicating the need for additional data in this case

The Mitochondrial Genome of Toxocara canis

Toxocara canis (Ascaridida: Nematoda), which parasitizes (at the adult stage) the small intestine of canids, can be transmitted to a range of other mammals, including humans, and can cause the disease toxocariasis. Despite its significance as a pathogen, the genetics, epidemiology and biology of this parasite remain poorly understood. In addition, the zoonotic potential of related species of Toxocara, such as T. cati and T. malaysiensis, is not well known. Mitochondrial DNA is known to provide genetic markers for investigations in these areas, but complete mitochondrial genomic data have been lacking for T. canis and its congeners. In the present study, the mitochondrial genome of T. canis was amplified by long-range polymerase chain reaction (long PCR) and sequenced using a primer-walking strategy. This circular mitochondrial genome was 14162 bp and contained 12 protein-coding, 22 transfer RNA, and 2 ribosomal RNA genes consistent for secernentean nematodes, including Ascaris suum and Anisakis simplex (Ascaridida). The mitochondrial genome of T. canis provides genetic markers for studies into the systematics, population genetics and epidemiology of this zoonotic parasite and its congeners. Such markers can now be used in prospecting for cryptic species and for exploring host specificity and zoonotic potential, thus underpinning the prevention and control of toxocariasis in humans and other hosts