Small RNA pathways act at the front line of defence against transposable elements across the Eukaryota. In animals, Piwi interacting small RNAs (piRNAs) are a crucial arm of this defence. However, the evolutionary relationships among piRNAs and other small RNA pathways targeting transposable elements are poorly resolved. To address this question we sequenced small RNAs from multiple, diverse nematode species, producing the first phylum-wide analysis of how small RNA pathways evolve. Surprisingly, despite their prominence in Caenorhabditis elegans and closely related nematodes, piRNAs are absent in all other nematode lineages. We found that there are at least two evolutionarily distinct mechanisms that compensate for the absence of piRNAs, both involving RNA-dependent RNA polymerases (RdRPs). Whilst one pathway is unique to nematodes, the second involves Dicer-dependent RNA-directed DNA methylation, hitherto unknown in animals, and bears striking similarity to transposon-control mechanisms in fungi and plants. Our results highlight the rapid, context-dependent evolution of small RNA pathways and suggest piRNAs in animals may have replaced an ancient eukaryotic RNA-dependent RNA polymerase pathway to control transposable elements.
A survey of the nematode phylum reveals loss of the Piwi/piRNA pathway in several lineages, but RNA-dependent RNA polymerases control transposable elements in its absence.
Transposable elements are segments of DNA that have the ability to copy themselves independently of the host genome and thus pose a severe threat to the integrity of the genome. Organisms have evolved mechanisms to restrict the spread of transposable elements, with small RNA molecules being one of the most important defense mechanisms. In animals, the predominant small RNA transposon-silencing mechanism is the piRNA pathway, which appears to be widely conserved. However, little is known about how small RNA pathways that target transposons evolve. In order to study this question we investigated small RNA pathways across the nematode phylum, using a well-studied model organism—the nematode Caenorhabditis elegans—as the starting point. Surprisingly we found that the piRNA pathway has been completely lost in all groups of nematodes bar those most closely related to C. elegans. This finding raises the intriguing question of how these nematodes are able to control transposable element mobilization without piRNAs. We discovered that there are other small RNA pathways that target transposable elements in these nematodes, employing RNA-dependent RNA polymerases in order to make small RNAs antisense to transposable elements. Intriguingly, the most ancient of these mechanisms, found in the most basal nematodes, is a Dicer-dependent RNA-directed DNA methylation pathway. This pathway shares strong similarity to transposon-silencing mechanisms in plants and fungi, suggesting that it might have been present in an ancient common ancestor of all eukaryotes. Our results highlight the rapid evolution of small RNA pathways and demonstrate the importance of examining molecular pathways in detail across a range of evolutionary distances.
microRNAs (miRNAs), a class of short, non-coding RNA can be found in a highly stable, cell-free form in mammalian body fluids. Specific miRNAs are secreted by parasitic nematodes in exosomes and have been detected in the serum of murine and dog hosts infected with the filarial nematodes Litomosoides sigmodontis and Dirofilaria immitis, respectively. Here we identify extracellular, parasite-derived small RNAs associated with Onchocerca species infecting cattle and humans.
Small RNA libraries were prepared from total RNA extracted from the nodule fluid of cattle infected with Onchocerca ochengi as well as serum and plasma from humans infected with Onchocerca volvulus in Cameroon and Ghana. Parasite-derived miRNAs were identified based on the criteria that sequences unambiguously map to hairpin structures in Onchocerca genomes, do not align to the human genome and are not present in European control serum.
A total of 62 mature miRNAs from 52 distinct pre-miRNA candidates were identified in nodule fluid from cattle infected with O. ochengi of which 59 are identical in the genome of the human parasite O. volvulus. Six of the extracellular miRNAs were also identified in sequencing analyses of serum and plasma from humans infected with O. volvulus. Based on sequencing analysis the abundance levels of the parasite miRNAs in serum or plasma range from 5 to 127 reads/per million total host miRNA reads identified, comparable to our previous analyses of Schistosoma mansoni and L. sigmodontis miRNAs in serum. All six of the O. volvulus miRNAs identified have orthologs in other filarial nematodes and four were identified in the serum of mice infected with L. sigmodontis.
We have identified parasite-derived miRNAs associated with onchocerciasis in cattle and humans. Our results confirm the conserved nature of RNA secretion by diverse nematodes. Additional species-specific small RNAs from O. volvulus may be present in serum based on the novel miRNA sequences identified in the nodule fluid. In our analyses comparison to European control serum illuminates the scope for false-positives, warranting caution in criteria that should be applied to identification of biomarkers of infection.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-015-0656-1) contains supplementary material, which is available to authorized users.
microRNAs; Extracellular RNA; Filarial nematode; Onchocerciasis; Host-pathogen
In mammalian systems RNA can move between cells via vesicles. Here we demonstrate that the gastrointestinal nematode Heligmosomoides polygyrus, which infects mice, secretes vesicles containing microRNAs (miRNAs) and Y RNAs as well as a nematode Argonaute protein. These vesicles are of intestinal origin and are enriched for homologues of mammalian exosome proteins. Administration of the nematode exosomes to mice suppresses Type 2 innate responses and eosinophilia induced by the allergen Alternaria. Microarray analysis of mouse cells incubated with nematode exosomes in vitro identifies Il33r and Dusp1 as suppressed genes, and Dusp1 can be repressed by nematode miRNAs based on a reporter assay. We further identify miRNAs from the filarial nematode Litomosoides sigmodontis in the serum of infected mice, suggesting that miRNA secretion into host tissues is conserved among parasitic nematodes. These results reveal exosomes as another mechanism by which helminths manipulate their hosts and provide a mechanistic framework for RNA transfer between animal species.
Mammalian cell-derived exosomes can carry RNA and proteins from cell to cell, but this mode of transport has not been shown in nematodes. Here the authors show that a gastrointestinal parasite secretes exosomes that transfer microRNAs to mammalian cells and regulate innate immunity.
Anguillicola crassus is a swim bladder nematode of eels. The parasite is native to the Asian eel Anguilla japonica, but was introduced to Europe and the European eel Anguilla anguilla in the early 1980s. A Taiwanese source has been proposed for this introduction. In the new host in the recipient area, the parasite appears to be more pathogenic. As a reason for these differences, genetically fixed differences in infectivity and development between Taiwanese and European A.crassus have been described and disentangled from plasticity induced by different host environments. To explore whether transcriptional regulation is involved in these lifecycle differences, we have analysed a “common garden”, cross infection experiment, using deep-sequencing transcriptomics. Surprisingly, in the face of clear phenotypic differences in life history traits, we identified no significant differences in gene expression between parasite populations or between experimental host species. From 120,000 SNPs identified in the transcriptome data we found that European A. crassus were not a genetic subset of the Taiwanese nematodes sampled. The loci that have the major contribution to the European-Taiwanese population differentiation show an enrichment of synonymous and non-coding polymorphism. This argues against positive selection in population differentiation. However, genes involved in protein processing in the endoplasmatic reticulum membrane and genes bearing secretion signal sequences were enriched in the set of genes most differentiated between European and Taiwanese A. crassus. These genes could be a source for the phenotypically visible genetically fixed differences between European and Taiwanese A. crassus.
Anguillicola crassus; SNPs; Population differentiation; Gene expression; Transcriptome; Discriminant analysis of principal components (DAPC); Anguilla; Eel; Invasive parasite; Anguillicoloides crassus
Transposable elements can be categorised into DNA and RNA elements based on their mechanism of transposition. Tyrosine recombinase elements (YREs) are relatively rare and poorly understood, despite sharing characteristics with both DNA and RNA elements. Previously, the Nematoda have been reported to have a substantially different diversity of YREs compared to other animal phyla: the Dirs1-like YRE retrotransposon was encountered in most animal phyla but not in Nematoda, and a unique Pat1-like YRE retrotransposon has only been recorded from Nematoda. We explored the diversity of YREs in Nematoda by sampling broadly across the phylum and including 34 genomes representing the three classes within Nematoda. We developed a method to isolate and classify YREs based on both feature organization and phylogenetic relationships in an open and reproducible workflow. We also ensured that our phylogenetic approach to YRE classification identified truncated and degenerate elements, informatively increasing the number of elements sampled. We identified Dirs1-like elements (thought to be absent from Nematoda) in the nematode classes Enoplia and Dorylaimia indicating that nematode model species do not adequately represent the diversity of transposable elements in the phylum. Nematode Pat1-like elements were found to be a derived form of another Pat1-like element that is present more widely in animals. Several sequence features used widely for the classification of YREs were found to be homoplasious, highlighting the need for a phylogenetically-based classification scheme. Nematode model species do not represent the diversity of transposable elements in the phylum.
Motivation: The Oxford Nanopore MinION device represents a unique sequencing technology. As a mobile sequencing device powered by the USB port of a laptop, the MinION has huge potential applications. To enable these applications, the bioinformatics community will need to design and build a suite of tools specifically for MinION data.
Results: Here we present poRe, a package for R that enables users to manipulate, organize, summarize and visualize MinION nanopore sequencing data. As a package for R, poRe has been tested on Windows, Linux and MacOSX. Crucially, the Windows version allows users to analyse MinION data on the Windows laptop attached to the device.
Availability and implementation: poRe is released as a package for R at http://sourceforge.net/projects/rpore/. A tutorial and further information are available at https://sourceforge.net/p/rpore/wiki/Home/
Supplementary information: Supplementary data are available at Bioinformatics online.
Wolbachia are common endosymbionts of terrestrial arthropods, and are also found in nematodes: the animal-parasitic filaria, and the plant-parasite Radopholus similis. Lateral transfer of Wolbachia DNA to the host genome is common. We generated a draft genome sequence for the strongyloidean nematode parasite Dictyocaulus viviparus, the cattle lungworm. In the assembly, we identified nearly 1 Mb of sequence with similarity to Wolbachia. The fragments were unlikely to derive from a live Wolbachia infection: most were short, and the genes were disabled through inactivating mutations. Many fragments were co-assembled with definitively nematode-derived sequence. We found limited evidence of expression of the Wolbachia-derived genes. The D. viviparus Wolbachia genes were most similar to filarial strains and strains from the host-promiscuous clade F. We conclude that D. viviparus was infected by Wolbachia in the past, and that clade F-like symbionts may have been the source of filarial Wolbachia infections.
Bovine lungworms are economically important nematode parasites of cattle. We have sequenced the genome of the bovine lungworm to provide information for drug and vaccine discovery. Within the lungworm genome we found extensive evidence of an ancient association between the lungworm and a bacterium called Wolbachia. The lungworm Wolbachia is now a “fossil” in the genome, but tells of an ancient infection. Association between lungworms, and related nematode worms, and Wolbachia was not known previously. We have used the lungworm Wolbachia sequence to explore the history of nematode-Wolbachia interactions, particularly the jumping of these symbionts between arthropods and nematodes.
Next-generation sequencing (NGS) technologies have dramatically expanded the breadth of genomics. Genome-scale data, once restricted to a small number of biomedical model organisms, can now be generated for virtually any species at remarkable speed and low cost. Yet non-model organisms often lack a suitable reference to map sequence reads against, making alignment-based quality control (QC) of NGS data more challenging than cases where a well-assembled genome is already available. Here we show that by generating a rapid, non-optimized draft assembly of raw reads, it is possible to obtain reliable and informative QC metrics, thus removing the need for a high quality reference. We use benchmark datasets generated from control samples across a range of genome sizes to illustrate that QC inferences made using draft assemblies are broadly equivalent to those made using a well-established reference, and describe QC tools routinely used in our production facility to assess the quality of NGS data from non-model organisms.
Illumina sequencing; de novo assembly; quality control; insert size; PCR duplicates; mate pair
Root knot nematodes (RKN) can infect most of the world’s agricultural crop species and are among the most important of all plant pathogens. As yet however we have little understanding of their origins or the genomic basis of their extreme polyphagy. The most damaging pathogens reproduce by obligatory mitotic parthenogenesis and it has been suggested that these species originated from interspecific hybridizations between unknown parental taxa. We have sequenced the genome of the diploid meiotic parthenogen Meloidogyne floridensis, and use a comparative genomic approach to test the hypothesis that this species was involved in the hybrid origin of the tropical mitotic parthenogen Meloidogyne incognita. Phylogenomic analysis of gene families from M. floridensis, M. incognita and an outgroup species Meloidogyne hapla was carried out to trace the evolutionary history of these species’ genomes, and we demonstrate that M. floridensis was one of the parental species in the hybrid origins of M. incognita. Analysis of the M. floridensis genome itself revealed many gene loci present in divergent copies, as they are in M. incognita, indicating that it too had a hybrid origin. The triploid M. incognita is shown to be a complex double-hybrid between M. floridensis and a third, unidentified, parent. The agriculturally important RKN have very complex origins involving the mixing of several parental genomes by hybridization and their extreme polyphagy and success in agricultural environments may be related to this hybridization, producing transgressive variation on which natural selection can act. It is now clear that studying RKN variation via individual marker loci may fail due to the species’ convoluted origins, and multi-species population genomics is essential to understand the hybrid diversity and adaptive variation of this important species complex. This comparative genomic analysis provides a compelling example of the importance and complexity of hybridization in generating animal species diversity more generally.
Genome sequencing; Phylogenomics; Meloidogyne incognita; Meloidogyne hapla; Meloidogyne floridensis; Comparative genomics; Hybrid speciation
The genetics of development in the nematode Caenorhabditis elegans has been described in exquisite detail. The phylum Nematoda has two classes: Chromadorea (which includes C. elegans) and the Enoplea. While the development of many chromadorean species resembles closely that of C. elegans, enoplean nematodes show markedly different patterns of early cell division and cell fate assignment. Embryogenesis of the enoplean Romanomermis culicivorax has been studied in detail, but the genetic circuitry underpinning development in this species has not been explored.
We generated a draft genome for R. culicivorax and compared its gene content with that of C. elegans, a second enoplean, the vertebrate parasite Trichinella spiralis, and a representative arthropod, Tribolium castaneum. This comparison revealed that R. culicivorax has retained components of the conserved ecdysozoan developmental gene toolkit lost in C. elegans. T. spiralis has independently lost even more of this toolkit than has C. elegans. However, the C. elegans toolkit is not simply depauperate, as many novel genes essential for embryogenesis in C. elegans are not found in, or have only extremely divergent homologues in R. culicivorax and T. spiralis. Our data imply fundamental differences in the genetic programmes not only for early cell specification but also others such as vulva formation and sex determination.
Despite the apparent morphological conservatism, major differences in the molecular logic of development have evolved within the phylum Nematoda. R. culicivorax serves as a tractable system to contrast C. elegans and understand how divergent genomic and thus regulatory backgrounds nevertheless generate a conserved phenotype. The R. culicivorax draft genome will promote use of this species as a research model.
Nematode; Genome; Evolution; Development; Caenorhabditis; Mermithida; Romanomermis
Generating the raw data for a de novo genome assembly project for a target eukaryotic species is relatively easy. This democratization of access to large-scale data has allowed many research teams to plan to assemble the genomes of non-model organisms. These new genome targets are very different from the traditional, inbred, laboratory-reared model organisms. They are often small, and cannot be isolated free of their environment – whether ingested food, the surrounding host organism of parasites, or commensal and symbiotic organisms attached to or within the individuals sampled. Preparation of pure DNA originating from a single species can be technically impossible, but assembly of mixed-organism DNA can be difficult, as most genome assemblers perform poorly when faced with multiple genomes in different stoichiometries. This class of problem is common in metagenomic datasets that deliberately try to capture all the genomes present in an environment, but replicon assembly is not often the goal of such programs. Here we present an approach to extracting, from mixed DNA sequence data, subsets that correspond to single species’ genomes and thus improving genome assembly. We use both numerical (proportion of GC bases and read coverage) and biological (best-matching sequence in annotated databases) indicators to aid partitioning of draft assembly contigs, and the reads that contribute to those contigs, into distinct bins that can then be subjected to rigorous, optimized assembly, through the use of taxon-annotated GC-coverage plots (TAGC plots). We also present Blobsplorer, a tool that aids exploration and selection of subsets from TAGC-annotated data. Partitioning the data in this way can rescue poorly assembled genomes, and reveal unexpected symbionts and commensals in eukaryotic genome projects. The TAGC plot pipeline script is available from https://github.com/blaxterlab/blobology, and the Blobsplorer tool from https://github.com/mojones/Blobsplorer.
next-generation sequencing; metagenomics; assembly; parasites; symbionts; commensals; contaminants
Chromatin diminution is the programmed elimination of specific DNA sequences during development. It occurs in diverse species, but the function(s) of diminution and the specificity of sequence loss remain largely unknown. Diminution in the nematode Ascaris suum occurs during early embryonic cleavages and leads to the loss of germline genome sequences and the formation of a distinct genome in somatic cells. We found that ~43 Mb (~13%) of genome sequence is eliminated in A. suum somatic cells, including ~12.7 Mb of unique sequence. The eliminated sequences and location of the DNA breaks are the same in all somatic lineages from a single individual, and between different individuals. At least 685 genes are eliminated. These genes are preferentially expressed in the germline and during early embryogenesis. We propose that diminution is a mechanism of germline gene regulation that specifically removes a large number of genes involved in gametogenesis and early embryogenesis.
Next-generation DNA sequencing technologies have made it possible to generate transcriptome data for novel organisms quickly and cheaply, to the extent that the effort required to annotate and publish a new transcriptome is greater than the effort required to sequence it. Often, following publication, details of the annotation effort are only available in summary form, hindering subsequent exploitation of the data. To promote best-practice in annotation and to ensure that data remain accessible, we have written afterParty, a web application that allows users to assemble, annotate and publish novel transcriptomes using only a web browser.
afterParty is a robust web application that implements best-practice transcriptome assembly, annotation, browsing, searching, and visualization. Users can turn a collection of reads (from Roche 454 chemistry) or assembled contigs (from any sequencing chemistry, including Illumina Solexa RNA-Seq) into a searchable, browsable transcriptome resource and quickly make it publicly available. Contigs are functionally annotated based on similarity to known sequences and protein domains. Once assembled and annotated, transcriptomes derived from multiple species or libraries can be compared and searched. afterParty datasets can either be created using the existing afterParty server, or using local instances that can be built easily using a virtual machine. afterParty includes powerful visualization tools for transcriptome dataset exploration and uses a flexible annotation architecture which will allow additional types of annotation to be added in the future.
afterParty's main use case scenario is one in which a working biologist has generated a large volume of transcribed sequence data and wishes to turn it into a useful resource that has some durability. By reducing the effort, bioinformatics skills, and computational resources needed to annotate and publish a transcriptome, afterParty will facilitate the annotation and sharing of sequence data that would otherwise remain unavailable. A typical metazoan transcriptome containing several tens of thousands of contigs can be annotated in a few minutes of interactive time and a few days of computational time.
Transcriptome; Assembly; Annotation
Wolbachia, endosymbiotic bacteria of the order Rickettsiales, are widespread in arthropods but also present in nematodes. In arthropods, A and B supergroup Wolbachia are generally associated with distortion of host reproduction. In filarial nematodes, including some human parasites, multiple lines of experimental evidence indicate that C and D supergroup Wolbachia are essential for the survival of the host, and here the symbiotic relationship is considered mutualistic. The origin of this mutualistic endosymbiosis is of interest for both basic and applied reasons: How does a parasite become a mutualist? Could intervention in the mutualism aid in treatment of human disease? Correct rooting and high-quality resolution of Wolbachia relationships are required to resolve this question. However, because of the large genetic distance between Wolbachia and the nearest outgroups, and the limited number of genomes so far available for large-scale analyses, current phylogenies do not provide robust answers. We therefore sequenced the genome of the D supergroup Wolbachia endosymbiont of Litomosoides sigmodontis, revisited the selection of loci for phylogenomic analyses, and performed a phylogenomic analysis including available complete genomes (from isolates in supergroups A, B, C, and D). Using 90 orthologous genes with reliable phylogenetic signals, we obtained a robust phylogenetic reconstruction, including a highly supported root to the Wolbachia phylogeny between a (A + B) clade and a (C + D) clade. Although we currently lack data from several Wolbachia supergroups, notably F, our analysis supports a model wherein the putatively mutualist endosymbiotic relationship between Wolbachia and nematodes originated from a single transition event.
Wolbachia; phylogenomics; mutualism; Litomosoides sigmodontis; endosymbiosis
The left-right asymmetry of snails, including the direction of shell coiling, is determined by the delayed effect of a maternal gene on the chiral twist that takes place during early embryonic cell divisions. Yet, despite being a well-established classical problem, the identity of the gene and the means by which left-right asymmetry is established in snails remain unknown. We here demonstrate the power of new genomic approaches for identification of the chirality gene, “D”. First, heterozygous (Dd) pond snails Lymnaea stagnalis were self-fertilised or backcrossed, and the genotype of more than six thousand offspring inferred, either dextral (DD/Dd) or sinistral (dd). Then, twenty of the offspring were used for Restriction-site-Associated DNA Sequencing (RAD-Seq) to identify anonymous molecular markers that are linked to the chirality locus. A local genetic map was constructed by genotyping three flanking markers in over three thousand snails. The three markers lie either side of the chirality locus, with one very tightly linked (<0.1 cM). Finally, bacterial artificial chromosomes (BACs) were isolated that contained the three loci. Fluorescent in situ hybridization (FISH) of pachytene cells showed that the three BACs tightly cluster on the same bivalent chromosome. Fibre-FISH identified a region of greater that ∼0.4 Mb between two BAC clone markers that must contain D. This work therefore establishes the resources for molecular identification of the chirality gene and the variation that underpins sinistral and dextral coiling. More generally, the results also show that combining genomic technologies, such as RAD-Seq and high resolution FISH, is a robust approach for mapping key loci in non-model systems.
Summary: High-quality draft genomes are now easy to generate, as sequencing and assembly costs have dropped dramatically. However, building a user-friendly searchable Web site and database for a newly annotated genome is not straightforward. Here we present Badger, a lightweight and easy-to-install genome exploration environment designed for next generation non-model organism genomes.
Availability: Badger is released under the GPL and is available at http://badger.bio.ed.ac.uk/. We show two working examples: (i) a test dataset included with the source code, and (ii) a collection of four filarial nematode genomes.
Linking behavioural phenotypes to their underlying genotypes is crucial for uncovering the mechanisms that underpin behaviour and for understanding the origins and maintenance of genetic variation in behaviour. Recently, interest has begun to focus on the transcriptome as a route for identifying genes and gene pathways associated with behaviour. For many behavioural traits studied at the phenotypic level, we have little or no idea of where to start searching for “candidate” genes: the transcriptome provides such a starting point. Here we consider transcriptomic changes associated with oviposition in the parasitoid wasp Nasonia vitripennis. Oviposition is a key behaviour for parasitoids, as females are faced with a variety of decisions that will impact offspring fitness. These include choosing between hosts of differing quality, as well as making decisions regarding clutch size and offspring sex ratio. We compared the whole-body transcriptomes of resting or ovipositing female Nasonia using a “DeepSAGE” gene expression approach on the Illumina sequencing platform. We identified 332 tags that were significantly differentially expressed between the two treatments, with 77% of the changes associated with greater expression in resting females. Oviposition therefore appears to focus gene expression away from a number of physiological processes, with gene ontologies suggesting that aspects of metabolism may be down-regulated during egg-laying. Nine of the most abundant differentially expressed tags showed greater expression in ovipositing females though, including the genes purity-of-essence (associated with behavioural phenotypes in Drosophila) and glucose dehydrogenase (GLD). The GLD protein has been implicated in sperm storage and release in Drosophila and so provides a possible candidate for the control of sex allocation by female Nasonia during oviposition. Oviposition in Nasonia therefore clearly modifies the transcriptome, providing a starting point for the genetic dissection of oviposition.
In the last decade, many diverse RNAi (RNA interference) pathways have been discovered that mediate gene silencing at epigenetic, transcriptional and post-transcriptional levels. The diversity of RNAi pathways is inherently linked to the evolution of Ago (Argonaute) proteins, the central protein component of RISCs (RNA-induced silencing complexes). An increasing number of diverse Agos have been identified in different species. The functions of most of these proteins are not yet known, but they are generally assumed to play roles in development, genome stability and/or protection against viruses. Recent research in the nematode Caenorhabditis elegans has expanded the breadth of RNAi functions to include transgenerational epigenetic memory and, possibly, environmental sensing. These functions are inherently linked to the production of secondary siRNAs (small interfering RNAs) that bind to members of a clade of WAGOs (worm-specific Agos). In the present article, we review briefly what is known about the evolution and function of Ago proteins in eukaryotes, including the expansion of WAGOs in nematodes. We postulate that the rapid evolution of WAGOs enables the exceptional functional plasticity of nematodes, including their capacity for parasitism.
Argonaute; helminth; microRNA (miRNA); nematode; RNA interference (RNAi); small interfering RNA (siRNA); Ago, Argonaute; ALG, Ago-like gene; At, Arabidopsis thaliana; CSR, chromosome segregation- and RNAi-deficient; miRNA, microRNA; piRNA, piwi-interacting RNA; PRG, Piwi-related gene; RdRP, RNA-dependent RNA polymerase; RDE, RNAi-defective; RISC, RNA-induced silencing complex; RNAi, RNA interference; siRNA, small interfering RNA; WAGO, worm-specific Ago
Anguillicola crassus is an economically and ecologically important parasitic nematode of eels. The native range of A. crassus is in East Asia, where it infects Anguilla japonica, the Japanese eel. A. crassus was introduced into European eels, Anguilla anguilla, 30 years ago. The parasite is more pathogenic in its new host than in its native one, and is thought to threaten the endangered An. anguilla across its range. The molecular bases for the increased pathogenicity of the nematodes in their new hosts is not known.
A reference transcriptome was assembled for A. crassus from Roche 454 pyrosequencing data. Raw reads (756,363 total) from nematodes from An. japonica and An. anguilla hosts were filtered for likely host contaminants and ribosomal RNAs. The remaining 353,055 reads were assembled into 11,372 contigs of a high confidence assembly (spanning 6.6 Mb) and an additional 21,153 singletons and contigs of a lower confidence assembly (spanning an additional 6.2 Mb). Roughly 55% of the high confidence assembly contigs were annotated with domain- or protein sequence similarity derived functional information. Sequences conserved only in nematodes, or unique to A. crassus were more likely to have secretory signal peptides. Thousands of high quality single nucleotide polymorphisms were identified, and coding polymorphism was correlated with differential expression between individual nematodes. Transcripts identified as being under positive selection were enriched in peptidases. Enzymes involved in energy metabolism were enriched in the set of genes differentially expressed between European and Asian A. crassus.
The reference transcriptome of A. crassus is of high quality, and will serve as a basis for future work on the invasion biology of this important parasite. The polymorphisms identified will provide a key tool set for analysis of population structure and identification of genes likely to be involved in increased pathogenicity in European eel hosts. The identification of peptidases under positive selection is a first step in this programme.
Heliconius butterflies represent a recent radiation of species, in which wing pattern divergence has been implicated in speciation. Several loci that control wing pattern phenotypes have been mapped and two were identified through sequencing. These same gene regions play a role in adaptation across the whole Heliconius radiation. Previous studies of population genetic patterns at these regions have sequenced small amplicons. Here, we use targeted next-generation sequence capture to survey patterns of divergence across these entire regions in divergent geographical races and species of Heliconius. This technique was successful both within and between species for obtaining high coverage of almost all coding regions and sufficient coverage of non-coding regions to perform population genetic analyses. We find major peaks of elevated population differentiation between races across hybrid zones, which indicate regions under strong divergent selection. These ‘islands’ of divergence appear to be more extensive between closely related species, but there is less clear evidence for such islands between more distantly related species at two further points along the ‘speciation continuum’. We also sequence fosmid clones across these regions in different Heliconius melpomene races. We find no major structural rearrangements but many relatively large (greater than 1 kb) insertion/deletion events (including gain/loss of transposable elements) that are variable between races.
Heliconius; colour pattern; divergence; target enrichment; speciation; genomic islands
The evolutionary importance of hybridization and introgression has long been debated1. We used genomic tools to investigate introgression in Heliconius, a rapidly radiating genus of neotropical butterflies widely used in studies of ecology, behaviour, mimicry and speciation2-5 . We sequenced the genome of Heliconius melpomene and compared it with other taxa to investigate chromosomal evolution in Lepidoptera and gene flow among multiple Heliconius species and races. Among 12,657 predicted genes for Heliconius, biologically important expansions of families of chemosensory and Hox genes are particularly noteworthy. Chromosomal organisation has remained broadly conserved since the Cretaceous, when butterflies split from the silkmoth lineage. Using genomic resequencing, we show hybrid exchange of genes between three co-mimics, H. melpomene, H. timareta, and H. elevatus, especially at two genomic regions that control mimicry pattern. Closely related Heliconius species clearly exchange protective colour pattern genes promiscuously, implying a major role for hybridization in adaptive radiation.
The cestode Echinococcus granulosus - the agent of cystic echinococcosis, a zoonosis affecting humans and domestic animals worldwide - is an excellent model for the study of host-parasite cross-talk that interfaces with two mammalian hosts. To develop the molecular analysis of these interactions, we carried out an EST survey of E. granulosus larval stages. We report the salient features of this study with a focus on genes reflecting physiological adaptations of different parasite stages.
We generated ∼10,000 ESTs from two sets of full-length enriched libraries (derived from oligo-capped and trans-spliced cDNAs) prepared with three parasite materials: hydatid cyst wall, larval worms (protoscoleces), and pepsin/H+-activated protoscoleces. The ESTs were clustered into 2700 distinct gene products. In the context of the biology of E. granulosus, our analyses reveal: (i) a diverse group of abundant long non-protein coding transcripts showing homology to a middle repetitive element (EgBRep) that could either be active molecular species or represent precursors of small RNAs (like piRNAs); (ii) an up-regulation of fermentative pathways in the tissue of the cyst wall; (iii) highly expressed thiol- and selenol-dependent antioxidant enzyme targets of thioredoxin glutathione reductase, the functional hub of redox metabolism in parasitic flatworms; (iv) candidate apomucins for the external layer of the tissue-dwelling hydatid cyst, a mucin-rich structure that is critical for survival in the intermediate host; (v) a set of tetraspanins, a protein family that appears to have expanded in the cestode lineage; and (vi) a set of platyhelminth-specific gene products that may offer targets for novel pan-platyhelminth drug development.
This survey has greatly increased the quality and the quantity of the molecular information on E. granulosus and constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic analyses focused on cestodes and platyhelminths.
Cestodes are a neglected group of platyhelminth parasites, despite causing chronic infections to humans and domestic animals worldwide. We used Echinococcus granulosus as a model to study the molecular basis of the host-parasite cross-talk during cestode infections. For this purpose, we carried out a survey of the genes expressed by parasite larval stages interfacing with definitive and intermediate hosts. Sequencing from several high quality cDNA libraries provided numerous insights into the expression of genes involved in important aspects of E. granulosus biology, e.g. its metabolism (energy production and antioxidant defences) and the synthesis of key parasite structures (notably, the one exposed to humans and livestock intermediate hosts). Our results also uncovered the existence of an intriguing set of abundant repeat-associated non-protein coding transcripts that may participate in the regulation of gene expression in all surveyed stages. The dataset now generated constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic studies focused on cestodes and platyhelminths. In particular, the detailed characterization of a range of newly discovered genes will contribute to a better understanding of the biology of cestode infections and, therefore, to the development of products allowing their efficient control.
The heartworm Dirofilaria immitis is an important parasite of dogs. Transmitted by mosquitoes in warmer climatic zones, it is spreading across southern Europe and the Americas at an alarming pace. There is no vaccine, and chemotherapy is prone to complications. To learn more about this parasite, we have sequenced the genomes of D. immitis and its endosymbiont Wolbachia. We predict 10,179 protein coding genes in the 84.2 Mb of the nuclear genome, and 823 genes in the 0.9-Mb Wolbachia genome. The D. immitis genome harbors neither DNA transposons nor active retrotransposons, and there is very little genetic variation between two sequenced isolates from Europe and the United States. The differential presence of anabolic pathways such as heme and nucleotide biosynthesis hints at the intricate metabolic interrelationship between the heartworm and Wolbachia. Comparing the proteome of D. immitis with other nematodes and with mammalian hosts, we identify families of potential drug targets, immune modulators, and vaccine candidates. This genome sequence will support the development of new tools against dirofilariasis and aid efforts to combat related human pathogens, the causative agents of lymphatic filariasis and river blindness.—Godel, C., Kumar, S., Koutsovoulos, G., Ludin, P., Nilsson, D., Comandatore, F., Wrobel, N., Thompson, M., Schmid, C. D., Goto, S., Bringaud, F., Wolstenholme, A., Bandi, C., Epe, C., Kaminsky, R., Blaxter, M., Mäser, P. The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets.
comparative genomics; filaria; transposon; Wolbachia