Nicola Palmieri

The life cycle of Drosophila orphan genes

Orphans are genes restricted to a single phylogenetic lineage and emerge at high rates. While this predicts an accumulation of genes, the gene number has remained remarkably constant through evolution. This paradox has not yet been resolved. Because orphan genes have been mainly analyzed over long evolutionary time scales, orphan loss has remained unexplored. Here we study the patterns of orphan turnover among close relatives in the Drosophila obscura group. We show that orphans are not only emerging at a high rate, but that they are also rapidly lost. Interestingly, recently emerged orphans are more likely to be lost than older ones. Furthermore, highly expressed orphans with a strong male-bias are more likely to be retained. Since both lost and retained orphans show similar evolutionary signatures of functional conservation, we propose that orphan loss is not driven by high rates of sequence evolution, but reflects lineage-specific functional requirements. DOI: http://dx.doi.org/10.7554/eLife.01311.001

Evaluation of Different Reference Based Annotation Strategies Using RNA-Seq – A Case Study in Drososphila pseudoobscura

RNA-Seq is a powerful tool for the annotation of genomes, in particular for the identification of isoforms and UTRs. Nevertheless, several software tools exist and no standard strategy to obtain a reliable annotation is yet established. We tested different combinations of the most commonly used reference-based alignment tools (TopHat, GSNAP) in combination with two frequently used reference-based assemblers (Cufflinks, Scripture) and evaluated the potential of RNA-Seq to improve the annotation of Drosophila pseudoobscura. While GSNAP maps a higher proportion of reads, TopHat resulted in a more accurate annotation when used in combination with Cufflinks. Scripture had the lowest sensitivity. Interestingly, after subsampling to the same coverage for GSNAP and TopHat, we find that both mappers have similar performance, implying that the advantage of TopHat is mainly an artifact of the lower coverage. Overall, we observed a low concordance among the different approaches tested both at junction and isoform levels. Using data from both sexes of two adult strains of D. pseudoobscura we detected alternative splicing for about 30% of the FlyBase multiple-exon genes. Moreover, we extended the boundaries for 6523 genes (about 40%). We annotated 669 new genes, 45% of them with splicing evidence. Most of the new genes are located on unassembled contigs, reflecting their incomplete annotation. Finally, we identified 99 additional new genes that are not represented in the current genome contigs of D. pseudoobscura, probably due to location in genomic regions that are difficult to assemble (e.g. heterochromatic regions).

Genome assembly and annotation of a Drosophila simulans strain from Madagascar.

Drosophila simulans is a close relative of the genetic model D. melanogaster. Its worldwide distribution in combination with the absence of segregating chromosomal inversions makes this species an increasingly attractive model to study the molecular signatures of adaptation in natural and experimental populations. In an effort to improve the genomic resources for D. simulans, we assembled and annotated the genome of a strain originating from Madagascar (M252), the ancestral range of D. simulans. The comparison of the M252 genome to other available D. simulans assemblies confirmed its high quality, but also highlighted genomic regions that are difficult to assemble with NGS data. The annotation of M252 provides a clear improvement with alternative splicing for 52% of the multiple‐exon genes, UTRs for 70% of the genes, 225 novel genes and 781 pseudogenes being reported. We anticipate that the M252 genome will be a valuable resource for many research questions.

Genomic Resources Notes Accepted 1 August 2014–30 September 2014

This article documents the public availability of (i) transcriptome sequence data, assembly and annotation, and single nucleotide polymorphisms (SNPs) for the cone snail Conus miliaris; (ii) a set of SNP markers for two biotypes from the Culex pipiens mosquito complex; (iii) transcriptome sequence data, assembly and annotation for the mountain fly Drosophila nigrosparsa; (iv) transcriptome sequence data, assembly and annotation and SNPs for the Neotropical toads Rhinella marina and R. schneideri; and (v) partial genomic sequence assembly and annotation for 35 spiny lizard species (Genus Sceloporus).

Y Chromosome Uncovers the Recent Oriental Origin of Modern Stallions

The Y chromosome directly reflects male genealogies, but the extremely low Y chromosome sequence diversity in horses has prevented the reconstruction of stallion genealogies [1, 2]. Here, we resolve the first Y chromosome genealogy of modern horses by screening 1.46 Mb of the male-specific region of the Y chromosome (MSY) in 52 horses from 21 breeds. Based on highly accurate pedigree data, we estimated the de novo mutation rate of the horse MSY and showed that various modern horse Y chromosome lineages split much later than the domestication of the species. Apart from few private northern European haplotypes, all modern horse breeds clustered together in a roughly 700-year-old haplogroup that was transmitted to Europe by the import of Oriental stallions. The Oriental horse group consisted of two major subclades: the Original Arabian lineage and the Turkoman horse lineage. We show that the English Thoroughbred MSY was derived from the Turkoman lineage and that English Thoroughbred sires are largely responsible for the predominance of this haplotype in modern horses.

Cystoisospora suis - A Model of Mammalian Cystoisosporosis

Cystoisospora suis is a coccidian species that typically affects suckling piglets. Infections occur by oral uptake of oocysts and are characterized by non-hemorrhagic transient diarrhea, resulting in poor weight gain. Apparently, primary immune responses to C. suis cannot readily be mounted by neonates, which contributes to the establishment and rapid development of the parasite, while in older pigs age-resistance prevents disease development. However, the presence of extraintestinal stages, although not unequivocally demonstrated, is suspected to enable parasite persistence together with the induction and maintenance of immune response in older pigs, which in turn may facilitate the transfer of C. suis-specific factors from sow to offspring. It is assumed that neonates are particularly prone to clinical disease because infections with C. suis interfere with the establishment of the gut microbiome. Clostridia have been especially inferred to profit from the altered intestinal environment during parasite infection. New tools, particularly in the area of genomics, might illustrate the interactions between C. suis and its host and pave the way for the development of new control methods not only for porcine cystoisosporosis but also for other mammalian Cystoisospora infections. The first reference genome for C. suis is under way and will be a fertile ground to discover new drugs and vaccines. At the same time, the establishment and refinement of an in vivo model and an in vitro culture system, supporting the complete life cycle of C. suis, will underpin the functional characterization of the parasite and shed light on its biology and control.

The genome of the protozoan parasite Cystoisospora suis and a reverse vaccinology approach to identify vaccine candidates

Graphical abstract

Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein

BackgroundThe genome of the apicomplexan parasite Cystoisospora suis (syn. Isospora suis) has recently been sequenced and annotated, opening the possibility for the identification of novel therapeutic targets against cystoisosporosis. It was previously proposed that a 42xa0kDa uncharacterized merozoite protein, encoded by gene CSUI_005805, might be a relevant vaccine candidate due to its high immunogenic score, high expression level and species-specificity as determined in silico.MethodsThe 1170xa0bp coding sequence of the CSUI_005805 gene was PCR amplified and cloned into the bacterial expression vector pQE-31. The specificity of the expressed recombinant protein was evaluated in an immunoblot, and relative levels of expression in different developmental stages and subcellular localization were determined by quantitative real-time PCR and indirect immunofluorescence assay, respectively.ResultsThe CSUI_005805 gene encoded for a 389 amino acid protein containing a histidine-rich region. Quantitative RT-PCR showed that CSUI_005805 was differentially expressed during the early development of C. suis in vitro, with higher transcript levels in merozoites compared to sporozoites. The recombinant protein was specifically recognized by sera from chicken immunized with recombinant CSUI_005805 protein and sera from piglets experimentally infected with C. suis, all of which suggested that despite prokaryotic expression, the recombinant CSUI_005805 protein maintained antigenic determinants and could elicit an immune response in the host. Immunofluorescence labelling and confocal microscopy revealed localization primarily at the surface of the parasite.ConclusionsThe results suggest that CSUI_005805 is highly expressed in merozoites and might thus be critical for their survival and establishment inside host cells. Owing to its specificity, localization and expression pattern, CSUI_005805 could be exploited as an attractive candidate for alternative control strategies against C. suis such as vaccines.

Comparison of an injectable toltrazuril-gleptoferron (Forceris®) and an oral toltrazuril (Baycox®) + injectable iron dextran for the control of experimentally induced piglet cystoisosporosis

BackgroundCystoisospora suis causes diarrhoeal disease and reduced weight gain in suckling piglets, and a toltrazuril-based oral suspension is available for treatment. Recently a combinatorial product with toltrazuril plus iron has been developed for parenteral application. In this study we compared the efficacy of the injectable product with the oral suspension against experimentally induced piglet cystoisosporosis.MethodsIn a randomised controlled study, three groups of piglets (n = 10–13) were treated either with a fixed dose of 45 mg toltrazuril + 200 mg gleptoferron i.m. per piglet (Forceris®) on the second day of life (study day 2; SD 2) or with 20 mg toltrazuril/kg body weight as an oral suspension (Baycox® 5%) on SD 4 or left untreated (Control group). The Baycox® and the Control group received 200 mg of iron dextran/piglet on SD 2. All piglets were infected with 1000 sporulated C. suis oocysts on SD 3. Faecal samples were taken daily from SD 7 to SD 20 to determine faecal consistency, oocyst shedding and other diarrhoeal pathogens. Body weight was recorded on SD 1 and then weekly until SD 29. Animals were observed daily for general health and after treatment for possible adverse events.ResultsIn the Control group all animals shed oocysts for 3.1 days on average and all animals showed diarrhoea for an average of five days. Excretion peaked on SD 9 (max. 48,618 oocysts per gram of faeces). Treatment with Forceris® completely suppressed oocyst excretion. In the Baycox® group, low levels of excretion could be detected. Diarrhoea was reduced to single piglets in the treated groups. Body weight development was reduced in the Control group compared to the treated groups. Enteropathogenic bacteria (Escherichia coli, Clostridium perfringens) could be detected. All parameters related to oocyst excretion, faecal consistency and weight gain were significantly improved in the treated groups compared to the Control group without significant differences between the treated groups. Both products were safe to use.ConclusionsTreatment with both the injectable (Forceris®) and the oral (Baycox®) formulation of toltrazuril in the prepatent period were safe and highly effective against experimental infection with C. suis in newborn piglets.

Morphological and molecular identification of nasopharyngeal bot fly larvae infesting red deer (Cervus elaphus) in Austria.

Nasopharyngeal myiases are caused by larvae of bot flies (Diptera: Oestridae), which have evolved a high specificity for their hosts. Bot flies (nu2009=u2009916) were collected from 137 (57.6xa0%) out of 238 red deer (Cervus elaphus) hunted in Vorarlberg and Tyrol (Western Austria). After being stored in 75xa0% ethanol, larvae were identified to species level and developmental stage using morphological and morphometric keys. Larvae were also molecularly characterized by polymerase chain reaction (PCR) amplification and partial sequencing of the mitochondrial cytochrome oxidase subunit I gene. Morphological and molecular analysis allowed identification of larvae as Cephenemyia auribarbis and Pharyngomyia picta. Genetic variations were also examined within the specimens collected in both geographical locations.