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var genes encode Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens. These highly diverse antigens are displayed on the surface of infected erythrocytes and play a critical role in immune evasion and sequestration of infected erythrocytes. Studies of var expression using non-leukocyte-depleted blood are challenging because of the predominance of host genetic material and lack of conserved var segments. Our goal was to enrich for parasite RNA, allowing de novo assembly of var genes and detection of expressed novel variants. We used two overall approaches: (i) enriching for total mRNA in the sequencing library preparations and (ii) enriching for parasite RNA with a custom capture array based on Roche’s SeqCap EZ enrichment system. The capture array was designed with probes based on the whole 3D7 reference genome and an additional >4,000 full-length var gene sequences from other P. falciparum strains. We tested each method on RNA samples from Malian children with severe or uncomplicated malaria infections. All reads mapping to the human genome were removed, the remaining reads were assembled de novo into transcripts, and from these, var-like transcripts were identified and annotated. The capture array produced the longest maximum length and largest numbers of var gene transcripts in each sample, particularly in samples with low parasitemia. Identifying the most-expressed var gene sequences in whole-blood clinical samples without the need for extensive processing or generating sample-specific reference genome data is critical for understanding the role of PfEMP1s in malaria pathogenesis. IMPORTANCE Malaria parasites display antigens on the surface of infected red blood cells in the human host that facilitate attachment to blood vessels, contributing to the severity of infection. These antigens are highly variable, allowing the parasite to evade the immune system. Identifying these expressed antigens is critical to understanding the development of severe malarial disease. However, clinical samples contain limited amounts of parasite genetic material, a challenge for sequencing efforts further compounded by the extreme diversity of the parasite surface antigens. We present a method that enriches for these antigen sequences in clinical samples using a custom capture array, requiring minimal processing in the field. While our results are focused on the malaria parasite Plasmodium falciparum, this approach has broad applicability to other highly diverse antigens from other parasites and pathogens such as those that cause giardiasis and leishmaniasis.

As countries work towards malaria elimination, it is important to monitor imported cases to prevent reestablishment of local transmission. The Plasmodium falciparum Pfs47 gene has strong geographic population structure, because only those parasites with Pfs47 haplotypes compatible with the mosquito vector species in a given continent are efficiently transmitted. Analysis of 4,971 world-wide Pfs47 sequences identified two SNPs (at 707 and 725 bp) as sufficient to establish the likely continent of origin of P. falciparum isolates. Pfs47 sequences from Africa, Asia, and the New World presented more that 99% frequency of distinct combinations of the SNPs 707 and 725 genotypes. Interestingly, Papua New Guinea Pfs47 sequences have the highest diversity in SNPs 707 and 725. Accurate and reproducible High-Resolution Melting (HRM) assays were developed to genotype Pfs47 SNPs 707 and 725 in laboratory and field samples, to assess the geographic origin and risk of local transmission of imported P. falciparum malaria cases., Molina-Cruz et al. analyzed 4,971 sequences of the Plasmodium falciparum Pfs47 gene and identified a combination of two SNP haplotypes highly correlated with geographic origin and transmission potential by mosquito vectors. A simple High-Resolution Melting RT-PCR assay was developed to genotype these SNPs and establish the likely continent of origin and transmission risk of P. falciparum isolates.

Babesia bovis is a hemoprotozoan parasite of cattle that has a complex life cycle within vertebrate and invertebrate hosts. In the mammalian host, B. bovis undergoes asexual reproduction while in the tick midgut, gametes are induced, fuse, and form zygotes. The zygote infects tick gut epithelial cells and transform into kinetes that are released into the hemolymph and invade other tick tissues such as the ovaries, resulting in transovarial transmission to tick offspring. To compare gene regulation between different B. bovis life stages, we collected parasites infecting bovine erythrocytes and tick hemolymph. Total RNA samples were isolated, and multiplexed libraries sequenced using paired-end 100 cycle reads of a HiSeq 2500. The data was normalized using the TMM method and analysed for significant differential expression using the generalized linear model likelihood ratio test (GLM LRT) in edgeR. To validate our datasets, ten genes were selected using NormFinder. Genes that had no significant fold change between the blood and tick stages in the RNA-Seq datasets were tested by quantitative PCR to determine their suitability as "housekeeping" genes. The normalized RNA-Seq data revealed genes upregulated during infection of the mammalian host or tick vector and six upregulated genes were validated by quantitative PCR. These datasets can help identify useful targets for controlling bovine babesiosis.

Theileria parva is an economically important, intracellular, tick-transmitted parasite of cattle. A live vaccine against the parasite is effective against challenge from cattle-transmissible T. parva but not against genotypes originating from the African Cape buffalo, a major wildlife reservoir, prompting the need to characterize genome-wide variation within and between cattle- and buffalo-associated T. parva populations. Here, we describe a capture-based target enrichment approach that enables, for the first time, de novo assembly of nearly complete T. parva genomes derived from infected host cell lines. This approach has exceptionally high specificity and sensitivity and is successful for both cattle- and buffalo-derived T. parva parasites. De novo genome assemblies generated for cattle genotypes differ from the reference by ~54K single nucleotide polymorphisms (SNPs) throughout the 8.31 Mb genome, an average of 6.5 SNPs/kb. We report the first buffalo-derived T. parva genome, which is ~20 kb larger than the genome from the reference, cattle-derived, Muguga strain, and contains 25 new potential genes. The average non-synonymous nucleotide diversity (πN) per gene, between buffalo-derived T. parva and the Muguga strain, was 1.3%. This remarkably high level of genetic divergence is supported by an average Wright’s fixation index (FST), genome-wide, of 0.44, reflecting a degree of genetic differentiation between cattle- and buffalo-derived T. parva parasites more commonly seen between, rather than within, species. These findings present clear implications for vaccine development, further demonstrated by the ability to assemble nearly all known antigens in the buffalo-derived strain, which will be critical in design of next generation vaccines. The DNA capture approach used provides a clear advantage in specificity over alternative T. parva DNA enrichment methods used previously, such as those that utilize schizont purification, is less labor intensive, and enables in-depth comparative genomics in this apicomplexan parasite., Author summary An estimated 50 million cattle in sub-Saharan Africa are at risk of the deadly livestock disease East coast fever (ECF), caused by the parasite Theileria parva, which imposes tremendous economic hardship on smallholder farmers. An existing ECF vaccine protects against strains circulating among cattle, but not against T. parva derived from African Cape buffalo, its main wildlife carrier. Understanding this difference in protective efficacy requires characterization of the genetic diversity in T. parva strains associated with each mammalian host, a goal that has been hindered by the proliferation of T. parva in nucleated host cells, with much larger genomes. Here we adapted a sequence capture approach to target the whole parasite genome, enabling enrichment of parasite DNA over that of the host. Choices in protocol development resulted in nearly 100% parasite genome specificity and sensitivity, making this approach the most successful yet to generate T. parva genome sequence data in a high-throughput manner. The analyses uncovered a degree of genetic differentiation between cattle- and buffalo-derived genotypes that is akin to levels more commonly seen between species. This approach, which will enable an in-depth T. parva population genomics study from cattle and buffalo in the endemic regions, can easily be adapted to other intracellular pathogens.

Individuals acquire immunity to clinical malaria after repeated Plasmodium falciparum infections. This immunity to disease is thought to reflect the acquisition of a repertoire of responses to multiple alleles in diverse parasite antigens. In previous studies, we identified polymorphic sites within individual antigens that are associated with parasite immune evasion by examining antigen allele dynamics in individuals followed longitudinally. Here we expand this approach by analyzing genome-wide polymorphisms using whole genome sequence data from 140 parasite isolates representing malaria cases from a longitudinal study in Malawi and identify 25 genes that encode likely targets of naturally acquired immunity and that should be further characterized for their potential as vaccine candidates.

Saline migrants into freshwater habitats constitute among the most destructive invaders in aquatic ecosystems throughout the globe. However, the evolutionary and physiological mechanisms underlying such habitat transitions remain poorly understood. To explore the mechanisms of freshwater adaptation and distinguish between adaptive (evolutionary) and acclimatory (plastic) responses to salinity change, we examined genome-wide patterns of gene expression between ancestral saline and derived freshwater populations of the Eurytemora affinis species complex, reared under two different common-garden conditions (0 versus 15 PSU). We found that evolutionary shifts in gene expression (between saline and freshwater inbred lines) showed far greater changes and were more widespread than acclimatory responses to salinity (0 versus 15 PSU). Most notably, 30-40 genes showing evolutionary shifts in gene expression across the salinity boundary were associated with ion transport function, with inorganic cation transmembrane transport forming the largest Gene Ontology category. Of particular interest was the sodium transporter, the Na+ /H+ antiporter (NHA) gene family, which was discovered in animals relatively recently. Thirty key ion regulatory genes, such as NHA paralogue #7, demonstrated concordant evolutionary and plastic shifts in gene expression, suggesting the evolution of ion transporter function and plasticity during rapid invasions into novel salinities. Moreover, freshwater invasions were associated with the evolution of reduced plasticity in the freshwater population, again for the same key ion transporters, consistent with the predicted evolution of canalization following adaptation to stressful conditions. Our results have important implications for understanding evolutionary and physiological mechanisms of range expansions by some of the most widespread invaders in aquatic habitats.

Background Encoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes, resulting in immune evasion and severe malaria disease. Sequencing and assembling the 40–60 var gene complement for individual infections has been notoriously difficult, impeding molecular epidemiological studies and the assessment of particular var elements as subunit vaccine candidates. Methods We developed and validated a novel algorithm, Exon-Targeted Hybrid Assembly (ETHA), to perform targeted assembly of var gene sequences, based on a combination of Pacific Biosciences and Illumina data. Results Using ETHA, we characterized the repertoire of var genes in 12 samples from uncomplicated malaria infections in children from a single Malian village and showed them to be as genetically diverse as vars from isolates from around the globe. The gene var2csa, a member of the var family associated with placental malaria pathogenesis, was present in each genome, as were vars previously associated with severe malaria. Conclusion ETHA, a tool to discover novel var sequences from clinical samples, will aid the understanding of malaria pathogenesis and inform the design of malaria vaccines based on PfEMP1. ETHA is available at: https://sourceforge.net/projects/etha/. Electronic supplementary material The online version of this article (doi:10.1186/s13073-017-0422-4) contains supplementary material, which is available to authorized users.

Background The apicomplexan parasite Theileria parva causes a livestock disease called East coast fever (ECF), with millions of animals at risk in sub-Saharan East and Southern Africa, the geographic distribution of T. parva. Over a million bovines die each year of ECF, with a tremendous economic burden to pastoralists in endemic countries. Comprehensive, accurate parasite genome annotation can facilitate the discovery of novel chemotherapeutic targets for disease treatment, as well as elucidate the biology of the parasite. However, genome annotation remains a significant challenge because of limitations in the quality and quantity of the data being used to inform the location and function of protein-coding genes and, when RNA data are used, the underlying biological complexity of the processes involved in gene expression. Here, we apply our recently published RNAseq dataset derived from the schizont life-cycle stage of T. parva to update structural and functional gene annotations across the entire nuclear genome. Results The re-annotation effort lead to evidence-supported updates in over half of all protein-coding sequence (CDS) predictions, including exon changes, gene merges and gene splitting, an increase in average CDS length of approximately 50 base pairs, and the identification of 128 new genes. Among the new genes identified were those involved in N-glycosylation, a process previously thought not to exist in this organism and a potentially new chemotherapeutic target pathway for treating ECF. Alternatively-spliced genes were identified, and antisense and multi-gene family transcription were extensively characterized. Conclusions The process of re-annotation led to novel insights into the organization and expression profiles of protein-coding sequences in this parasite, and uncovered a minimal N-glycosylation pathway that changes our current understanding of the evolution of this post-translational modification in apicomplexan parasites.

BackgroundPlasmodium falciparum(Pf) whole-organism sporozoite vaccines have provided excellent protection against controlled human malaria infection (CHMI) and naturally transmitted heterogeneous Pf in the field. Initial CHMI studies showed significantly higher durable protection against homologous than heterologous strains, suggesting the presence of strain-specific vaccine-induced protection. However, interpretation of these results and understanding of their relevance to vaccine efficacy (VE) have been hampered by the lack of knowledge on genetic differences between vaccine and CHMI strains, and how these strains are related to parasites in malaria endemic regions.MethodsWhole genome sequencing using long-read (Pacific Biosciences) and short-read (Illumina) sequencing platforms was conducted to generatede novogenome assemblies for the vaccine strain, NF54, and for strains used in heterologous CHMI (7G8 from Brazil, NF166.C8 from Guinea, and NF135.C10 from Cambodia). The assemblies were used to characterize sequence polymorphisms and structural variants in each strain relative to the reference Pf 3D7 (a clone of NF54) genome. Strains were compared to each other and to clinical isolates from South America, Sub-Saharan Africa, and Southeast Asia.ResultsWhile few variants were detected between 3D7 and NF54, we identified tens of thousands of variants between NF54 and the three heterologous strains both genome-wide and within regulatory and immunologically important regions, including in pre-erythrocytic antigens that may be key for sporozoite vaccine-induced protection. Additionally, these variants directly contribute to diversity in immunologically important regions of the genomes as detected throughin silicoCD8+T cell epitope predictions. Of all heterologous strains, NF135.C10 consistently had the highest number of unique predicted epitope sequences when compared to NF54, while NF166.C8 had the lowest. Comparison to global clinical isolates revealed that these four strains are representative of their geographic region of origin despite long-term culture adaptation; of note, NF135.C10 is from an admixed population, and not part of recently-formed drug resistant subpopulations present in the Greater Mekong Sub-region.ConclusionsThese results are assisting the interpretation of VE of whole-organism vaccines against homologous and heterologous CHMI, and may be useful in informing the choice of strains for inclusion in region-specific or multi-strain vaccines.

Protective immunity induced by the infective sporozoite stage of Theileria parva indicates a potential role for antibodies directed against conserved serologically reactive regions of the major sporozoite surface antigen p67 in vaccination to control the parasite. We have examined the allelic variation and determined the extent of B cell epitope polymorphism of the gene encoding p67 among field isolates originating from cattle exposed to infected ticks in the Marula area of the rift valley in central Kenya where the African cape buffalo (Syncerus caffer) and cattle co-graze. In the first of two closely juxtaposed epitope sequences in the central region of the p67 protein, an in-frame deletion of a seven-amino acid segment results in a truncation that was observed in parasites derived from cattle that co-grazed with buffalo. In contrast, the variation in the second epitope was primarily due to nonsynonymous substitutions, resulting in relatively low overall amino acid conservation in this segment of the protein. We also observed polymorphism in the region of the protein adjacent to the two defined epitopes, but this was not sufficient to provide statistically significant evidence for positive selection. The data indicates that B cell epitopes previously identified within the p67 gene are polymorphic within the Marula field isolates. Given the complete sequence identity of the p67 gene in all previously characterized T. parva isolates that are transmissible between cattle by ticks, the diversity observed in p67 from the Marula isolates in combination with the clinical reaction of the infected cattle is consistent with them originating from ticks that had acquired T. parva from buffalo.

doi: 10.1099/mgen.0.000122.001. As sequencing technologies have evolved, the tools to analyze these sequences have made similar advances. However, for multi-species samples, we observed important and adverse differences in alignment specificity and computation time for bwa- mem (Burrows–Wheeler aligner-maximum exact matches) relative to bwa-aln. Therefore, we sought to optimize bwa-mem for alignment of data from multi-species samples in order to reduce alignment time and increase the specificity of alignments. In the multi-species cases examined, there was one majority member (i.e. Plasmodium falciparum or Brugia malayi) and one minority member (i.e. human or the Wolbachia endosymbiont wBm) of the sequence data. Increasing bwa-mem seed length from the default value reduced the number of read pairs from the majority sequence member that incorrectly aligned to the reference genome of the minority sequence member. Combining both source genomes into a single reference genome increased the specificity of mapping, while also reducing the central processing unit (CPU) time. In Plasmodium, at a seed length of 18 nt, 24.1 % of reads mapped to the human genome using 1.7±0.1 CPU hours, while 83.6 % of reads mapped to the Plasmodium genome using 0.2±0.0 CPU hours (total: 107.7 % reads mapping; in 1.9±0.1 CPU hours). In contrast, 97.1 % of the reads mapped to a combined Plasmodium–human reference in only 0.7±0.0 CPU hours. Overall, the results suggest that combining all references into a single reference database and using a 23 nt seed length reduces the computational time, while maximizing specificity. Similar results were found for simulated sequence reads from a mock metagenomic data set. We found similar improvements to computation time in a publicly available human-only data set.

Background VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, mediates the binding of P. falciparum-infected erythrocytes to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally acquired immunity and, as such, is a leading vaccine candidate against placental malaria. This protein is very polymorphic and technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time consuming and costly, and that performs poorly when applied to clinical samples that are commonly polyclonal. A next-generation sequencing platform, Pacific Biosciences (PacBio), offers an alternative approach to overcome these issues. Methods PCR primers were designed that target a 5 kb segment in the 5′ end of var2csa and the resulting amplicons were sequenced using PacBio sequencing. The primers were optimized using two laboratory strains and were validated on DNA from 43 clinical samples, extracted from dried blood spots on filter paper or from cryopreserved P. falciparum-infected erythrocytes. Sequence reads were assembled using the SMRT-analysis ConsensusTools module. Results Here, a PacBio sequencing-based approach for recovering a segment encoding the majority of VAR2CSA’s extracellular region is described; this segment includes the totality of the first four domains in the 5′ end of var2csa (~5 kb), from clinical malaria samples. The feasibility of the method is demonstrated, showing a high success rate from cryopreserved samples and more limited success from dried blood spots stored at room temperature, and characterized the genetic variation of the var2csa locus. Conclusions This method will facilitate a detailed analysis of var2csa genetic variation and can be adapted to sequence other hypervariable P. falciparum genes. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-1976-8) contains supplementary material, which is available to authorized users.

Background Amplified copy number in the plasmepsin II/III genes within Plasmodium falciparum has been associated with decreased sensitivity to piperaquine. To examine this association and test whether additional loci might also contribute, we performed a genome-wide association study of ex vivo P. falciparum susceptibility to piperaquine. Methods Plasmodium falciparum DNA from 183 samples collected primarily from Cambodia was genotyped at 33716 genome-wide single nucleotide polymorphisms (SNPs). Linear mixed models and random forests were used to estimate associations between parasite genotypes and piperaquine susceptibility. Candidate polymorphisms were evaluated for their association with dihydroartemisinin-piperaquine treatment outcomes in an independent dataset. Results Single nucleotide polymorphisms on multiple chromosomes were associated with piperaquine 90% inhibitory concentrations (IC90) in a genome-wide analysis. Fine-mapping of genomic regions implicated in genome-wide analyses identified multiple SNPs in linkage disequilibrium with each other that were significantly associated with piperaquine IC90, including a novel mutation within the gene encoding the P. falciparum chloroquine resistance transporter, PfCRT. This mutation (F145I) was associated with dihydroartemisinin-piperaquine treatment failure after adjusting for the presence of amplified plasmepsin II/III, which was also associated with decreased piperaquine sensitivity. Conclusions Our data suggest that, in addition to plasmepsin II/III copy number, other loci, including pfcrt, may also be involved in piperaquine resistance.

Chemosensory-related gene (CRG) families have been studied extensively in insects, but their evolutionary history across the Arthropoda had remained relatively unexplored. Here, we address current hypotheses and prior conclusions on CRG family evolution using a more comprehensive data set. In particular, odorant receptors were hypothesized to have proliferated during terrestrial colonization by insects (hexapods), but their association with other pancrustacean clades and with independent terrestrial colonizations in other arthropod subphyla have been unclear. We also examine hypotheses on which arthropod CRG family is most ancient. Thus, we reconstructed phylogenies of CRGs, including those from new arthropod genomes and transcriptomes, and mapped CRG gains and losses across arthropod lineages. Our analysis was strengthened by including crustaceans, especially copepods, which reside outside the hexapod/branchiopod clade within the subphylum Pancrustacea. We generated the first high-resolution genome sequence of the copepod Eurytemora affinis and annotated its CRGs. We found odorant receptors and odorant binding proteins present only in hexapods (insects) and absent from all other arthropod lineages, indicating that they are not universal adaptations to land. Gustatory receptors likely represent the oldest chemosensory receptors among CRGs, dating back to the Placozoa. We also clarified and confirmed the evolutionary history of antennal ionotropic receptors across the Arthropoda. All antennal ionotropic receptors in E. affinis were expressed more highly in males than in females, suggestive of an association with male mate-recognition behavior. This study is the most comprehensive comparative analysis to date of CRG family evolution across the largest and most speciose metazoan phylum Arthropoda.

Babesia microti, a tick-transmitted, intraerythrocytic protozoan parasite circulating mainly among small mammals, is the primary cause of human babesiosis. While most cases are transmitted by Ixodes ticks, the disease may also be transmitted through blood transfusion and perinatally. A comprehensive analysis of genome composition, genetic diversity, and gene expression profiling of seven B. microti isolates revealed that genetic variation in isolates from the Northeast United States is almost exclusively associated with genes encoding the surface proteome and secretome of the parasite. Furthermore, we found that polymorphism is restricted to a small number of genes, which are highly expressed during infection. In order to identify pathogen-encoded factors involved in host-parasite interactions, we screened a proteome array comprised of 174 B. microti proteins, including several predicted members of the parasite secretome. Using this immuno-proteomic approach we identified several novel antigens that trigger strong host immune responses during the onset of infection. The genomic and immunological data presented herein provide the first insights into the determinants of B. microti interaction with its mammalian hosts and their relevance for understanding the selective pressures acting on parasite evolution.

Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives.

The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia.P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently.The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10(-12)). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas.K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.

Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF). ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer), and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR) in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1) or the host (hypoxanthine phosphoribosyltransferase 1, hprt1) are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field.

The human malaria parasite Plasmodium vivax is responsible for 25-40% of the approximately 515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.

Background There are no commercially available vaccines against human protozoan parasitic diseases, despite the success of vaccination-induced long-term protection against infectious diseases. East Coast fever, caused by the protist Theileria parva, kills one million cattle each year in sub-Saharan Africa, and contributes significantly to hunger and poverty in the region. A highly effective, live, multi-isolate vaccine against T. parva exists, but its component isolates have not been characterized. Here we sequence and compare the three component T. parva stocks within this vaccine, the Muguga Cocktail, namely Muguga, Kiambu5 and Serengeti-transformed, aiming to identify genomic features that contribute to vaccine efficacy. Results We find that Serengeti-transformed, originally isolated from the wildlife carrier, the African Cape buffalo, is remarkably and unexpectedly similar to the Muguga isolate. The 420 detectable non-synonymous SNPs were distributed among only 53 genes, primarily subtelomeric antigens and antigenic families. The Kiambu5 isolate is considerably more divergent, with close to 40,000 SNPs relative to Muguga, including >8,500 non-synonymous mutations distributed among >1,700 (42.5 %) of the predicted genes. These genetic markers of the component stocks can be used to characterize the composition of new batches of the Muguga Cocktail. Conclusions Differences among these three isolates, while extensive, represent only a small proportion of the genetic variation in the entire species. Given the efficacy of the Muguga Cocktail in inducing long-lasting protection against infections in the field, our results suggest that whole-organism vaccines against parasitic diseases can be highly efficacious despite considerable genome-wide differences relative to the isolates against which they protect., BMC Genomics, 16, ISSN:1471-2164

We report the genome sequence of Theileria parva , an apicomplexan pathogen causing economic losses to smallholder farmers in Africa. The parasite chromosomes exhibit limited conservation of gene synteny with Plasmodium falciparum , and its plastid-like genome represents the first example where all apicoplast genes are encoded on one DNA strand. We tentatively identify proteins that facilitate parasite segregation during host cell cytokinesis and contribute to persistent infection of transformed host cells. Several biosynthetic pathways are incomplete or absent, suggesting substantial metabolic dependence on the host cell. One protein family that may generate parasite antigenic diversity is not telomere-associated.

To determine how population structure of the host species affects the spread of transposable elements and to assess the strength of selection acting on different structural regions, we sequenced P elements from strains of Drosophila willistoni and Drosophila sturtevanti sampled from across the distributions of these species. Elements from D. sturtevanti exhibited considerable sequence variation, and similarity among them was correlated to geographic distance between collection sites. By contrast, all D. willistoni elements sampled were essentially identical (π < 0.2%) and exhibited patterns typical of a recent population expansion. While the canonical P elements sampled from D. sturtevanti appear to be long-time residents in that species, a rapid expansion of a very young canonical P-element lineage is suggested in D. willistoni, overcoming barriers such as large geographical distances and moderate levels of population subdivision. Between-species comparisons reveal selective constraints on P-element evolution, as indicated by significantly different substitution rates in noncoding, silent, and replacement sites. Most remarkably, in addition to replacement sites, selection pressure appears to be strong in the first and third introns and in the 3′ and 5′ flanking regions.

Mariner transposable elements encoding a D,D34D motif-bearing transposase are characterized by their pervasiveness among, and exclusivity to, animal phyla. To date several hundred sequences have been obtained from taxa ranging from cnidarians to humans, only two of which are known to be functional. Related transposons have been identified in plants and fungi, but their absence among protists is noticeable. Here, we identify and characterize Tvmar1, the first representative of the mariner family to be found in a species of protist, the human parasite Trichomonas vaginalis. This is the first D,D34D element to be found outside the animal kingdom, and its inclusion in the mariner family is supported by both structural and phylogenetic analyses. Remarkably, Tvmar1 has all the hallmarks of a functional element, and has recently expanded to several hundred copies in the genome of T. vaginalis. Our results show that a new potentially active mariner has been found, which belongs to a distinct mariner lineage, and has successfully invaded a non-animal, single-celled organism. The considerable genetic distance between Tvmar1 and other mariners may have valuable implications for the design of new, high-efficiency vectors to be used in transfection studies in protists.

We have analyzed the alignment of a long homologous region of the human and baboon genomes (approximately 1.5 Mb). We show that the frequency of gaps between aligned segments decreases slowly with gap length, indicating that several successive nucleotides are often deleted or inserted in one event. By contrast, runs of consecutive mismatches decrease rapidly in frequency with increasing length, following an exponential distribution, indicating that nucleotides are mostly substituted one at a time. Nucleotide substitutions are clumped at the scales of10 and 1000-10,000 nucleotides, but show almost no aggregation at the scales of10-100 and over approximately 50,000 nucleotides. Apparently, two rather different factors make the substitution rate not exactly uniform along the DNA sequence. Comparison of regions of very similar genomes that are approximately selectively neutral makes it possible to study spontaneous mutation at a new level of resolution.

High throughput sequencing has accelerated the determination of genome sequences for thousands of human infectious disease pathogens and dozens of their vectors. The scale and scope of these data are enabling genotype-phenotype association studies to identify genetic determinants of pathogen virulence and drug/insecticide resistance, and phylogenetic studies to track the origin and spread of disease outbreaks. To maximize the utility of genomic sequences for these purposes, it is essential that metadata about the pathogen/vector isolate characteristics be collected and made available in organized, clear, and consistent formats. Here we report the development of the GSCID/BRC Project and Sample Application Standard, developed by representatives of the Genome Sequencing Centers for Infectious Diseases (GSCIDs), the Bioinformatics Resource Centers (BRCs) for Infectious Diseases, and the U.S. National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), informed by interactions with numerous collaborating scientists. It includes mapping to terms from other data standards initiatives, including the Genomic Standards Consortium’s minimal information (MIxS) and NCBI’s BioSample/BioProjects checklists and the Ontology for Biomedical Investigations (OBI). The standard includes data fields about characteristics of the organism or environmental source of the specimen, spatial-temporal information about the specimen isolation event, phenotypic characteristics of the pathogen/vector isolated, and project leadership and support. By modeling metadata fields into an ontology-based semantic framework and reusing existing ontologies and minimum information checklists, the application standard can be extended to support additional project-specific data fields and integrated with other data represented with comparable standards. The use of this metadata standard by all ongoing and future GSCID sequencing projects will provide a consistent representation of these data in the BRC resources and other repositories that leverage these data, allowing investigators to identify relevant genomic sequences and perform comparative genomics analyses that are both statistically meaningful and biologically relevant.

Sequences homologous to the P element of Drosophila melanogaster were previously identified in Drosophila mediopunctata, a member of the tripunctata group, subgenus Drosophila. We report here that the P element is present in about three to five copies in the D. mediopunctata genome. While one of the insertion sites appears to be fixed, others may be polymorphic, indicating relatively recent P element activity. Phylogenetic analysis revealed that the D. mediopunctata element belongs to the canonical subfamily of P elements and that divergence of the D. mediopunctata element from other members of this subfamily ranges from 2% to 5% at the nucleotide level. This is the first report of a canonical P element outside the subgenus Sophophora. Based primarily on the striking incongruence between P element and host species phylogenies, the presence of a canonical P element in D. mediopunctata is most likely explained by horizontal transfer between species.

We induced mutations in Drosophila melanogaster males by treating them with 21.2 mm ethyl methanesulfonate (EMS). Nine quantitative traits (developmental time, viability, fecundity, longevity, metabolic rate, motility, body weight, and abdominal and sternopleural bristle numbers) were measured in outbred heterozygous F3 (viability) or F2 (all other traits) offspring from the treated males. The mean values of the first four traits, which are all directly related to the life history, were substantially affected by EMS mutagenesis: the developmental time increased while viability, fecundity, and longevity declined. In contrast, the mean values of the other five traits were not significantly affected. Rates of recessive X-linked lethals and of recessive mutations at several loci affecting eye color imply that our EMS treatment was equivalent to ∼100 generations of spontaneous mutation. If so, our data imply that one generation of spontaneous mutation increases the developmental time by 0.09% at 20° and by 0.04% at 25°, and reduces viability under harsh conditions, fecundity, and longevity by 1.35, 0.21, and 0.08%, respectively. Comparison of flies with none, one, and two grandfathers (or greatgrandfathers, in the case of viability) treated with EMS did not reveal any significant epistasis among the induced mutations.

The roles of selection and horizontal transfer in the evolution of the canonical subfamily of P: elements were studied in the saltans and willistoni species groups of the genus Drosophila (subgenus Sophophora). We estimate that the common ancestor of the canonical P: subfamily dates back 2-3 Myr at the most, despite the much older age (more than 40 Myr) of the P: family as a whole. The evolution of the canonical P: subfamily is characterized by weak selection at nonsynonymous sites. These sites have evolved at three quarters the rate of synonymous sites, in which no selective constraints were detected. Their recent horizontal transfer best explains the high degree of similarity among canonical P: elements from the saltans and willistoni species groups. These results are consistent with a model of P:-element evolution in which selective constraints are imposed at the time of horizontal transfer. Furthermore, it is estimated that the spread and diversification of the canonical subfamily involved a minimum of 11 horizontal transfer events among the 18 species surveyed within the past 3 Myr. The presence of multiple P: subfamilies in the saltans and willistoni species groups is likely to be the result of multiple invasions that have previously swept through these taxa in a succession of horizontal transfer events. These results suggest that horizontal transfer among eukaryotes might be more common than anticipated.

The use of transposable elements (TEs) as genetic drive mechanisms was explored using Drosophila melanogaster as a model system. Alternative strategies, employing autonomous and nonautonomous P element constructs were compared for their efficiency in driving the ry+ allele into populations homozygous for a ry- allele at the genomic rosy locus. Transformed flies were introduced at 1%, 5%, and 10% starting frequencies to establish a series of populations that were monitored over the course of 40 generations, using both phenotypic and molecular assays. The transposon-borne ry+ marker allele spread rapidly in almost all populations when introduced at 5% and 10% seed frequencies, but 1% introductions frequently failed to become established. A similar initial rapid increase in frequency of the ry+ transposon occurred in several control populations lacking a source of transposase. Constructs carrying ry+ markers also increased to moderate frequencies in the absence of selection on the marker. The results of Southern and in situ hybridization studies indicated a strong inverse relationship between the degree of conservation of construct integrity and transposition frequency. These finding have relevance to possible future applications of transposons as genetic drive mechanisms.

Babesia microti is the primary causative agent of human babesiosis, an emerging pathogen that causes a malaria-like illness with possible fatal outcome in immunocompromised patients. The genome sequence of the B. microti R1 strain was reported in 2012 and revealed a distinct evolutionary path for this pathogen relative to that of other apicomplexa. Lacking from the first genome assembly and initial molecular analyses was information about the terminal ends of each chromosome, and both the exact number of chromosomes in the nuclear genome and the organization of the mitochondrial genome remained ambiguous. We have now performed various molecular analyses to characterize the nuclear and mitochondrial genomes of the B. microti R1 and Gray strains and generated high-resolution Whole Genome maps. These analyses show that the genome of B. microti consists of four nuclear chromosomes and a linear mitochondrial genome present in four different structural types. Furthermore, Whole Genome mapping allowed resolution of the chromosomal ends, identification of areas of misassembly in the R1 genome, and genomic differences between the R1 and Gray strains, which occur primarily in the telomeric regions. These studies set the stage for a better understanding of the evolution and diversity of this important human pathogen.

Apicomplexa are protist parasites of tremendous medical and economic importance, causing millions of deaths and billions of dollars in losses each year. Apicomplexan-related diseases may be controlled via inhibition of essential enzymes. Ribonucleotide reductase (RNR) provides the only de novo means of synthesizing deoxyribonucleotides, essential precursors for DNA replication and repair. RNR has long been the target of antibacterial and antiviral therapeutics. However, targeting this ubiquitous protein in eukaryotic pathogens may be problematic unless these proteins differ significantly from that of their respective host. The typical eukaryotic RNR enzymes belong to class Ia, and the holoenzyme consists minimally of two R1 and two R2 subunits (α2β2). We generated a comparative, annotated, structure-based, multiple-sequence alignment of R2 subunits, identified a clade of R2 subunits unique to Apicomplexa, and determined its phylogenetic position. Our analyses revealed that the apicomplexan-specific sequences share characteristics with both class I R2 and R2lox proteins. The putative radical-harboring residue, essential for the reduction reaction by class Ia R2-containing holoenzymes, was not conserved within this group. Phylogenetic analyses suggest that class Ia subunits are not monophyletic and consistently placed the apicomplexan-specific clade sister to the remaining class Ia eukaryote R2 subunits. Our research suggests that the novel apicomplexan R2 subunit may be a promising candidate for chemotherapeutic-induced inhibition as it differs greatly from known eukaryotic host RNRs and may be specifically targeted. Electronic supplementary material The online version of this article (doi:10.1007/s00239-013-9583-y) contains supplementary material, which is available to authorized users.

The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum -specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia.

Human cryptosporidiosis is caused primarily by Cryptosporidium hominis, C. parvum and C. meleagridis. To accelerate research on parasites in the genus Cryptosporidium, we generated annotated, draft genome sequences of human C. hominis isolates TU502_2012 and UKH1, C. meleagridis UKMEL1, also isolated from a human patient, and the avian parasite C. baileyi TAMU-09Q1. The annotation of the genome sequences relied in part on RNAseq data generated from the oocyst stage of both C. hominis and C. baileyi. The genome assembly of C. hominis is significantly more complete and less fragmented than that available previously, which enabled the generation of a much-improved gene set for this species, with an increase in average gene length of 500 bp relative to the protein-encoding genes in the 2004 C. hominis annotation. Our results reveal that the genomes of C. hominis and C. parvum are very similar in both gene density and average gene length. These data should prove a valuable resource for the Cryptosporidium research community., The release of the draft genome sequence, and corresponding annotation, of Cryptosporidium baileyi, C. hominis isolates TU502_2012 and UKH1, and C. meleagridis, will accelerate research on Cryptosporidium parasites.

Human cryptosporidiosis, caused primarily by Cryptosporidium hominis and a subset of Cryptosporidium parvum, is a major cause of moderate-to-severe diarrhea in children under 5 years of age in developing countries and can lead to nutritional stunting and death. Cryptosporidiosis is particularly severe and potentially lethal in immunocompromised hosts. Biological and technical challenges have impeded traditional vaccinology approaches to identify novel targets for the development of vaccines against C. hominis, the predominant species associated with human disease. We deemed that the existence of genomic resources for multiple species in the genus, including a much-improved genome assembly and annotation for C. hominis, makes a reverse vaccinology approach feasible. To this end, we sought to generate a searchable online resource, termed C. hominis gene catalog, which registers all C. hominis genes and their properties relevant for the identification and prioritization of candidate vaccine antigens, including physical attributes, properties related to antigenic potential and expression data. Using bioinformatic approaches, we identified ∼400 C. hominis genes containing properties typical of surface-exposed antigens, such as predicted glycosylphosphatidylinositol (GPI)-anchor motifs, multiple transmembrane motifs and/or signal peptides targeting the encoded protein to the secretory pathway. This set can be narrowed further, e.g. by focusing on potential GPI-anchored proteins lacking homologs in the human genome, but with homologs in the other Cryptosporidium species for which genomic data are available, and with low amino acid polymorphism. Additional selection criteria related to recombinant expression and purification include minimizing predicted post-translation modifications and potential disulfide bonds. Forty proteins satisfying these criteria were selected from 3745 proteins in the updated C. hominis annotation. The immunogenic potential of a few of these is currently being tested.Database URL: http://cryptogc.igs.umaryland.edu.

Background Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites. Results The known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp. Conclusions The EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence. T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms.

BackgroundTheileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8+ T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8+ T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8+ T-cell epitopes, and to analyse the sequences for evidence of selection.Methodology/Principal FindingsPartial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (~12%) in Tp1 and in 320 positions (~61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle.Conclusions/SignificanceThe results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.

Background Parasites in the genus Theileria cause lymphoproliferative diseases in cattle, resulting in enormous socio-economic losses. The availability of the genome sequences and annotation for T. parva and T. annulata has facilitated the study of parasite biology and their relationship with host cell transformation and tropism. However, the mechanism of transcriptional regulation in this genus, which may be key to understanding fundamental aspects of its parasitology, remains poorly understood. In this study, we analyze the evolution of non-coding sequences in the Theileria genome and identify conserved sequence elements that may be involved in gene regulation of these parasitic species. Results Intergenic regions and introns in Theileria are short, and their length distributions are considerably right-skewed. Intergenic regions flanked by genes in 5'-5' orientation tend to be longer and slightly more AT-rich than those flanked by two stop codons; intergenic regions flanked by genes in 3'-5' orientation have intermediate values of length and AT composition. Intron position is negatively correlated with intron length, and positively correlated with GC content. Using stringent criteria, we identified a set of high-quality orthologous non-coding sequences between T. parva and T. annulata, and determined the distribution of selective constraints across regions, which are shown to be higher close to translation start sites. A positive correlation between constraint and length in both intergenic regions and introns suggests a tight control over length expansion of non-coding regions. Genome-wide searches for functional elements revealed several conserved motifs in intergenic regions of Theileria genomes. Two such motifs are preferentially located within the first 60 base pairs upstream of transcription start sites in T. parva, are preferentially associated with specific protein functional categories, and have significant similarity to know regulatory motifs in other species. These results suggest that these two motifs are likely to represent transcription factor binding sites in Theileria. Conclusion Theileria genomes are highly compact, with selection seemingly favoring short introns and intergenic regions. Three over-represented sequence motifs were independently identified in intergenic regions of both Theileria species, and the evidence suggests that at least two of them play a role in transcriptional control in T. parva. These are prime candidates for experimental validation of transcription factor binding sites in this single-celled eukaryotic parasite. Sequences similar to two of these Theileria motifs are conserved in Plasmodium hinting at the possibility of common regulatory machinery across the phylum Apicomplexa.

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated “gene dumps” and, perhaps, simultaneously, as “gene factories”., Author Summary Aspergillus is an extremely diverse genus of filamentous ascomycetous fungi (molds) found ubiquitously in soil and decomposing vegetation. Being supreme opportunists, aspergilli have adapted to overcome various chemical, physical, and biological stresses found in heterogeneous environments. While most species in the genus are saprophytes, a surprising number are able to infect wounded plants and animals. Remarkably, the allergic human host also responds abnormally to the aspergilli with lung and sinus disease. The advent of immunosuppressive agents and other medical advances have created a large worldwide pool of human hosts susceptible to some Aspergillus species, including the world's most harmful mold and the causative agent of invasive aspergillosis, Aspergillus fumigatus. In this study, we have used the power of comparative genomics to gain insight into genetic mechanisms that may contribute to the metabolic versatility and pathogenicity of this important human pathogen. Comparison of the genomes of two A. fumigatus clinical isolates and two closely related, but rarely pathogenic species showed that their genomes contain several large isolate- and species-specific chromosomal islands. The metabolic capabilities encoded by these highly labile regions are likely to contribute to their rapid adaptation to heterogeneous environments such as soil or a living host.

Background: High gene numbers in plant genomes reflect polyploidy and major gene duplication events. Oryza sativa, cultivated rice, is a diploid monocotyledonous species with a ~390 Mb genome that has undergone segmental duplication of a substantial portion of its genome. This, coupled with other genetic events such as tandem duplications, has resulted in a substantial number of its genes, and resulting proteins, occurring in paralogous families. Results: Using a computational pipeline that utilizes Pfam and novel protein domains, we characterized paralogous families in rice and compared these with paralogous families in the model dicotyledonous diploid species, Arabidopsis thaliana. Arabidopsis, which has undergone genome duplication as well, has a substantially smaller genome (~120 Mb) and gene complement compared to rice. Overall, 53% and 68% of the non-transposable element-related rice and Arabidopsis proteins could be classified into paralogous protein families, respectively. Singleton and paralogous family genes differed substantially in their likelihood of encoding a protein of known or putative function; 26% and 66% of singleton genes compared to 73% and 96% of the paralogous family genes encode a known or putative protein in rice and Arabidopsis, respectively. Furthermore, a major skew in the distribution of specific gene function was observed; a total of 17 Gene Ontology categories in both rice and Arabidopsis were statistically significant in their differential distribution between paralogous family and singleton proteins. In contrast to mammalian organisms, we found that duplicated genes in rice and Arabidopsis tend to have more alternative splice forms. Using data from Massively Parallel Signature Sequencing, we show that a significant portion of the duplicated genes in rice show divergent expression although a correlation between sequence divergence and correlation of expression could be seen in very young genes. Conclusion: Collectively, these data suggest that while co-regulation and conserved function are present in some paralogous protein family members, evolutionary pressures have resulted in functional divergence with differential expression patterns., National Science Foundation (U.S.). Plant Genome Research Program (DBI-0321538), National Science Foundation (U.S.) (DBI-0321437)

We describe the genome sequence of the protist Trichomonas vaginalis , a sexually transmitted human pathogen. Repeats and transposable elements comprise about two-thirds of the ∼160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins may exemplify adaptations of the parasite during its transition to a urogenital environment. The genome sequence predicts previously unknown functions for the hydrogenosome, which support a common evolutionary origin of this unusual organelle with mitochondria.

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.

A comparison of gene content and genome architecture of Trypanosoma brucei, Trypanosoma cruzi , and Leishmania major , three related pathogens with different life cycles and disease pathology, revealed a conserved core proteome of about 6200 genes in large syntenic polycistronic gene clusters. Many species-specific genes, especially large surface antigen families, occur at nonsyntenic chromosome-internal and subtelomeric regions. Retroelements, structural RNAs, and gene family expansion are often associated with syntenic discontinuities that—along with gene divergence, acquisition and loss, and rearrangement within the syntenic regions—have shaped the genomes of each parasite. Contrary to recent reports, our analyses reveal no evidence that these species are descended from an ancestor that contained a photosynthetic endosymbiont.

We analysed the distribution of transposable elements (TEs) in 100 aligned pairs of orthologous intergenic regions from the mouse and human genomes. Within these regions, conserved segments of high similarity between the two species alternate with segments of low similarity. Identifiable TEs comprise 40-60% of segments of low similarity. Within such segments, a particular copy of a TE found in one species has no orthologue in the other. Overall, TEs comprise only approximately 20 % of conserved segments. However, TEs from two families, MIR and L2, are rather common within conserved segments. Statistical analysis of the distributions of TEs suggests that a majority of the MIR and L2 elements present in murine intergenic regions have human orthologues. These elements must have been present in the common ancestor of human and mouse and have remained under substantial negative selection that prevented their divergence beyond recognition. If so, recruitment of MIR- and L2-derived sequences to perform a function that increases host fitness is rather common, with at least two such events per host gene. The central part of the MIR consensus sequence is over-represented in conserved segments given its background frequency in the genome, suggesting that it is under the strongest selective constraint.

Species of malaria parasite that infect rodents have long been used as models for malaria disease research. Here we report the whole-genome shotgun sequence of one species, Plasmodium yoelii yoelii, and comparative studies with the genome of the human malaria parasite Plasmodium falciparum clone 3D7. A synteny map of 2,212 P. y. yoelii contiguous DNA sequences (contigs) aligned to 14 P. falciparum chromosomes reveals marked conservation of gene synteny within the body of each chromosome. Of about 5,300 P. falciparum genes, more than 3,300 P. y. yoelii orthologues of predominantly metabolic function were identified. Over 800 copies of a variant antigen gene located in subtelomeric regions were found. This is the first genome sequence of a model eukaryotic parasite, and it provides insight into the use of such systems in the modelling of Plasmodium biology and disease.

Background For three decades the Mutator system was thought to be exclusive of plants, until the first homolog representatives were characterized in fungi and in early-diverging amoebas earlier in this decade. Results Here, we describe and characterize four families of Mutator-like elements in a new eukaryotic group, the Parabasalids. These T richomonas v aginalis Mu tator- l ike e lements, or TvMULEs, are active in T. vaginalis and patchily distributed among 12 trichomonad species and isolates. Despite their relatively distinctive amino acid composition, the inclusion of the repeats TvMULE1, TvMULE2, TvMULE3 and TvMULE4 into the Mutator superfamily is justified by sequence, structural and phylogenetic analyses. In addition, we identified three new TvMULE-related sequences in the genome sequence of Candida albicans. While TvMULE1 is a member of the MuDR clade, predominantly from plants, the other three TvMULEs, together with the C. albicans elements, represent a new and quite distinct Mutator lineage, which we named TvCaMULEs. The finding of TvMULE1 sequence inserted into other putative repeat suggests the occurrence a novel TE family not yet described. Conclusion These findings expand the taxonomic distribution and the range of functional motif of MULEs among eukaryotes. The characterization of the dynamics of TvMULEs and other transposons in this organism is of particular interest because it is atypical for an asexual species to have such an extreme level of TE activity; this genetic landscape makes an interesting case study for causes and consequences of such activity. Finally, the extreme repetitiveness of the T. vaginalis genome and the remarkable degree of sequence identity within its repeat families highlights this species as an ideal system to characterize new transposable elements.

Background Introns are under less selection pressure than exons, and consequently, intronic sequences have a higher rate of gain and loss than exons. In a number of plant species, a large portion of the genome has been segmentally duplicated, giving rise to a large set of duplicated genes. The recent completion of the rice genome in which segmental duplication has been documented has allowed us to investigate intron evolution within rice, a diploid monocotyledonous species.