Project description:Programmed DNA elimination is a developmentally regulated process leading to the reproducible loss of specific genomic sequences. DNA elimination occurs in unicellular ciliates and a variety of metazoans, including invertebrates and vertebrates. In metazoa, DNA elimination typically occurs in somatic cells during early development, leaving the germline genome intact. Reference genomes for metazoa that undergo DNA elimination are not available. Here, we generated germline and somatic reference genome sequences of the DNA eliminating pig parasitic nematode Ascaris suum and the horse parasite Parascaris univalens. In addition, we carried out in-depth analyses of DNA elimination in the parasitic nematode of humans, Ascaris lumbricoides, and the parasitic nematode of dogs, Toxocara canis. Our analysis of nematode DNA elimination reveals that in all species, repetitive sequences (that differ among the genera) and germline-expressed genes (approximately 1000-2000 or 5%-10% of the genes) are eliminated. Thirty-five percent of these eliminated genes are conserved among these nematodes, defining a core set of eliminated genes that are preferentially expressed during spermatogenesis. Our analysis supports the view that DNA elimination in nematodes silences germline-expressed genes. Over half of the chromosome break sites are conserved between Ascaris and Parascaris, whereas only 10% are conserved in the more divergent T. canis. Analysis of the chromosomal breakage regions suggests a sequence-independent mechanism for DNA breakage followed by telomere healing, with the formation of more accessible chromatin in the break regions prior to DNA elimination. Our genome assemblies and annotations also provide comprehensive resources for analysis of DNA elimination, parasitology research, and comparative nematode genome and epigenome studies.
Project description:Parascaris univalens is an ascaridoid nematode of equids. Little is known about its epidemiology and population genetics in domestic and wild horse populations. PCR-based methods are suited to support studies in these areas, provided that reliable genetic markers are used. Recent studies have shown that mitochondrial (mt) genomic markers are applicable in such methods, but no such markers have been defined for P. univalens.Mt genome regions were amplified from total genomic DNA isolated from P. univalens eggs by long-PCR and sequenced using Illumina technology. The mt genome was assembled and annotated using an established bioinformatic pipeline. Amino acid sequences inferred from all protein-encoding genes of the mt genomes were compared with those from other ascaridoid nematodes, and concatenated sequences were subjected to phylogenetic analysis by Bayesian inference.The circular mt genome was 13,920 bp in length and contained two ribosomal RNA, 12 protein-coding and 22 transfer RNA genes, consistent with those of other ascaridoids. Phylogenetic analysis of the concatenated amino acid sequence data for the 12 mt proteins showed that P. univalens was most closely related to Ascaris lumbricoides and A. suum, to the exclusion of other ascaridoids.This mt genome representing P. univalens now provides a rich source of genetic markers for future studies of the genetics and epidemiology of this parasite and its congener, P. equorum. This focus is significant, given that there is no published information on the specific prevalence and distribution of P. univalens infection in domestic and wild horse populations.
Project description:P-glycoproteins (Pgp) have been proposed as contributors to the widespread macrocyclic lactone (ML) resistance in several nematode species including a major pathogen of foals, Parascaris univalens. Using new and available RNA-seq data, ten different genomic loci encoding Pgps were identified and characterized by transcriptome-guided RT-PCRs and Sanger sequencing. Phylogenetic analysis revealed an ascarid-specific Pgp lineage, Pgp-18, as well as two paralogues of Pgp-11 and Pgp-16. Comparative gene expression analyses in P. univalens and Caenorhabditis elegans show that the intestine is the major site of expression but individual gene expression patterns were not conserved between the two nematodes. In P. univalens, PunPgp-9, PunPgp-11.1 and PunPgp-16.2 consistently exhibited the highest expression level in two independent transcriptome data sets. Using RNA-Seq, no significant upregulation of any Pgp was detected following in vitro incubation of adult P. univalens with ivermectin suggesting that drug-induced upregulation is not the mechanism of Pgp-mediated ML resistance. Expression and functional analyses of PunPgp-2 and PunPgp-9 in Saccharomyces cerevisiae provide evidence for an interaction with ketoconazole and ivermectin, but not thiabendazole. Overall, this study established reliable reference gene models with significantly improved annotation for the P. univalens Pgp repertoire and provides a foundation for a better understanding of Pgp-mediated anthelmintic resistance.