Limits...
Complete mitochondrial genomes and nuclear ribosomal RNA operons of two species of Diplostomum (Platyhelminthes: Trematoda): a molecular resource for taxonomy and molecular epidemiology of important fish pathogens.

Brabec J, Kostadinova A, Scholz T, Littlewood DT - Parasit Vectors (2015)

Bottom Line: Mt protein-coding genes and nuclear rRNA genes were subjected to phylogenetic analysis by maximum likelihood and the resulting topologies compared.Analyses of the mitogenomic data invariably recovered the Diplostomidae as a sister lineage of the order Plagiorchiida rather than as a basal lineage of the Diplostomida as inferred in rDNA phylogenies; this was concordant with the mt gene order of Diplostomum spp. exhibiting closer match to the conserved gene order of the Plagiorchiida.Complete sequences of the mt genome and rRNA operon of two species of Diplostomum provide a valuable resource for novel genetic markers for species delineation and large-scale molecular epidemiology and disease ecology studies based on the most accessible life-cycle stages of eye flukes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Parasitology, Biology Centre of the Czech Academy of Sciences and Faculty of Science, University of South Bohemia, Branišovská 31, 370 05, České Budějovice, Czech Republic. brabcak@paru.cas.cz.

ABSTRACT

Background: The genus Diplostomum (Platyhelminthes: Trematoda: Diplostomidae) is a diverse group of freshwater parasites with complex life-cycles and global distribution. The larval stages are important pathogens causing eye fluke disease implicated in substantial impacts on natural fish populations and losses in aquaculture. However, the problematic species delimitation and difficulties in the identification of larval stages hamper the assessment of the distributional and host ranges of Diplostomum spp. and their transmission ecology.

Methods: Total genomic DNA was isolated from adult worms and shotgun sequenced using Illumina MiSeq technology. Mitochondrial (mt) genomes and nuclear ribosomal RNA (rRNA) operons were assembled using established bioinformatic tools and fully annotated. Mt protein-coding genes and nuclear rRNA genes were subjected to phylogenetic analysis by maximum likelihood and the resulting topologies compared.

Results: We characterised novel complete mt genomes and nuclear rRNA operons of two closely related species, Diplostomum spathaceum and D. pseudospathaceum. Comparative mt genome assessment revealed that the cox1 gene and its 'barcode' region used for molecular identification are the most conserved regions; instead, nad4 and nad5 genes were identified as most promising molecular diagnostic markers. Using the novel data, we provide the first genome wide estimation of the phylogenetic relationships of the order Diplostomida, one of the two fundamental lineages of the Digenea. Analyses of the mitogenomic data invariably recovered the Diplostomidae as a sister lineage of the order Plagiorchiida rather than as a basal lineage of the Diplostomida as inferred in rDNA phylogenies; this was concordant with the mt gene order of Diplostomum spp. exhibiting closer match to the conserved gene order of the Plagiorchiida.

Conclusions: Complete sequences of the mt genome and rRNA operon of two species of Diplostomum provide a valuable resource for novel genetic markers for species delineation and large-scale molecular epidemiology and disease ecology studies based on the most accessible life-cycle stages of eye flukes.

No MeSH data available.


Related in: MedlinePlus

Schematic alignment of linearised mt genomes of Diplostomum spathaceum and D. pseudospathaceum. Outline arrows indicate the direction of transcription of protein-coding and rRNA genes, hairline arrows indicate the position of tRNA genes (see Additional file 4: Table S1 for the single letter abbreviations). Solid horizontal lines in the grey central area depict average nucleotide identities of individual protein- and rRNA-coding regions (the ‘barcode’ region of cox1 defined by Folmer primers indicated by a dotted line); NC non-coding region
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4477422&req=5

Fig1: Schematic alignment of linearised mt genomes of Diplostomum spathaceum and D. pseudospathaceum. Outline arrows indicate the direction of transcription of protein-coding and rRNA genes, hairline arrows indicate the position of tRNA genes (see Additional file 4: Table S1 for the single letter abbreviations). Solid horizontal lines in the grey central area depict average nucleotide identities of individual protein- and rRNA-coding regions (the ‘barcode’ region of cox1 defined by Folmer primers indicated by a dotted line); NC non-coding region

Mentions: Following the assembly, identity and position of individual mt protein- and tRNA-coding regions were determined using a suite of bioinformatics tools. MITOS [20] was used to reveal the position of mt protein-coding and rRNA genes, whereas tRNAscan-SE web server [21] together with ARWEN [22] were employed to localise tRNA genes and reconstruct their secondary structures. Exact boundaries of the mt protein-coding genes were then confirmed by alignment of inferred amino acid sequences with those of mt genomes of Trichobilharzia regenti (NC_009680), Fasciola hepatica (NC_002546) and Schistosoma japonicum (NC_002544), using E-INS-i aligning algorithm of MAFFT [23] implemented in Geneious. Identical aligning strategy was also used to create pairwise alignment of the whole-length of mt genomes of D. spathaceum and D. pseudospathaceum (Fig. 1).Fig. 1


Complete mitochondrial genomes and nuclear ribosomal RNA operons of two species of Diplostomum (Platyhelminthes: Trematoda): a molecular resource for taxonomy and molecular epidemiology of important fish pathogens.

Brabec J, Kostadinova A, Scholz T, Littlewood DT - Parasit Vectors (2015)

Schematic alignment of linearised mt genomes of Diplostomum spathaceum and D. pseudospathaceum. Outline arrows indicate the direction of transcription of protein-coding and rRNA genes, hairline arrows indicate the position of tRNA genes (see Additional file 4: Table S1 for the single letter abbreviations). Solid horizontal lines in the grey central area depict average nucleotide identities of individual protein- and rRNA-coding regions (the ‘barcode’ region of cox1 defined by Folmer primers indicated by a dotted line); NC non-coding region
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4477422&req=5

Fig1: Schematic alignment of linearised mt genomes of Diplostomum spathaceum and D. pseudospathaceum. Outline arrows indicate the direction of transcription of protein-coding and rRNA genes, hairline arrows indicate the position of tRNA genes (see Additional file 4: Table S1 for the single letter abbreviations). Solid horizontal lines in the grey central area depict average nucleotide identities of individual protein- and rRNA-coding regions (the ‘barcode’ region of cox1 defined by Folmer primers indicated by a dotted line); NC non-coding region
Mentions: Following the assembly, identity and position of individual mt protein- and tRNA-coding regions were determined using a suite of bioinformatics tools. MITOS [20] was used to reveal the position of mt protein-coding and rRNA genes, whereas tRNAscan-SE web server [21] together with ARWEN [22] were employed to localise tRNA genes and reconstruct their secondary structures. Exact boundaries of the mt protein-coding genes were then confirmed by alignment of inferred amino acid sequences with those of mt genomes of Trichobilharzia regenti (NC_009680), Fasciola hepatica (NC_002546) and Schistosoma japonicum (NC_002544), using E-INS-i aligning algorithm of MAFFT [23] implemented in Geneious. Identical aligning strategy was also used to create pairwise alignment of the whole-length of mt genomes of D. spathaceum and D. pseudospathaceum (Fig. 1).Fig. 1

Bottom Line: Mt protein-coding genes and nuclear rRNA genes were subjected to phylogenetic analysis by maximum likelihood and the resulting topologies compared.Analyses of the mitogenomic data invariably recovered the Diplostomidae as a sister lineage of the order Plagiorchiida rather than as a basal lineage of the Diplostomida as inferred in rDNA phylogenies; this was concordant with the mt gene order of Diplostomum spp. exhibiting closer match to the conserved gene order of the Plagiorchiida.Complete sequences of the mt genome and rRNA operon of two species of Diplostomum provide a valuable resource for novel genetic markers for species delineation and large-scale molecular epidemiology and disease ecology studies based on the most accessible life-cycle stages of eye flukes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Parasitology, Biology Centre of the Czech Academy of Sciences and Faculty of Science, University of South Bohemia, Branišovská 31, 370 05, České Budějovice, Czech Republic. brabcak@paru.cas.cz.

ABSTRACT

Background: The genus Diplostomum (Platyhelminthes: Trematoda: Diplostomidae) is a diverse group of freshwater parasites with complex life-cycles and global distribution. The larval stages are important pathogens causing eye fluke disease implicated in substantial impacts on natural fish populations and losses in aquaculture. However, the problematic species delimitation and difficulties in the identification of larval stages hamper the assessment of the distributional and host ranges of Diplostomum spp. and their transmission ecology.

Methods: Total genomic DNA was isolated from adult worms and shotgun sequenced using Illumina MiSeq technology. Mitochondrial (mt) genomes and nuclear ribosomal RNA (rRNA) operons were assembled using established bioinformatic tools and fully annotated. Mt protein-coding genes and nuclear rRNA genes were subjected to phylogenetic analysis by maximum likelihood and the resulting topologies compared.

Results: We characterised novel complete mt genomes and nuclear rRNA operons of two closely related species, Diplostomum spathaceum and D. pseudospathaceum. Comparative mt genome assessment revealed that the cox1 gene and its 'barcode' region used for molecular identification are the most conserved regions; instead, nad4 and nad5 genes were identified as most promising molecular diagnostic markers. Using the novel data, we provide the first genome wide estimation of the phylogenetic relationships of the order Diplostomida, one of the two fundamental lineages of the Digenea. Analyses of the mitogenomic data invariably recovered the Diplostomidae as a sister lineage of the order Plagiorchiida rather than as a basal lineage of the Diplostomida as inferred in rDNA phylogenies; this was concordant with the mt gene order of Diplostomum spp. exhibiting closer match to the conserved gene order of the Plagiorchiida.

Conclusions: Complete sequences of the mt genome and rRNA operon of two species of Diplostomum provide a valuable resource for novel genetic markers for species delineation and large-scale molecular epidemiology and disease ecology studies based on the most accessible life-cycle stages of eye flukes.

No MeSH data available.


Related in: MedlinePlus