An integrated pipeline for next generation sequencing and annotation of the complete mitochondrial genome of the giant intestinal fluke, Fasciolopsis buski (Lankester, 1857) Looss, 1899.

Helminths include both parasitic nematodes (roundworms) and platyhelminths (trematode and cestode flatworms) that are abundant, and are of clinical importance. The genetic characterization of parasitic flatworms using advanced molecular tools is central to the diagnosis and control of infections. Although the nuclear genome houses suitable genetic markers (e.g., in ribosomal (r) DNA) for species identification and molecular characterization, the mitochondrial (mt) genome consistently provides a rich source of novel markers for informative systematics and epidemiological studies. In the last decade, there have been some important advances in mtDNA genomics of helminths, especially lung flukes, liver flukes and intestinal flukes. Fasciolopsis buski, often called the giant intestinal fluke, is one of the largest digenean trematodes infecting humans and found primarily in Asia, in particular the Indian subcontinent. Next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation within a short span of time. Herein, we describe a high-throughput sequencing and bioinformatics pipeline for mt genomics for F. buski that emphasizes the utility of short read NGS platforms such as Ion Torrent and Illumina in successfully sequencing and assembling the mt genome using innovative approaches for PCR primer design as well as assembly. We took advantage of our NGS whole genome sequence data (unpublished so far) for F. buski and its comparison with available data for the Fasciola hepatica mtDNA as the reference genome for design of precise and specific primers for amplification of mt genome sequences from F. buski. A long-range PCR was carried out to create an NGS library enriched in mt DNA sequences. Two different NGS platforms were employed for complete sequencing, assembly and annotation of the F. buski mt genome. The complete mt genome sequences of the intestinal fluke comprise 14,118 bp and is thus the shortest trematode mitochondrial genome sequenced to date. The noncoding control regions are separated into two parts by the tRNA-Gly gene and don't contain either tandem repeats or secondary structures, which are typical for trematode control regions. The gene content and arrangement are identical to that of F. hepatica. The F. buski mtDNA genome has a close resemblance with F. hepatica and has a similar gene order tallying with that of other trematodes. The mtDNA for the intestinal fluke is reported herein for the first time by our group that would help investigate Fasciolidae taxonomy and systematics with the aid of mtDNA NGS data. More so, it would serve as a resource for comparative mitochondrial genomics and systematic studies of trematode parasites.

Utility of divergent domains of 28S ribosomal RNA in species discrimination of paramphistomes (Trematoda: Digenea: Paramphistomoidea).

Among the digenetic trematodes, paramphistomes are known to be the causative agent of "amphistomiasis" or the stomach fluke disease of domestic and wild animals, mainly ruminants. The use of 28S (divergent domains) and 18S rRNA for phylogenetic inference is significantly warranted for these flukes since it is as yet limited to merely the exploration of the second internal transcribed spacer (ITS2) region. The present study intended to explore the divergent domains (D1-D3) of 28S rRNA and simultaneously equate the phylogenetic information with 18S rRNA in paramphistomes. Divergence of the 28S rRNA domains was evident amongst the divergent (D) domains, where D1 domain emerged as the most variable and D2, the most robust domain, since the latter could provide a higher resolution of the species. D2 was the only domain that comprised compensatory mutations in the helices of its structural constraints; this domain is thus well suited for species distinction and may be considered a potential DNA barcode complementary to mitochondrial DNA. 28S (D1 + D2 + D3) rRNA provided a significant resolution of the taxa corroborating with the taxonomy of these flukes and thus proved to be more robust as a phylogenetic marker for lower levels than 18S rRNA. Phylogenetic inferences of paramphitomes are still scarcely explored; additional data from other taxa belonging to this family may estimate better the biodiversity of these flukes.

Secondary structure analysis of ITS2 in the rDNA of three Indian paramphistomid species found in local livestock.

Of paramphistomid trematodes, three species viz., Homalogaster paloniae, Calicophoron calicophorum and Orthocoelium streptocoelium are commonly prevalent in bovine hosts in Northeast India. The aim of the present study was to genetically characterise these species using rDNA second internal transcribed spacer (ITS2) so as to supplement the morphological criteria substantiated by molecular findings. The annotated ITS2 region from H. paloniae, C. calicophorum and O. streptocoelium were found to be 289 bp, 288 bp and 288 bp long, respectively. On comparison, the Indian isolates of the three species were observed to have a maximum identity of 99% with each of their respective counterparts from Japan. The secondary structure models were inferred using minimum free energy modelling algorithms. The paramphistomes displayed the typical four helix ITS2 secondary structure and differed from each other due to minor nucleotide differences. The consensus ITS2 secondary structure model revealed the presence of conservative motifs GACGAGGGUG and GCGGUAGAGUC in helix III. Monophyly is well supported for a clade consisting of the Japanese and Indian paramphistomes with significant bootstrap values.