A Portrait of the Transcriptome of the Neglected Trematode, Fasciola gigantica—Biological and Biotechnological Implications

Abstract

Fasciola gigantica (Digenea) is an important foodborne trematode that causes liver fluke disease (fascioliasis) in mammals, including ungulates and humans, mainly in tropical climatic zones of the world. Despite its socioeconomic impact, almost nothing is known about the molecular biology of this parasite, its interplay with its hosts, and the pathogenesis of fascioliasis. Modern genomic technologies now provide unique opportunities to rapidly tackle these exciting areas. The present study reports the first transcriptome representing the adult stage of F. gigantica (of bovid origin), defined using a massively parallel sequencing-coupled bioinformatic approach. From >20 million raw sequence reads, >30,000 contiguous sequences were assembled, of which most were novel. Relative levels of transcription were determined for individual molecules, which were also characterized (at the inferred amino acid level) based on homology, gene ontology, and/or pathway mapping. Comparisons of the transcriptome of F. gigantica with those of other trematodes, including F. hepatica, revealed similarities in transcription for molecules inferred to have key roles in parasite-host interactions. Overall, the present dataset should provide a solid foundation for future fundamental genomic, proteomic, and metabolomic explorations of F. gigantica, as well as a basis for applied outcomes such as the development of novel methods of intervention against this neglected parasite.

A Deep Exploration of the Transcriptome and “Excretory/Secretory” Proteome of Adult Fascioloides magna

“…Total RNA was extracted from three individual adults of F. magna using TriPure reagent (Roche, Switzerland) and DNase I-treated (25). RNA amounts were estimated spectrophotometrically (NanoDrop Technologies, USA), and RNA integrity was verified using a 2100 BioAnalyser (Agilent, Santa Clara, CA).…”

“…Polyadenylated (poly(A)ϩ) RNA was purified from 10 g of total RNA using Sera-mag oligo(dT) beads, fragmented to a length of 100 to 500 bp, reversetranscribed using random hexamers, and end-repaired and adaptorligated according to the manufacturer's protocol (Illumina). Ligated products of ϳ300 bp were excised from agarose and PCR-amplified (15 cycles) (25). Products were cleaned using a MinElute column (Qiagen, Netherlands) and paired-end sequenced on a Genome Analyzer II (Illumina) (33), according to the manufacturer's protocols.…”

“…The remaining sequences (99%) were used to construct a de Bruijn graph using a k-mer value of 43 bp. Because de novo transcriptome assemblies of short-read sequence libraries can potentially lead to an underrepresentation of members of large families of molecules characterized by a high degree of sequence similarity, such as the cathepsins (25), reads with a high degree of sequence homology (98% nucleotide identity) to the conserved cathepsin propeptide inhibitor domain (I29; 177 nucleotides, nt) of a cathepsin L sequence from F. magna (GenBank protein accession number ACG50798; nucleotide EU877764.1) were used to generate contigs from the paired-end sequence datasets employing the PRICE program (http://derisilab.ucsf.edu). Eight cycles of contig extension were performed, with each new cycle using contigs (Ͼ500 nt in length) generated from the previous assembly as a template.…”

“…In addition, despite major efforts in studies aimed at developing novel intervention strategies against liver flukes (16 -23), there is still a paucity of information on host-parasite interactions at the molecular level. Recent studies (3,24,25) have provided the first insights into the molecular biology of fasciolids by exploring the transcriptomes of the adult stages of both Fa. hepatica and Fa.…”

“…hepatica and Fa. gigantica (3,25) and of the composition of the excretory/ secretory (ES) 1 products from both the juvenile and adult stages of Fa. hepatica (24).…”