| Background:The larval stage of Taenia multiceps, a global cestode, encysts in the central nervous system (CNS) of sheep and other livestock. This frequently leads to their death and huge socioeconomic losses, especially in developing countries. This parasite can also cause zoonotic infections in humans, but has been largely neglected due to a lack of diagnostic techniques and studies. Expression levels of specific genes, differential splicing, allele-specific expression of transcripts can be accurately determined by RNA-Seq experiments to address many biological-related issues. MicroRNAs (miRNAs) have important roles in the post-transcriptional regulation of a large number of animal genes by imperfectly binding target mRNAs. Recent developments in next-generation sequencing provide an opportunity to explore the transcriptome and microRNA of T. multiceps. Methodology/Principal Findings:[Transcriptome analyze]:We obtained a total of 31,282 unigenes (mean length 920 bp) using Illumina paired-end sequencing technology and a new Trinity de novo assembler without a referenced genome. Individual transcription molecules were determined by sequence-based annotations and/or domain-based annotations against public databases (Nr, UniprotKB/Swiss-Prot, COG, KEGG, UniProtKB/TrEMBL, InterPro and Pfam). We identified 26,110 (83.47%) unigenes and inferred 20,896 (66.8%) coding sequences (CDS). Further comparative transcripts analysis with other cestodes (Taenia pisiformis, Taenia solium, Echincoccus granulosus and Echincoccus multilocularis) and intestinal parasites (Trichinella spiralis, Ancylostoma caninum and Ascaris suum) showed that 5,100 common genes were shared among three Taenia tapeworms,261 conserved genes were detected among five Taeniidae cestodes, and 109 common genes were found in four zoonotic intestinal parasites. Some of the common genes were genes required for parasite survival, involved in parasite-host interactions. In addition, we amplified two full-length CDS of unigenes from the common genes using RT-PCR.MicroRNAs identification]:We obtained 12.8 million high quality raw reads from T. multiceps small RNA library using Illumina sequencing technology. The raw reads dataset was deposited in GEO database of NCBI with the accession number:GSE35647. A total of 796 conserved miRNA families (containing 1,006 miRNAs) from 170,888 unique miRNAs were characterized using miRBase (Release 17.0). Based on the genome of E. multilocularis, we predicted 44 candidate miRNAs, and two of them were multi-located in E. multilocularis genome. Here, we selected three conserved miRNA/miRNA* (antisense strand) duplexes at random and amplified their corresponding precursors using a PCR-based method. Furthermore,20 novel candidate miRNA precursors were verified by genomic PCR. Among these, six corresponding T. multiceps miRNAs are considered specific for Taeniidae because no homologs were found in other species annotated in miRBase. In addition,181,077 target sites within T. multiceps transcriptome were predicted for 20 newly identified miRNAs. The MFEI formula was used to select novel miRNAs with MFEI>0.85. However,13 low-confidence novel miRNAs determined using the MFEI formula could be validated by PCR-based method and meeting the secondary structure and the required MFE.Conclusions/Significance:This study provides an extensive transcriptome and large-scale miRNAs investigation of the adult stage of T. multiceps. This transcriptome dataset forms a substantial public information platform to achieve a fundamental understanding of the biology of T. multiceps, and helps in the identification of drug targets and parasite-host interaction studies. This miRNA dataset provides a substantial platform for improving our understanding of the molecular regulation of T. multiceps development. Our results demonstrate that comparative transcript analyze is worthwhile, whereas MFEI method is not a good fit to select confidence candidate miRNA for T. multiceps. |
