Identification, characterization and target gene analysis of testicular microRNAs in the oriental fruit fly Bactrocera dorsalis

MicroRNAs (miRNAs) are small noncoding RNAs that regulate various diverse biological processes includ- ing insect spermatogenesis. The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive horticultural pests in East Asia and the Pacific region. Although developmental miRNA profiles of B. dorsa- lis have enriched our knowledge, specific testicular miRNAs in this dipteran species are unexplored. In this study, we identified miRNAs from B. dorsalis testes by deep sequencing, which provided an over- view of miRNA expression during spermatogenesis. Small RNA libraries were constructed from the testes of fully mature (FM), immature (IM) and middle-aged (MA) adult flies of B. dorsalis. Small RNA sequencing and data analysis revealed 172 known and 78 novel miRNAs amongst these libraries. Pairwise compari- sons of libraries led to the identification of 24, 15 and 14 differentially expressed miRNAs in FM vs. IM, FM vs. MA and IM vs. MA insects, respectively. Using a bioinformatic approach, we predicted 124 target genes against the 13 most differentially expressed miRNAs. Furthermore, the expression patterns of six randomly selected miRNAs (from the 13 most differentially expressed miRNAs) and their putative target genes (from the 124 predicted target genes) were ana- lysed in the testis of B. dorsalis by quantitative real- time PCR, which showed that out of six, four tested miRNAs-mRNAs had an inverse expression pattern and are probably co-regulated. This study is the first comparative profile of the miRNA transcriptome in three developmental stages of the testis, and pro- vides a useful resource for further studies on the role of miRNAs in spermatogenesis in B. dorsalis.