Identification of conserved and novel microRNAs in Manduca sexta and their possible roles in the regulation of immunity-related gene expression

The tobacco hornworm Manduca sexta has served as a model for insect biochemical and physiological research. However, the knowledge on posttranscriptional regulation of gene expression by microRNAs is still rudimentary in this species. Our previous study identified 163 conserved and 13 novel microRNAs in M. sexta, most of which are present at low levels in pupae. To identify more M. sexta microRNAs and examine their possible roles in the expression regulation of immunity-related genes, we constructed four small RNA libraries using fat body and hemocytes from naïve or bacteria-injected larvae and obtained 32.9 million reads of 18-31 nucleotides by Illumina sequencing. In the dataset, there were two newly identified conserved microRNAs and nine conserved variants. We also found seven novel microRNAs and one cluster of those. mse-miR-1b, predicted as the anti-sense microRNA of mse-miR-1, and putative mse-miR-929 in the previous study, were confirmed in the tissue small RNA samples. mse-miR-281-star, mse-miR-965-star, mse-miR-31-star, mse-miR-9a-star, mse-miR-9b-star, and mse-miR-2a-star were present at higher levels than their respective mature strands. We also observed abundance changes in microRNAs before and after the immune challenge. Based on the quantitative data of mRNA levels in control and induced fat body and hemocytes as well as the results of microRNA target site analysis, we suggest that certain microRNAs and microRNA*s regulate gene expression of some pattern recognition receptors, prophenoloxidase activation cascade components, serpins, and members of the conserved intracellular signaling pathways (Toll, IMD, JAK-STAT, and MAPK-JNK-p38). In summary, this work enriches our knowledge on M. sexta microRNAs and how some of them may regulate immunity-related gene expression.