Background: Previous work showed that miRNAs play key roles in the regulation of metamorphosis in the hemimetabolan species Blattella germanica. To gain insight about which miRNAs might be important, we have constructed two miRNA libraries, one of the penultimate, pre-metamorphic nymphal instar (N5) and the other of the last, metamorphic nymphal instar (N6).
Results: High throughput sequencing gave 61 canonical miRNAs present in the N5 and N6 libraries, although at different proportions in each. Comparison of both libraries led to the identification of three and 37 miRNAs significantly more expressed in N5 and N6 respectively. Twelve of these 40 miRNAs were then investigated further by qRT-PCR and results indicated that miR-252-3p was well expressed in N5 but not in N6, whereas let-7-5p, miR-100-5p and miR-125-5p showed the reverse pattern. 20-Hydroxyecdysone (20E) tended to stimulate miRNA expression, whereas juvenile hormone (JH) inhibited the 20E stimulatory effect. Expression of let-7, miR-100 and miR-125 was increased by 20E, which has also been observed in D. melanogaster. The only miRNA that was inhibited by 20E was miR-252-3p. The involvement of let-7, miR-100 and miR-125 in metamorphosis has been demonstrated in other insects. Depletion of miR-252-3p caused growth and developmental delays, which suggests that this miRNA is involved in regulating these processes prior to metamorphosis.
Conclusions: The comparative analysis of miRNA libraries from pre-metamorphic (N5) and metamorphic stages (N6) of B. germanica proved to be a useful tool to identify miRNAs with roles in hemimetabolan metamorphosis. Three miRNAs emerged as important factors in the metamorphic stage (N6): let-7-5p, miR-100-5p and miR-125-5p, whereas miR-252-3p appears to be important in the pre-metamorphic stage (N5).