Expression of pri-ame-<i>mir-375</i>, pri-ame-<i>mir-263b</i>, and pri-ame-<i>mir-34</i> in the oocytes and during early embryonic development of <i>Apis mellifera</i>.

<p><i>In situ</i> hybridization (A-B, D, F, H-K, and M) and PCR assay (N) showing a positive reaction for all tested mRNAs in the early phases of development, thus indicating the synthesis of miRNAs. In the embryos, the reaction was localized around the nuclei, and the PCR assay revealed the expression of pri-miRNAs in all of the analyzed phases. In the PCR assay, U6 was used as a positive control (additional qPCR experiments confirmed these results; data not shown). The presence of nuclei in the early embryo was determined using DAPI staining (C, E, G, and L). (A, D, E, I and M) 200 μm scale bar and 10X objective; (B, C, F, G, K and L) 100 μm scale bar and 20X objective; (B, C, F, G, K and L) 50 μm scale bar and 40X objective on the Olympus BX61 microscope. The negative controls are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146447#pone.0146447.s012" target="_blank">S4 Fig</a>.