Expression profile of certain mRNAs and miRNA of each class in the early embryogenesis of <i>Apis mellifera</i>.

<p>(A) Profile of mRNAs belonging to the three different classes of genes: I, II, and III. Examples of mRNAs from class I, II and III are plotted in a separated graphic for the haploid and diploid embryos. (B) Profile of miRNAs belonging to class I, II, and III. Note the differential expression and arm use among the embryo types (such as ame-<i>miR-184-3p</i> and <i>5p</i> of class II and ame-<i>miR-263-5p</i> of class II in haploids but class III in diploids). The total number of identified mRNAs for each class is as follows: class I, 667 in diploid and 475 in haploid embryos; class II, 2680 in diploid and 475 in haploid embryos; class III, 263 in diploid and 147 in haploid embryos. The total number of miRNAs for each class is as follows: class I, 90 in diploid and 72 in haploid embryos; class II, 23 in diploid and 24 in haploid embryos; class III, 37 in diploid and 67 in haploid embryos. Overall, the distribution demonstrates specific gene expression dynamics for mRNAs and miRNA in both types of embryos. The Expression and DEGseq output of the mRNAs and miRNAs plotted here are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146447#pone.0146447.s005" target="_blank">S5 Table</a>.</p

Predicted miRNA-target interactions for the diploid (A and B) and haploid (C and D) embryos of <i>Apis mellifera</i> during the early steps of embryogenesis.

<p>(A and C) Complete network and (B and D) reduced networks showing the predicted miRNA-target interactions and focusing on specific genes. Different colors and shapes identify class I, class II and class III miRNAs and genes. Note that the networks denote specific interactions for haploid and diploid embryos.</p

Location of <i>zelda</i> expression in the embryos of <i>A</i>. <i>mellifera</i> according to <i>in situ</i> hybridization.

<p>(A) <i>Zelda</i> expression in a mature oocyte with stronger staining at the anterior pole. (B, C, and D) Embryo within 2 h of development. (C and D in detail) Close to dividing nuclei positive reaction indicating foci of <i>zelda</i>. (E and F) Embryo with 6 h of development showing (in detail) the expression of <i>zelda</i> in the cleavage nuclei at the anterior pole. (G) <i>zelda</i> detected in 18–24 h embryos during the blastoderm stage. The gene product was concentrated in the central region of the embryo. (H) <i>zelda</i> detected in the blastodermic cells in 32–38 h embryos at the beginning of gastrulation. (I and J) Embryo at 42–48 h and (K) 68–72 h of development showing <i>zelda</i> expression in the primordial cells of the nervous system. Asterisk (*) indicates the ventral nerve cord in J and K. (L and M) Haploid embryos at 0–2 h of development showing <i>zelda</i> as an anterior-posterior stained strip along the embryo (Arrow in M).(A, B, G, M, L, and E): 200 μm scale bar, objective 10X; (E, J, and M): 100 μm scale bar, objective 20X on an Olympus BX61 microscope; (C, D, and F): 50 μm scale bar, 40X objective; (C): DAPI staining. 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>.</p

<i>Apis mellifera</i> developmental phases used for sample collection and RNA sequencing.

<p>Mature oocytes were collected directly from queen ovaries and age-controlled haploid and diploid embryos (at 0–2, 0–6 and 18–24 h after egg laying) from queenright or queenless <i>Apis mellifera</i> colonies (arrows).</p

Diagram showing the number of mRNAs with the predicted TAGteam motif in the diploid and haploid mRNAs and examples of putative transcripts detected during the zygotic genome activation of <i>A</i>. <i>mellifera</i>.

<p>Putative TAGteam motif of <i>A</i>. <i>mellifera</i> localized in mRNAs with a zygotic expression profile (class II and class III) in the diploid and haploid embryos. Certain mRNAs were involved in patterning (<i>run</i> and <i>cact)</i>, cellularization (CG1124 and <i>gl</i>) and sex determination (<i>run</i> and <i>tra2</i>). The expression values of these mRNAs are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146447#pone.0146447.s011" target="_blank">S3 Fig</a>.</p

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>.</p

Nutritionally Driven Differential Gene Expression Leads to Heterochronic Brain Development in Honeybee Castes

<div><p>The differential feeding regimes experienced by the queen and worker larvae of the honeybee <i>Apis mellifera</i> shape a complex endocrine response cascade that ultimately gives rise to differences in brain morphologies. Brain development analyzed at the morphological level from the third (L3) through fifth (L5) larval instars revealed an asynchrony between queens and workers. In the feeding phase of the last larval instar (L5F), two well-formed structures, pedunculi and calyces, are identifiable in the mushroom bodies of queens, both of which are not present in workers until a later phase (spinning phase, L5S). Genome-wide expression analyses and normalized transcript expression experiments monitoring specific genes revealed that this differential brain development starts earlier, during L3. Analyzing brains from L3 through L5S1 larvae, we identified 21 genes with caste-specific transcription patterns (e.g., <i>APC-4, GlcAT-P, fax, kr-h1</i> and <i>shot</i>), which encode proteins that are potentially involved in the development of brain tissues through controlling the cell proliferation rate (APC4, kr-h1) and fasciculation (GlcAT-P, fax, and shot). <i>Shot</i>, whose expression is known to be required for axon extension and cell proliferation, was found to be transcribed at significantly higher levels in L4 queens compared with worker larvae. Moreover, the protein encoded by this gene was immunolocalized to the cytoplasm of cells near the antennal lobe neuropiles and proximal to the Kenyon cells in the brains of L4 queens. In conclusion, during the larval period, the brains of queens are larger and develop more rapidly than workers’ brains, which represents a developmental heterochrony reflecting the effect of the differential feeding regime of the two castes on nervous system development. Furthermore, this differential development is characterized by caste-specific transcriptional profiles of a set of genes, thus pointing to a link between differential nutrition and differential neurogenesis via genes that control cell proliferation and fasciculation.</p></div

Characteristics of the primers used in the RT-qPCR assays.

<p>Characteristics of the primers used in the RT-qPCR assays.</p

<i>In situ</i> hybridization to detect <i>shot</i> expression in the brains of <i>A.</i><i>mellifera</i> workers and queens.

<p>Confocal images of brain of 4^th instar honeybee queens show <i>shot</i> mRNA throughout the entire organ (<b>A, D, G</b>). No mRNA was detected in worker brains in the 4^th instar (<b>B, E, H</b>), and expression only appeared in the 5^th stage (<b>C, F, I</b>). Green: shot mRNA; blue: DAPI. Note the <i>shot</i> mRNA surrounding nuclei.</p

Shot immunostaining during the 4^th larval instar of <i>A.</i><i>mellifera</i> castes.

<p>Triple labeling with DAPI (A, B), phalloidin staining (<b>C, D</b>) and anti-shot (mAbRod1; <b>E, F</b>). (<b>G</b>) Shot protein was immunolocalized to the cytoplasm of cells near the antennal lobe neuropiles (arrowhead) and proximal to the Kenyon cells in the brains of L4 queens (arrow). (<b>H</b>) As observed for mRNA, Shot protein was absent in worker brains in the 4^th larval instar. Confocal plane of a queen brain (<b>I</b>) showing Shot staining in the cytoplasm of cells (arrows and arrowheads).</p