Fig 1.
qRT-PCR analysis of mKast expression in various worker body parts.
(A) The amounts of mKast transcript in the worker brains, heads without brains, thoraxes and abdomens were determined by qRT-PCR and normalized with that of Rpl32 transcript. The Steel-Dwass test was used for statistical analysis. Different letters (a, b and c) indicate statistical differences (p < 0.01). Data are shown as mean ± SEM (n = 7). (B) The amounts of mKast transcript in the worker brains and retinas were determined by qRT-PCR and normalized with that of Rpl32 transcript. Welch’s t-test was used for statistical analysis. Data with a significant difference are indicated with ** (p < 0.01). Data are shown as mean ± SEM (n = 5).
Fig 2.
qRT-PCR and in situ hybridization analyses of mKast expression in pupal brains during metamorphosis.
Upper arrow indicates progression of the developmental stages of workers from egg (left) through larva, prepupa (PP), pupa to adult (right). L1 to L5 indicate first to fifth larval instar, respectively. P1 to P9 indicate pupal stages: 1 to 9 days after puparium formation, respectively. Developmental stages analyzed by in situ hybridization (P3 and P7) are boxed. (A) The amounts of mKast transcript in the brains of workers at different developmental stages (L5, P3, P7 and adult) were determined by qRT-PCR and normalized to gapdh transcript amounts. The Steel-Dwass test was used for statistical analysis. Different letters (a, b and c) indicate statistically significant differences (p < 0.05). Data are shown as mean ± SEM (for L5, P3, P5 and adult samples, n = 6, 5, 5 and 7, respectively). (B) In situ hybridization analysis of mKast in the brains of pupae at the P3 and P7 stages. Upper and lower panels indicate results with antisense and sense probes, respectively. Left and right panels indicate brains of pupae at the P3 and P7 stages, respectively. mKast signals are detectable in several brain regions of pupa at the P7 stage with antisense probe. Red arrows indicate signals at the MB mKCs and red arrowheads indicate signals in the other brain regions. MB, mushroom body; OL, optic lobe; AL, antennal lobe. Scale bars indicate 200 μm. Note that retina is removed from our specimen. There is a huge variance in adult brain mKast levels, because gapdh expression, which was used to normalize mKast expression, for one of the seven adult brain samples was very small. The raw data for quantification of mKast and gapdh transcripts of the seven samples are provided in S2 Table.
Fig 3.
Immunoblotting analysis with affinity purified anti-mKast antibodies.
(A) SDS-polyacrylamide gel electrophoresis of the purified recombinant mKast protein. The band corresponding to recombinant mKast is indicated by a red arrowhead. The positions of molecular mass markers are indicated at the left of the lane with kDa. (B) Immunoblotting analysis of the worker brain homogenates using affinity purified anti-mKast antibodies (left) or normal IgG (right). The band corresponding to mKast is indicated by a red arrowhead.
Fig 4.
Fluorescent immunohistochemical analysis of worker brain sections using anti-mKast antibodies.
(A) Schematic drawing of a worker head (sagittal view). Outline of the brain is indicated by orange lines. Areas that comprise neuropiles are shown in yellow. Blue broken vertical lines indicate location of coronal brain sections B-H. Ant, antenna: Ma, mandible; He, head; Th, thorax. A, P, D and V indicate anterior, posterior, dorsal and ventral, respectively. (B-H) Photomicrographs of immunohistochemical sections at different depth B-H. Left panels; schematic drawings of the distribution of mKast-like immunoreactivity (red dots) in the coronal brain sections B-H indicated in (A). Boxes a-i, see below. Ca, MB calyx; α, MB vertical lobe; β, MB medial lobe; La, lamina; Me, medulla; Lo, lobula; CB, central body; AL, antennal lobe; SOG, subesophageal ganglion; MNC, medial neurosecretory cell; LNC, lateral neurosecretory cell. Photomicrographs from left to right; serial 10μm coronal brain sections stained using affinity purified anti-mKast antibodies (leftmost photos), the same section stained with DAPI (2nd photos), merged images of antibody-staining and DAPI-staining (3rd photos), and the serial sections stained with normal guinea pig IgG and DAPI as a negative control (right panels), respectively. Red and blue signals indicate mKast-like immunoreactivity analyzed with anti-mKast antibodies and nuclear staining with DAPI, respectively. (a-i) Magnified views of merged images of antibody-staining and DAPI-staining (3rd photos of the above photomicrographs) of brain regions corresponding to boxes a-i shown in the left schematic drawings. mKast-like immunoreactivities detected at the outer surface of the OL lobula (a), LNC somata localized beneath the lateral MB calyx (b and c), MNC somata localized between the medial MB calyces (d), somata of neurons located at the outer surface of the ALs (e), putative projections to ALs (f), somata of neurons localized at the outer surface of SOG (g), inside of the MB calyces (h and i), are indicated by white arrowheads. Red and blue signals indicate mKast-like immunoreactivities and nuclear staining with DAPI, respectively. mKast-like immunoreactivities detected at the outer surface of the OL lobula are surrounded by a white broken line (a). Scale bars indicate 200 μm in panels (B-H) and 100 μm in panels (a-i), respectively. Note that the DAPI signals are weak in Fig 4B, probably because DAPI staining was not enough in these panels. Also note that only representative positive signals are indicated by white arrowheads in panels (a-i), because many cells with positive signals are clustered in these panels.
Fig 5.
In situ hybridization analysis of mKast in adult worker brain.
(A and D) Schematic drawings of the anterior and posterior coronal brain sections used for in situ hybridization analysis. (B, C) and (E, F) Magnified images of the results of in situ hybridization analysis of mKast that correspond to the boxed areas in (A) and (D) with antisense (B and E) or sense probes (C and F). Note that the sections in (A-C) contain regions corresponding to the posterior ALs and anterior SOGs, whereas the sections in (D-F) contain regions corresponding to SOGs. Red arrowheads in panels (B) and (E) indicate mKast expression. AL, antennal lobe; SOG, subesophageal ganglion. Scale bars indicate 200 μm.