Fig 1.
Specificity of the anti-HEX 110 antibody.
(A) Larval fat body extracts (source of hexamerins) were incubated with the specific antibody for HEX 110 immunoprecipitation. The immunoprecipitate (Ip) was used as samples in SDS-PAGE stained with Coomassie blue (Cb) and western blot (Wb). The HEX 110 band position is indicated (the 45 kDa band is the anti-HEX 110 antibody present in the Ip). (B) Mass spectrogram of the Coomassie blue-stained HEX 110 band shown in A. The peaks 1 to 5 correspond to HEX 110 amino acid stretches (listed at the right); peaks 6–9 do not correspond to HEX 110 polypeptides. (C) The HEX 110 amino acid sequence as deduced from the fully sequenced cDNA [2] (GenBank accession number ABU92559). The five amino acid stretches identified by mass spectrometry are marked in red (overlapped amino acids are shown in blue). The amino acid sequence used for anti-HEX 110 synthesis (epitope) is marked by a rectangle. (D) Native PAGE stained with Coomassie blue (Cb) and western blot (Wb) of a partially purified fraction of larval haemolymph (source of hexamerins) obtained by ultracentrifugation (Utc). The western blot using the anti-HEX 110 antibody shows a band migrating at the 669 kDa position, thus suggesting the homohexameric nature of the native HEX 110 and confirming the antibody specificity. (E) SDS-PAGE stained with Coomassie blue (Cb) and western blot (Wb) using total haemolymph. The western blot revealed with anti-HEX 110 antibody shows specifically the HEX 110 subunit band. (F) SDS-PAGE stained with Coomassie blue (Cb) and western blot (Wb) using total haemolymph. The western blot was revealed with pre-immune serum as a negative control. M = molecular mass marker.
Fig 2.
Ovariole structure and stages characterization.
The ovariole comprises the anterior terminal filament, germarium and developing follicles (vitellarium) as indicated at the right. The oocytes are marked in yellow. The ovarioles are categorized according to the degree of development in stages S0, S1, S2, S3 and S4. Stage S0: presence of initial oocytes (cystocytes) and precursors of nurse and somatic cells in the germarium. Stage S1: an oocyte accompanied by differentiated nurse cells is positioned at the basal region of the ovariole. Stage S2: a slight constriction between the basal oocyte and accompanying nurse cells marks the beginning of oocyte chamber and nurse cell chamber separation; the basal oocyte is surrounded by follicle cells. Stage S3: a sequence of developing oocytes and nurse cell chambers is evident in the ovariole; the larger oocyte at the basal region is well-separated from the nurse cell chamber. Stage S4: presence of several developing oocytes and nurse cell chambers through the ovariole; a quasi-fully or a fully developed oocyte is evident at the most basal region. The classification of the maturing oocytes in stages 1 to 7 (stage 6 is not shown) was based in Wilson et al. [44].
Fig 3.
Immunolocalization of HEX 110 in the inactive ovarioles (stage S0) of queenright newly-emerged workers.
(A-C) Part of the terminal filament (at the top) and upper region of the germarium (at the bottom). The dashed lines highlight the shape of the ovariole that appears folded in the images. (D-F) Lower region of the germarium. (A and D) DAPI-stained cell nuclei (blue). Initial oocytes (cystocytes) (Oc) are identified in D. (B and E) HEX 110 foci (green) in the cytoplasm (arrows) and nucleus (arrowheads) of germarium cells evidenced with anti-HEX 110/Alexa-Fluor 488. (C, F) Merged images: arrows and arrowheads point to the same cytoplasmic and nuclear HEX 110 foci seen in B and E.
Fig 4.
Immunolocalization of HEX 110 in ovarioles (stage S1) of nurse workers from a queenright colony.
HEX 110 foci (green) were detected with anti-HEX 110/Alexa-Fluor 488. Propidium iodide (red) stains DNA and RNA and was used to highlight the nuclei. (A) Upper region of the germarium of two ovarioles (separated by dashed lines) showing germ- and somatic cell precursors containing cytoplasmic (arrows) and nuclear (arrowheads) HEX 110 foci. (B) Lower region of the germarium showing initial oocytes (cystocytes) (Oc) and nurse- and follicle cell precursors, these still morphologically undistinguishable from each other. (C) The same germarium region showing cytoplasmic (arrows) and nuclear (arrowheads) HEX 110 foci. (D) A follicle at the basal region of the ovariole showing a developing oocyte (Oc, stage 1, see Fig 2), and nurse cells (Nc). Oocyte nucleus (N) and cytoplasm (c) are indicated. Small follicle cells (Fc) are seen around the oocyte and nurse cell chamber. (E) The same follicle shown in D. Arrows point to cytoplasmic HEX 110 foci. The arrowhead points to nuclear HEX 110 foci in a follicle cell within the nurse cell chamber.
Fig 5.
Colocalization of HEX 110 and fibrillarin, a nucleolar protein marker, in the active ovarioles (stage S3) of queenless workers.
(A) Terminal filament of an ovariole stained with anti-Fibrillarin/Alexa-Fluor 594 (red foci). (B) The same image showing HEX 110 foci (green) identified with anti-HEX 110/Alexa-Fluor 488. (C) The merged image: anti-Fibrillarin/Alexa-Fluor 594 (red foci) and anti-HEX 110/Alexa-Fluor 488 (green foci). The insert highlights costained HEX 110/fibrillarin (yellow foci pointed by arrows). The area delimited by the dashed rectangle was used for estimation of Manders colocalization coefficients, M1 (red) and M2 (green), estimated as 0.3814 and 0.3462, respectively (red and green signals amounted to 37.45% and 27.11% of the selected area, respectively). (D) The same image added with DAPI-labeled nuclei (blue). The longer arrows point to HEX 110 foci in the cell cytoplasm. The insert highlights costained HEX 110/fibrillarin (yellow foci pointed by shorter arrows) and costained nucleolar DNA/fibrillarin (pink foci pointed by arrowheads). (E) A developing follicle stained with anti-Fibrillarin/Alexa-Fluor 594 (red foci) shows multiple nucleoli in the nurse cells (Nc) and a single nucleolus in the follicle cells (Fc) surrounding the oocyte (stage 2, see Fig 2) at the bottom of the figure. (F) The same image showing HEX 110 foci (green) identified with anti-HEX 110/Alexa-Fluor 488. (G) The merged image: anti-Fibrillarin/Alexa-Fluor 594 (red foci), anti-HEX 110/Alexa-Fluor 488 (green foci). The insert (nurse cell) highlights costained HEX 110/fibrillarin (yellow foci pointed by arrows). The area delimited by the dashed rectangle was used for estimation of Manders colocalization coefficients, M1 (red) and M2 (green), resulting in values of 0.6423 and 0.2203, respectively (red and green signals amounted to 79.83% and 28.36% of the delimited area, respectively). (H) The same image added with DAPI-labeled nuclei (blue). The insert (nurse cell) highlights costained HEX 110/fibrillarin (yellowish foci pointed by short arrows) and costained nucleolar DNA/fibrillarin (pink foci pointed by arrowheads). The longer arrow in this insert points to HEX 110 foci (green) in the cytoplasm of a nurse cell.
Fig 6.
Immunolocalization of HEX 110 in the queen active ovarioles (stage S4).
(A-C) Part of the terminal filament (Tf) and upper region of the germarium (G) stained with (A) propidium iodide (red), which stains RNA and DNA, or with (B) anti-HEX 110/Alexa-Fluor 488; (C) merged image. Arrowheads point to nucleolar HEX 110/propidium iodide foci (yellowish green). Arrows point to cytoplasmic HEX 110 foci (green). The area delimited by the dashed rectangle was used for estimation of Manders colocalization coefficients, M1 (red) and M2 (green), resulting in values of 0.2902 and 0.2208, respectively (red and green signals amounted to 20.33% and 16.38% of the delimited area, respectively). (D-F) Developing oocyte (Oc, stage 3–4, see Fig 2), nurse cells (Nc) in the nurse cell chamber and follicle cells (Fc). (D) propidium iodide. (E) anti-HEX 110/Alexa-Fluor 488. (F) Merged image showing yellow foci (arrowheads) indicating HEX 110/propidium iodide colocalization (and thus, HEX 110/RNA or DNA association) in the nuclei or the nurse cells (Nc) (in the basal region of the nurse cell chamber) and in the follicle cells (Fc). Arrows point to cytoplasmic HEX 110 foci. The area delimited by a dashed square was used for estimation of Manders colocalization coefficients, M1 (red) and M2 (green), resulting in values of 0.4014 and 0.2404, respectively (red and green signals amounted to 35.19% and 22.33% of the delimited area, respectively). (G-I) Magnification of part of a follicle showing nurse cells (Nc) stained with (G) anti-Fibrillarin/Alexa-Fluor 594 (red foci) or (H) anti-HEX 110/Alexa-Fluor 488 (green foci) plus DAPI (blue). The yellow foci in the cells of the merged (I) image (arrowheads) identify nucleoli and confirm their presence in the basal region of the nurse cell chamber (as seen in F image). Follicle cells (Fc) and part of an oocyte (Oc) are also visible in H and I images. (J-L) Merged HEX 110/propidium iodide-stained images. (J) Oocyte (Oc, stage 5, see Fig 2) involved by somatic epithelial follicle cells (Fc) and separated from the nurse cell chamber. HEX 110 foci (green) are mainly seen in the cytoplasm of follicle cells (Fc) and in the nurse cell (Nc) chamber (arrows). The propidium iodide red-stained oocyte (Oc) reflects its large amount of cytoplasmic RNA. (K) The quasi-mature oocyte (Oc, stage 7, see Fig 2) surrounded by follicle cells (Fc) in the lower portion of the ovariole. HEX 110 foci are evident in the cytoplasm of follicle cells (Fc) and in the peripheral cytoplasm of the oocyte (arrows). Arrowheads point to colocalized HEX 110/propidium iodide (yellow foci). (L) Magnification of part of the follicular epithelium and ooplasm. HEX 110 foci are mainly evident in the follicle cell (Fc) cytoplasm and ooplasm (arrows, green foci). In the nuclei of the follicle cells, HEX 110 clearly colocalized with propidium iodide (Fig 6L, yellow foci pointed by arrowhead).
Fig 7.
Immunolocalization of HEX 110 in RNase A-treated ovarioles (stage S2) of queenright nurse workers.
(A) Control ovariole non-incubated with RNase A. HEX 110 green foci (labeled with anti-HEX 110/Alexa-Fluor 488) are abundant in the nucleus (arrowheads) and cytoplasm (arrows) of nurse (Nc) and follicle (Fc) cells surrounding the oocyte (stage 2, see Fig 2). (B) Ovariole incubated with RNase A shows scarce HEX 110 foci (arrows). DAPI-staining (blue) in B highlights the small nuclei of follicle cells (Fc) that surround the oocyte (stage 1, see Fig 2) and are interspersed between the large nurse cell (Nc) nuclei. Quantification of pixels intensity in the selected areas (dashed rectangles) of the images obtained from RNase A-treated and untreated ovarioles showed lower intensity in RNase A-treated (Mean: 17.29; Min/Max gray values: 0/255; IntDen: 575.51) than in untreated controls (Mean: 29.39; Min/Max: 0/255; IntDen: 978.45).
Fig 8.
Colocalization of HEX 110 and pyronin Y, a RNA-specific stain, in the ovarioles (stage S2) of queenright nurse workers.
Upper (A-D) and lower (E-H) regions of the germarium. Developing follicles immediately below the germarium (I-L) and at the botton of the ovariole (M-P). Oc: oocyte; Fc: follicle cell; Nc: nurse cell. Arrowheads and arrows show nuclear and cytoplasmic foci, respectively, identified with anti-HEX 110/Alexa-Fluor 488 (green foci) or pyronin Y (red foci). (D, H, L, P) The yellowish/yellow foci indicate HEX 110/RNA colocalization in the nucleoli of follicle (Fc) and nurse (Nc) cells, but not in the oocyte (Oc) nucleolus. (L) HEX 110 colocalizes with RNA in the periphery of the stage 1-oocyte cytoplasm (Oc, arrow). The estimated M1 (red) and M2 (green) Manders colocalization coefficients for the large area delimited by dashed lines were 0.4971 and 0.4399, respectively (red and green signals amounted to 51.59% and 44.92% of the selected large area). For the small area delimited by a dashed square, the M1 and M2 coefficients were 0.3434 and 0.2567, respectively (red and green signals amounted to 40.36% and 30.94% of the small area, respectively). (P) HEX 110 appears in the nucleus of the stage 2-oocyte (Oc) at the base of the ovariole (nuclear green foci pointed by arrowhead), but is not colocalized with RNA (nuclear red foci pointed by arrowhead). HEX 110 colocalized with RNA in the nucleoli of nurse cells (Nc): the M1 and M2 values for the nurse cell nuclear area enclosed by the dashed square corresponded to 0.2883 and 0.2624, respectively (red and green signals amounted to 35.99% and 35.11%, respectively).