sNPFR immunoreactive cluster c9 is only detected in the posterior lateral protocerebrum of mediums and minors (posterior view).

<p>(A) Schematic representation of the location of c5 (central) and c9 (lateral). Images in the top panel show the right hemisphere and the bottom images, the left one. In minor worker brains cluster c9 is detected near the optic lobe, symmetrically on both sides of the brain (B, G). Also, c9 is present in medium worker brains (C, H). In majors, cluster c9 is undetectable (D, I). No fluorescent signal was observed in negative controls, with pre-immune serum (E, F) or antigen-preabsorbed antibodies (J). Lo: lobula.</p

Schematic representation of the sNPFR immunolocalization in the brain and SEG of all worker subcastes.

<p>Anterior (top panel) and posterior (bottom panel) views of the brain show different cell clusters expressing the sNPFR. (A, D) represent the localization in majors; (B, E) in mediums and (C, F) in minors. Dashed-empty circles indicate the cells can be observed faintly from the anterior view but are located deeper in the brain; purple checkered-filled circles indicate the same, but when the brain is seen from the posterior side. Within clusters the depth of certain cells may vary. The brain orientation is indicated by the direction of the arrows shown in the top left corner of each subpanel: A =  anterior, P =  posterior, D =  dorsal, V =  ventral L =  left, R =  right.</p

Cluster c5 is immunolocalized at the superior edge of the antennal lobe in all subcastes.

<p>Cluster c5 could be observed in the brain of majors (A), medium (B) and minor (C) workers more clearly from the anterior view but also from the posterior view, slightly deeper in the brain. A schematic representation is shown over a Nomarsky image of a medium worker brain from the anterior view in D. Cluster c5 is most often represented by two cells, which are symmetrically located at the superior edge of the ALo. In the posterior view of the brain in E, c5 is depicted using checkered dots; purple solid dots correspond to cluster c9. Cluster c5 in majors is shown in detail in F (left), G (right); notice that in those images c5 is composed of three cells. H (left), I (right) show a detail of c5 in mediums, and the same is shown in J (left), K (right) for minors. No fluorescent signal was observed in negative controls with pre-immune serum (L) or with the antigen-preabsorbed antibody (M). Left or right refers to the brain hemisphere shown.</p

The superior protocerebrum exhibits differential sNPFR immunoreactive neurons among subcastes (anterior brain view).

<p>A: Nomarsky image of a minor worker brain showing the location of clusters c7 (center) and c2 (lateral). In majors, clusters c7 (B, center), and c2 (C, right; D, left) were not detected. E: shows c7 in the brain of a medium worker (detail in F); however, cluster c2 normally under the lCa is not observed (G, left; I, right). In minors, c7 is clearly visible close to the mCa (H, detail in K) and c2 is located under the right (L) and left lCa (J). The lack of fluorescent signal on the left lCa of a minor worker brain treated with pre-absorbed antibody as negative control is shown in (M), and both brain hemispheres are shown in (N). No fluorescent signal was observed for the pre-immune negative control (O). mCa: median calyces, lCa: lateral calyces, smP: superior medial protocerebrum. Left or right refers to the brain hemisphere shown.</p

Brain immunolocalization of the sNPFR in worker subcastes from colonies without brood and comparisons with those with brood.

<p>(A–H) Schematic of the location of cell clusters expressing the sNPFR in all subcastes, comparing colonies with and without brood. Areas in orange boxes enclose specific brain areas for comparison. Dashed white circles correspond to the expected location of immunoreactive cells in colonies with brood. A–D, G and H show the receptor signal in the posterior brain; E and F show the anterior brain view. In colonies without brood cluster c7 became undetectable both in minor (H, I) and medium workers (D, K); compare with C, G. Additionally, cluster c2 became undetectable in minors from these colonies (F, J). In the posterior view, cluster c9 also became undetectable in minors (H, and insets in M, N) and mediums (D, L, inset). Cluster c14 remained present in minor (O, P) and medium workers (Q, R), but cluster c15 became undetectable in these workers, and only observed in majors, however, composed by lower number of immunoreactive cells (S, T) in comparison to majors in colonies with brood. Cluster c12 also is undetectable in majors (B, and in S, lower right corner) when brood is absent.</p

Comparison of the head widths (H.W.) among worker subcastes in fire ants.

<p>Majors are considered the biggest workers, minors are considered the smallest, and mediums are intermediate in size between majors and minors. The heads of a major worker (left; 0.93–1.46 mm H.W.), a medium worker (center; 0.73–0.92 mm H.W.) and a minor worker (right; ≤ 0.72 mm H.W.) are shown.</p

Common and subcaste-differential sNPFR immunoreactive clusters are present in the posterior brain and SEG.

<p>A schematic of all possible clusters detected in the posterior SEG across subcastes are shown in (A) over a Nomarsky image of a major. In all subcastes clusters c13 (top arrows in B), c15 (B, bottom arrowheads) and c16 (B, center) are present. Clusters c12 and c14 are differentially detected among subcastes. (B) In majors, c12 (dashed inset, arrow) is present but c14, that should be located internal to c15, is not detected. A detail of c15 in the right hemisphere of majors is shown in (C) and left, in (D). Medium and minors exhibit all clusters except c12, but although c14 and c15 are present, they have reduced cell number with respect to those in majors (compare D with H and M). In these two subcastes c14 and c15 could be observed from both sides of the brain. (E) Medium worker, clusters c13 to c16; details in F (c13), G (c16), H (c14, c15, left) and I (c14, c15, right). In minors, c13 is shown in (J), and in both brain hemispheres, in (K). (L) Brain of minor; sometimes cells in c16 are not perfectly distributed in a cross-like pattern (compare with G). (N) Distribution of clusters c13 to c16 in minors. Clusters c14 and c15 are show in M (left) and O (right). No fluorescent signal was observed in negative controls with pre-immune serum (P) or antigen-preabsorbed antibodies (Q).</p

Number of sNPFR immunoreactive cells found in the brain of worker subcastes from colonies with and without brood and comparison to those reported previously for queens.

<p>Footnotes: Names of clusters in capital letters correspond to those in the queen, but cell numbers under minor, mediums and majors correspond to worker clusters in similar position to those clusters found in queens and that we identified with small letter c throughout the manuscript. Note that clusters only present in the midline of the brain, and therefore not symmetrically distributed are: C1, C6, C12 and c16; clusters c13 through c16 are exclusively found in workers, but c14 is absent in majors.</p>(1)<p>The number of cells per cluster in one brain/SEG hemisphere is indicated only for clusters that show a symmetrical distribution; in workers, numbers separated by a hyphen indicate the range in the number of cells observed in different individuals. N/A refers to clusters in the midline of the brain which cell number is only indicated in the total number per brain column.</p>(2)<p>From Lu <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083966#pone.0083966-Lu1" target="_blank">[28]</a>.</p>(3)<p>The percentage decrease in the cell number range per subcastes in colonies without brood was calculated with respect to the respective range in cell numbers in colonies with brood.</p><p>(a) are queen exclusive clusters;</p><p>(b) minors and queen exclusive;</p><p>(c) is a cluster common to all females (all worker subcastes and queen);</p><p>(d) common to mediums, minors and queens;</p><p>(e) queen and majors exclusive. In workers, clusters labeled c or f are immunostained regardless of the presence or absence of brood (C5 and c13, c14, c16). In workers, clusters with asterisks change in cell number depending on the presence or absence of brood; c15 is the only worker exclusive cluster that responds to the absence of brood (g).</p

Cluster c16 is located near trachea in the posterior brain.

<p>This cluster is observed in all subcastes, generally located at the center of the SEG, with 5 cells distributed in a cross-like fashion. Sometimes, the location of each individual cell can vary, and they could be observed near, or perhaps in association with trachea, as shown here in a minor brain.</p

Detection of <i>Solenopsis invicta</i> short neuropeptide F receptor (<i>Solin</i> sNPFR) in stably transformed CHO-K1 cell line <i>Si</i>sNPFR-C6E8.

<p>The HA-tagged <i>Solin</i> sNPFR was detected by fluorescence immunocytochemistry using an anti-HA-tag antibody. The cell line names are shown on the left panel, and the antibody used labels the top of the first column. For each row, the images on the center show the nuclear staining with DAPI (blue) of the same cells on the left, and those on the right are merged images of the two previous. The HA-tag (red signal) is detected in <i>Solin</i> sNPFR cells (A–C) but not in the vector-only transformed cells (D–F). Rhimi-CAP_2b-R cells (G–I) showing red signal were used as positive controls for the HA-tag labeling. No red signal was detected in <i>Solin</i> sNPFR cells incubated with pre-immune rabbit serum (J–L). An anti-α-tubulin antibody was used as positive control for the labeling of a cytoplasmic structural protein (M–O); the red pattern is different than for the HA-tag (contrast A and G to M). Scale bar, 50 µm.</p