Oxytocin/vasopressin-like peptide inotocin regulates cuticular hydrocarbon synthesis and water balancing in ants

Abstract

Oxytocin/vasopressin-like peptides are important regulators of physiology and social behavior in vertebrates. However, the function of inotocin, the homologous peptide in arthropods, remains largely unknown. Here, we show that the level of expression of inotocin and inotocin receptor are correlated with task allocation in the ant Camponotus fellah Both genes are up-regulated when workers age and switch tasks from nursing to foraging. in situ hybridization revealed that inotocin receptor is specifically expressed in oenocytes, which are specialized cells synthesizing cuticular hydrocarbons which function as desiccation barriers in insects and for social recognition in ants. dsRNA injection targeting inotocin receptor, together with pharmacological treatments using three identified antagonists blocking inotocin signaling, revealed that inotocin signaling regulates the expression of cytochrome P450 4G1 (CYP4G1) and the synthesis of cuticular hydrocarbons, which play an important role in desiccation resistance once workers initiate foraging.

Keywords: behavioral tracking; cuticular hydrocarbon; division of labor; oxytocin/vasopressin-like peptide; social insect.

Fig. 1.

Relative expression of int and intR in different castes and tissues of C. japonicus. Relative expression (mean ± SEM) of int (A and C) and intR (B and D) in the head, thorax, and abdomen of mated queens (red), virgin queens (pink), males (blue), and workers (green) is shown. The expression of each gene was scaled by the average value in each graph. Values are therefore not comparable between graphs. Number of individuals are shown in each box. The relative expression levels of each gene were tested with two-way ANOVA. Groups differing significantly (P < 0.05) are marked with different letters. Samples shown in A and B were collected in 2016 and samples in C and D in 2015. We did not collect any mated queens in 2015.

Fig. 2.

Correlation between the expression of inotocin signaling and task allocation. (A) Setup of the C. fellah colonies. (B) Age-dependent division of labor in one representative colony. Nurses are shown in pink and foragers in light blue. (C and D) Box plots of the relative expression levels of int in the head (C) and intR in the abdomen (D) for each age class. Number of individuals is shown in each box. (E) The time spent in the food region and in the nest and distance moved was quantified over 6 d in the tracking system for one representative box. (F–H) Relationship between the behavioral parameters and int expression. (I–K) Relationship between the behavioral parameters and intR expression. Different colors indicate the colony of origin of workers. R² and P values are shown in the top left of the graphs. The correlation between the expression of int or intR and behavioral parameters were tested with a generalized linear mixed model (GLMM). ns, P > 0.05; ***P < 0.001.

Fig. 3.

The expression of int in neural tissues and intR in the fat body plus oenocytes. (A) Fluorescence in situ hybridization using a specific int antisense probe (shown in magenta). Arrowheads indicate the pair of neurons labeled with int mRNA in the subesophageal zone (SEZ, outlined with white dotted line). (B and C) Magnified image of each int mRNA-positive neuron (shown in magenta in A) with stained nucleus using DAPI (shown in green). (D and E) Immunohistochemistry for anti-int antibody (shown in magenta) and anti-synapsin antibody (shown in green) in SEZ (D) and protocerebrum (E). Arrowheads indicate cytoplasmic expression of anti-int immunoreactivity. Arrows indicate the neurons positive with anti-int antibody in SEZ (D) and protocerebrum (E), in which brain compartments are labeled as mushroom body (MB), medial superior protocerebrum (SMP), and vertical lobe (vL). (F) Schematic presentation from central to peripheral nervous system in workers. (G–J) One pair of neurons labeled with anti-int antibody from the subesophageal zone to the ventral nerve cord (G), the prothoracic (H), mesothoracic and metathoracic (I), and abdominal ganglia (J). (K) Distribution of endogenous inotocin peptide tested with unpaired two-tailed t test. ***P < 0.001. (L) qRT-PCR for the intR in the digestive tract (DT) and fat body plus oenocytes (FB+OE). The expression of intR mRNA was tested with GLMM with Benjamini–Hochberg post hoc test. ns, P > 0.05; ***P < 0.001. (M) in situ hybridization using an intR antisense probe (Left) and sense probe (Right) in the dissected fat body plus oenocytes. Oenocytes are indicated with open arrows and trophocytes (the lipid-storing cells of the fat body) with filled arrows. [Scale bars, 100 µm (A, D, E, and G–J) and 20 µm (B, C, and M).]

Fig. 4.

Expression of CYP4G1 correlating with that of intR. (A) qRT-PCR data for CYP4G1 expression in the digestive tract (DT) and fat body plus oenocytes (FB+OE) for 1- and 6-mo-old individuals. ns, P > 0.05; ***P < 0.001 (GLMM with Benjamini–Hochberg post hoc tests). (B) The CYP4G1 protein is expressed in the fat body plus oenocytes (FB+OE), but not in the digestive tract (DT). The band at 63 kDa (shown with red arrowhead in B, Left) was not detected with the anti-CYP4G1 antibody after the absorption with its antigen peptide (B, Right). (C) The CYP4G1 protein (magenta) is specifically present in the oenocytes (round-shaped nucleus stained with Hoechst; light blue), but not in the trophocytes (irregular-shaped nucleus and lipid droplets labeled with BODIPY; green). (Scale bar, 50 µm.) (D) Correlation between the expression of CYP4G1 and intR in the abdomen. R² and P values are shown on the lower right of the graph. ***P < 0.001. (E) Feeding of dsRNA targeting intR leads to a significant decrease in the level of expression of intR (magenta; unpaired two-tailed t test, **P < 0.01) compared with feeding of dsRNA targeting GFP (gray). Number of individuals is shown in each box. (F) Relationship between the expression levels of intR and CYP4G1 in the treatment with dsRNA targeting GFP (gray) and intR (magenta). R² and P values for each treatment are shown on the lower right of the graph. **P < 0.01; ***P < 0.001. (G) CYP4G1 expression is significantly down-regulated with feeding dsRNA targeting intR (magenta; unpaired two-tailed t test) compared with feeding of dsRNA targeting GFP (gray). Number of individuals is shown in each box.

Fig. 5.

Chemical screening to identify the inhibitor against inotocin signaling. (A) Relative fluorescence units (RFU) of Fluo-4 probe in response to inotocin, oxytocin, and vasopressin in CHO cells expressing inotocin receptor. Inotocin receptor is activated by inotocin (black line; EC₅₀ = 1.1 nM) and oxytocin (blue line; EC₅₀ = 41.6 µM), but not by vasopressin (red line). (B) Atosiban blocks the activation of inotocin signaling (IC₅₀ = 599 µM). A concentration of 5 nM inotocin was used as an approximate EC₈₀ concentration. Data represent the mean ± SEM (n = 3). (C) Competition curve of specific binding of 10 nM labeled inotocin ligand with increasing concentrations of atosiban (1.95 µM to 2 mM) measured by TR-FRET. Data represent the mean ± SEM of a representative experiment (n = 3). TR-FRET ratio = (665-nm acceptor signal/620-nm donor signal) × 10,000. (D) Scatter plot showing the inhibition rate of each compound in the first screening. The red line indicates a 50% inhibition. (E) Scatter plot showing the Z′ factor for each plate in the first screening. All Z′ factors in each plate are >0.5 indicated by the red line. (F) Inhibition rate against inotocin and oxytocin signaling are plotted for each of the 902 compounds showing >50% inhibition rate in the first screening. Compounds showing <0.5 ratio for oxytocin inhibition rate versus inotocin inhibition rate are in the pink-shaded region. (G) Structure of three compounds, A, B, and C. (H) Dose-dependent inhibition against inotocin signaling of compound (comp.) A (IC₅₀ = 6.8 µM; pink line), comp. B (IC₅₀ = 2.6 µM; orange line), and comp. C (IC₅₀; no data; green line). Data represent the mean ± SEM of a representative experiment (n = 4). RFU indicates the activation of inotocin receptor calculated from Fluo-4 intensity.

Fig. 6.

Relationship between inotocin signaling, hydrocarbon synthesis, and desiccation resistance. (A) CYP4G1 expression (qRT-PCR) for comp. A-, B-, and C-treated and control 4-mo-old workers. ns, P > 0.05; *P < 0.05 (GLMM with Dunnett post hoc tests). (B–D) The quantities of normal (B), branched (C), and total alkanes (D) in comp. B-treated and control >5-mo-old workers. **P < 0.01; ***P < 0.001. (E) qRT-PCR for CYP4G1 in comp. B-fed and control 4-mo-old workers. Number of individuals is shown in each box. The data were analyzed with GLMMs. (F) Survival curves for control (gray) and comp. B-treated (orange) ants in 55% RH (solid line) or 85% RH (dashed line). Sample sizes are as follows; n = 28 for ctrl_55% and comp. B_55%, and n = 35 for ctrl_85% and comp. B_85%.