Oxytocin-like signaling in ants influences metabolic gene expression and locomotor activity

Abstract

Ants are emerging model systems to study cellular signaling because distinct castes possess different physiologic phenotypes within the same colony. Here we studied the functionality of inotocin signaling, an insect ortholog of mammalian oxytocin (OT), which was recently discovered in ants. In Lasius ants, we determined that specialization within the colony, seasonal factors, and physiologic conditions down-regulated the expression of the OT-like signaling system. Given this natural variation, we interrogated its function using RNAi knockdowns. Next-generation RNA sequencing of OT-like precursor knock-down ants highlighted its role in the regulation of genes involved in metabolism. Knock-down ants exhibited higher walking activity and increased self-grooming in the brood chamber. We propose that OT-like signaling in ants is important for regulating metabolic processes and locomotion.-Liutkevičiūtė, Z., Gil-Mansilla, E., Eder, T., Casillas-Pérez, B., Di Giglio, M. G., Muratspahić, E., Grebien, F., Rattei, T., Muttenthaler, M., Cremer, S., Gruber, C. W. Oxytocin-like signaling in ants influences metabolic gene expression and locomotor activity.

Keywords: G protein–coupled receptor; inotocin; insect; vasopressin; vasotocin.

Figure 1

Structure and pharmacology of the L. neglectus inotocin signaling system. A) Chemical structure of the Lasius inotocin peptide (CLITNCPRG-NH₂). B) Illustration of the typical 7-transmembrane (TM) GPCR architecture of the IR from L. neglectus. Amino acid sequence differences to the L. niger receptor (GenBank: A0A1L2FSX9) are presented via 1-letter code, residue number, and approximate position (P₁₃S, M₁₅₅V, T₂₀₅M, I₂₃₄V, N₂₅₁V, and V₂₆₉I), and receptor domains are labeled (N-terminal domain; ICL, intracellular loops; ECL, extracellular loops; C-terminal domain). C, D) Concentration-dependent receptor activation was measured by inositol-phosphate 1 (IP1) (C) and luciferase reporter (D) assay via activation of the cAMP-response element to determine Gq- and Gs-dependent signaling of inotocin, respectively, in HEK293 cells transiently expressing the L. neglectus IR. The error bars represent the mean ± sem from 3 independent experiments. Potency (EC₅₀) was determined by nonlinear regression (sigmoidal, 3 parameters, fixed Hill slope of 1) and normalized to the percentage of maximal activation. E) Saturation radioligand binding was performed on membrane preparations (10 μg) transiently expressing IR from L. neglectus and specific binding (pmol of [³H]inotocin/mg of membrane protein) was determined as difference between nonspecific and total binding (n = 2). Affinity (K_d) was determined by nonlinear regression using a 1-site binding equation. F) HEK293 cells transiently expressing a GFP-tagged version of the L. neglectus IR localizes mainly at the plasma membrane (green). Cell nuclei were stained with DAPI (blue).

Figure 2

Quantification of mRNA transcripts of the inotocin signaling system in L. neglectus. Expression of IR and IP in different development stages and castes (A), parts of the body (B), and organs (only receptor) (C) was determined by qPCR. At least 3 biologic replicates are represented as a dot plot with a mean ± sem. A) Eggs (n = 10–30) and larvae (n = 3–6) were pooled, and pupa and different adults were analyzed as 1 animal per biologic replicate. B) Workers (n = 8–15) and males (n = 5–9) were pooled, whereas only 1 queen was used per biologic sample of head, thorax, and abdomen. C) Organs (n = 4–8) of different animals for workers and males and queens (n = 1–2) were pooled together in every biologic sample. “Mated queen” corresponds to queens that mated at least 2 mo prior to sampling. Dufour’s gland and poison gland of the abdomen were analyzed together (termed “gland”). The gland of the head corresponds to postpharyngeal gland in workers and mandibular glands in males, respectively, which is a predominant gland in each case. The rest of the head is defined as head without brain and postpharyngeal (workers) or mandibular (males) gland. The reproductive system of males corresponds to the testis, seminal vesicles, and ejaculatory duct; the reproductive system of queens and workers corresponds only to ovaries. M, mated; v, virgin.

Figure 3

Localization of the inotocin peptide in L. neglectus by immunostaining. A) Ant brain with the 2 immunoreactive somata in the SEG. A, anterior; L, lateral. B) Magnification of SEG. C) Abdominal ganglia of the ventral nerve cord (from top starting with the petiole ganglion). Scale bars, 100 μm (A, C) and 50 μm (B). DAPI nuclear staining is shown in blue; inotocin peptide staining is shown in green. For further information, refer to Supplemental Fig. 4.

Figure 4

Expression profiles of the IP and IR of L. neglectus during different seasons (A), in foragers vs. nurses (B), and under starvation (only precursor) (C). Results are represented as dot blots with means ± SEM. A) Ants from summer- and winter-conditioned colonies were collected at the same time (n = 13–21, Student’s t test). B) Ants were collected from foraging area (foragers) and brood chamber (nurses) from the same nest (n = 20, Student’s t test). C) Relative expression ratio of a precursor before (d 0), during (d 2), and after (d 3 and 9) the starvation period of 48 h (n = 17–20). See detailed statistical analyses in Supplemental Table 5.

Figure 5

Altered gene expression of IP-KD L. neglectus workers. mRNA sequencing: GO term enrichment analyses of differentially expressed transcripts. Statistically significant GO terms (P < 0.01) are shown, which were reduced using the Revigo online tool (semantic similarity was measured using the Lin method; only GO terms covering 2 or more transcripts were used) (72). A full list of statistically significant GO terms (P < 0.01) is shown in Supplemental Table 7. Black bars correspond to biologic process, gray bars correspond to molecular functions, and white bars correspond to cellular components. ^#The full GO term is “oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen,” A, activity; B, binding; MP, metabolic process; str, structural.

Figure 6

Physiology of IP-KD L. neglectus workers. A) Behavior in a group experiment: walking and self-grooming scores of 5 ants in a group were measured every 5 min during 2 h outside and inside brood chamber (n = 12). B–D) Walking assays. Single ant walking activity on a dish was monitored for 60 min and is presented as walking time (B), distance (C), and number of walking episodes (D) [control (ctrl), n = 20; IP-KD, n = 19]. E) Eating experiment. After a starvation period of 3.5 h, food was introduced, and the percentage of feeding ants and ants being near the food was calculated (ctrl, n = 88; IP-KD, n = 89). Data are presented as dot plots and median with interquartile range. Generalized linear mixed models (A–D) or Mann-Whitney U test (E) were used. A detailed description of statistical analyses is provided in Supplemental Table 3. *P value reflects treatment effect over all data.

Figure 7

Summary of the functional role of inotocin signaling in Lasius ants. *P = 0.066.