GoKawiilGeneral ant husbandry and maintenance
Ooceraea biroi colonies of clonal line B81 were housed in 0.6-quart ClickClack boxes with an approximately 3-cm-deep plaster of Paris floor. Colonies were kept in a climate-controlled environmental room at around 25 °C and around 60% humidity, and the plaster floor was kept damp by regularly adding water. O. biroi colonies cycle between a reproductive phase during which colonies contain eggs and pupae and ants do not forage, and a foraging phase when larvae are present and ants forage for food27. An entire colony cycle takes around 5 weeks. Towards the end of the reproductive phase, eggs hatch into larvae. The foraging phase begins when larvae enter the third instar about 1 week later, coinciding with the emergence of the previous cohort of callow workers. During the foraging phase, which lasts around 2 weeks, colonies are composed entirely of workers and third and/or fourth instar larvae. In this phase, colonies were fed three times per week with frozen fire ant (Solenopsis invicta) brood. At the time of feeding, the plaster floor was briefly cleaned with Q-tips soaked in 10% bleach, and the plaster was then watered.
Ant maintenance for experiments
Naturally cycling O. biroi colonies contain several generations of adults. During the foraging phase, the youngest generation of ants are easily recognizable by their lightly melanized cuticle for up to 6–7 days after they eclose from pupae. For the primary and secondary neuropeptide screens, 300–500 6-to-7-day-old ants were separated from their colonies into new boxes together with larvae in an approximately 1:1 ratio and aged to 12 days old. At that time, they were tested in the screens. For all other experiments, we generated age-matched colonies of ants by seeding new colonies with 300–500 6-to-7-day-old ants and larvae in an approximately 1:1 ratio. At each subsequent foraging phase, the newly eclosed callow ants were removed and transferred into new colonies with larvae in an approximately 1:1 ratio. This procedure was repeated for every foraging phase in every colony. Ants in all resulting colonies were aged up to 5 months old. This resulted in a regular supply of age-matched ants that were 12 days old, and 1, 2, 3, 4 and 5 months old, as well as larvae for experiments. All ants and larvae used in experiments came from colonies in the foraging phase when the larvae were in the fourth instar, and 12–16 days after callow workers had eclosed. For one week before behavioural experiments, ant colonies were fed every other day with frozen S. invicta brood stained with 0.5% weight/volume bromophenol blue (Sigma Aldrich; B5525) in autoclaved reverse-osmosis (RO) water. This stain accumulates in the larval gut, increasing the visual contrast and thus facilitating automated tracking (see below). Ant colonies were last fed 24 h before behavioural experiments.
Neuropeptidome annotation
Lists of neuropeptide precursor protein sequences were compiled from literature focusing on insects31,32,33,34,35,36,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101, as well as from a broad survey of neuropeptides across metazoan phyla23. We identified putative O. biroi neuropeptide precursor proteins by a BLASTp homology search of the compiled lists against the O. biroi non-redundant protein sequences (nr) database using default settings. Precursor proteins with high degrees of certainty were retrieved from additional insect species, with a focus on Hymenoptera. Homology of candidate O. biroi neuropeptide precursor proteins to query sequences was confirmed through reciprocal BLASTp on the nr database of the query sequence species. Signal peptides were identified using SignalP 6.0 (https://dtu.biolib.com/SignalP-6). Neuropeptides were annotated manually by identification of stereotypical proteolytic cleavage site motifs (RR, RK, KR and KK) and alignment to homologous neuropeptides from representative insect species (Supplementary Tables 1–3). Post-translational modifications were predicted by comparison with annotations of homologous insect neuropeptides in the UniProtKB database (https://www.uniprot.org). These annotations were performed in 2021 and reflect the neuropeptidome literature at that time.
Synthesis, storage and preparation of neuropeptides
Neuropeptides of 35 amino acids or fewer in size were synthesized including known modifications such as amidation and cyclization. For the neuropeptide screen treatment control, a 31-amino-acid neuropeptide, DH31, was synthesized with a biotin tag at the Proteomics Resource Center at Rockefeller University and provided lyophilized at 15% purity. Lyophilized DH31–biotin was resuspended in pure dimethyl sulfoxide (DMSO) to 30 mM concentration, aliquoted and stored at −80 °C before use. For the primary round of screening, neuropeptides were synthesized at the Proteomics Resource Center at Rockefeller University and provided lyophilized at 15–92% purity. We opted for this low level of purity of neuropeptides in the primary screen because of the large number of peptides and the costs associated with achieving high-purity preparations. This implies, however, that the rates of false positives in this screen might have been high, which we then mitigated by a stringent secondary screen using high-purity neuropeptides (see below). Lyophilized neuropeptides were stored for up to 4 months at −80 °C before resuspension. Stock solutions were made by resuspending lyophilized neuropeptides in pure DMSO to 2 mM–100 mM, depending on solubility, and then aliquoted and stored at −20 °C for up to 6 months before use. For the experiment to test whether the size range of our neuropeptide screening library could penetrate the cuticle, 30 mM DH31–biotin stock solution was diluted in autoclaved RO water to a final concentration of 1 mM DH31–biotin and 1% DMSO. The validation was then performed by soaking ants in biotinylated neuropeptides, dissecting out their brains and treating the brains with fluorescent-dye-conjugated streptavidin before performing quantitative confocal microscopy. For the primary neuropeptide screen, stock solutions of neuropeptides were diluted in autoclaved, RO-purified water to a final concentration of 1 mM neuropeptides and 1% DMSO. Control solutions were 1% DMSO in autoclaved RO water.
For the secondary round of dose–response analysis screening, neuropeptides were synthesized by Bio-Synthesis and provided lyophilized at higher than 95% purity. Stock solutions were made by resuspending lyophilized neuropeptides to a concentration of 10 mM in pure DMSO, and then aliquoted and stored at −20 °C for up to 4 months before use. For all experiments, fresh aliquots of stock solutions were diluted to avoid freeze–thaw cycles that compromise protein integrity. For the secondary screen, stock solutions were diluted to test concentrations of 0.1 mM, 0.3 mM, 1 mM and 3 mM neuropeptides in 1% DMSO and autoclaved RO water. For the repeat of the NPF dose–response experiment, stock solution of NPF was diluted to 0.003 mM, 0.01 mM, 0.03 mM, 0.1 mM, 0.3 mM, 1 mM and 3 mM NPF in 1% DMSO. Control solutions were 1% DMSO in autoclaved RO water.
Colony experiment
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