Persistent antennal dueling behavior results in social dominance and reproductive succession in Harpegnathos saltator workers. (A) Schematic of the worker-to-gamergate transition and tissue sampling at different time points. In a queenless colony, nonreproductive workers labeled with a unique color code initiate an antennal dueling battle among each other. The duelers eventually display queenlike phenotypes (egg-laying and nonworker behaviors) after 50 d of the dueling tournament. Three different tissues, including the nonvisual brain, abdominal fat body, and ovary, were collected for RNA-seq experiments at three different time points: prior to the transition setup, and on day 3 and day 10 after the beginning of the dueling tournament. (B) Two groups of workers were classified based on the antennal dueling activity during the worker-togamergate transition. Hierarchical clustering analysis of the dueling activity over 50 d after initiation of the dueling tournament classifies workers into high-and low-dueling clusters. A heat map displays average dueling activity of each 5-d window for over a 50-d period. Total numbers of egg-laying events observed in high-and low-dueling individuals from three independent colonies are shown in the right panel. The number of high-and low-dueling individuals (n) are represented in magenta and cyan, respectively. (C ) Average numbers of egg-laying events per individual in the high-and low-dueling groups during the 50-d period. (Magenta) High-dueling; (cyan) low-dueling. ( * * * * ) P < 0.0001, Mann-Whitney test. N = 20, 57, respectively, Bars and errors represent mean ± SEM. (D) Differential patterns of antennal dueling activity and egg-laying events in worker and gamergate castes. Worker (W) and gamergate (G) fates were behaviorally and physiologically determined after the 50 d of the dueling tournament. Antennal dueling activities of W and G (5-d average) over the 50-d period. (E) Cumulative numbers of the observed egg-laying events in workers (W) and gamergates (G) over the 50-d period. (W) Black; (G) gray. N = 3 colonies. Bars and errors represent mean ± SEM.

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... et al. 2018). However, upon death or waning reproductive output of the queen, H. saltator workers quickly become involved in a dueling tournament to determine which workers will become the reproductives of the colony ( Liebig et al. 1999). H. saltator workers duel with each other by trading strikes with their antennae (antennal dueling) ( Fig. 1A; Supplemental Movie S1), and the tournament is often performed for months. Individuals with high dueling activities eventually become gamergates or pseudoqueens, as it takes several gamergates that share social dominance hierarchies to replace the larger senescent/deceased queen Sasaki et al. 2016). Gamergates show extensive adult ...

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... or gamergates. The older workers, which forage and perform other colony maintenance tasks, were introduced to maintain the colony health and were not analyzed as they did not contribute significantly to dueling. All workers were uniquely painted with a threecolor-code system, allowing us to distinguish each individual and trace its activity ( Fig. 1A; see the Materials and Methods). During an adaptation period of 5-7 d following the colony setup, workers huddled in the colony nest and groomed each other. This period of peace was disrupted when groups of workers began to duel. The first observation of dueling was denoted as day 1 in order to keep the time scales consistent between ...

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... over this 50-d period from the three colonies showed that there are two clearly distinct groups of workers: high-and low-dueling individuals. High-dueling workers actively and constantly duel (trading strikes with their antennae) during the caste switch, while individuals with no or only transient dueling are considered as low-dueling workers (Fig. 1B). Colony 1 consisted of seven high-dueling and 20 low-dueling individuals, colony 2 had six high and 19 low duelers, and colony 3 had seven high duelers and 18 low duelers (Fig. 1B); few animals (three, five, and five, respectively) died during the tournament in the three colonies. The total number of egg-laying events was significantly ...

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... duel (trading strikes with their antennae) during the caste switch, while individuals with no or only transient dueling are considered as low-dueling workers (Fig. 1B). Colony 1 consisted of seven high-dueling and 20 low-dueling individuals, colony 2 had six high and 19 low duelers, and colony 3 had seven high duelers and 18 low duelers (Fig. 1B); few animals (three, five, and five, respectively) died during the tournament in the three colonies. The total number of egg-laying events was significantly higher in the high-dueling group compared with that of the low-dueling group (Fig. 1B,C). Each high-dueling individual laid ∼6.8 eggs in total, while the low duelers laid 0.3 egg ...

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... colony 2 had six high and 19 low duelers, and colony 3 had seven high duelers and 18 low duelers (Fig. 1B); few animals (three, five, and five, respectively) died during the tournament in the three colonies. The total number of egg-laying events was significantly higher in the high-dueling group compared with that of the low-dueling group (Fig. 1B,C). Each high-dueling individual laid ∼6.8 eggs in total, while the low duelers laid 0.3 egg on average during the 50-d period (Fig. 1C). These findings highlight that the well-known association between antennal dueling behavior and reproductive potential in H. saltator is very robust (Hölldobler and Wilson 2008;Sasaki et al. ...

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... five, and five, respectively) died during the tournament in the three colonies. The total number of egg-laying events was significantly higher in the high-dueling group compared with that of the low-dueling group (Fig. 1B,C). Each high-dueling individual laid ∼6.8 eggs in total, while the low duelers laid 0.3 egg on average during the 50-d period (Fig. 1C). These findings highlight that the well-known association between antennal dueling behavior and reproductive potential in H. saltator is very robust (Hölldobler and Wilson 2008;Sasaki et al. ...

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... Workers performed nest cleaning and cricket hunting/foraging while gamergates continued antennal dueling and kept laying eggs. In addition, gamergates showed slow locomotion inside the nest and often held an upright posture on a brood pile, whereas workers were outside of the nest and displayed submissive posture near gamergates (Supplemental Fig. S1A). We then quantified the daily antennal dueling activity from day 1 to 50: The overall dueling activity of gamergates was dramatically higher than that of workers (Fig. 1D), consistent with the dueling classification obtained from the hierarchical clustering analysis (Fig. 1B). During days 1-5 of the dueling tournament, members of both ...

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... inside the nest and often held an upright posture on a brood pile, whereas workers were outside of the nest and displayed submissive posture near gamergates (Supplemental Fig. S1A). We then quantified the daily antennal dueling activity from day 1 to 50: The overall dueling activity of gamergates was dramatically higher than that of workers (Fig. 1D), consistent with the dueling classification obtained from the hierarchical clustering analysis (Fig. 1B). During days 1-5 of the dueling tournament, members of both castes joined the dueling, but the gamergates showed higher dueling activity relative to workers. Individuals that would become gamergates increased their dueling activity ...

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... the nest and displayed submissive posture near gamergates (Supplemental Fig. S1A). We then quantified the daily antennal dueling activity from day 1 to 50: The overall dueling activity of gamergates was dramatically higher than that of workers (Fig. 1D), consistent with the dueling classification obtained from the hierarchical clustering analysis (Fig. 1B). During days 1-5 of the dueling tournament, members of both castes joined the dueling, but the gamergates showed higher dueling activity relative to workers. Individuals that would become gamergates increased their dueling activity and reached the peak during the day 6-10 period, whereas the dueling activity of workers decreased (Fig. ...

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... (Fig. 1B). During days 1-5 of the dueling tournament, members of both castes joined the dueling, but the gamergates showed higher dueling activity relative to workers. Individuals that would become gamergates increased their dueling activity and reached the peak during the day 6-10 period, whereas the dueling activity of workers decreased (Fig. 1D). Later on, gamergates showed intensive dueling activity that persisted for at least another 45 d, whereas workers rapidly diminished their dueling activity after 5 d and subsequently rarely joined the battle (Fig. 1D). After 10 d, gamergates often laid eggs, but it was a rare event in workers (Fig. 1E). In summary, our behavioral ...

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... their dueling activity and reached the peak during the day 6-10 period, whereas the dueling activity of workers decreased (Fig. 1D). Later on, gamergates showed intensive dueling activity that persisted for at least another 45 d, whereas workers rapidly diminished their dueling activity after 5 d and subsequently rarely joined the battle (Fig. 1D). After 10 d, gamergates often laid eggs, but it was a rare event in workers (Fig. 1E). In summary, our behavioral observations confirmed that initial antennal dueling is a robust behavioral marker of the gamergate fate and that the tournament peaks as early as 5-10 d after its ...

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... the dueling activity of workers decreased (Fig. 1D). Later on, gamergates showed intensive dueling activity that persisted for at least another 45 d, whereas workers rapidly diminished their dueling activity after 5 d and subsequently rarely joined the battle (Fig. 1D). After 10 d, gamergates often laid eggs, but it was a rare event in workers (Fig. 1E). In summary, our behavioral observations confirmed that initial antennal dueling is a robust behavioral marker of the gamergate fate and that the tournament peaks as early as 5-10 d after its ...

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... several studies have shown an overall correlation between dueling behavior and gamergate development ( Peeters et al. 2000;Sasaki et al. 2016), the temporal dynamics of the dueling battle had remained unexplored. According to our behavioral observations, the peak of the dueling tournament arises at its very early stages (Fig. 1B,D). We therefore examined the early dueling dynamics of the worker-to-gamergate transition. First, we performed clustering analysis using the dueling data on days 1-7 in each colony with two main clusters of high-and low-dueling individuals ( Fig. 2A; Supplemental Fig. S1B). Because of the dynamics and the colony instability in the early ...

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... therefore examined the early dueling dynamics of the worker-to-gamergate transition. First, we performed clustering analysis using the dueling data on days 1-7 in each colony with two main clusters of high-and low-dueling individuals ( Fig. 2A; Supplemental Fig. S1B). Because of the dynamics and the colony instability in the early stages of colony re-establishment, some true workers (as defined after day 50) initially displayed high dueling activity. ...

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... 7 d postinitiation of the tournament, while the remaining 18 were categorized as low duelers. Six out of the seven high duelers subsequently became true gamergates, while all of the low duelers remained workers (Fig. 2A). The results in the two other colonies showed similar early divergence between high-and low-dueling patterns (Supplemental Fig. S1B). We also checked cumulative early dueling data over the first 7 d and performed statistical analysis to determine the earliest time we could identify significant differences between the dueling activities of workers and gamergates: Gamergates showed a significant increase in cumulative dueling activity within the first 5 d and ...

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... predicted gamergates; and specificity, the ratio of true to predicted workers ( Fig. 2C; Supplemental Table S1; Supplemental Fig. S1C). We found that based on the first week of dueling data, the unsupervised clustering identified future gamergates with 86% accuracy on day 3 postinitiation of the dueling tournament (Fig. 2C). The predictability of gamergates (sensitivity) and workers (specificity) increased every day and reached its peak on day 5 (Fig. 2C). Thus, ...

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... laying is first observed at day 10 (Fig. 1E). In accordance, we identified thousands of ovarian DEGs at day 10 but none at day 3 when the ovary was still inactive (Fig. 3A ′′ ). Among the 3740 ovarian DEGs at day 10, approximately half (1897 DEGs) were down-regulated, and the rest (1843 DEGs) were up-regulated in the activated ovaries (Fig. 3A ′′ , ...

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... within the first few days of the dueling tournament. Future gamergates show significantly increased dueling activity compared with the workers, allowing us to predict the gamergate fate with 86% accuracy at 3 d postinitiation of dueling (Fig. 2C). Activation of the ovary of the future gamergates is rapid, with egg laying being apparent at day 10 (Fig. 1E). The ovary consists of eight ovarioles in total. At day 3 postdueling initiation, the dueler's ovary is partially activated and occasionally develops a yolky oocyte in one of the ovarioles. By day 10, prospective gamergates have developed yolky oocytes in approximately three of the eight ovaries (Supplemental Fig. S2B). Dueling ...

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... of eight ovarioles in total. At day 3 postdueling initiation, the dueler's ovary is partially activated and occasionally develops a yolky oocyte in one of the ovarioles. By day 10, prospective gamergates have developed yolky oocytes in approximately three of the eight ovaries (Supplemental Fig. S2B). Dueling activity peaks during days 6-10 ( Fig. 1D), followed by a decrease that corresponds to an increase in egg-laying events after day 10 ( Fig. 1E), implying that the cost of social dominance is swiftly reinvested in reproduction after the re-establishment of social structure. Nevertheless, dueling has not yet ended, and the prospective gamergates constantly duel to show and ...

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... and occasionally develops a yolky oocyte in one of the ovarioles. By day 10, prospective gamergates have developed yolky oocytes in approximately three of the eight ovaries (Supplemental Fig. S2B). Dueling activity peaks during days 6-10 ( Fig. 1D), followed by a decrease that corresponds to an increase in egg-laying events after day 10 ( Fig. 1E), implying that the cost of social dominance is swiftly reinvested in reproduction after the re-establishment of social structure. Nevertheless, dueling has not yet ended, and the prospective gamergates constantly duel to show and maintain their social dominance, suggesting a well-balanced energy investment between reproduction and ...

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... and eventually remained as workers, suggesting that the antennal dueling is not always a winner-winner scenario, at least during the unstable social structure in the early transition. Once duelers (immature gamergates) reach a mature stage and fully develop their production of queenlike pheromones, dueling activity considerably decreases (Fig. 1D). The other workers (subordinates) in the colony recognize the mature gamergates as the reproductives and do not challenge them ( Liebig et al. 1999Liebig et al. , 2000, and their reproductive capacity is repressed by the pheromones of the mature gamergates ( Ghaninia et al. 2017). Other social interactions (for example, dominant ...

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... of each individual based on its dueling activity and egg laying. Any high-dueling individual that laid eggs at least twice was classified as a gamergate. Low-dueling ants with less than two egg-laying events were considered as workers. Other cast-specific behaviors such as cricket hunting, trash collection, and dominant behavior (Supplemental Fig. S1A) were also observed and considered for further validation by gene expression. The egg-laying events were scored together with the dueling behavior and only observed within a specific time window during the daytime in each video. Not all the events were ...