Pheromone trail following is not modulated by previous visit to food location, distance travelled, or travel direction in the ant Lasius niger

Pheromone trail following is not modulated by previous visit to food location, distance travelled, or travel direction in the ant Lasius niger | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Pheromone trail following is not modulated by previous visit to food location, distance travelled, or travel direction in the ant Lasius niger Laure-Anne Poissonnier, Delia Winter, Federico Federico-Javier Olivera-Rodriguez, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7630446/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Ant foraging and recruitment often relies heavily on pheromone trails, and ants modulate pheromone trail deposition strategically. Ants are also known to modulate trail following depending on their own private information, such as the known location of the nest and their knowledge of food sources in the environment. Here we ask how a series of important context variables; distance from the nest, direction of travel, and prior food discovery—affects the fidelity of pheromone trail following in the black garden ant Lasius niger . Using both an open arena assay and a binary Y-maze design, we evaluated whether ants adjust their trail-following behaviour. Ants exhibited robust and consistent trail-following behaviour across all conditions. Surprisingly, we found no significant modulation by distance, travel direction, or recent experience. However, we observed that path length before finding a pheromone trail significantly influenced walking patterns such as meander, speed, and total distance travelled. These findings suggest that while L. niger trail-following behaviour is remarkably stable across contexts, locomotory traits remain plastic and context-sensitive. The decoupling of pheromone response from internal or external conditions highlights the need for further investigation into the mechanisms regulating individual foraging decisions in social insects. Ants Pheromone Decision-making Information use Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Pheromones are used ubiquitously by insects, for a variety of purpose, such as attracting mates or signalling a danger. Social insects stand out in the complexity of their pheromonal communication, where they are deployed for a wide range of purposes. Among those, perhaps the most studied are the pheromone trails of ants, which serves as both an attractant, inducing workers to start foraging, and as an orientator, giving the direction of the food source (Czaczkes et al., 2015 ). In some ant species, pheromone trails can lead to the mobilisation of thousands of workers in a couple of minutes, in a self-organised process called mass recruitment (Wilson, 1962a ). While this system is highly efficient at recruiting a high number of individuals to a food source, it might require modulation to adapt to changes in the environment (such as the discovery of a higher quality food source). The flexibility (or lack thereof) of the trail pheromone system has been relatively well studied (reviewed in Czaczkes et al., 2015 ). At the collective level, many ant species have been shown to recruit more strongly to higher quality food sources (Frizzi et al., 2018 ; Hangartner, 1970 ; Jackson & Chaline, 2007 ; Jaffe & Howse, 1979 ; Verhaeghe, 1982 ; Wilson, 1962a ). Ants also recruit more strongly to larger food sources which they fail to move alone (Czaczkes & Ratnieks, 2012 ; Robson & Traniello, 1998 ; Schatz et al., 1997 ). At the individual level, pheromone deposition increases with starvation and with food quality (Hangartner, 1969 ). Foragers have been shown to increase the number of pheromone depositions per distance (Beckers et al., 1992 ), and they might also be able to modify the quantity per deposition (Hangartner, 1970 ). Since these pioneer experiments, evidence has accumulated to show that the modulation of pheromone trail is driven by a very broad range of factors, related to the state and experience of the individual, the state of the colony, and the environment and state of the recruitment trail. In the ant Lasius niger , foragers must ingest a critical minimum volume to start the recruitment process (Mailleux et al., 2000 ), and this critical volume is larger in starved colonies (Mailleux et al., 2006 ). Previous experience also modulates pheromone laying: ants expecting high-quality food reduce recruitment to a moderate-quality food source relative to ants which expected a moderate-quality food source (Wendt et al., 2019 ). The objective food quality being held constant, pheromone deposition can also be modulated according to its perceived (subjective) value, such as the food source requiring more effort to reach (Czaczkes et al., 2018 ), being an expected flavour (Oberhauser & Czaczkes, 2018 ), or being presented all together, as opposed to in multiple smaller resource units (De Agrò et al., 2022 ). However, other findings in the same species show that recruiters lay trail marks of equal intensity, whatever the number of food sources visited (Mailleux et al., 2003 ). Ants can deploy pheromone deposition strategically, in order to improve navigation on hard-to-learn paths (Czaczkes et al., 2013 ) or when it is dark (Jones et al., 2019 ), or when especially close to a food source (Czaczkes et al., 2024 ; Devigne & Detrain, 2006 ). In addition to modulation of the trail deposition, foragers also have the possibility to vary their propensity to follow the pheromone signals. Outside of trail pheromones, the response to alarm pheromone is stronger in older individuals in four ant species(Hart et al., 2024 ; Norman et al., 2017 ; Pokorny et al., 2020 ) and the honeybee (Robinson, 1987 ). A recent study in the clonal raider ant revealed that this is due to some areas of the antennal lobe becoming more sensitized (Hart et al., 2024 ). Older Myrmica rubra workers follow pheromone trails more accurately (Cammaerts-Tricot & Verhaeghe, 1974 ). Pheromone following can be flexible and context dependant: ants can ignore pheromone trails in favour of following route memories, depending on the relative strength of the trail and the memory (Almeida et al., 2018 ; Fourcassie & Beugnon, 1988 ; Grüter et al., 2011 ; Harrison et al., 1989 ). However, ants primed to expect the pheromone to lead to a better food source than the one they have been previously exploiting follow pheromone trails over route memories (Czaczkes et al., 2019 ). Ants can even learn to ignore pheromone when it consistently leads to unrewarded locations, although they cannot learn to avoid pheromone trails (Wenig et al., 2021 ). This could be explained by the fact that ignoring a cue is more challenging than associating it positively or negatively with a stimulus. Surprisingly, internal states have often not been found to modulate trail following. Whether an ant is a naïve scout, has been recruited, or has previously found food, did not influence their probability of following a trail pheromone on a T-maze (Czaczkes et al., 2017 ). Some species, such as Monomorium pharaonis , seem to have specialised ‘pathfinders’ which can find faint trails, follow them, and reactivate them (Jackson et al., 2006 a). However, by contrast no evidence could be found for specialised trail ignorers in the ant Lasius niger (Koch & Czaczkes, 2021 ). Ants thus appear to have the ability to modulate trail following, but do not do so in all circumstances in which they might be expected to. To get a deeper understanding of this question, examining pheromone following behaviour in a more complex setting than classical experiments may be helpful, allowing the scoring of more subtle behaviour than a simple binary response of the decision to follow or not the pheromone. In this study, we thus investigated the effect of context on the decision of an ant to follow a pheromone trail, but we also investigated how close the ants stayed to the trail in an open arena, and examined the ants’ path characteristics. We explored the effects of distance travelled prior to encountering a trail in an open arena. We also measured the effects of travel direction and prior food discovery on trail-following in a more traditional Y-maze design. We hypothesize that ants that are further away from their nests will pay a higher cost for becoming lost, and thus should attend to pheromone trails more. Similarly, we hypothesised that naïve ants heading away from the nest should follow pheromone trails with a higher fidelity than experienced ants which have already located a food source and are returning to it. Methods Ant collection and care Lasius niger workers were collected on campus at the University of Regensburg. They were housed in plastic boxes with plaster of Paris nests and floor, and fed 0.5M sugar water solution ad libitum , and chopped cockroaches three times a week. Each colony was kept for at least a week in lab before being tested, in order to give them time to acclimatize. Colonies were queenless and comprised of ca. 500–1500 workers. The ants were starved 3 or 4 days prior to the experiments to ensure motivation to forage and explore. Data were collected between the 27th March and 5th of April 2024 for experiment 1, and between the 30th May and 12th of June 2023 for experiment 2. Pheromone solution Artificial pheromone was created by dissecting and macerating the trail pheromone glands (hindgut) of 4 individuals in 1 mL of Dichloromethane (DCM). 6µl of such a solution over 10 cm results in a strong but ecologically sensible pheromone trail (Thienenet al., 2014 ). Experiment 1 – Does distance from the nest on an outward journey impacts pheromone following and exploration behaviour in an open arena? The objective of the first experiment was to evaluate whether ants are more inclined to follow a pheromone trail encountered when they have already travelled far from their nests, compared to ants that have travelled only a short distance from the nest. In addition, we measured how path length and pheromone presence affected the exploration of the open arena. - Experimental procedure : A single forager was allowed to walk onto a piece of paper and placed on either a short path − 20 cm long (and 1cm wide) straight runway, or a 100cm long runway. At the end of the runway, a toothpick allowed them to climb vertically via a hole onto the centre of a paper-covered A4 platform (21x30cm). A solvent (DCM) or pheromone trail (ca 36 µl over 30cm) was present, crossing the platform lengthwise and parallel to the path the ant came from (see Fig. 1 ). DCM and pheromone treatments were alternated between each ant. Ants’ movement was video recorded until it reached the boarder of the arena. Ants were discarded if they fell off the path (34/143 ants), crawled under the arena or took more than 20 minutes to reach the arena (1/143 ants). The setup was cleaned with ethanol and a fresh sheet of paper placed between each ant. Trials were video recorded from above. N = 26 ants per condition. - Analysis : Ants were tracked with AnimalTA (Chiara et al., 2023 ). For each video, we manually recorded the coordinates of the 4 corners of the A4 arena, which gave us a scale factor to calculate the area 1cm on each side of the middle of the paper. All analyses were carried out in R (4.4.1)(R Foundation for Statistical Computing, 2024 ) via Rstudio (2024.12.0). We used linear mixed models to explore the effects of path length and pheromone trail presence on 5 variables of ant movement, using the lmerTest package (Kuznetsova A, 2017 ), with colony ID added as a random effect. The variables were as followed: the proportion of time spent in the central zone 1cm on each side of the pheromone (or solvent) trail (see Fig. 1 ), the average speed, the total distance travelled, the meander of the path, and the time to reach on of the arena’s edges. Meander was calculated by animalTA as “the average of the change in direction (turning angle) divided by the distance travelled for each frame (25 per second), including only data where the ant was moving”. Model fit checks were performed using the DHARMa package (Hartig, 2024 ). Except for speed, all data was to be log transformed to improve model fit. Experiment 2 – Does direction of travel and of food location impact pheromone following in a binary decision? The aim of the second experiment was to ask whether ants are more inclined to follow pheromone trails when they are heading outwards in search of food, compared to when they are returning to their colonies. We additionally compared pheromone following of ants that have recently found a food source (a drop of 0.8M sucrose solution) and were either returning satiated to the nest, or going outwards again in order to relocate the food source. - Experimental procedure : The experimental procedure begins with the lowering of a bridge leading to the Y-maze. The ants experienced one of 4 conditions, all of which involve a Y-maze with a pheromone trail (6µl solution over 10cm) on one arm, and 6µl DCM on the other: (A) Outwards naïve: The ant walked outwards from the colony and was presented with a Y-maze. (B) Outwards experienced: The ant walked outwards from the colony via a straight runway and found food (0.8M sucrose) at the end of it. The ant was marked with paint while feeding and allowed to return to its colony. Once the ant had shared food with its nestmates, it was allowed to return to the setup, and was presented with a Y-maze. (C) Return experienced: The ant walked outwards from the colony via a straight bridge and found food. It was marked with paint while feeding and allowed to return to share food in the colony. The ant returned to the food source and while eating the straight runway was replaced by a Y-maze, and the ant allowed to return nestwards via the Y-maze. (D) Return naïve: The ant walked outwards from the colony via a straight runway and did not find any source of food at the end. While on a platform at the end of the runway, the runway is replaced with a Y-maze, and the ant allowed to return nestwards via the Y-maze. Each protocol was carried out with the pheromone placed alternatively on the left or right arm of the Y-maze. Ants that did not reach the Y-maze, the food source, or the end of the straight runway in less than ten minutes were discarded. All overlays were removed, and the Y-maze, both straight runways, and the bridge were cleaned with 70% ethanol between each visit. - Analysis : To test the effect of travel direction and experience on pheromone following we run a linear model and attempted to predict whether the ant would follow a pheromone trail (1) or not (0) by the interaction of travel direction (outwards or nestwards) and experience (naïve or experienced), with the addition of the pheromone-marked side as another predictive variable, and colony ID as a random effect, varying by intercept and slope. A binomial error family was used with the package glmmTMB. N = 99 or 100 per condition. Results The entire datasets for each experiment can be found as supplements and on OSF (link to experiments dataset). The entire analysis code and output can be found as supplement S2 and on OSF (link to code). Experiment 1 – Does distance from the nest on an outward journey impacts pheromone following and exploration behaviour in an open arena? Before exploring the effects of pheromone on movement data, we will assess whether the pheromone solution induced trail following in the ants. Indeed, the pheromone solution induced the ants to spend more time in the central zone 1 cm on each side of the pheromone trail the middle of the arena (Estimate = 0.18, p-value < 0.01, see also Fig. 4 ). There was no effect of path length (Estimate = 0.01462, p-value = 0.76) or interaction of path length and pheromone (Estimate = 0.01962, p-value = 0.77) on the time spent in this zone: ants who had travelled further away from their nests were not more likely to follow a trail they encountered on their path than ants which encountered a pheromone trail closer to their nests. Walking speed was marginally impacted by path length (Estimate = -2.93, p-value = 0.096), with ants coming from the short path (mean speed 23,47mm/sec) tending to be slower than ants coming from the long one (mean speed 26mm/sec). Surprisingly, the presence of pheromone did not increase walking speed. There was no effect of the interaction between path length and pheromone presence. Meander was also affected by path length (Estimate = 0.6768, p-value = 0.036), pheromone (Estimate = 0.75, p-value = 0.02), and of the interaction between the two (Estimate = -1.0828, p-value = 0.018): When no pheromone was present, ants coming from the long path walked in a more linear trajectory, but the reverse was found when pheromone was present (Fig. 4 ). Path length affected time to reach the boarder of the arena (Estimate = 0.62902, p-value < 0.01), ants coming from the short path took twice as long to exit the platform, likely due to their less linear trajectories and higher meander values noted previously. Pheromone did not have an effect, however the interaction was almost significant (Estimate = -2.926, p-value = 0.096), with the time to reach one of the arena’s edges not being different anymore when pheromone was present, as ants were following the trail to the edge of the paper. Distance travelled was significantly impacted by path length (Estimate = 0.52, p-value < 0.01): When no pheromone was present, ants coming from the short path travelled almost twice as much as the ones coming from the long path before reaching an edge of the arena (on average 501mm vs 266mm, Fig. 2 ). Interestingly, this difference was reduced by the presence of the pheromone trail: the interaction between path length and pheromone was significant (Estimate = 0.34627, p-value = 0.04). As an additional exploration of the data, we plotted the coordinates at which the ants exited the arena (Fig. 3 ). When pheromone trail was present, more ants exited the arena where the trail reached the edge of the paper. It seems that ants coming from the long path exited less from the right edge. Experiment 2 – Do ants follow pheromone trails more accurately when returning to the nest? There was no effect of travel direction, experience, or the interaction of travel direction and experience, on pheromone following accuracy (all Χ 2 0.54, see Fig. 5 ). The side of the Y maze on which pheromone was placed did not affect pheromone following (Χ2 = 2.19, P = 0.14). Overall, 87.4% of ants followed the pheromone trail (348 / 398). Discussion The trail following fidelity of Lasius niger appears to be quite resistant to task state, travel direction, and distance from the nest. We expected more pheromone following further from the nest, as the further from the nest, the more errors can accumulate and the more costly it is to get lost. We also expected more pheromone following going outwards than inwards for naïve ants because returning ants have information (where is nest) and outgoing ants do not know where food will be found. However, we found that pheromone following in a Y-maze did not differ between outward and return trips to the nest, nor was it affected by whether food had been encountered on the previous trip. Similarly, Czaczkes et al. ( 2017 ) reported that L. niger foragers in a T-maze showed no change in pheromone-following behaviour regardless of whether they were naïve, recruited, or experienced. Several factors may explain this absence of effect. One possibility is that the ants had no strong incentive to favour one branch over the other, since they had not experienced the alternative branch as unrewarding. Another explanation could be that traversing the few centimetres of maze imposed little energetic cost, reducing the need to discriminate between branches. Perhaps had there been some conflict (pheromone pointing further away from the nest-food axis, for example), or a more complex path, we would have seen differences between treatments, as outgoing experienced foragers rely on memory more than outgoing naïve ants. In addition, there is some evidence that ants can monitor their own uncertainty (Czaczkes & Heinze, 2015 ; Merkle & Wehner, 2010 ) and the uncertainty in their path integration system (Merkle & Wehner, 2010 ). Since the ants were restricted to a linear runway, they may not have accumulated sufficient noise in their path integration system to cause a detectable change in behaviour, and there may not be a strong cost of not following the pheromone. In order to complement the Y-maze experiments, in this study we also looked at trail following in an open arena, which could allow us to detect more subtle differences in trail following that might not be apparent in the Y-maze. In the open arena, we found that distance travelled before encountering the pheromone trail did not affect the following accuracy of the trail. In an older study in Solenopsis saevissima , deviation of trail following did not vary with distance along a 10 cm long trail in (Wilson, 1962b ), pointing to similarities with L. niger . However, we did find effects of the distance travelled on the walking behaviour of the foragers, with ants having walked a longer way having a more linear trajectory. Oddly, this effect was reversed when pheromone was present, but we wonder if this is a real effect or simply due to chance. Could the lack of effect of distance travelled be due to ants not being able to perceive the difference between 20 and 100cm? It appears more likely that pheromone following responds to different rules than pheromone deposition. Indeed, in the same species and with the same path length (20 vs 100cm), we found that foragers deposited more pheromone to food sources that were more distant from their nest (Czaczkes et al., 2024 ), a finding which replicated previous similar findings using a different setup (Devigne & Detrain, 2006 ). Ants are therefore able to detect differences in the range of distance we used. Why would pheromone information provision rules differ from pheromone following rules, when both have the same goal? Perhaps the costs of not depositing pheromone and not following pheromone are under different constraints. For instance, if the ant decides not to lay pheromone, the direct costs at the individual level might be minimal, as the ant can still remember the way to the food. However, the costs could be high at the collective level, as other ants will not be directed to the food source, which could be quickly monopolised by another colony instead. On the other hand, the decision not to follow the pheromone could affect the individual level more directly, through the costs of exploring unknown territory or getting lost for example, leading to less flexibility in pheromone following. This study demonstrates a remarkable robustness in trail pheromone following decisions (c. 85%) in an ant, which is not due to ceiling effects, and unlikely to be due to limitations on detection. This replicability in pheromone following proportion is echoed in previous studies too. Taken together, it is clear that pheromone following probability is tightly controlled, and not set at 100%. The question which remains is: why is this, and do these results translate to other contexts and uses of pheromone? Would ants which are recruiting to a nest site after their nest has been destroyed follow pheromone trails with greater fidelity? Or perhaps would starving colonies follow pheromone trails with more fidelity, given that finding other food sources seems unlikely? A system with a stable proportion of pheromone followers and some variation in the pheromone laying might allow the colony to have a balance between flexibility and quick efficient recruitment to a food source, while relying on simple rules at the individual level. The flexibility of pheromone following in a variety of contexts outside foraging for sucrose awaits further exploration. Declarations Declarations We declare no competing interests Acknowledgements: ໿L-A. P. was supported by an Alexander von Humboldt Postdoctoral Fellowship (1223114) at the beginning of the project, then by a Postdoctoral Equal Opportunities Fellowship from the University of Regensburg. T.J.C was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Project No. 462101190. References Almeida NG, De, Camargo RDS, Forti LC, Lopes JFS (2018) Hierarchical establishment of information sources during foraging decision-making process involving Acromyrmex subterraneus. Biology Ecol Divers 62(1):36–39. https://doi.org/10.1016/J.RBE.2017.11.006 Beckers R, Deneubourg JL, Goss S (1992) Trail laying behaviour during food recruitment in the ant Lasius niger (L). Insectes Sociaux 39(1):59–72. https://doi.org/10.1007/BF01240531 Cammaerts-Tricot MC, Verhaeghe JC (1974) Ontogenesis of trail pheromone production and trail following behaviour in the workers of Myrmica rubra L. (Formicidae). Insectes Sociaux 21(3):275–282. https://doi.org/10.1007/BF02226918 Chiara V, Kim S-Y, Animal GE, Cacti T (2023) AnimalTA: A highly flexible and easy-to-use program for tracking and analysing animal movement in different environments. Methods Ecol Evol 14(7):1699–1707. https://doi.org/10.1111/2041-210X.14115 Czaczkes TJ, Beckwith JJ, Horsch A-L, Hartig F (2019) The multi-dimensional nature of information drives prioritization of private over social information in ants. Proceedings of the Royal Society B: Biological Sciences , 286 (1909), 20191136. https://doi.org/10.1098/rspb.2019.1136 Czaczkes TJ, Brandstetter B, di Stefano I, Heinze J (2018) Greater effort increases perceived value in an invertebrate (Lasius niger). J Comp Psychol 132(2):200–209. https://doi.org/10.1037/COM0000109 Czaczkes TJ, Castorena M, Schürch R, Heinze J (2017) Pheromone trail following in the ant Lasius niger: high accuracy and variability but no effect of task state. Physiol Entomol 42(1):91–97. https://doi.org/10.1111/PHEN.12174 Czaczkes TJ, Grüter C, Ellis L, Wood E, Ratnieks FLW (2013) Ant foraging on complex trails: Route learning and the role of trail pheromones in Lasius niger. J Exp Biol 216(2):188–197. https://doi.org/10.1242/JEB.076570/257938 . /AM/ANT-FORAGING-ON-COMPLEX-TRAILS-ROUTE-LEARNING-AND Czaczkes TJ, Grüter C, Ratnieks FLW (2015) Trail Pheromones: An Integrative View of Their Role in Social Insect Colony Organization. Ann Rev Entomol 60(1):581–599. https://doi.org/10.1146/annurev-ento-010814-020627 Czaczkes TJ, Heinze J (2015) Ants adjust their pheromone deposition to a changing environment and their probability of making errors. Proceedings of the Royal Society B: Biological Sciences , 282 (1810). https://doi.org/10.1098/RSPB.2015.0679 Czaczkes TJ, Olivera-Rodriguez F-J, Poissonnier L-A (2024) Ants (Lasius niger) deposit more pheromone close to food sources and further from the nest but do not attempt to update erroneous pheromone trails. Insectes Sociaux 71:367–376. https://doi.org/10.1007/S00040-024-00995-Y Czaczkes TJ, Ratnieks FLW (2012) Pheromone trails in the Brazilian ant Pheidole oxyops: Extreme properties and dual recruitment action. Behav Ecol Sociobiol 66(8):1149–1156. https://doi.org/10.1007/S00265-012-1367-7/FIGURES/5 De Agrò M, Matschunas C, Czaczkes TJ (2022) Bundling and segregation affect pheromone deposition, but not choice, in an ant. ELife , 11 . https://doi.org/10.7554/ELIFE.79314 Devigne C, Detrain C (2006) How does food distance influence foraging in the ant Lasius niger: The importance of home-range marking. Insectes Sociaux 53(1):46–55. https://doi.org/10.1007/S00040-005-0834-9/METRICS Fourcassie V, Beugnon G (1988) How do red wood ants orient when foraging in a three dimensional system? Insectes Sociaux 35(1):92–105 Frizzi F, Talone F, Santini G (2018) Modulation of trail laying in the ant Lasius neglectus (Hymenoptera: Formicidae) and its role in the collective selection of a food source. Ethology 124(12):870–880. https://doi.org/10.1111/ETH.12821 Grüter C, Czaczkes TJ, Ratnieks FLW (2011) Decision making in ant foragers (Lasius niger) facing conflicting private and social information. Behav Ecol Sociobiol 65(2):141–148. https://doi.org/10.1007/s00265-010-1020-2 Hangartner W (1969) Structure and variability of the individual odor trail in Solenopsis geminata Fabr. (Hymenoptera, Formicidae). Z Für Vergleichende Physiologie 62(1):111–120. https://doi.org/10.1007/BF00298046/METRICS Hangartner W (1970) Control of pheromone quantity in odor trails of the ant Acanthomyops interjectus MAYR. Experientia 26:664–665. https://doi.org/https://doi.org/10.1007/BF01898753 Harrison JF, Fewell JH, Stiller TM, Breed MD (1989) Effects of experience on use of orientation cues in the giant tropical ant. Animal Behaviour , 37 (PART 5), 869–871. https://doi.org/10.1016/0003-3472(89)90076-6 Hart T, Lopes LE, Frank DD, Kronauer DJC (2024) Pheromone representation in the ant antennal lobe changes with age. Curr Biol 34(14):3233–3240e4. https://doi.org/10.1016/j.cub.2024.05.031 Hartig F (2024) Residual Diagnostics for Hierarchical (Multi-Level / Mixed) Regression Models [R package DHARMa version 0.4.7]. In CRAN: Contributed Packages . Comprehensive R Archive Network (CRAN). https://doi.org/10.32614/CRAN.PACKAGE.DHARMA Jackson D, Chaline N (2007) Modulation of pheromone trail strength with food quality in Pharaoh’s ant, Monomorium pharaonis. Anim Behav 74(3):463–470. https://doi.org/10.1016/J.ANBEHAV.2006.11.027 Jackson DE, Martin SJ, Holcombe M, Ratnieks FLW (2006) Longevity and detection of persistent foraging trails in Pharaoh’s ants, Monomorium pharaonis (L). Anim Behav 71:351–359. https://doi.org/10.1016/j.anbehav.2005.04.018 Jaffe K, Howse PE (1979) The mass recruitment system of the leaf cutting ant, Atta cephalotes (L.). Animal Behaviour , 27 (PART 3), 930–939. https://doi.org/10.1016/0003-3472(79)90031-9 Jones S, Czaczkes TJ, Gallager AJ, Oberhauser FB, Gourlay E, Bacon JP (2019) Copy when uncertain: lower light levels increase trail pheromone depositing and reliance on pheromone trails in ants. Anim Behav 156:87–95. https://doi.org/10.1016/j.anbehav.2019.08.007 Koch A, Czaczkes TJ (2021) No specialist pheromone-ignoring ants in Lasius niger. Ecol Entomol 46(3):677–680. https://doi.org/10.1111/EEN.12995 Kuznetsova A, B. P. C. R (2017) lmerTest Package: Tests in Linear Mixed Effects Models. J Stat Softw 82(13):1–26 Mailleux AC, Deneubourg JL, Detrain C (2003) Regulation of ants’ foraging to resource productivity. Proceedings of the Royal Society of London. Series B: Biological Sciences , 270 (1524), 1609–1616. https://doi.org/10.1098/RSPB.2003.2398 Mailleux A-C, Deneubourg J-L, Detrain C (2000) How do ants assess food volume? Anim Behav 59(5):1061–1069. https://doi.org/10.1006/anbe.2000.1396 Mailleux A-C, Detrain C, Deneubourg J-L (2006) Starvation drives a threshold triggering communication. J Exp Biol 209:4224–4229. https://doi.org/10.1242/jeb.02461 Merkle T, Wehner R (2010) Desert ants use foraging distance to adapt the nest search to the uncertainty of the path integrator. Behav Ecol 21(2):349–355. https://doi.org/10.1093/BEHECO/ARP197 Norman VC, Butterfield T, Drijfhout F, Tasman K, Hughes WOH (2017) Alarm Pheromone Composition and Behavioral Activity in Fungus-Growing Ants. J Chem Ecol 43(3):225–235. https://doi.org/10.1007/S10886-017-0821-4/FIGURES/4 Oberhauser FB, Czaczkes TJ (2018) Tasting the unexpected: disconfirmation of expectations leads to lower perceived food value in an invertebrate. Biol Lett 14(9). https://doi.org/10.1098/RSBL.2018.0440 Pokorny T, Sieber LM, Hofferberth JE, Bernadou A, Ruther J (2020) Age-dependent release of and response to alarm pheromone in a ponerine ant. J Exp Biol 223(6). https://doi.org/10.1242/JEB.218040/VIDEO-1 R Foundation for Statistical Computing (2024) R Core Team. _R: A Language and Environment for Statistical Computing (4.4.1) Robinson GE (1987) Modulation of alarm pheromone perception in the honey bee: evidence for division of labor based on hormonall regulated response thresholds. J Comp Physiol A 160(5):613–619. https://doi.org/10.1007/BF00611934/METRICS Robson SK, Traniello JF (1998) Resource assessment, recruitment behavior, and organization of cooperative prey retrieval in the ant Formica schaufussi (Hymenoptera: Formicidae). J Insect Behav 11(1):1–22. http://link.springer.com/article/ 10.1023/A:1020859531179 Schatz B, Lachaud JP, Beugnon G (1997) Graded recruitment and hunting strategies linked to prey weight and size in the ponerine ant Ectatomma ruidum. Behav Ecol Sociobiol 40(6):337–349. https://doi.org/10.1007/S002650050350/METRICS Thienen W, Von, Metzler D, Choe DH, Witte V (2014) Pheromone communication in ants: A detailed analysis of concentration-dependentdecisions in three species. Behav Ecol Sociobiol 68(10):1611–1627. https://doi.org/10.1007/S00265-014-1770-3/TABLES/2 Verhaeghe JC (1982) Food recruitment in Tetramorium impurum (Hymenoptera: Formicidae). Insectes Sociaux 29(1):67–85. https://doi.org/10.1007/BF02224528/METRICS Wendt S, Strunk KS, Heinze R, Roider J, A., Czaczkes TJ (2019) Positive and negative incentive contrasts lead to relative value perception in ants . https://doi.org/10.7554/eLife.45450.001 Wenig K, Bach R, Czaczkes TJ (2021) Hard limits to cognitive flexibility: Ants can learn to ignore but not avoid pheromone trails. J Exp Biol 224(11). https://doi.org/10.1242/JEB.242454/261714 . /AM/HARD-LIMITS-TO-COGNITIVE-FLEXIBILITY-ANTS-CAN Wilson EO (1962a) Chemical communication among workers of the fire ant Solenopsis saevissima (Fr. Smith) 1. The Organization of Mass-Foraging. Anim Behav 10(1–2):134–147. https://doi.org/10.1016/0003-3472(62)90141-0 Wilson EO (1962b) Chemical communication among workers of the fire ant Solenopsis saevissima (Fr. Smith) 2. An information analysis of the odour trail. Anim Behav 10(1–2):148–158. https://doi.org/10.1016/0003-3472(62)90142-2 Supplementary Files Code.rmd DataPheromoneFollowing.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revisions Needed 19 Dec, 2025 Reviewers agreed at journal 09 Nov, 2025 Reviewers invited by journal 30 Oct, 2025 Editor assigned by journal 20 Sep, 2025 First submitted to journal 19 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7630446","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":529774149,"identity":"2c3f7288-c36c-4a20-bd98-644a9be76777","order_by":0,"name":"Laure-Anne Poissonnier","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-2264-6964","institution":"University of Regensburg: Universitat Regensburg","correspondingAuthor":true,"prefix":"","firstName":"Laure-Anne","middleName":"","lastName":"Poissonnier","suffix":""},{"id":529774150,"identity":"151a7f6e-ae80-42c4-846f-3b37cecf2aac","order_by":1,"name":"Delia Winter","email":"","orcid":"","institution":"University of Regensburg: Universitat Regensburg","correspondingAuthor":false,"prefix":"","firstName":"Delia","middleName":"","lastName":"Winter","suffix":""},{"id":529774151,"identity":"bc6b3c3b-cc4c-4c0f-a70d-c15c63f958a7","order_by":2,"name":"Federico Federico-Javier Olivera-Rodriguez","email":"","orcid":"","institution":"University of Regensburg: Universitat Regensburg","correspondingAuthor":false,"prefix":"","firstName":"Federico","middleName":"Federico-Javier","lastName":"Olivera-Rodriguez","suffix":""},{"id":529774152,"identity":"ae761b5d-8a65-4598-a8d4-e5d52cc9681d","order_by":3,"name":"Cosmina Werneke","email":"","orcid":"","institution":"University of Regensburg: Universitat Regensburg","correspondingAuthor":false,"prefix":"","firstName":"Cosmina","middleName":"","lastName":"Werneke","suffix":""},{"id":529774153,"identity":"009c088e-a8fd-4475-a6be-6e06d25f2e8c","order_by":4,"name":"Tomer J. Czaczkes","email":"","orcid":"","institution":"University of Regensburg: Universitat Regensburg","correspondingAuthor":false,"prefix":"","firstName":"Tomer","middleName":"J.","lastName":"Czaczkes","suffix":""}],"badges":[],"createdAt":"2025-09-16 12:30:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7630446/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7630446/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93966443,"identity":"3755b353-cb5c-4ba1-bb60-c740f38bd58e","added_by":"auto","created_at":"2025-10-20 18:58:19","extension":"xml","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9641,"visible":true,"origin":"","legend":"","description":"","filename":"insoINSOD2500138.xml","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/6c30a514483e7f4f205a92df.xml"},{"id":93966448,"identity":"df449931-0e15-4516-a033-3b84fcb67dc8","added_by":"auto","created_at":"2025-10-20 18:58:19","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1041,"visible":true,"origin":"","legend":"","description":"","filename":"INSOD25001383043.go.xml","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/4cd148f16ac611d844019172.xml"},{"id":93966962,"identity":"8a44edd8-fc2b-43f4-acce-fbccd775237c","added_by":"auto","created_at":"2025-10-20 19:14:19","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":905,"visible":true,"origin":"","legend":"","description":"","filename":"INSOD2500138Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/40d4e8e43b5b9bee0dfb6f5f.xml"},{"id":93966632,"identity":"aa175740-6f18-4646-a3b5-a508fbc528cf","added_by":"auto","created_at":"2025-10-20 19:06:19","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100983,"visible":true,"origin":"","legend":"","description":"","filename":"INSOD25001380enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/50844d25efb808bf373d6a14.xml"},{"id":93966627,"identity":"b6c82ee3-7a84-4012-823c-e89dc7cd9f7f","added_by":"auto","created_at":"2025-10-20 19:06:19","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":38133,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/2dd4de2cf0f8b63db62908f5.png"},{"id":93966456,"identity":"32ebe4e9-4d08-46d0-abf0-7ff656d62ae8","added_by":"auto","created_at":"2025-10-20 18:58:19","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115844,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/45e329d5a38c04af68f63e9c.png"},{"id":93966628,"identity":"d17a52cc-0398-40e5-9115-0af8b51c8c09","added_by":"auto","created_at":"2025-10-20 19:06:19","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":43020,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/d2937f236b03e9c31d54767a.png"},{"id":93966453,"identity":"f9aa243e-12da-48ee-be40-edc82f8d33f0","added_by":"auto","created_at":"2025-10-20 18:58:19","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":250062,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/c8bda2b70432842e23c4ce0c.png"},{"id":93966961,"identity":"0553dec2-3b3a-4b09-89ef-66919b2d8421","added_by":"auto","created_at":"2025-10-20 19:14:19","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":41208,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/0af3645512aa275d9bb4665f.png"},{"id":93966458,"identity":"ef0ba113-f632-477c-be51-a8e1dfca650f","added_by":"auto","created_at":"2025-10-20 18:58:19","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100092,"visible":true,"origin":"","legend":"","description":"","filename":"INSOD25001380structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/4e03026cfc7d7fcd0415a843.xml"},{"id":93966460,"identity":"374374f5-5dd4-4958-a223-3e4f68c5c9cb","added_by":"auto","created_at":"2025-10-20 18:58:19","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107640,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/3a3da0a76653bd95570ebbd6.html"},{"id":93966445,"identity":"36d50d4c-6aba-4dd8-80ce-60c7760a896a","added_by":"auto","created_at":"2025-10-20 18:58:19","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":59215,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental setup for experiment 1, investigating the effect of path distance on pheromone following accuracy. Ants are placed at the end of the runway (short 20cm shown, long 100cm not shown), traverse the runway, climb up the central toothpick, and emerge onto the arena, covered with disposable paper and either a pheromone trail (shown) or a solvent control. Area is 297 x 210mm (A4).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/600da391e75a78f034a0603a.jpeg"},{"id":93966633,"identity":"24bdbc7d-72bc-4d6b-8e56-18327bb874d6","added_by":"auto","created_at":"2025-10-20 19:06:19","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":334030,"visible":true,"origin":"","legend":"\u003cp\u003eMean values of 5 movement metrics, +- 95% confidence interval for the mean, for 4 conditions: the ants arrived on the platform from a short path (20cm long), or a long path (60cm long), and either solvent or a pheromone trail was present on the middle of the arena. Units are mm and seconds. N=26 ants per condition.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/9eb6ce32c81d6b334051bdef.jpeg"},{"id":93966446,"identity":"36a37edd-1f8b-4565-99b7-9ef7104222fa","added_by":"auto","created_at":"2025-10-20 18:58:19","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":180572,"visible":true,"origin":"","legend":"\u003cp\u003eExit coordinates of ants per condition, each dot representing an individual ant. The top panels represents where the ants reached one of the A4 arena edges when no pheromone trail was applied. The bottom panels represent the same data for ants that encountered a pheromone trail deposited in a horizontal line crossing the entrance point. The trail was parallel to the path the ants were coming from. The left panels show ants which travelled 20 cm to reach the entrance point (short path), the right panels ants which travelled 60 cm to reach the entrance point.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/0a6173d20458d9eda5021ec6.jpeg"},{"id":93966626,"identity":"66ce743e-a910-456f-b535-c07ef5fb4571","added_by":"auto","created_at":"2025-10-20 19:06:19","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":902205,"visible":true,"origin":"","legend":"\u003cp\u003eAll coordinates of all ants in each condition (long path of 60cm or short path of 20cm, pheromone or control DMC horizontal line drawn in the middle of the A4 paper).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/8115e1184998cb078485bef7.jpeg"},{"id":93966631,"identity":"26a1c6ac-08a2-4b8b-ab24-59b943c525fb","added_by":"auto","created_at":"2025-10-20 19:06:19","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":222149,"visible":true,"origin":"","legend":"\u003cp\u003eProportion of \u003cem\u003eL. niger\u003c/em\u003e foragers that followed pheromone in a Y-maze where one branch presented pheromone, and the other the solvent only. The choices of the ant were recorded in 4 different situations. The Y-maze was presented either on the way out of the nest - Outwards, or on the way back to the nest – Return. In addition, the ants either experienced a food reward - Fed, or did not - naïve condition. The red diamonds represent the mean, and the error bars are 95 bootstrap confidence intervals. N=99 or 100 ants per condition.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/8f7d1c6cad7480ea6adeabf1.jpeg"},{"id":93966963,"identity":"9d12b0ac-fe00-49aa-8abe-b1502c7cac5c","added_by":"auto","created_at":"2025-10-20 19:14:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2159808,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/297d3ae6-daa7-4dfa-8c5b-b67b4019315c.pdf"},{"id":93966625,"identity":"39f44050-dc3a-4075-b4d8-42bda5ed48b1","added_by":"auto","created_at":"2025-10-20 19:06:19","extension":"rmd","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12778,"visible":true,"origin":"","legend":"","description":"","filename":"Code.rmd","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/8867d467218f801ec324f5bc.rmd"},{"id":93966450,"identity":"4061efc2-8763-4708-9dfb-b86e94f0540b","added_by":"auto","created_at":"2025-10-20 18:58:19","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":77500,"visible":true,"origin":"","legend":"","description":"","filename":"DataPheromoneFollowing.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7630446/v1/6ed82dcf144fc4d63cdaf00a.xlsx"}],"financialInterests":"","formattedTitle":"Pheromone trail following is not modulated by previous visit to food location, distance travelled, or travel direction in the ant Lasius niger","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePheromones are used ubiquitously by insects, for a variety of purpose, such as attracting mates or signalling a danger. Social insects stand out in the complexity of their pheromonal communication, where they are deployed for a wide range of purposes. Among those, perhaps the most studied are the pheromone trails of ants, which serves as both an attractant, inducing workers to start foraging, and as an orientator, giving the direction of the food source (Czaczkes et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In some ant species, pheromone trails can lead to the mobilisation of thousands of workers in a couple of minutes, in a self-organised process called mass recruitment (Wilson, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1962a\u003c/span\u003e). While this system is highly efficient at recruiting a high number of individuals to a food source, it might require modulation to adapt to changes in the environment (such as the discovery of a higher quality food source).\u003c/p\u003e\u003cp\u003eThe flexibility (or lack thereof) of the trail pheromone system has been relatively well studied (reviewed in Czaczkes et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). At the collective level, many ant species have been shown to recruit more strongly to higher quality food sources (Frizzi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hangartner, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Jackson \u0026amp; Chaline, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Jaffe \u0026amp; Howse, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Verhaeghe, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Wilson, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1962a\u003c/span\u003e). Ants also recruit more strongly to larger food sources which they fail to move alone (Czaczkes \u0026amp; Ratnieks, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Robson \u0026amp; Traniello, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Schatz et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). At the individual level, pheromone deposition increases with starvation and with food quality (Hangartner, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1969\u003c/span\u003e). Foragers have been shown to increase the number of pheromone depositions per distance (Beckers et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), and they might also be able to modify the quantity per deposition (Hangartner, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). Since these pioneer experiments, evidence has accumulated to show that the modulation of pheromone trail is driven by a very broad range of factors, related to the state and experience of the individual, the state of the colony, and the environment and state of the recruitment trail. In the ant \u003cem\u003eLasius niger\u003c/em\u003e, foragers must ingest a critical minimum volume to start the recruitment process (Mailleux et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), and this critical volume is larger in starved colonies (Mailleux et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Previous experience also modulates pheromone laying: ants expecting high-quality food reduce recruitment to a moderate-quality food source relative to ants which expected a moderate-quality food source (Wendt et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The objective food quality being held constant, pheromone deposition can also be modulated according to its perceived (subjective) value, such as the food source requiring more effort to reach (Czaczkes et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), being an expected flavour (Oberhauser \u0026amp; Czaczkes, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), or being presented all together, as opposed to in multiple smaller resource units (De Agr\u0026ograve; et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, other findings in the same species show that recruiters lay trail marks of equal intensity, whatever the number of food sources visited (Mailleux et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Ants can deploy pheromone deposition strategically, in order to improve navigation on hard-to-learn paths (Czaczkes et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) or when it is dark (Jones et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), or when especially close to a food source (Czaczkes et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Devigne \u0026amp; Detrain, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition to modulation of the trail deposition, foragers also have the possibility to vary their propensity to follow the pheromone signals. Outside of trail pheromones, the response to alarm pheromone is stronger in older individuals in four ant species(Hart et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Norman et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pokorny et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and the honeybee (Robinson, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). A recent study in the clonal raider ant revealed that this is due to some areas of the antennal lobe becoming more sensitized (Hart et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Older \u003cem\u003eMyrmica rubra\u003c/em\u003e workers follow pheromone trails more accurately (Cammaerts-Tricot \u0026amp; Verhaeghe, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1974\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePheromone following can be flexible and context dependant: ants can ignore pheromone trails in favour of following route memories, depending on the relative strength of the trail and the memory (Almeida et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Fourcassie \u0026amp; Beugnon, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Gr\u0026uuml;ter et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Harrison et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). However, ants primed to expect the pheromone to lead to a better food source than the one they have been previously exploiting follow pheromone trails over route memories (Czaczkes et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Ants can even learn to ignore pheromone when it consistently leads to unrewarded locations, although they cannot learn to avoid pheromone trails (Wenig et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This could be explained by the fact that ignoring a cue is more challenging than associating it positively or negatively with a stimulus. Surprisingly, internal states have often not been found to modulate trail following. Whether an ant is a na\u0026iuml;ve scout, has been recruited, or has previously found food, did not influence their probability of following a trail pheromone on a T-maze (Czaczkes et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Some species, such as \u003cem\u003eMonomorium pharaonis\u003c/em\u003e, seem to have specialised \u0026lsquo;pathfinders\u0026rsquo; which can find faint trails, follow them, and reactivate them (Jackson et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003ea). However, by contrast no evidence could be found for specialised trail ignorers in the ant \u003cem\u003eLasius niger\u003c/em\u003e (Koch \u0026amp; Czaczkes, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAnts thus appear to have the ability to modulate trail following, but do not do so in all circumstances in which they might be expected to. To get a deeper understanding of this question, examining pheromone following behaviour in a more complex setting than classical experiments may be helpful, allowing the scoring of more subtle behaviour than a simple binary response of the decision to follow or not the pheromone. In this study, we thus investigated the effect of context on the decision of an ant to follow a pheromone trail, but we also investigated how close the ants stayed to the trail in an open arena, and examined the ants\u0026rsquo; path characteristics. We explored the effects of distance travelled prior to encountering a trail in an open arena. We also measured the effects of travel direction and prior food discovery on trail-following in a more traditional Y-maze design. We hypothesize that ants that are further away from their nests will pay a higher cost for becoming lost, and thus should attend to pheromone trails more. Similarly, we hypothesised that na\u0026iuml;ve ants heading away from the nest should follow pheromone trails with a higher fidelity than experienced ants which have already located a food source and are returning to it.\u003c/p\u003e"},{"header":"Methods","content":"\n\u003ch3\u003eAnt collection and care\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eLasius niger\u003c/em\u003e workers were collected on campus at the University of Regensburg. They were housed in plastic boxes with plaster of Paris nests and floor, and fed 0.5M sugar water solution \u003cem\u003ead libitum\u003c/em\u003e, and chopped cockroaches three times a week. Each colony was kept for at least a week in lab before being tested, in order to give them time to acclimatize. Colonies were queenless and comprised of ca. 500\u0026ndash;1500 workers. The ants were starved 3 or 4 days prior to the experiments to ensure motivation to forage and explore. Data were collected between the 27th March and 5th of April 2024 for experiment 1, and between the 30th May and 12th of June 2023 for experiment 2.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePheromone solution\u003c/h2\u003e\u003cp\u003eArtificial pheromone was created by dissecting and macerating the trail pheromone glands (hindgut) of 4 individuals in 1 mL of Dichloromethane (DCM). 6\u0026micro;l of such a solution over 10 cm results in a strong but ecologically sensible pheromone trail (Thienenet al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eExperiment 1 \u0026ndash; Does distance from the nest on an outward journey impacts pheromone following and exploration behaviour in an open arena?\u003c/span\u003e\u003c/p\u003e\u003cp\u003eThe objective of the first experiment was to evaluate whether ants are more inclined to follow a pheromone trail encountered when they have already travelled far from their nests, compared to ants that have travelled only a short distance from the nest. In addition, we measured how path length and pheromone presence affected the exploration of the open arena.\u003c/p\u003e\u003cp\u003e\u003cem\u003e- Experimental procedure\u003c/em\u003e: A single forager was allowed to walk onto a piece of paper and placed on either a short path \u0026minus;\u0026thinsp;20 cm long (and 1cm wide) straight runway, or a 100cm long runway. At the end of the runway, a toothpick allowed them to climb vertically via a hole onto the centre of a paper-covered A4 platform (21x30cm). A solvent (DCM) or pheromone trail (ca 36 \u0026micro;l over 30cm) was present, crossing the platform lengthwise and parallel to the path the ant came from (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). DCM and pheromone treatments were alternated between each ant. Ants\u0026rsquo; movement was video recorded until it reached the boarder of the arena. Ants were discarded if they fell off the path (34/143 ants), crawled under the arena or took more than 20 minutes to reach the arena (1/143 ants). The setup was cleaned with ethanol and a fresh sheet of paper placed between each ant. Trials were video recorded from above. N\u0026thinsp;=\u0026thinsp;26 ants per condition.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e- Analysis\u003c/em\u003e: Ants were tracked with AnimalTA (Chiara et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For each video, we manually recorded the coordinates of the 4 corners of the A4 arena, which gave us a scale factor to calculate the area 1cm on each side of the middle of the paper. All analyses were carried out in R (4.4.1)(R Foundation for Statistical Computing, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) via Rstudio (2024.12.0). We used linear mixed models to explore the effects of path length and pheromone trail presence on 5 variables of ant movement, using the lmerTest package (Kuznetsova A, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), with colony ID added as a random effect. The variables were as followed: the proportion of time spent in the central zone 1cm on each side of the pheromone (or solvent) trail (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the average speed, the total distance travelled, the meander of the path, and the time to reach on of the arena\u0026rsquo;s edges. Meander was calculated by animalTA as \u0026ldquo;the average of the change in direction (turning angle) divided by the distance travelled for each frame (25 per second), including only data where the ant was moving\u0026rdquo;. Model fit checks were performed using the DHARMa package (Hartig, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Except for speed, all data was to be log transformed to improve model fit.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eExperiment 2 \u0026ndash; Does direction of travel and of food location impact pheromone following in a binary decision?\u003c/span\u003e\u003c/p\u003e\u003cp\u003eThe aim of the second experiment was to ask whether ants are more inclined to follow pheromone trails when they are heading outwards in search of food, compared to when they are returning to their colonies. We additionally compared pheromone following of ants that have recently found a food source (a drop of 0.8M sucrose solution) and were either returning satiated to the nest, or going outwards again in order to relocate the food source.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003e- Experimental procedure\u003c/em\u003e: The experimental procedure begins with the lowering of a bridge leading to the Y-maze. The ants experienced one of 4 conditions, all of which involve a Y-maze with a pheromone trail (6\u0026micro;l solution over 10cm) on one arm, and 6\u0026micro;l DCM on the other:\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e(A) Outwards na\u0026iuml;ve: The ant walked outwards from the colony and was presented with a Y-maze.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e(B) Outwards experienced: The ant walked outwards from the colony via a straight runway and found food (0.8M sucrose) at the end of it. The ant was marked with paint while feeding and allowed to return to its colony. Once the ant had shared food with its nestmates, it was allowed to return to the setup, and was presented with a Y-maze.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e(C) Return experienced: The ant walked outwards from the colony via a straight bridge and found food. It was marked with paint while feeding and allowed to return to share food in the colony. The ant returned to the food source and while eating the straight runway was replaced by a Y-maze, and the ant allowed to return nestwards via the Y-maze.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e(D) Return na\u0026iuml;ve: The ant walked outwards from the colony via a straight runway and did not find any source of food at the end. While on a platform at the end of the runway, the runway is replaced with a Y-maze, and the ant allowed to return nestwards via the Y-maze.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eEach protocol was carried out with the pheromone placed alternatively on the left or right arm of the Y-maze. Ants that did not reach the Y-maze, the food source, or the end of the straight runway in less than ten minutes were discarded. All overlays were removed, and the Y-maze, both straight runways, and the bridge were cleaned with 70% ethanol between each visit.\u003c/p\u003e\u003cp\u003e\u003cem\u003e- Analysis\u003c/em\u003e: To test the effect of travel direction and experience on pheromone following we run a linear model and attempted to predict whether the ant would follow a pheromone trail (1) or not (0) by the interaction of travel direction (outwards or nestwards) and experience (na\u0026iuml;ve or experienced), with the addition of the pheromone-marked side as another predictive variable, and colony ID as a random effect, varying by intercept and slope. A binomial error family was used with the package glmmTMB. N\u0026thinsp;=\u0026thinsp;99 or 100 per condition.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe entire datasets for each experiment can be found as supplements and on OSF (link to experiments dataset). The entire analysis code and output can be found as supplement S2 and on OSF (link to code).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eExperiment 1 \u0026ndash; Does distance from the nest on an outward journey impacts pheromone following and exploration behaviour in an open arena?\u003c/span\u003e\u003c/p\u003e\u003cp\u003eBefore exploring the effects of pheromone on movement data, we will assess whether the pheromone solution induced trail following in the ants. Indeed, the pheromone solution induced the ants to spend more time in the central zone 1 cm on each side of the pheromone trail the middle of the arena (Estimate\u0026thinsp;=\u0026thinsp;0.18, p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01, see also Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). There was no effect of path length (Estimate\u0026thinsp;=\u0026thinsp;0.01462, p-value\u0026thinsp;=\u0026thinsp;0.76) or interaction of path length and pheromone (Estimate\u0026thinsp;=\u0026thinsp;0.01962, p-value\u0026thinsp;=\u0026thinsp;0.77) on the time spent in this zone: ants who had travelled further away from their nests were not more likely to follow a trail they encountered on their path than ants which encountered a pheromone trail closer to their nests.\u003c/p\u003e\u003cp\u003eWalking speed was marginally impacted by path length (Estimate = -2.93, p-value\u0026thinsp;=\u0026thinsp;0.096), with ants coming from the short path (mean speed 23,47mm/sec) tending to be slower than ants coming from the long one (mean speed 26mm/sec). Surprisingly, the presence of pheromone did not increase walking speed. There was no effect of the interaction between path length and pheromone presence.\u003c/p\u003e\u003cp\u003eMeander was also affected by path length (Estimate\u0026thinsp;=\u0026thinsp;0.6768, p-value\u0026thinsp;=\u0026thinsp;0.036), pheromone (Estimate\u0026thinsp;=\u0026thinsp;0.75, p-value\u0026thinsp;=\u0026thinsp;0.02), and of the interaction between the two (Estimate = -1.0828, p-value\u0026thinsp;=\u0026thinsp;0.018): When no pheromone was present, ants coming from the long path walked in a more linear trajectory, but the reverse was found when pheromone was present (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePath length affected time to reach the boarder of the arena (Estimate\u0026thinsp;=\u0026thinsp;0.62902, p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01), ants coming from the short path took twice as long to exit the platform, likely due to their less linear trajectories and higher meander values noted previously. Pheromone did not have an effect, however the interaction was almost significant (Estimate = -2.926, p-value\u0026thinsp;=\u0026thinsp;0.096), with the time to reach one of the arena\u0026rsquo;s edges not being different anymore when pheromone was present, as ants were following the trail to the edge of the paper.\u003c/p\u003e\u003cp\u003eDistance travelled was significantly impacted by path length (Estimate\u0026thinsp;=\u0026thinsp;0.52, p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01): When no pheromone was present, ants coming from the short path travelled almost twice as much as the ones coming from the long path before reaching an edge of the arena (on average 501mm vs 266mm, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Interestingly, this difference was reduced by the presence of the pheromone trail: the interaction between path length and pheromone was significant (Estimate\u0026thinsp;=\u0026thinsp;0.34627, p-value\u0026thinsp;=\u0026thinsp;0.04).\u003c/p\u003e\u003cp\u003eAs an additional exploration of the data, we plotted the coordinates at which the ants exited the arena (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). When pheromone trail was present, more ants exited the arena where the trail reached the edge of the paper. It seems that ants coming from the long path exited less from the right edge.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eExperiment 2 – Do ants follow pheromone trails more accurately when returning to the nest?\u003c/h3\u003e\n\u003cp\u003eThere was no effect of travel direction, experience, or the interaction of travel direction and experience, on pheromone following accuracy (all Χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.38, all P\u0026thinsp;\u0026gt;\u0026thinsp;0.54, see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The side of the Y maze on which pheromone was placed did not affect pheromone following (Χ2\u0026thinsp;=\u0026thinsp;2.19, P\u0026thinsp;=\u0026thinsp;0.14). Overall, 87.4% of ants followed the pheromone trail (348 / 398).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe trail following fidelity of \u003cem\u003eLasius niger\u003c/em\u003e appears to be quite resistant to task state, travel direction, and distance from the nest. We expected more pheromone following further from the nest, as the further from the nest, the more errors can accumulate and the more costly it is to get lost. We also expected more pheromone following going outwards than inwards for na\u0026iuml;ve ants because returning ants have information (where is nest) and outgoing ants do not know where food will be found.\u003c/p\u003e\u003cp\u003eHowever, we found that pheromone following in a Y-maze did not differ between outward and return trips to the nest, nor was it affected by whether food had been encountered on the previous trip. Similarly, Czaczkes et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported that \u003cem\u003eL. niger\u003c/em\u003e foragers in a T-maze showed no change in pheromone-following behaviour regardless of whether they were na\u0026iuml;ve, recruited, or experienced. Several factors may explain this absence of effect. One possibility is that the ants had no strong incentive to favour one branch over the other, since they had not experienced the alternative branch as unrewarding. Another explanation could be that traversing the few centimetres of maze imposed little energetic cost, reducing the need to discriminate between branches. Perhaps had there been some conflict (pheromone pointing further away from the nest-food axis, for example), or a more complex path, we would have seen differences between treatments, as outgoing experienced foragers rely on memory more than outgoing na\u0026iuml;ve ants. In addition, there is some evidence that ants can monitor their own uncertainty (Czaczkes \u0026amp; Heinze, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Merkle \u0026amp; Wehner, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and the uncertainty in their path integration system (Merkle \u0026amp; Wehner, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Since the ants were restricted to a linear runway, they may not have accumulated sufficient noise in their path integration system to cause a detectable change in behaviour, and there may not be a strong cost of not following the pheromone.\u003c/p\u003e\u003cp\u003eIn order to complement the Y-maze experiments, in this study we also looked at trail following in an open arena, which could allow us to detect more subtle differences in trail following that might not be apparent in the Y-maze. In the open arena, we found that distance travelled before encountering the pheromone trail did not affect the following accuracy of the trail. In an older study in \u003cem\u003eSolenopsis saevissima\u003c/em\u003e, deviation of trail following did not vary with distance along a 10 cm long trail in (Wilson, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1962b\u003c/span\u003e), pointing to similarities with \u003cem\u003eL. niger\u003c/em\u003e. However, we did find effects of the distance travelled on the walking behaviour of the foragers, with ants having walked a longer way having a more linear trajectory. Oddly, this effect was reversed when pheromone was present, but we wonder if this is a real effect or simply due to chance.\u003c/p\u003e\u003cp\u003eCould the lack of effect of distance travelled be due to ants not being able to perceive the difference between 20 and 100cm? It appears more likely that pheromone following responds to different rules than pheromone deposition. Indeed, in the same species and with the same path length (20 \u003cem\u003evs\u003c/em\u003e 100cm), we found that foragers deposited more pheromone to food sources that were more distant from their nest (Czaczkes et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), a finding which replicated previous similar findings using a different setup (Devigne \u0026amp; Detrain, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Ants are therefore able to detect differences in the range of distance we used.\u003c/p\u003e\u003cp\u003eWhy would pheromone information provision rules differ from pheromone following rules, when both have the same goal? Perhaps the costs of not depositing pheromone and not following pheromone are under different constraints. For instance, if the ant decides not to lay pheromone, the direct costs at the individual level might be minimal, as the ant can still remember the way to the food. However, the costs could be high at the collective level, as other ants will not be directed to the food source, which could be quickly monopolised by another colony instead. On the other hand, the decision not to follow the pheromone could affect the individual level more directly, through the costs of exploring unknown territory or getting lost for example, leading to less flexibility in pheromone following.\u003c/p\u003e\u003cp\u003eThis study demonstrates a remarkable robustness in trail pheromone following decisions (c. 85%) in an ant, which is not due to ceiling effects, and unlikely to be due to limitations on detection. This replicability in pheromone following proportion is echoed in previous studies too. Taken together, it is clear that pheromone following probability is tightly controlled, and not set at 100%. The question which remains is: why is this, and do these results translate to other contexts and uses of pheromone? Would ants which are recruiting to a nest site after their nest has been destroyed follow pheromone trails with greater fidelity? Or perhaps would starving colonies follow pheromone trails with more fidelity, given that finding other food sources seems unlikely? A system with a stable proportion of pheromone followers and some variation in the pheromone laying might allow the colony to have a balance between flexibility and quick efficient recruitment to a food source, while relying on simple rules at the individual level. The flexibility of pheromone following in a variety of contexts outside foraging for sucrose awaits further exploration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclarations\u003c/h2\u003e\u003cp\u003eWe declare no competing interests\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e\u003cp\u003e໿L-A. P. was supported by an Alexander von Humboldt Postdoctoral Fellowship (1223114) at the beginning of the project, then by a Postdoctoral Equal Opportunities Fellowship from the University of Regensburg. T.J.C was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Project No. 462101190.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlmeida NG, De, Camargo RDS, Forti LC, Lopes JFS (2018) Hierarchical establishment of information sources during foraging decision-making process involving Acromyrmex subterraneus. Biology Ecol Divers 62(1):36\u0026ndash;39. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.RBE.2017.11.006\u003c/span\u003e\u003cspan address=\"10.1016/J.RBE.2017.11.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBeckers R, Deneubourg JL, Goss S (1992) Trail laying behaviour during food recruitment in the ant Lasius niger (L). Insectes Sociaux 39(1):59\u0026ndash;72. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF01240531\u003c/span\u003e\u003cspan address=\"10.1007/BF01240531\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCammaerts-Tricot MC, Verhaeghe JC (1974) Ontogenesis of trail pheromone production and trail following behaviour in the workers of Myrmica rubra L. (Formicidae). Insectes Sociaux 21(3):275\u0026ndash;282. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF02226918\u003c/span\u003e\u003cspan address=\"10.1007/BF02226918\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChiara V, Kim S-Y, Animal GE, Cacti T (2023) AnimalTA: A highly flexible and easy-to-use program for tracking and analysing animal movement in different environments. Methods Ecol Evol 14(7):1699\u0026ndash;1707. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/2041-210X.14115\u003c/span\u003e\u003cspan address=\"10.1111/2041-210X.14115\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCzaczkes TJ, Beckwith JJ, Horsch A-L, Hartig F (2019) The multi-dimensional nature of information drives prioritization of private over social information in ants. \u003cem\u003eProceedings of the Royal Society B: Biological Sciences\u003c/em\u003e, \u003cem\u003e286\u003c/em\u003e(1909), 20191136. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rspb.2019.1136\u003c/span\u003e\u003cspan address=\"10.1098/rspb.2019.1136\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCzaczkes TJ, Brandstetter B, di Stefano I, Heinze J (2018) Greater effort increases perceived value in an invertebrate (Lasius niger). J Comp Psychol 132(2):200\u0026ndash;209. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1037/COM0000109\u003c/span\u003e\u003cspan address=\"10.1037/COM0000109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCzaczkes TJ, Castorena M, Sch\u0026uuml;rch R, Heinze J (2017) Pheromone trail following in the ant Lasius niger: high accuracy and variability but no effect of task state. Physiol Entomol 42(1):91\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/PHEN.12174\u003c/span\u003e\u003cspan address=\"10.1111/PHEN.12174\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCzaczkes TJ, Gr\u0026uuml;ter C, Ellis L, Wood E, Ratnieks FLW (2013) Ant foraging on complex trails: Route learning and the role of trail pheromones in Lasius niger. J Exp Biol 216(2):188\u0026ndash;197. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/JEB.076570/257938\u003c/span\u003e\u003cspan address=\"10.1242/JEB.076570/257938\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/AM/ANT-FORAGING-ON-COMPLEX-TRAILS-ROUTE-LEARNING-AND\u003c/span\u003e\u003cspan address=\"http:///AM/ANT-FORAGING-ON-COMPLEX-TRAILS-ROUTE-LEARNING-AND\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCzaczkes TJ, Gr\u0026uuml;ter C, Ratnieks FLW (2015) Trail Pheromones: An Integrative View of Their Role in Social Insect Colony Organization. Ann Rev Entomol 60(1):581\u0026ndash;599. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-ento-010814-020627\u003c/span\u003e\u003cspan address=\"10.1146/annurev-ento-010814-020627\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCzaczkes TJ, Heinze J (2015) Ants adjust their pheromone deposition to a changing environment and their probability of making errors. \u003cem\u003eProceedings of the Royal Society B: Biological Sciences\u003c/em\u003e, \u003cem\u003e282\u003c/em\u003e(1810). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/RSPB.2015.0679\u003c/span\u003e\u003cspan address=\"10.1098/RSPB.2015.0679\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCzaczkes TJ, Olivera-Rodriguez F-J, Poissonnier L-A (2024) Ants (Lasius niger) deposit more pheromone close to food sources and further from the nest but do not attempt to update erroneous pheromone trails. Insectes Sociaux 71:367\u0026ndash;376. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S00040-024-00995-Y\u003c/span\u003e\u003cspan address=\"10.1007/S00040-024-00995-Y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCzaczkes TJ, Ratnieks FLW (2012) Pheromone trails in the Brazilian ant Pheidole oxyops: Extreme properties and dual recruitment action. Behav Ecol Sociobiol 66(8):1149\u0026ndash;1156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S00265-012-1367-7/FIGURES/5\u003c/span\u003e\u003cspan address=\"10.1007/S00265-012-1367-7/FIGURES/5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDe Agr\u0026ograve; M, Matschunas C, Czaczkes TJ (2022) Bundling and segregation affect pheromone deposition, but not choice, in an ant. \u003cem\u003eELife\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7554/ELIFE.79314\u003c/span\u003e\u003cspan address=\"10.7554/ELIFE.79314\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDevigne C, Detrain C (2006) How does food distance influence foraging in the ant Lasius niger: The importance of home-range marking. Insectes Sociaux 53(1):46\u0026ndash;55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S00040-005-0834-9/METRICS\u003c/span\u003e\u003cspan address=\"10.1007/S00040-005-0834-9/METRICS\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFourcassie V, Beugnon G (1988) How do red wood ants orient when foraging in a three dimensional system? Insectes Sociaux 35(1):92\u0026ndash;105\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFrizzi F, Talone F, Santini G (2018) Modulation of trail laying in the ant Lasius neglectus (Hymenoptera: Formicidae) and its role in the collective selection of a food source. Ethology 124(12):870\u0026ndash;880. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/ETH.12821\u003c/span\u003e\u003cspan address=\"10.1111/ETH.12821\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGr\u0026uuml;ter C, Czaczkes TJ, Ratnieks FLW (2011) Decision making in ant foragers (Lasius niger) facing conflicting private and social information. Behav Ecol Sociobiol 65(2):141\u0026ndash;148. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00265-010-1020-2\u003c/span\u003e\u003cspan address=\"10.1007/s00265-010-1020-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHangartner W (1969) Structure and variability of the individual odor trail in Solenopsis geminata Fabr. (Hymenoptera, Formicidae). Z F\u0026uuml;r Vergleichende Physiologie 62(1):111\u0026ndash;120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00298046/METRICS\u003c/span\u003e\u003cspan address=\"10.1007/BF00298046/METRICS\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHangartner W (1970) Control of pheromone quantity in odor trails of the ant Acanthomyops interjectus MAYR. Experientia 26:664\u0026ndash;665. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1007/BF01898753\u003c/span\u003e\u003cspan address=\"10.1007/BF01898753\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHarrison JF, Fewell JH, Stiller TM, Breed MD (1989) Effects of experience on use of orientation cues in the giant tropical ant. \u003cem\u003eAnimal Behaviour\u003c/em\u003e, \u003cem\u003e37\u003c/em\u003e(PART 5), 869\u0026ndash;871. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0003-3472(89)90076-6\u003c/span\u003e\u003cspan address=\"10.1016/0003-3472(89)90076-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHart T, Lopes LE, Frank DD, Kronauer DJC (2024) Pheromone representation in the ant antennal lobe changes with age. Curr Biol 34(14):3233\u0026ndash;3240e4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cub.2024.05.031\u003c/span\u003e\u003cspan address=\"10.1016/j.cub.2024.05.031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHartig F (2024) Residual Diagnostics for Hierarchical (Multi-Level / Mixed) Regression Models [R package DHARMa version 0.4.7]. In \u003cem\u003eCRAN: Contributed Packages\u003c/em\u003e. Comprehensive R Archive Network (CRAN). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.32614/CRAN.PACKAGE.DHARMA\u003c/span\u003e\u003cspan address=\"10.32614/CRAN.PACKAGE.DHARMA\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJackson D, Chaline N (2007) Modulation of pheromone trail strength with food quality in Pharaoh\u0026rsquo;s ant, Monomorium pharaonis. Anim Behav 74(3):463\u0026ndash;470. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ANBEHAV.2006.11.027\u003c/span\u003e\u003cspan address=\"10.1016/J.ANBEHAV.2006.11.027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJackson DE, Martin SJ, Holcombe M, Ratnieks FLW (2006) Longevity and detection of persistent foraging trails in Pharaoh\u0026rsquo;s ants, Monomorium pharaonis (L). Anim Behav 71:351\u0026ndash;359. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.anbehav.2005.04.018\u003c/span\u003e\u003cspan address=\"10.1016/j.anbehav.2005.04.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJaffe K, Howse PE (1979) The mass recruitment system of the leaf cutting ant, Atta cephalotes (L.). \u003cem\u003eAnimal Behaviour\u003c/em\u003e, \u003cem\u003e27\u003c/em\u003e(PART 3), 930\u0026ndash;939. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0003-3472(79)90031-9\u003c/span\u003e\u003cspan address=\"10.1016/0003-3472(79)90031-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJones S, Czaczkes TJ, Gallager AJ, Oberhauser FB, Gourlay E, Bacon JP (2019) Copy when uncertain: lower light levels increase trail pheromone depositing and reliance on pheromone trails in ants. Anim Behav 156:87\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.anbehav.2019.08.007\u003c/span\u003e\u003cspan address=\"10.1016/j.anbehav.2019.08.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoch A, Czaczkes TJ (2021) No specialist pheromone-ignoring ants in Lasius niger. Ecol Entomol 46(3):677\u0026ndash;680. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/EEN.12995\u003c/span\u003e\u003cspan address=\"10.1111/EEN.12995\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKuznetsova A, B. P. C. R (2017) lmerTest Package: Tests in Linear Mixed Effects Models. J Stat Softw 82(13):1\u0026ndash;26\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMailleux AC, Deneubourg JL, Detrain C (2003) Regulation of ants\u0026rsquo; foraging to resource productivity. \u003cem\u003eProceedings of the Royal Society of London. Series B: Biological Sciences\u003c/em\u003e, \u003cem\u003e270\u003c/em\u003e(1524), 1609\u0026ndash;1616. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/RSPB.2003.2398\u003c/span\u003e\u003cspan address=\"10.1098/RSPB.2003.2398\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMailleux A-C, Deneubourg J-L, Detrain C (2000) How do ants assess food volume? Anim Behav 59(5):1061\u0026ndash;1069. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/anbe.2000.1396\u003c/span\u003e\u003cspan address=\"10.1006/anbe.2000.1396\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMailleux A-C, Detrain C, Deneubourg J-L (2006) Starvation drives a threshold triggering communication. J Exp Biol 209:4224\u0026ndash;4229. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/jeb.02461\u003c/span\u003e\u003cspan address=\"10.1242/jeb.02461\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMerkle T, Wehner R (2010) Desert ants use foraging distance to adapt the nest search to the uncertainty of the path integrator. Behav Ecol 21(2):349\u0026ndash;355. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/BEHECO/ARP197\u003c/span\u003e\u003cspan address=\"10.1093/BEHECO/ARP197\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNorman VC, Butterfield T, Drijfhout F, Tasman K, Hughes WOH (2017) Alarm Pheromone Composition and Behavioral Activity in Fungus-Growing Ants. J Chem Ecol 43(3):225\u0026ndash;235. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S10886-017-0821-4/FIGURES/4\u003c/span\u003e\u003cspan address=\"10.1007/S10886-017-0821-4/FIGURES/4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOberhauser FB, Czaczkes TJ (2018) Tasting the unexpected: disconfirmation of expectations leads to lower perceived food value in an invertebrate. Biol Lett 14(9). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/RSBL.2018.0440\u003c/span\u003e\u003cspan address=\"10.1098/RSBL.2018.0440\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePokorny T, Sieber LM, Hofferberth JE, Bernadou A, Ruther J (2020) Age-dependent release of and response to alarm pheromone in a ponerine ant. J Exp Biol 223(6). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/JEB.218040/VIDEO-1\u003c/span\u003e\u003cspan address=\"10.1242/JEB.218040/VIDEO-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eR Foundation for Statistical Computing (2024) \u003cem\u003eR Core Team. _R: A Language and Environment for Statistical Computing\u003c/em\u003e (4.4.1)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRobinson GE (1987) Modulation of alarm pheromone perception in the honey bee: evidence for division of labor based on hormonall regulated response thresholds. J Comp Physiol A 160(5):613\u0026ndash;619. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00611934/METRICS\u003c/span\u003e\u003cspan address=\"10.1007/BF00611934/METRICS\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRobson SK, Traniello JF (1998) Resource assessment, recruitment behavior, and organization of cooperative prey retrieval in the ant Formica schaufussi (Hymenoptera: Formicidae). J Insect Behav 11(1):1\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://link.springer.com/article/\u003c/span\u003e\u003cspan address=\"http://link.springer.com/article/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1023/A:1020859531179\u003c/span\u003e\u003cspan address=\"10.1023/A:1020859531179\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchatz B, Lachaud JP, Beugnon G (1997) Graded recruitment and hunting strategies linked to prey weight and size in the ponerine ant Ectatomma ruidum. Behav Ecol Sociobiol 40(6):337\u0026ndash;349. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S002650050350/METRICS\u003c/span\u003e\u003cspan address=\"10.1007/S002650050350/METRICS\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThienen W, Von, Metzler D, Choe DH, Witte V (2014) Pheromone communication in ants: A detailed analysis of concentration-dependentdecisions in three species. Behav Ecol Sociobiol 68(10):1611\u0026ndash;1627. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S00265-014-1770-3/TABLES/2\u003c/span\u003e\u003cspan address=\"10.1007/S00265-014-1770-3/TABLES/2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVerhaeghe JC (1982) Food recruitment in Tetramorium impurum (Hymenoptera: Formicidae). Insectes Sociaux 29(1):67\u0026ndash;85. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF02224528/METRICS\u003c/span\u003e\u003cspan address=\"10.1007/BF02224528/METRICS\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWendt S, Strunk KS, Heinze R, Roider J, A., Czaczkes TJ (2019) \u003cem\u003ePositive and negative incentive contrasts lead to relative value perception in ants\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7554/eLife.45450.001\u003c/span\u003e\u003cspan address=\"10.7554/eLife.45450.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWenig K, Bach R, Czaczkes TJ (2021) Hard limits to cognitive flexibility: Ants can learn to ignore but not avoid pheromone trails. J Exp Biol 224(11). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/JEB.242454/261714\u003c/span\u003e\u003cspan address=\"10.1242/JEB.242454/261714\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/AM/HARD-LIMITS-TO-COGNITIVE-FLEXIBILITY-ANTS-CAN\u003c/span\u003e\u003cspan address=\"http:///AM/HARD-LIMITS-TO-COGNITIVE-FLEXIBILITY-ANTS-CAN\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWilson EO (1962a) Chemical communication among workers of the fire ant Solenopsis saevissima (Fr. Smith) 1. The Organization of Mass-Foraging. Anim Behav 10(1\u0026ndash;2):134\u0026ndash;147. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0003-3472(62)90141-0\u003c/span\u003e\u003cspan address=\"10.1016/0003-3472(62)90141-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWilson EO (1962b) Chemical communication among workers of the fire ant Solenopsis saevissima (Fr. Smith) 2. An information analysis of the odour trail. Anim Behav 10(1\u0026ndash;2):148\u0026ndash;158. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0003-3472(62)90142-2\u003c/span\u003e\u003cspan address=\"10.1016/0003-3472(62)90142-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ants, Pheromone, Decision-making, Information use","lastPublishedDoi":"10.21203/rs.3.rs-7630446/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7630446/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnt foraging and recruitment often relies heavily on pheromone trails, and ants modulate pheromone trail deposition strategically. Ants are also known to modulate trail following depending on their own private information, such as the known location of the nest and their knowledge of food sources in the environment. Here we ask how a series of important context variables; distance from the nest, direction of travel, and prior food discovery\u0026mdash;affects the fidelity of pheromone trail following in the black garden ant \u003cem\u003eLasius niger\u003c/em\u003e. Using both an open arena assay and a binary Y-maze design, we evaluated whether ants adjust their trail-following behaviour. Ants exhibited robust and consistent trail-following behaviour across all conditions. Surprisingly, we found no significant modulation by distance, travel direction, or recent experience. However, we observed that path length before finding a pheromone trail significantly influenced walking patterns such as meander, speed, and total distance travelled. These findings suggest that while \u003cem\u003eL. niger\u003c/em\u003e trail-following behaviour is remarkably stable across contexts, locomotory traits remain plastic and context-sensitive. The decoupling of pheromone response from internal or external conditions highlights the need for further investigation into the mechanisms regulating individual foraging decisions in social insects.\u003c/p\u003e","manuscriptTitle":"Pheromone trail following is not modulated by previous visit to food location, distance travelled, or travel direction in the ant Lasius niger","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-20 18:58:14","doi":"10.21203/rs.3.rs-7630446/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-12-19T08:49:44+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-11-10T01:48:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-30T16:27:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-20T09:31:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Insectes Sociaux","date":"2025-09-19T05:30:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a2ea0b4d-8186-4568-a927-30871835b621","owner":[],"postedDate":"October 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T20:47:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-20 18:58:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7630446","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7630446","identity":"rs-7630446","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}