Differential responses to chemical cues correlate with task performance in ant foragers

Detrain Claire *1, Pereira Hugo*1 and Fourcassié Vincent2

*These two authors contributed equally to the study

1. Service d’écologie sociale, Université Libre de Bruxelles, 50 avenue F Roosevelt, 1050 Bruxelles, Belgium

2. Centre de Recherche sur la Cognition Animale CRCA), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, France

Corresponding Author: C. Detrain Email :cdetrain@ulb.ac.be Phone : +32 2 650 5529

Funding: Hugo Pereira was financially supported by a Belgian PhD grant from the F.R.I.A. (Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture). Claire Detrain is Research Director from the Belgian National Fund for Scientific Research (F.N.R.S).

Abstract

Division of labor in social insects has been explained by response threshold models which are based on differential responses to task-specific stimuli. In the present study, we argue that other types of stimuli, such as location-related cues, which are correlated with but not directly linked to task performance, may be significant. Using the black garden ant Lasius niger as a model, we focused on three groups of workers that perform extranidal tasks: 1°) scouts, which explore new and unmarked areas remote from the nest, 2°) patrollers, which are not recruited by other ants, but nonetheless walk outside in the nest vicinity and 3°) recruits, which are temporary foragers whose exit is triggered by recruitment. We used standardized tests to investigate, in a context-independent way, whether differences in task performance by these three groups could be correlated to intrinsic differences in their responsiveness to trail pheromone or to nest-related stimuli such as the presence of nestmates or colony odor. Overall, we found that the task profile of workers was correlated neither with their tendency to explore unmarked areas nor with their social attraction to nestmates. Scouts showed a lower attraction to colony odor and lower scores of trail following than recruits. Conversely, recruits were more attracted to colony chemical cues and showed lower response threshold to trail pheromone. Patrollers displayed behaviors between those of recruits and scouts. Our study thus shows that differences in ant responsiveness to location-related cues and recruitment trails contribute to the regulation of extranidal tasks in ants.

Keywords: ants, foraging, division of labor, social cues, colonial marking, trail pheromone

Significance Statement

Work organization in insect societies has been explained by models of differential response thresholds to stimuli that are directly linked to the performance of specific tasks. However, the task performance of individuals could also depend on their responsiveness to social and location-related cues that are correlated but not directly linked to these tasks. Using standardized context-independent tests, we found that scout ants, which explore unknown areas, display a lower responsiveness to colony area marking and trail pheromone. Conversely, recruits, which forage outside the nest only during food exploitation, are the most attracted to nestmates, colony area marking and trail pheromone. Patrollers display intermediate levels of attraction. Location-related cues could therefore play a role in determining the level of task performance by ants and should be taken into consideration in threshold models of task allocation.

1. Introduction

Division of labor describes the process by which individuals within a group perform only a subset of tasks, thus presumably allowing the whole group to achieve an overall increase in productivity and work efficiency (Oster and Wilson 1978; Gordon 2016). This process is epitomized in eusocial insects in which tasks are distributed and allocated to individuals that differ in behavior and, in some cases, in size and morphology. The basis for the division of labor in these insects is the behavioral variability observed between individuals within colonies. This variability can produce marked differences in the way work is organized by whole colonies and it is considered as one of the adaptive group-level features of insect societies (for a review see Jeanson and Weidenmüller 2014). Several studies have described the different mechanisms that generate this variability. In eusocial insects, the propensity of an individual to perform a task is known to be determined by or correlated with several factors including morphology (Powell 2016), physiology (Robinson 2009; Robinson et al. 2012), body size (Schwander et al. 2015), age (Seid and Traniello 2006), genetic background (Waddington et al. 2011; Schwander et al. 2005) or individual experience (Ravary et al. 2007; Bernadou et al. 2018). Moreover, in most social insects, individuals can change task throughout their lifetime. Typically, young workers carry out safer tasks, such as brood care inside the nest, while older individuals perform more hazardous tasks outside the nest, such as foraging and defense (Oster and Wilson 1978; Moroń et al. 2008; Mersch et al. 2013; Giraldo and Traniello 2014). In some ant species, however, age does not determine the tasks performed by the workers but the breadth of their behavioral repertoire (Seid and Traniello 2006). The transition from inside to outside tasks is most often associated with deep changes in gene expression (Whitfield et al. 2003; Oettler et al 2015; Bockhoven et al 2017), juvenile hormone titers (Dolezal et al. 2012, Norman and Hughes 2016), biogenic amine levels in the brain (Wnuk et al. 2011, Muscedere et al. 2012), exocrine gland activity (Holldobler and Wilson 1990), internal anatomy (Muscedere et al. 2011), ovary development (Dolezal 2013), or fat content (Bernadou et al. 2015; Silberman et al. 2016). Along with these changes, the brain and olfactory organs may also undergo modifications as individuals age (Kühn-Bühlmann and Wehner 2006; Gronenberg et al. 1996; Muscedere and Traniello 2012; Giraldo et al. 2016).

Studies on the origins of inter-individual variability in social insects have been complemented by others aiming to understand how work is organized among the hundreds to thousands of individuals that form the insect society. Many theoretical studies -and to a lesser extent empirical ones- have been developed using a framework based on response threshold models. According to these models, variation in task performance between individuals arises from differences in the internal response thresholds to task-specific stimuli (see e.g. Bonabeau et al 1996, Beshers and Fewell 2001). In essence, these models allow division of labor to be explained as follows: individuals with a low threshold for a given task start performing the task, which, as a consequence reduces the average stimulus intensity and thus makes other nestmates with higher thresholds less likely to carry out the task. These models are now widely accepted as the main paradigm for the organization of work in insect societies since they account for the task specialization observed at the individual level as well as for the task flexibility and resilience observed at the colony level. All these models implicitly assume that individuals are in the right place and at the right time to respond in an appropriate way to the stimuli associated with the task they are specialized in. However, although spatial proximity is a prerequisite for task performance, it is still unclear the extent to which the task performance profile of workers is correlated to intrinsic differences in their responsiveness to factors that may determine their spatial position.

In this study, we used the common garden ant Lasius niger as a model organism, and focused our research on the diversity of tasks that can be performed by ants outside their nest. Several studies found that distinct groups of workers can perform separated extranidal tasks (Oettler and Johnson 2008, Tschinkel 2012, Gordon 2016, D’Eustacchio et al. 2019 but see Robson and Traniello 2002). These extranidal tasks include the management of colony waste, patrolling the colony foraging area, searching for new food sources as well as retrieving food items at the end of recruitment trails. We investigated whether inter-individual variability in the performance of these tasks can be correlated to intrinsic differences between individuals linked, not only to their boldness but also to their level of response to the social and chemical cues that may influence their spatial location. The term “boldness” will be used hereafter only to describe the tendency of workers to leave the nest and to walk over new unmarked areas rather than to indicate a behavioral syndrome (Wright et al 2019). To investigate potential intrinsic differences between workers in boldness and/or responsiveness to spatially-related cues, we ran context-independent tests on individual workers and correlated their behavioral responses to their performance of extranidal tasks. Context-independent tests in ants have hitherto mainly be used to assess behaviors related to boldness, alarm and nestmate recognition (Chapman et al. 2011; Norman et al. 2014; Larsen et al. 2016). In addition to being the first attempt to differentiate behavioral phenotypes within workers performing extranidal tasks, the novelty of the present study is to propose a set of context-independent behavioral assays that include the sensitivity to social and chemical cues related to extranidal tasks. Workers were tested individually under context-independent standardized conditions in order to limit biases in motivation and in behavioral responses that may be due to confounding factors. We quantified to what extent individuals 1) spontaneously explored and walked over unknown areas (boldness test), 2) were sensitive to social cues such as the presence of nestmates (sociality test), 3) were attracted by nest-related cues such as areas marked with colony odor (colony odor test) and 4) are sensitive to the trail pheromone laid by their nestmates (trail-following test). Given that inter-colony differences in exploratory behavior can arise from differences in boldness of colony members (Wright et al 2019), we assumed that the group of scouts could be differentiated from the two other groups by their individual boldness traits. Whilst at first sight, boldness tests bear no relation to responsiveness to location-related cues, these traits are interdependent since boldness requires leaving the safety of the nest and facing unknown areas that are not marked with colony odor. Tests of attraction to nestmates and to area-marking aimed to measure the reluctance of ants to move away from nearby nestmates and hence to forage far from the nest. The propensity of workers to be mobilized outside the nest by recruiting stimuli was assessed by their response threshold to trail pheromone and by their efficiency at following artificial trails of known concentration.

We anticipated that scouts, which have to stay outside the nest for longer periods of time and further away from the nest, would be bolder and less responsive to nest-related cues. Conversely, recruits, which leave the nest only occasionally during food exploitation and whose harvesting efficiency depends on their trail-following accuracy, would be more responsive to both nest-related cues and trail pheromone.

By focusing on intrinsic differences in boldness and/or responsiveness to cues related to spatial location of individuals, these behavioral experiments will contribute to drawing up a more complete picture of the mechanisms underlying the task-performance profile of workers.

2. Material and methods

2.1. Species studied and rearing conditions.

Experiments were run on ants from five colonies of L. niger collected in September 2015 on the campus of the University of Brussels (50.5◦N et 4.2◦ E, Belgium). All colonies were queenless and contained around 500 workers with brood. We placed each colony in a plastic box (27 X 14 X 9cm) that was used as a foraging area and whose walls were coated with Fluon in order to prevent ants from escaping. Four test tubes covered with a transparent red acetate film and with a water reservoir plugged with a cotton wool at their end, were provided as nesting sites to each ant colony. Ants had access ad libitum to several test tubes that were filled with pure water or with a 0.3M sucrose solution. In addition, they were fed twice a week with four mealworm larvae (Tenebrio molitor) cut into pieces. The experimental room was lighted according to a 12:12 L:D regime and was maintained at a temperature around 21°C and a humidity level of 50%.

2.2 Worker groups studied.

We focused our study on three groups of workers involved in extranidal tasks: 1°) scout ants, which spontaneously exit the nest to search for new food sources and thus are susceptible to explore new areas, 2°) recruits, which exit the nest after being recruited by a successful scout ant and which stay outside the nest mainly during the collective exploitation of food sources and 3°) patrollers, which are regularly observed outside the nest, independent of any recruitment process, working on the waste pile, drinking water or walking in a seemingly aimless way.

To identify scout ants, we began by connecting three new areas (143 cm² each) to the foraging area of each studied colonies. These new areas were free of any chemical marking but had a droplet of 1M sucrose solution deposited in their center. The first 10 individuals which explored one of these three areas and drank at the food source were considered as scout ants and (if not yet marked) were marked. The experiment was repeated every day for five days.

To identify the recruits, we starved ants for 3 days and then elicited a food recruitment event in each of the five studied colonies. First, we connected a small elevated platform (4 X 4 cm) to the foraging area of each colony by a bridge (9 X 2 cm) and deposited in its center a droplet of sucrose solution (0.3 M). Then, once food recruitment was launched by the first ant returning to the nest, we selected and marked the first 20 individuals that were recruited by gently seizing them with a forceps while they started climbing up the bridge.

Finally, we considered as patrollers the individuals that were wandering outside the nest, seemingly aimless, and which had not been previously marked as scouts or recruits. The patrollers could be observed at various locations outside the nest, such as close to the water tubes, waste piles or food sources.

In each group of ants, the workers were chilled at -10°C for 20 seconds and then were individually marked with two dots of enamel paint (Edding TM) on their abdomen with a unique 2-colour code. The color of the first dot, close to the ant’s petiole was the same for all workers of the same group, while the color of the second dot allowed to identify each individual within the group. In total, we individually marked 129 individuals that underwent at least one of the behavioral bioassays described below. Ant marking and the whole series of bioassays that followed, were conducted in several batches of at least 12 day-duration. Among the 129 marked individuals, 14 scout ants, 12 recruits and 11 patrollers underwent the whole series of bioassays as they had kept their paint marks intact in all tests, i.e. for at least 12 successive days (sample size detailed in supplementary file 1).

2.3. Behavioral bioassays

The two factors that could influence the affiliation of the workers to one of the three functional groups defined above (scouts, recruits, patrollers) are their motivation to stay outside the nest and explore new areas and their responsiveness to nest-related and social stimuli. We thus ran four behavioral bioassays. The first aimed to test the propensity of individuals to explore a new unmarked area, i.e. their boldness, the second at testing their attraction to social cues emanating directly from nestmates, the third at testing their attraction to colony odor and the fourth at testing their response to trail pheromone.

For each behavioral assay, the marked individuals of the three functional groups that were tested, were collected by either picking them up on the colony foraging area or by gently dragging them out from the nest tubes. The bioassays were carried out in random order on each batch of marked individuals. All bioassays were video recorded using a digital camera (Logitech Pro HD C920). Based on preliminary experiments, we adapted the timings of each bioassay so that 1°) all ants were calmed down before being tested and 2°) the number of censured data was limited. The last point was obtained by adjusting the duration of video recordings to the average duration of the observed behavior.

2.3.1. Boldness test

The experimental setup used in this test was adapted from Chapman et al. (2011) (as shown in supplementary file 2). The assays were conducted in a squared box (20 X 20 x 5 cm) whose floor was cleaned with ethanol to remove any chemical markers and which was connected by a small tube to a refuge. The refuge consisted in a small Petri dish (4cm diameter) covered with a red transparent sheet and whose floor was covered with a paper marked with the odor of the ant colony. At the beginning of an assay, the tested ant was carefully placed inside the refuge while the connection with the experimental area was blocked by a small cotton plug. The ant was allowed to calm down in the refuge for one minute and was then given access to the experimental area by removing the plug. Once the ant had entered the area, we filmed its behavior during 5 minutes and we measured the time it spent exploring the area before returning to the refuge (‘exploration’ index). We also calculated the ratio of the time spent by the ant near the walls of the box (within a 5 mm distance) over the time it spent in the center of the experimental area (‘thigmotaxis’ index). Indeed, refraining from the strong tendency to follow walls and entering open space are common criteria used to assess individual boldness in vertebrates (e.g. Wagle et al.2017) but also in ants (e.g. Chapman et al 2011) and other insects (Besson & Martin, 2005). Therefore, we assumed that bolder individuals would stay more time outside the refuge (high values of exploration index) and would spend less time close to the box walls (low values of thigmotaxis index). In total, 114 ants were tested in this assay: 34 scouts, 38 patrollers and 45 recruits.

2.3.2. Social attraction to nestmates

Tests of social attraction were carried out in a rectangular elongated box (18x3x2 cm) one end of which was connected to a 1 X 3 cm chamber (as shown in supplementary file 3). This chamber was separated from the box by a metallic grid that prevented ants from passing but that still allowed antennal contacts between ants on each side of the grid. At the beginning of a test, a single ant was introduced in the box, close to the chamber which contained ten workers belonging to the same colony. We then allowed the ants to calm down for one minute. During this initial phase, the ant placed in the box had the opportunity to antennate with its nestmates present in the chamber. Furthermore, a removable barrier prevented it from going further than 2 cm away from the grid, thus ensuring that antennal contacts really took place between nestmates across the grid.

After this initial phase of antennal contacts, we removed the barrier and we filmed the tested individual for 3 min. The level of social attraction was assessed by the amount of time the ant remained in the vicinity of its nestmates, i.e. stayed at a distance of at most 5 mm from the grid. Furthermore, we assessed the propensity of the ant to move towards locations that were remote from its nestmates by measuring the latency time for the ant to reach the half of the box opposite to the chamber. As a control, we also measured these two variables in a second set of experiments with the chamber being left empty. Thus, each ant was tested twice, with the order of experimental condition (with and without nestmates) randomized, before being replaced in its colony. In total, 108 ants were tested: 33 scouts, 34 patrollers and 41 recruits.

2.3.3. Attraction to colony odor

Ants were tested in a Y-shaped setup consisting in a starting branch (15 X 0.7 cm) connected to two branches of equal length (5 X 0.7 cm each) separated by a 60° angle (as shown in supplementary file 4). The starting branch and one branch of the setup were covered with a piece of filter paper free of any chemical markers while the other branch was covered with a piece of filter paper that was marked with the odor of the colony of the tested ant. This colonial marking was obtained by leaving about ten pieces of paper (5 X 0.7 cm each) in front of the test tubes housing the colonies 24h prior to each experiment. This way walking ants passively laid footprints over the filter paper and thereby marked it with colony-specific chemical compounds (Devigne and Detrain 2002; Lenoir et al. 2009). After twenty-four hours, ants walked on the papers as frequently as in the rest of the foraging area, which suggests that they no longer considered the papers as novel areas and thus that the papers were sufficiently marked. The experiment started by gently depositing an ant behind a removable wall on the first 2 cm of the starting branch and by allowing it to calm down for one minute. We then removed the wall to give the ant access to the Y-maze and we observed its choice between the control and the colony-marked branch. The ant was considered as having chosen one branch as soon as it walked on it over a distance of at least 1 cm. Each ant was tested successively 6 times in the setup and was assigned a score ranging from 0 to 6 depending on the number of choices it made towards the colony-marked branch. Around five minutes elapsed between each of the six successive trials. Furthermore, in order to limit the biases due to a possible side preference of the ant, the chemically marked paper was alternatively put on either side of the Y-maze. After each test, we replaced the paper overlay in order to remove any trail pheromone that could have been laid by the ant. In total, 99 ants were tested: 31 scouts, 33 patrollers and 35 recruits.

2.3.4. Response to trail pheromone

Forty rectal glands, which are known to produce the trail pheromone in the ant L. niger (Morgan 2009), were dissected under binocular microscope and were put in 0.5 mL dichloromethane. This stock solution subsequently underwent several dilutions by adding the appropriate volumes of dichloromethane solvent. Based on a previous study on L. niger trail-following responses (Von Thienen et al. 2014), we used the following concentrations of glandular extracts: 0.008 gland/50μL; 0.040 gland/50μL; 0.080 gland/50μL; 0.400 gland/50μL; 0.800 gland/50μL. Trail following experiments were carried out in a Petri dish (13.5 cm diameter) whose floor was covered with a disk of filter paper (as shown in supplementary file 5). Before starting the experiment, we drew a circular trail (5.5 cm diameter) on the paper disk on the bottom of the Petri dish with a Hamilton needle delivering 50μL of glandular extract and placed a small ring (diameter: 2cm, height: 3 cm) in the center of the Petri dish. Then, three workers that were randomly drawn out of each colony, were gently deposited inside the ring where they were allowed to calm down for one minute. For each test made with an artificial trail, we tried as much as possible to get groups of workers composed of one individual from each of three functional groups. The different trail concentrations were tested in a random order. As soon as the ring was removed, we recorded the trail-following response of the ants for five minutes. Knowing that the lifetime of L.niger trail pheromone was about 50 minutes (Beckers et al. 1993), the trail evaporation was negligible over the course of one bioassay. An ant was considered as following the trail as long as it walked within a 1 cm-wide annulus centered on it. A trail-following score was assigned to each ant by counting the total number of 10° arcs it followed during its first three passages in the trail annulus. Each ant was tested with each of the five concentrations of glandular extracts as well as with the dichloromethane solvent. The order of the tests was drawn randomly, with around 5 minutes elapsing between successive tests. For the most concentrated trails that triggered the highest responses (i.e. at 0.4 and 0.8 glands), we checked for possible biases due to some ant-induced following behavior. We found that the percentages of arcs travelled by an ant while following another (at a distance of less than two 10°arcs) represented only 1.7% and 2.4% of the total number of arcs followed by ants on trails made with 0.4 and a 0.8 glands respectively. In total, 80 ants were tested for each trail concentrations: 24 scouts, 25 patrollers and 31 recruits.

2.4. Data analysis

It was not possible to record and analyze data blind because our study involved animals specifically marked according to their functional group. Since none of the data met the condition of normality, we used non-parametric tests with a significance threshold of 0.05. We used a Kruskall Wallis test to compare the behavioral responses of ants between groups. When the test was significant, we used Nemenyi post-hoc tests to make pairwise comparisons between groups. We used a Wilcoxon Signed Rank test in the social attraction and trail-following bioassays to compare how the same individual ant behaved under the control and the experimental condition. For each group of ants, we used Wilcoxon tests to compare the trail-following scores of ant individuals between each bioassay with a trail made with rectal gland extracts and the control bioassay with a trail made with dichloromethane solvent. The response threshold of the ants was defined as the trail concentration at which the test became significant. The survival curves of the proportion of ants still following the trail were compared between groups by using Wald test.

All data were analyzed and the graphics generated with R software 3.2.0. All data values in the result section are expressed as medians. We used the subscript Sc, Pat and Rec for the group of scouts, patrollers and recruits respectively.

2.5. Ethical note

No licences or permits were required for this research. We collected ant colonies with care in the field and we provided them with suitable nesting sites, food and water, thus minimizing any adverse impact on their welfare. After the experiments, we kept ant colonies in the laboratory and reared them until their natural death.

2.5. Data availability

The data set generated and/or analyzed during the current study are available in the Zenodo repository, https://zenodo.org/record/2583520#.XH5GrShKhhE

3. Results

3.1.Boldness test

Scouts, patrollers and recruits were equally likely to explore a new area as they spent the same amount of time on the experimental area before returning to the refuge (fig. 1: duration of exploration: mSc= 109s, mPat=135s, mRec=124s; Kruskal-Wallis, NSc=34, NPat=38, NRec=45 ; H= 0.48; df=2; p=0.79). Likewise, individuals of the three groups explored the new area in a similar way. All ants spent most of the time walking close to the box walls instead of exploring its center and thus had values of thigmotactic index that were much higher than 1. The level of thigmotaxis was similar between groups (ratios: mSc= 1.77, mPat=2.29, mRec=1.68; Kruskal-Wallis, NSc=34, NPat =38, NREC=45; H= 1.86; ; df=2; p=0.39)

3.2. Social attraction to nestmates

Whatever the group, ants spent significantly more time close to the grid when the end chamber hosted nestmates than when it was empty (fig 2; Wilcoxon tests comparing the social attraction index to the theoretical value of 0: Scouts: W=429; df=1; p<0.007; Patrollers: W=481; df=1; p<0.007; Recruits: W= 764; df=1; p < 0.001). In fact, while ants spent only around 20 seconds in contact with the grid of the empty chamber (msc=18 s; mpat=25.6 s; mrec=16.4 s), they remained much longer (up to one minute) when nestmates were present in the chamber on the other side of the grid (msc=55.2 s; mpat=44.8 s; mrec=62.4 s). Furthermore, recruits were less prone to move away from their nestmates as their latency times before they reached the opposite half of the box were significantly higher in tests with nestmates than without nestmates (without nestmate: mrec=9.40 s; with nestmates: mrec=12.6s; Wilcoxon test, W= 662.5; df=1; p= 0.003). For patrollers and scouts, these latency times were not significantly different between the two conditions (without nestmates: msc=12.40 s, mpat=12.40 s; with nestmates msc=15.8 s, mpat=15.9 s; Wilcoxon tests: W= 344; df=1; p=0.130 and W= 370; df=1; p=0.22 for scouts and patrollers respectively). Although the ants always preferred to stay close to the chamber that hosted nestmates, we found no significant differences between the three functional groups in their level of social attraction. (fig 2; Kruskal Wallis test on social attraction index, H=1.8 ; df=2 ; p=0.41). Likewise, the increase in the latency time to reach the opposite side of the box was of the same order of magnitude in all functional groups (Kruskal Wallis, H=1.38 df=2, p=0.51).

3.3. Attraction to colony odor

Ants of the three groups differed in their attraction score towards an area marked with their own colony odor (fig 3: Kruskal-Wallis, H=8.39; df=2; p=0.015). Indeed, recruits showed significantly higher scores of chemotactic attraction (median: m Rec=4) than individuals from the two other groups (median mSC=3, mPat=3,) (Nemenyi Post hoc test comparing scores of Recruits vs Scouts, p=0.041; Recruits vs Patrollers; p=0.043). The scores obtained by scouts and patrollers were not significantly different (Nemenyi post hoc test p=0.998). No self-reinforcement seemed to occur over the six successive replicates of the test. Indeed, the percentage of ants choosing the marked branch did not statistically differ between the first and the sixth replicate ( test: 2.84, df =1, p=0.09).

3.4. Response to trail pheromone

Patrollers and recruits showed lower response thresholds to trail pheromone than scouts. When faced with trails made with 0.008 rectal gland/50μL extract, they followed it over distances significantly longer than when faced with trails made with dichloromethane solvent (table 1: patrollers: mDCM=9 arcs vs m0.008=15 arcs; Wilcoxon test: W = 67; df=1; p= 0.011; recruits: mDCM=7 arcs vs m0.008=13 arcs; Wilcoxon: W=115; df=1; p=0.046). By contrast, trails made with the same 0.008 gland concentration extract did not elicit a following response in scouts significantly different from that observed with trails made with the solvent (mDCM=9 arcs vs m0.008=10 arcs; Wilcoxon test: W=112.5; df=1; p=0.290). Scouts required trails made of at least 0.040 rectal gland/50μL extract in order to display trail-following responses significantly higher than those observed with trails made with solvent (table 1: scouts: mDCM=9 arcs vs m0.04=14 arcs; Wilcoxon test: W=45.5; df=1; p=0.034)

In addition to differences in their response threshold, scouts always showed the lowest trail following score for each tested concentration (table 1). At a concentration of 0.040 gland/50μL, the survival curves of the proportion of ants still following the trail was significantly steeper for scouts than for patrollers or recruits (figure 4; Wald test: df=2; p=0.011). At this trail concentration, scouts were actually about twice as likely to leave the trail as recruits or patrollers (table 2). The same trend holds for the other trail concentrations (data given in supplementary file 6) but the trail-following responses did not statistically differ between groups (table 1; Kruskall Wallis test: all p values >0.05 for comparisons across groups at a given trail concentration except p=0.047 for the 0.040 gland concentration extract).

4. Discussion

It is clear that inter-individual variability in task performance is the outcome of a complex interplay between the internal regulators of behavior (for a review see Jeanson and Weidenmuller 2014), the social interactions experienced by individuals (Gordon 2010, 2016; Mersch et al. 2013; Pamminger et al 2014) and the worksite location (Mersch et al. 2013; Pamminger et al 2014). It is also widely accepted that division of labor is based on the variability among individuals in their response thresholds to biologically relevant stimuli associated with specific tasks (Detrain and Pasteels 1991; Robinson 1992; Bonabeau et al.1996; Beshers and Fewell 2001; Jeanson and Weidenmüller 2014).

In our empirical study, we highlighted a differential responsiveness among workers to location-related chemical cues and to trail pheromone, which could contribute to the spatially structured allocation of foragers to patrolling, food exploitation and exploration of new areas.

Context-independent tests on ant individuals showed that responsiveness to colony odor allows us to distinguish recruits from the groups of patrollers and scouts. Indeed, recruits showed the highest level of attraction towards a substrate marked by their own colony odor what incidentally provides them with a location cue. Indeed, as these chemical markers are left passively on the substrate by walking ants, the amount of area-marking deposited at a given location provides ants with a proxy of the local density of congeners and of its covariate, the distance from the nest (Devigne and Detrain 2002; Devigne and Detrain 2006; Detrain and Deneubourg 2009). As a result, differences in the responsiveness of ants to area marking are expected to generate differences in the spatial location of workers. By being the most attracted to colony area marking, recruits are more likely to stay at locations that are regularly and frequently occupied by nestmates, such as the interior of the nest. From the perspective of task regulation at the colony level, the higher sensitivity of L.niger recruits to colony odor, makes them more likely to be locally aggregated inside the nest where the first steps of recruitment take place. These local aggregates of potential recruits could promote the emergence of cooperative behaviors such as a collective mobilization to exploit newly discovered food resources (Detrain et al. 1999). Our results suggest that density-related cues such as area marking may contribute to spatially organized work at the colony-level when being associated with differential responses of workers depending on their physical caste (minor VS major; Sempo et al. 2006) or behavioral profile (nurses VS foragers; Depickère et al. 2005),

As regards the scouts, even though they were significantly less attracted to colony odor than recruits, their lower responsiveness did not allow us to differentiate them from patrollers. Possible explanations to the higher propensity of scouts to explore new unknown areas would be that scouts are bolder and/or less eager to stay close to their nestmates. However, scouts and patrollers did not differ in their boldness traits since they showed a similar propensity to stay outside the nest and similar patterns of exploration of new areas. Likewise, the social attraction of scouts towards nestmates was of the same order of magnitude as the two other functional groups. A last hypothesis, although not tested in the present study, would be that scouts possess higher navigational abilities that allow them to explore locations that are remote from the nest without incurring the risk of being lost while homing.

We also found that L.niger scouts exhibited a higher response threshold to trail pheromone and a lower trail-following accuracy than the two other groups of foragers. We suspect that the lower abilities of scouts to perceive and follow chemical trails could make them more likely to forage away from the existing trail network and hence to discover new food sources on unexploited areas (Deneubourg et al. 1983). It remains to be tested whether such inter-individual differences of following responses observed on artificial trails are maintained or, conversely, mitigated over natural trails when ants are busy exploiting a food source and are highly motivated to reach the food target (as suggested by Czaczkes et al., 2017). Furthermore, it should be noted that, even in polymorphic ant species characterized by a well-differentiated system of caste polyethism, worker-size related responses to trail olfactory stimuli are not always easily explained in terms of division of labor. For instance, while the higher responses of Pheidole pallidula minors to trail stimuli are well correlated with their high foraging activity (Detrain and Pasteels 1991), conversely, the ability of different-sized workers of Pheidole rhea to detect trail pheromone does not align with their extranidal task performance (Gordon et al. 2018).

Put in a wider framework, our results also raise the question of whether differences in the performance of external tasks can be related to differences in the way workers process multiple sources of information, i.e. by making use of private navigational information such as their memory of topological cues or alternatively, by using social information such as the pheromone trail laid by nestmates (Grüter et al. 2011). The relative importance given to these two sources of information vary among ant species (Aron et al. 1993) and with the foraging experience of the ants (Grüter et al. 2011). Our study suggests that differences may also exist between groups of foragers with scouts giving less weight to social information than patrollers or recruits. Whether the lower response of scouts to the trail pheromone is due to differences in their sensory abilities or to experience-based changes in the type of cues that they use while foraging, still needs to be investigated.

As regards the patrollers, their behavioral responses in context-independent tests were between those observed for scouts and recruits. On the one hand, the low attraction of patrollers to social cues fits well with their propensity to walk outside the nest, even in the absence of any recruiting stimulus. On the other hand, their high trail-following response make patrollers as good candidates as recruits to contribute to the collective exploitation of food resources.

To conclude, we argue that colony area-marking as well as trail pheromone, which both provide indirect topological information to workers, contribute to spatially separate scouts from recruits as a result of their differential responsiveness to these socio-chemical cues. For example, one of the reasons why an ant behaves as recruit is that it tends to remain in the locations where the first phase of recruitment takes place (i.e. in populated areas marked with colony odor). By contrast, the lower sensitivity of scouts to nest-related chemical cues or to trail pheromone makes them more likely to depart from the spatial core of colony activities and to visit new areas that are still unexplored by the colony. This spatial structuring is a fundamental aspect for generating division of labor (Jeanson and Weidenmuller 2014, Pamminger et al. 2014) since the signals that trigger the response to a specific task are often available only at specific locations. The coupling of a lower response threshold to a task-associated stimulus and a higher responsiveness to cues related to locations specific to this task at the individual level would allow the emergence of a self-organized division of labor at the colony level.

As a word of caution, we would like to emphasize the fact that our experiments did not investigate causality in the relationship between task allocation and behavioral “phenotypes”. We can only speculate on which factors drive the intrinsic differences between the three functional groups of workers we studied. In social insects, age and/or individual experience is known to influence the sensitivity of individuals to task-related chemical cues, such as, for example, the repeated exposure to chemical cues that lower the response threshold of individuals to alarm pheromone (Norman et al. 2014). Likewise, together with a higher motivation to attack, the responsiveness of workers to non-nestmate odors develops whilst aging and changing tasks, from brood nursing to foraging.

Along with the changes in task, the brain and olfactory organs can also undergo alterations as individuals age (Withers et al. 1993; Gronenberg et al. 1996; Fahrbach et al.1998; Muscedere and Traniello 2012 but see Giraldo at al. 2016). We suggest a similar scenario of age polyethism among foraging ants. For instance, in the fire ant Solenopsis invicta, the forager population can be divided into a younger group of recruitable workers that wait for older scouts to activate them to help retrieve large food finds (Tschinkel 2011). On the basis of their life expectancy, recruits of Pogonomyrmex rugosus ants also seem to be younger than foragers (Oetler & Johnson 2008). Likewise, in the black garden ant L.niger, recruits might be rather young foragers whose response to colony-related cues, as well as to the trail pheromone, progressively decreases with age and/or with their individual experience outside the nest. Over time, these recruits might then become more likely to explore areas not occupied by their colony and thus act as scouts. Still to be investigated is the question of whether the weak behavioral response of scouts to area marking and to trail pheromone is related to a generalized lower ability to perceive chemical cues due to aging or to a shift of sensitivity towards other stimuli due to experience. The plasticity of olfactory sensitivity could make the spatial location of ant foragers change over time so that they face different task-related stimuli. As a result, this could generate heterogeneity in task performance and ultimately self-organized division of labor at the colony level.

5. Acknowledgments

Claire Detrain is a Research Director from the Belgian National Fund for Scientific Research (F.N.R.S) and Hugo Pereira is supported by a Belgian PhD grant from the F.R.I.A. (Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture). The authors warmly thank Nell Foster for proofreading the English as well as two anonymous referees and the editor for providing constructive comments on the manuscript.

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Figure Captions

Fig. 1: Boldness test. The thigmotaxis index represents the ratio of the time spent by ants close to the arena walls over the time spent in the center of the experimental area. The horizontal bar within the boxes represents the median, the upper and lower boundaries of the boxes represent respectively the 75th and 25th percentiles, whilst the whiskers extend to the smallest and largest values within 1.5 box lengths. The circles represent the outliers. Number of tested scouts (n=34), patrollers (n=38) and recruits (n=45).

Fig.2: Social attraction to nestmates. The social attraction index is given by the difference between the total time spent by an ant individual close to the grid when the end chamber was hosting nestmates and when this chamber was empty. We used a Wilcoxon test to compare the difference index for each functional group to the theoretical value of 0, which would be expected in the case of no social attraction towards nestmates. * : p<0.05; ** : p<0.01; *** p< 0.001. No significant difference was found in the social attraction index between the three functional groups (Kruskal-Wallis test, p=0.41). Number of tested scouts (n=33), patrollers (n=34), recruits (n=41). See figure 1 caption for other information about boxplots

Fig. 3: Attraction to colony odor. Attraction scores of individuals towards the Y-maze branch marked with homocolonial odor were calculated over six successive tests and thus varied from 0 to 6. Kruskall Wallis test followed by a post_hoc Nemenyi test for pairwise comparisons. Boxes with different letters are significantly different at a significance level of 0.05. Scouts (n=31), patrollers (n=33), recruits (n=35). See figure 1 caption for other information about boxplots

Fig. 4: Survival curves of the proportion of individual ants still following an artificial trail drawn with 50µL from a 0.04 rectal gland concentration for the groups of scouts (n=24 dashed line), patrollers (n=25 black line) and recruits (n=31 grey line)

Figure 1

Figure 2

Figure 3

Figure 4

Solvent

0.008 gland

0.040 gland

0.080 gland

0.400 gland

0.800 gland

Scouts

N=24

9 A

[6-12]

10 A

[7-16]

14 A

[10-23]

19 A

[12.5-25]

30.5 A

[18.5-66]

46.5 A

[25.5-111]

Patrollers

N= 25

9 A

[4-12]

15 A

[6-29-]

21 B

[11-35]

27 A

[12-46]

67 A

[18.5-112]

70 A

[37-120]

Recruits

N=31

7 A

[3-13]

13 A

[9-17.5-]

27 B

[12.5-34.5]

24 A

[13.5-44.5]

55 A

[25-101]

73 A

[53.5-119]

Kruskal-Wallis test

H=0.80

p=0.670

H=1.31 p=0.520

H=6.10 p=0.047

H=2.69 p=0.260

H=5.38 p=0.068

H=1.32 p=0.520

Table 1: Trail-following response. Median, first and third quartile value of trail-following scores are given. Within each group, pairwise comparisons were made between the trail-following score elicited by the solvent and each tested concentration of rectal gland with a Wilcoxon test. Trail following scores that were significantly higher than the response elicited by the solvent alone are indicated in bold. For each trail drawn with the same concentration of rectal gland, we compared the responses of the three groups of foragers by using a Kruskall Wallis test followed by a pairwise Nemenyi post-hoc test when significant. Values from the same column that share a common letter are not statistically different.

Variable

Estimate

+ SD

HR

Z score

P value

Recruits VS Patrollers

0.17

0.28

1.19

0.62

0.540

Recruits VS Scouts

0.84

0.29

2.32

2.91

0.004

Patrollers VS Scouts

0.67

0.30

1.96

2.27

0.023

Table 2: Effect of group affiliation on the trail-following response of ant foragers to a 0.040 rectal gland extract. Values of estimate parameters + standard deviation (SD), and hazard ratios (HR) of a Cox proportional hazard regression model are given. Z scores and the associated P-values compare the likelihood of an ant leaving the trail to the reference condition (given in bold).