Bundling and segregation affect pheromone deposition, but not choice, in an ant

  1. Massimo De Agrò  Is a corresponding author
  2. Chiara Matschunas
  3. Tomer J Czaczkes
  1. Animal Comparative Economics laboratory, Department of Zoology, University of Regensburg, Germany
  2. Department of Biology, University of Florence, Italy

Abstract

Behavioural economists have identified many psychological manipulations which affect perceived value. A prominent example of this is bundling, in which several small gains (or costs) are experienced as more valuable (or costly) than if the same total amount is presented together. While extensively demonstrated in humans, to our knowledge this effect has never been investigated in an animal, let alone an invertebrate. We trained individual Lasius niger workers to two of three conditions in which either costs (travel distance), gains (sucrose reward), or both were either bundled or segregated: (1) both costs and gains bundled, (2) both segregated, and (3) only gains segregated. We recorded pheromone deposition on the ants’ return trips to the nest as measure of perceived value. After training, we offer the ants a binary choice between odours associated with the treatments. While bundling treatment did not affect binary choice, it strongly influenced pheromone deposition. Ants deposited c. 80% more pheromone when rewards were segregated but costs bundled as compared with both costs and rewards being bundled. This pattern is further complicated by the pairwise experience each animal made, and which of the treatments it experiences first during training. This demonstrates that even insects are influenced by bundling effects. We propose that the deviation between binary choice and pheromone deposition in this case may be due to a possible linearity in distance perception in ants, while almost all other sensory perception in animals is logarithmic.

Editor's evaluation

This innovative study investigates the presence of biases in value perception in ants. The authors were able to show that the distribution of rewards and costs influences perceived reward value in ants, an effect that was observed for pheromone deposition but not choice behaviour.

https://doi.org/10.7554/eLife.79314.sa0

Introduction

Broadly speaking, when an animal must choose between options, it can employ one of three different strategies, characterized by different levels of precision: random choice; following a heuristic or rule of thumb; or comparison of outcome value and choice for the highest. Traditional economic theory, exemplified by expected utility theory, assumes (human) decision-makers are rational and perform strict value-based choice (Mankiw, 2011). The now well-established field of behavioural economics has vigorously pushed back against this idea, demonstrating that humans often make decisions which are not fully logical, economically rational, or ‘optimal’ (Camerer et al., 2011; Tversky and Kahneman, 1974), even when actively comparing options.

A major insight lying at the heart of behavioural economics is that value is perceived. This can lead directly to deviations from optimality: between the acquisition of the information and the evaluation of possible outcomes, something gets lost in translation. As has been well established for over a century by the study of pyschophysics, perception is non-linear, usually on a logarithmic scale (Gescheider, 1997). Value perception for humans is likewise non-linear, as famously stated by Kahneman and Tversky, and extensively demonstrated thereafter (Camerer, 2004; Kahneman and Tversky, 1979). Moreover, humans weigh losses more strongly than gains. Finally, value is relative, usually to an expectation or some sort of anchor, see Figure 1.

Simplified schematic of prospect theory from Kahneman and Tversky, 1979, with a graphical illustration of bundling and segregation.

On the x-axis, actual value of a gain or a loss (here exemplified with money). Perceived utility does not scale linearly with value, but logarithmically. Receiving a gain of €100 (segregated) twice will produce a level of ‘happiness’ of ‘2s’, more than the level ‘b’ perceived when receiving €200 all together (bundled). The same is true for ‘losing’ the same amounts, where two losses of €100 are felt stronger than a single one of €200.

The non-linear nature of perceived value results in many behavioural biases, a very prominent one being the bundling vs. segregation effect. Crucial to the current experiment, the perceived value of a compound item or option can be changed by presenting it either as one option (bundling) or as multiple small parts (segregation). Due to diminishing returns, bundling results in a weaker total sensation than segregation – either lower value for positive value options or lower cost for negative value options (see Figure 1). This is because more of the sensation occurs on the shallow part of the curve. This fact is regularly exploited by consumer psychologists and marketing experts, for example by bundling option when selling new cars: when spending €50,000 on a new car, spending €51,000 for the model with included sound system might not be experienced as painfully costly, even if, considered by itself, €1000 might be more than most people are willing to spend on a sound system for a car. Bundling and segregation have been extensively studied by consumer psychologists (Johnson et al., 1999; Naylor and Frank, 2001; Noone and Mattila, 2009).

As in the study of human economics, non-human animals have been treated and modelled as rational economic agents, leading to deep insights into animal behaviour via the optimal foraging theory framework (Davies, 2012; Emlen, 1966; MacArthur and Pianka, 1966; Pyke et al., 1977). However, often inspired directly by behavioural economic research on humans, predictable deviations from optimality and rationality have been described (Zentall, 2015). For example, pigeons, rats, and ants all show a preference for high-effort over low-effort associated options (Clement et al., 2000; Czaczkes et al., 2018a; Lydall et al., 2010), much as humans do (Norton et al., 2012). Similarly, much as in humans, the addition of an irrelevant option in an option set (a ‘decoy’) can change the preference structure in many animals, including birds, cats, bees, and ants (Bateson et al., 2002; Bateson et al., 2003; Parrish et al., 2015a; Sasaki and Pratt, 2011; Scarpi, 2011; Schuck-Paim et al., 2004; Shafir et al., 2002).

Ants and bees also show relative value perception, changing the perceived value of an option depending on their expectations (Bitterman, 1976; Couvillon and Bitterman, 1984; Wendt et al., 2019; Wendt and Czaczkes, 2020). An ant which is trained to expect a very sweet reward, for example, will be more likely to reject a moderate reward than an ant which was expecting a moderate one – a negative contrast. Likewise, ants expecting a very mild reward are more likely to accept a moderate reward than ants which were expecting that quality – a positive contrast (Wendt et al., 2019). These changes are also mirrored in the ants’ deposition of recruitment pheromone: ants deposit more pheromone to resources they perceive as higher quality (Beckers et al., 1993; Jackson and Châline, 2007), and indeed ants deposit more pheromone for moderate rewards if they had been expected poor quality, and less pheromone if they were expecting high quality. This demonstrates that a key aspect of prospect theory – relative value perception – is present in insects as well as in humans.

Crucially for the current experiment, there is evidence that social insects, especially ants, perceive value logarithmically. This is perhaps not surprising, given than logarithmic perception is a key assumption of the well-established Weber-Fechner law (Fechner, 1860; Weber, 1834), which describes the psychophysics of perception. Wendt et al., 2019, demonstrated a faster rise in food acceptance in the lower range of acceptance food qualities (e.g. from 0.1 to 0.3 molar) than in the higher range (e.g. from 0.5 to 1.5 molar). Recently, De Agrò et al., 2021, demonstrated that Lasius niger ants have a strong aversion to risky food sources (i.e. with fluctuating quality), which could be fully explained by logarithmic value perception, as predicted by prospect theory. Ants prefer a certain food source offering 0.55 M sucrose to one which fluctuates between 0.1 and 1.0, but if the options are logarithmically balanced (0.3 M vs. 0.1 or 0.9), ants are completely indifferent.

While the bundling vs. segregation effect is extremely well studied in humans, surprisingly, to our knowledge no attempt has been made to examine it in animals. In a related study, chimps were shown to prefer whole rewards (potato chips) to rewards which were broken into smaller pieces, even when the broken rewards had an higher absolute quantity (Parrish et al., 2015b). However, consumption of the whole and broken rewards took the same amount of time, and rewards were chosen before consumption could begin, so it is likely that both broken and whole rewards were considered as one unitary bundle.

In order to study bundling and segregation in ants, we segregated rewards spatially, using small sucrose drops along a linear runway. However, while this segregates rewards, it also segregates a potential cost – the walking distance to the reward. Thus, we first demonstrate that ants do indeed prefer closer to farther food sources, and thus that food distance is considered a cost. We then train individual ants to two of three treatments: rewards and costs bundled (‘bundled’), rewards and costs segregated (‘segregated all’), and rewards segregated but costs bundled (‘segregated reward’). We record pheromone depositions on the ant’s return from each treatment type, and then ask ants to choose between the pair of treatments they were trained on. We predicted that the ‘segregated reward’ treatment would be the preferred option, as bundling of costs should minimize their impact, and segregation of rewards should boost theirs. As we did not know the relative strength of the rewards and costs, we had no strong a priori predictions about the relative perception of ‘bundled’ and ‘segregated all’. However, as prospect theory predicts that losses are weighted more strongly than gains (Kahneman and Tversky, 1979), we had a weak expectation that ‘bundled’ would be perceived as slightly better than ‘all segregated’.

Materials and methods

Subjects

We used a total of 144 L. niger individuals, coming from 19 queen-less colony fragments, consisting of around 1000 ants each. Sample size was set a priori, based on the time and resources available (Lakens, 2022), while maintaining a minimum amount of ants per condition based on our previous experience with these types of experiment. Each fragment was collected from a different wild colony on the University of Regensburg campus. Workers from colony fragments forage, deposit pheromone, and learn well (Evison et al., 2008; Oberhauser et al., 2018). Each fragment was housed in a transparent plastic box (30 × 20 × 40 cm3), with a layer of plaster on the bottom. A circular plaster nest, 14 cm in diameter and 2 cm thick, was also provided. The colonies were kept at room temperature (21–25°C) and humidity (45–55%), on 12:12 light:dark cycle for around 9 months. Each colony was fed exclusively on 0.5 M sucrose solution ad libitum, and deprived of food 4 days prior to each test. Water was provided ad libitum and was always present.

Procedure

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All four experiments reported in this paper used a conditioning procedure described in Czaczkes, 2018c. The procedure was generally the same, with a few modifications dictated by the specific conditions.

For each tested subject the procedure started by connecting a drawbridge to the nest box. This bridge was composed of a 20 cm long, 1 cm wide, slanted section, one end of which laid on the plaster floor of the nest box. The other end led to a straight 10 cm long, 1 cm wide, runway section. Both of these were covered by unscented paper overlays. Depending on the visit, this bridge could lead either to a straight runway or to the stem of a Y-maze. In the first visit, multiple ants were allowed on the bridge. A 0.5 M sucrose solution drop was placed at the end of the bridge. The first ant to reach the drop and start drinking was marked on the abdomen with a dot of acrylic paint. The non-marked ants were gently returned to the nest box, while the marked one was allowed to drink to satiation, then allowed to return to the nest on her own. In the nest the marked ant performed trophallaxis (mouth to mouth food transfer) with nest-mates, and then returned to the bridge location ready for the following visit, which varied depending on the experiment being run.

Pilot experiment – Is increased food distance negatively perceived?

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As described in the Introduction, segregated rewards should be perceived as being of higher value than an equal-quality bundled alternative. However, this is also true for punishments. Since in our experiment we segregated rewards by placing them on different parts of a long runway (see next paragraph), we needed to test first whether increased travelled distance makes rewards less preferred.

The previously marked ant was allowed onto the bridge. This time, the bridge was attached to either a 25 or 75 cm long runway (systematically varied). This runway was covered with a scented paper overlay (either rose or lemon odour). Scenting was achieved by storing the overlays in a sealed plastic box with three drops of food flavouring for at least 24 hr. At the end of the runway, we placed a high-quality (1.5 M) sucrose solution drop, flavoured with the same smell as the runway (at a ratio of 1 µl food flavour per ml sucrose solution). The ant eventually found the drop, drank to satiation, and then went back to the nest. At this point, we discarded the scented overlays, in order to remove the deposited pheromone.

As soon as the ant unloaded, it was allowed back onto the bridge again. The bridge now connected to the reciprocal runway length (25 cm if the previous visit was to 75, and vice versa). This runway was covered with paper scented with a different smell to the previous visit. At the end of the runway, the ant again found a drop of 1.5 M sucrose solution, again flavoured to match the paper overlay. After drinking, the ant again allowed to return to the nest.

The same procedure was repeated another time. Thus, the ant experienced the long and the short runways twice each. For all visits, we measured the number of times the ant deposited pheromone on the scented runway, both the way towards the drop and the way back. Pheromone deposition in L. niger is a stereotyped behaviour, in which an ant pauses for c. 0.2 s, and curls its abdomen down, pressing it firmly onto the substrate (Figure 2A, Video 1). This behaviour is easily quantified by eye (Beckers et al., 1993). Pheromone deposition co-varies with the (perceived) value of a resource, with ants making more depositions for resources they consider to be high quality (Beckers et al., 1993; Wendt et al., 2019; Wendt and Czaczkes, 2020).

Description of the two dependent variables recorded.

In (A), a marked Lasius niger ant pauses and presses its abdomen to the runway, leaving pheromone mark. See Video 1. The number of pheromone depositions was counted. Photo : Julia Giehr. (B) shows a schematic representation of the Y-maze used for the binary choice test. Coming from the nest, the ant walks on an unscented runway, constituting the Y-maze stem, until it reaches the bifurcation. The two Y-maze arms were scented with two different odours, corresponding to the ones present during the previous visits and associated with the two experienced treatments. The bifurcation tapers in the middle to ensure that the ant senses both odours before making a choice.

Video 1
Pheromone deposition example.

After these four visits, the ant was allowed onto the bridge one last time. This time, the bridge was connected to the 10 cm long stem of a Y-maze (Figure 2B). The stem was covered with unscented paper, and tapered to a 2 mm wide point. Here, the two arms of the Y-maze started, also tapered, in order to ensure that the ant to contact both arms at the same time once at the end of the stem. One of the two arms was scented with the long runway odour, while the other was scented with the short runway odour. We noted on which of the two arms the ant ran for at least 2 cm (considered the ‘initial’ decision), and at the end of which of the two it arrived first (considered the ‘final’ decision). Once this happened, the ant was picked up with a piece of paper and moved back at the start of the stem to be retested. This way, we could test the ants’ preference three times. After the test, the ant was permanently removed from the colony.

A total of 24 ants from five different colonies was used for this experiment.

Main experiment – bundling vs. segregation

Having established that increased travel distance makes food sources less attractive (see Results), we proceeded with the main experiment.

The procedure is very similar to the pilot. Each marked ant was allowed onto the bridge, after which it was presented with a scented runway. This scented runway could correspond to one of three different treatments:

‘Segregated all’ (rewards and costs) (Figure 3A)

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The three possible experimental treatments.

Grey shapes represent the runway segments, each 25 cm long, 1 cm wide, tapering to 2 mm to ensure that ants encounter the sucrose drops. Big blue circles represent ad libitum 1.5 M sucrose solution, small circles represent 0.2 µl drops which the ants can drink, but will not satiate them. In (A) ‘segregated all’, both the costs (travel over the runways) and the rewards (drops of 1.5 M sucrose, blue circles) are segregated. In (B) ‘segregated rewards’, only the rewards are segregated. In (C) ‘bundled’ both the costs and the rewards are bundled.

The ant encountered a 75 cm long runway. Every 25 cm, the runway tapered to 2 mm in width with a 0.2 µl, 1.5 M, sucrose solution drop on the taper. After the drop, the runway widened again, until the next 25 cm taper. Here, the ant found a second drop, identical to the previously encountered one. The runway proceeded for a third 25 cm section, ending in a large drop. To avoid evaporation, as well as limiting the risk of the ant bypassing the rewards without noticing, the drops were delivered with a micropipette when the ant reached the designated position, rather than being placed prior to testing.

In this treatment, the reward (drops) was segregated into three different experiences, and as such its combined value should be perceived as higher than one being presented as a single one. The volume of the first two drops was selected in order to ensure that the ant would reach the third drop without becoming satiated after the first or the second one: The crop volume of L. niger foragers is under 1 µl (Mailleux et al., 2000), and ants which encounter such drops drink them and then continue walking forwards (Czaczkes et al., 2019). The last drop instead was much larger, to ensure that ants could drink to satiation, and thus avoiding other possible discounting effects, such as disliking not being completely satiated (Mailleux et al., 2006; Mailleux et al., 2005).

However, in this treatment also the cost is segregated: Rather than being experienced as a single 75 cm long runway, the ant encountered three 25 cm ones, interspersed by rewards. Thus, this condition is expected to enhance both the perceived value and the perceived cost.

‘Segregated reward’ (bundled costs) (Figure 3B)

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In this second treatment, the ant encountered a 75 cm long runway. At every 25 cm mark, a narrowing portion was present, but in this treatment no small sugar drops were provided. The narrowing of the paper was maintained to ensure consistency with the previous treatment. To assure consistency among treatments, the experimenter followed the same procedure of the ‘segregated all condition’: the micropipette was brought to the narrowing point at the end of the 25 cm runway when the ant got near it and the plunger depressed delivering no drop (i.e. a sham treatment). At the end of the 75 cm, two 0.2 µl, 1.5 M drops were presented in short succession, just 5 mm from each other. After another 5 mm, the third ad libitum drop was placed. Thus, in this treatment, the distance travelled (=cost) remained bundled, while the reward was still segregated.

‘Bundled’ (rewards and costs) (Figure 3C)

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In this third treatment, the ants encountered the same runway as above. However, instead of presenting three drops at the end, only the third ad libitum drop was offered. Thus, in this treatment, both the reward and the cost are bundled. The sham pipetting was also carried out in this treatment.

Pairwise training and a priori hypotheses

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We performed three different conditions, corresponding to the three different pairings of the three treatments (A vs. B, A vs. C, B vs. C). Forty ants were tested per condition, for a total of 120. Each ant would experience one of the three treatments for the first visit, associated with a distinct odour. On the subsequent one, the animal encountered a second treatment, associated with another odour. This procedure was repeated for four times, for a total of eight visits alternating between the two selected treatments. This way, each ant experienced four times each treatment. On the way back to the nest, we counted the pheromone deposited by each ant (Figure 2A) in each of the three runway sections. In the end, the ant was presented with a Y-maze, and had to choose between the two treatment odours. As for the pilot experiment, the Y-maze test (Figure 2B) was repeated three times, by picking up the ant immediately upon reaching the end of the chosen arm and placing it back at the start of the stem.

Broadly, we expect the following preference structure:

  • In condition 1 (B vs. C), the ‘segregated reward’ treatment should be preferred over the ‘bundled’ one. This is expected due to the introduced bundling effect.

  • In condition 2 (B vs. A), the ‘segregated reward’ treatment should be preferred over the ‘segregated all’ one. This is expected as the boosting of reward by segregation effect is identical, while the ‘segregated all’ condition also boost the cost.

  • In condition 3 (A vs. C), the ‘bundled’ treatment should be preferred over the ‘segregated all’ one. This is because costs are often more heavily weighted than gains (Lakshminarayanan et al., 2011; Tversky and Kahneman, 1981), and so boosting both rewards and costs by the same factor will tend to emphasize the costs more. Note that this was a tentative, and weak, expectation, as we had no a priori way of knowing how the cost of walking a set distance compares to a set sucrose reward.

Data analysis

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The entire statistical analysis code, including data handling, figure code, and analysis results, is presented in supplement ESM2. Raw data is available in supplement ESM1. All the statistical analyses were performed in R 4.1.2 (R Development Core Team, 2020). The packages readODS (Schutten et al., 2020) and reshape2 (Wickham, 2007) were used to load and prepare the data. We focused on two measures: the binomial choice at the last experimental visit, and the number of pheromone depositions during the training visits for the different options.

To analyse the former, we employed generalized mixed effect models with a binomial distribution using the package glmmTMB (Brooks et al., 2017; Magnusson et al., 2020). In every experiment, we included as predictors the choice order (first, second, or third visit to the Y-maze) and decision line (initial decision, passed the first 2 cm line; or final decision, reached the end of the arm). The ant identity nested in the colony of origin was included as a random effect. The goodness of fit was evaluated with the package ‘DHARMa’ (Hartig, 2018). We performed an analysis of deviance to observe the effect of the predictors using the package car (Fox and Weisberg, 2011), and then performed Bonferroni-corrected post hoc analysis on predictors that have an effect using the package emmeans (Lenth et al., 2020). Lastly, we computed effect sizes (Ben-Shachar et al., 2020).

For pheromone deposition we followed the same procedure. We employed GLMM with a Poisson error structure, varied into a Tweedie error structure when DHARMa testing suggested that as appropriate. In the pilot, we included direction (to the drop or back to the nest) and visit length (short or long) as predictors. For the other three conditions, we included runway section (nearest to the nest, middle, nearest to the end) and treatment of the visit (‘bundled’, ‘segregated all’, ‘segregated reward’). The ant identity nested in the colony of origin was included as a random intercept, with repeated visits as a random slope. The random effect was simplified by removing the repeated visits in models that failed to converge. We then followed up with analyses of deviance and post hoc analyses as well.

In the pilot experiment, we expected the ants to deposit more pheromone on the long runway, independently of their preference. All things being equal, a triple-length runway will offer triple the pheromone deposition time. To control for this bias, we multiplied the observed pheromone deposited on the short runway by three. However, L. niger are reported to deposit more pheromone nearer to the food source (Beckers et al., 1992). As such, a simple multiplication may have still not be fully appropriate as a comparison. The pheromone deposition for the pilot experiment is reported for completeness, but we thus advise caution in the interpretation of this data. This is not a problem for the three experimental conditions as the runway length remains fixed.

After analysis, the data was then passed onto a Python 3 (Van Rossum and Drake, 2009) environment using the package reticulate (Ushey et al., 2021), to produce graphs. To achieve this, we used the libraries pandas (Reback et al., 2020), numpy (Oliphant, 2006; van der Walt et al., 2011), matplotlib (Hunter, 2007), and seaborn (Waskom et al., 2017).

Examining the results of the aforementioned model, we discovered a possible contrast effect, as depending on the condition, the amount of pheromone deposited for the same treatment changed abruptly. It is indeed crucial to consider how the first experience of ants can have ripple effect on its subsequent decisions (De Agrò et al., 2021). To further examine this effect we remodelled the data including the first experienced treatment as a factor. We will present the results of the model that included and that did not include the first experienced treatment as a factor separately. The full analysis, and all the raw data, can be found in the supplements.

Results

Pilot experiment – cost of travelled distance

In the pilot experiment, the ants were asked to choose between two odours: one associated with a short runway and the other associated with a long one. We observed a 90% probability of the ants choosing the short-associated odour when encountering the Y-maze for the first time, significantly higher than chance level (GLMM post hoc: prob.=0.896, SE = 0.056, DF = 136, t=3.589, p=0.0014). The probability quickly dropped to chance level for the two subsequent visits (visit 2: prob.=0.547, SE = 0.124, DF = 136, t=0.376, p=1; visit 3: prob.=0.484, SE = 0.123, DF = 136, t=−0.129, p=1). This is generally to be expected in this type of experiment, as the lack of a reward and manipulation easily disrupts the ant decision.

The pheromone deposition analysis confirms this pattern, as indeed ants deposit double the amount of pheromone per unit length on the short runway over the long one (GLMM post hoc ratio = 2, SE = 0.231, DF = 185, t=5.973, p<0.0001).

Main experiment – bundling vs. segregation

Condition 1: ‘Segregated reward’ vs. ‘bundled’

In this experiment, we expected ants to prefer the ‘segregated reward’ treatment over the ‘bundled’ one due to bundling.

We observed an odd difference between the three subsequent tests on the Y-maze (GLMM ANODA, chi-square=9.5744, DF = 2, p=0.0083). Specifically, the ants showed no significant preference in the first visit (GLMM post hoc: prob.=0.516, SE = 0.0693, DF = 232, t=0.227, p=1) nor in the third (prob.=0.486, SE = 0.0693, DF = 232, t=−0.195, p=1). However, they significantly preferred the segregated option in the second visit (prob.=0.729, SE = 0.0.0596, DF = 232, t=3.279, p=0.0036). We consider this a false positive (see Discussion).

For the pheromone deposition (Figure 4), we observed no difference between the treatments (GLMM ANODA, chi-square=2.0487, DF = 1, p=0.1523). Here, we also observed a difference between the three sections (chi-square=107.2664, DF = 2, p<0.0001), and there was no effect of the interaction between the two other predictors (chi-square=0.6412, DF = 2, p=0.73). Specifically, there was no difference in the pheromone deposited for the ‘segregated reward’ option than for the ‘bundled’ one (GLMM post hoc: ratio = 0.71, SE = 0.1735, DF = 948, t=−1.399, p=0.6482). The ants deposited overall more pheromone on the section of the runway nearest the drop in respect to the second (ratio = 1.55, SE = 0.0955, DF = 948, t=7.124, p<0.0001) and in the second in respect to the third (ratio = 1.2, SE = 0.0842, DF = 948, t=2.599, p=0.038) section.

Modelled pheromone deposition for each treatment, across the three condition.

Y-axis: amount of deposited pheromone per runway section. Error bars represent standard error. In yellow, pheromone deposited in the ‘bundled’ vs. ‘segregated reward’ condition (n=40). The two treatments are not significantly different from each other (glmm post-hoc p=0.6482). In green, pheromone deposited in the ‘segregated all’ vs. ‘segregated reward’ condition (n=40). The two treatments are significantly different from each other (glmm post-hoc p<0.0001). In blue, pheromone deposited in the ‘segregated all’ vs. ‘bundled’ condition (n=40). The two treatments are significantly different from each other (glmm post-hoc p=0.022). All p-values are corrected for multiple testing.

Regarding the effect of the first encountered treatment (Figure 5), we observed an effect of the first encountered treatment (GLMM ANODA, chi-square=5.8514, DF = 1, p=0.01556). The effect disappears when looking at the post hoc (GLMM post hoc: ratio = 0.542, SE = 0.139, DF = 830, t=−2.382, p=0.1048), probably due to the weight of Bonferroni correction. Even if not significant, it seems that ants deposited slightly more pheromone for the segregated reward option when it was encountered first in respect to when it was encountered second (ratio = 0.354, SE = 0.141, DF = 830, t=−2.613, p=0.0549).

Modelled pheromone deposition for each treatment, across the three condition, including the influence of the first experienced option.

Y-axis: amount of deposited pheromone per runway section. Error bars represent standard error. n=40 for each condition. When ‘segregated reward’ is encountered first, in condition 2, the overall pheromone deposited is higher in respect to the ‘segregated all’ option (glmm post-hoc p=0.0022). In condition 3, when the ‘bundled’ option is encountered first, the overall pheromone deposited is lower in respect to the ‘segregated all’ option (glmm post-hoc p=0.0001).

Condition 2: ‘Segregated reward’ vs. ‘segregated all’

In this experiment we expected the ‘segregated reward’ treatment to be preferred over the ‘segregated all’ treatment.

We found no difference between subsequent Y-maze visits (GLMM ANODA: chi-square=3.1754, DF = 2, p=0.2044), and we observed no overall significant preference in the Y-maze test (GLMM post hoc: prob.=0.524, SE = 0.06, DF = 232, t=0.402, p=0.688).

Regarding pheromone deposition (Figure 4), we observed a difference between the treatments (GLMM ANODA, chi-square=60.7675, DF = 1, p<0.0001) and a difference between the three sections (chi-square=10.4118, DF = 2, p=0.0055). We did not observe a statistically significant effect of the interaction (chi-square=5.667, DF = 2, p=0.0588). Specifically, the ants deposited more pheromone for the ‘segregated reward’ option compared to the segregated cost one (GLMM post hoc: ratio = 0.555, SE = 0.0422, DF = 945, t=−7.746, p<0.0001). In the ‘segregated reward’ visits, but not the ‘segregated all’ visits, the ants deposited more pheromone on the section of the runway nearest the drop relative to the furthest one (ratio = 1.58, SE = 0.1809, DF = 945, t=3.995, p=0.0007).

Regarding the effect of the first encountered treatment (Figure 5), the ants deposited overall more pheromone when they encountered the ‘segregated reward’ option first, irrespective of the currently experienced treatment (GLMM post hoc: ratio = 0.472, SE = 0.0991, DF = 830, t=−3.576, p=0.002). We also observed that when the ‘segregated reward’ treatment was encountered first, the ant significantly preferred it to the ‘segregated all’ option (ratio = 0.296, SE = 0.1081, DF = 830, t=−3.333, p=0.0054) section. However, when they encountered the ‘segregated all’ treatment first the ants showed no difference (ratio = 0.703, SE = 0.2536, DF = 830, t=−0.976, p=1). Ants deposited more pheromone for the ‘segregated reward’ option when it was encountered first in respect to when it was encountered second (ratio = 0.306, SE = 0.1092, DF = 830, t=−3.318, p=0.0057).

Condition 3: ‘Segregated all’ vs. ‘bundled’

In this experiment, ‘bundled’ treatment was expected to be preferred over the ‘segregated all’ one.

The ants showed no overall significant preference for either treatment (GLMM post hoc prob.=0.466, SE = 0.122, DF = 232, t=−0.279, p=0.7803).

However, we saw large differences in pheromone deposition between the treatments (Figure 4). We found an effect of the two treatments (GLMM ANODA, chi-square=9.9822, DF = 1, p=0.00158), of the three runway sections (chi-square=24.606, DF = 2, p<0.0001) and of the interaction between those (chi-square=13.3183, DF = 2, p=0.00128). Specifically, ants deposited more pheromone in the ‘bundled’ visits than in the segregated cost ones (GLMM post hoc: ratio = 0.0505, SE = 0.1124, DF = 948, t=−3.07, p<0.0001). Moreover, in the ‘bundled’ visits, they deposited more pheromone on the runway section nearest the drop relative to the the middle section (ratio = 1.534, SE = 0.1427, t=4.6, p<0.0001) or the section nearest the bridge (ratio = 1.701, SE = 0.1626, DF = 948, t=5.557, p<0.0001). This pattern was not present in the segregated cost visits (first vs. second section: ratio = 1.055, SE = 0.1229, DF = 948, t=0.459, p=1; first vs. third section: ratio = 1.007, SE = 0.1161, DF = 948, t=−0.06, p=1).

Regarding the effect of the first encountered treatment (Figure 5), the ants deposited overall more pheromone when they encountered the ‘segregated all’ option first, irrespective of the currently experienced treatment (GLMM post hoc: ratio = 2.909, SE = 0.643, DF = 830, t=4.832, p<0.0001). We observed that when the ‘bundled’ treatment was encountered first, the ant significantly preferred it to the ‘segregated all’ option (ratio = 0.322, SE = 0.120, DF = 830, t=−3.032, p=0.015) section. Instead, when they encountered the ‘segregated all’ treatment first the ants showed no difference (ratio = 0.916, SE = 0.305 t=−0.263, p=1). Ants deposited more pheromone for the ‘segregated all’ option when it was encountered first in respect to when it was encountered second (ratio = 4.905, SE = 1.787, DF = 830, t=4.365, p=0.0001).

Discussion

Bundling and segregation treatment strongly affected the ants’ pheromone deposition, but not their choices. No consistent preference was found in the binary choices of the ants from the main study. While ants significantly preferred the segregated reward over the bundled reward in their second of three trials, we can conceive of no plausible biological or psychological reason for this to be so, and interpret this as a false positive. Thus, there is no evidence that bundling or segregation affect choice.

However, the pattern of pheromone deposition, which correlates strongly with perceived food quality in these and other ants (Beckers et al., 1993; Czaczkes et al., 2018b; Jackson and Châline, 2007; Wendt et al., 2019), was partially in line with our predictions: ants deposited most pheromone when the costs were bundled and rewards segregated, and less in the other two treatments (Figure 4). Our weaker prediction of ‘bundled’ presenting a higher deposition than ‘segregated all’ was also supported. Thus, pheromone deposition seems to be in line with predictions from classical behavioural economic and perception research (Camerer, 2004; Johnson et al., 1999; Kahneman and Tversky, 1979; Naylor and Frank, 2001; Noone and Mattila, 2009). Specifically, we observed more deposition for ‘bundled’ over ‘segregated all’, and for ‘segregated reward’ over ‘segregated all’. By contrast, the preference for ‘segregated reward’ over ‘bundled’ is not significant. This seems counter-intuitive, as the ‘bundled’ and ‘segregated reward’ options only differ in the boosting of a gain. Moreover, the contrast of ‘segregated all’ vs. ‘bundled’ – for which we had no strong a priori prediction as we had no idea how gains and losses are weighted – shows a very clear difference.

The observed result suggests two, non-mutually exclusive, interpretations: Either ants experience a strong segregation effect for losses, and a very mild one, if any, for gains; or our treatment failed to segregate rewards effectively. If the first interpretation is true, the ‘bundled’ and the ‘segregated reward’ treatments will be perceived as almost identical. In the contrast between ‘segregated all’ and ‘bundled’, the higher number of depositions for bundled makes sense, as the segregation of losses drives the preference completely. The higher deposition for ‘segregated reward’ over ‘segregated all’ remains equally clear, as the two options only differ in the realm of losses. If our second interpretation is correct, the three sucrose solution drops, especially in the ‘segregated reward’ condition, are still considered by the animal as a single (i.e. bundled) reward. In the ‘segregated all’ condition the separation between rewards seems more believable, however the results observed strongly suggest that the reward segregation have a very low effect on the perceived value, being overridden by the segregation of losses. It is impossible to disentangle these two options from the current data.

However, the pattern is somewhat more complicated, as we saw large differences in pheromone deposition for the same treatment, depending on the treatments they were paired with. For example, while ants encountering the ‘segregated rewards’ deposit twice as much pheromone as ‘segregated all’ when they are paired, for ‘segregated rewards’ they deposit much less than when paired with the ‘bundled’ treatment (see Figure 4). A similar pattern is seen with ‘bundled’, where more pheromone is deposited when paired with ‘segregated all’ and less when paired with ‘segregated reward’. One possibility is that these differences arise due to a series of contrast effects. This pattern is further complicated by a strong effect of first encountered treatment (see Figure 5). In every condition the effects strengthen when the preferred option is presented first, and drops to chance level when presented second. This suggest that the bias generated through the bundling process has a similar strength to the bias for the first experienced odour (Oberhauser, 2019), and thus they appear to counterbalance each other.

Bundling and segregation of options to modify perceived value in insects may have ecological implications, especially in plant-pollinator interactions. If, by segregation, the perceived value of a reward can be increased, plants may be selected to split rewards amongst multiple smaller flowers in an inflorescence, or flowers on a plant. Similarly, they may be selected to attempt to bundle costs, favouring multi-flower inflorescences, allowing insects to walk between flowers, over multiple small, separate flowers.

The finding that bundling and segregation affect ant value perception adds this behavioural economic effect to several others which have also been shown to affect perceived value in insects, including decoy effects (Sasaki and Pratt, 2011; Shafir et al., 2002; Tan et al., 2015), invested effort increasing perceived value (Czaczkes et al., 2018a), relative value perception (Bitterman, 1976; Couvillon and Bitterman, 1984; Wendt et al., 2019), and labelling effects (Hemingway and Muth, 2022; Wendt and Czaczkes, 2020). It is becoming clear that the underlying patterns driving value perception in insects are in many ways parallel to those of humans. This implies either an extremely early evolutionary origin of shared perceptual mechanisms resulting in shared psychophysical laws or convergent evolution on similarly effective systems. The finding of a bundling and segregation effect, alongside the way in which insects respond to differences between experienced and expected rewards, strongly implies that insects share the same broad value function shape as humans (see Figure 1). Thus, we would predict that any other behavioural economic effects in humans, which arise from this value function, to also be present in insects. Still missing is a demonstration of an inflection point – that is, like in humans, losses loom larger than gains for insects. That our results imply bundling to more strongly influence losses than gain (see above) suggests this is the case, but formal testing will be required.

The absence of a significant preference in the Y-maze test, contrasted with the clear effect appreciable in the pheromone deposition, is puzzling. We have to consider the possibility that processes often assumed to be tightly linked to each other are instead separate. In this specific case, choice in the Y-maze depends on memory formation, as the choice is made by mentally comparing two options previously experienced. Pheromone deposition instead happens immediately after experience, and does not require any mental comparison to be expressed.

It is possible that ants have simply failed to associate each odour with the reward treatment, and thus chooses randomly in the Y-maze. This seems highly unlikely, as many examples of similar experiments on the same species are available in the literature, where ants are demonstrated to be extremely capable learners, learning even complex multimodal associations with fewer exposures than used in the current study (Czaczkes and Kumar, 2020; De Agrò et al., 2020).

The most likely explanation to us is that while a difference in the value of options is perceived, the same difference is not recorded during memory formation. Scalar utility theory (Kacelnik and Brito e Abreu, 1998; Rosenström et al., 2016) is an influential framework that has been developed to describe decision making under uncertainty. This theory postulates that encoding neurons, responsible for the internal representation of values (i.e. quantity, quality, delay, etc.), have logarithmically spaced sensitivities and specificities. Prospect theory (see Introduction) also assumes a process based on a logarithmic function. Thus, both make very similar behavioural predictions, even though one concentrates on value perception, and the other on memory acquisition. However, they do not have to be identical. Most experiments recording both pheromone deposition and subsequent choice use food quality as the measured unit (De Agrò et al., 2021; Oberhauser and Czaczkes, 2018; Wendt et al., 2019). Logarithmic perception of quality is probably the reason a mismatch between choice and instantaneous value perception has eluded discovery. We believe that the mismatch produced in this experiment has to do with the type of cost chosen as treatment: distance travelled. As previously mentioned, our treatment seemed unable to produce a segregation effect for the reward, while showing a strong segregation effect for losses, that is, the travelled distance. It is reasonable to assume that ants perceive value logarithmically, in accordance with prospect theory (De Agrò et al., 2021). In other words, the overall perception of the experience is affected by the artefacts of a log curve, segregation effect included (see Figure 1). Being produced instantaneously, pheromone deposition is likely linked to this information stream. However, when registering distances in memory ants need to be extremely precise. They possess an internal step counter and a visual odometer, which allows them to judge distances and return successfully to the nest (Narendra, 2007; Wittlinger et al., 2006). Encoding steps logarithmically would be absolutely insufficient for accurate homing, given the need to already cope with errors introduced in the path integration process (Merkle et al., 2006; Merkle and Wehner, 2010; Müller and Wehner, 1988; Schwarz et al., 2011). For this, a linear, 1:1 correspondence is required. It is possible that distance is memorized linearly and in a separated stream from the value perception. When asked to choose, the ant can only compare the two memories of distances, which due to their linear nature are immune to the segregation effect (Figure 6). If the ant odometer truly is a rare example of linear perception, it would be an invaluable system for investigating information processing in insects, as it allows the roles of perception and post-perceptual processing to be disentangled.

Proposed model of perception and memory in light of economic theories.

Perception can be the same as the formed memory, but not necessarily. During a foraging bout, the ant perceives (p) gains (food quality, q) and losses (energy spent to reach, e), according to prospect theory (PT). Food quality seems to be registered into memory (m) in to the same scale, congruently with scalar utility theory (SUT). Distance travelled (d) represents a special case, as it requires precise memory in the context of ant navigation. As such, it may possess a dedicated, direct, and linear memorization circuit (md), like the step counter (SC). In our experiment, we failed to imprint a segregation effect into rewards greyed out boxes; see Results and Discussion, and as such all our options were equal in this realm. With costs perceived logarithmically, but memorized linearly, we would expect the results observed in this experiment.

The dissociation between immediate reaction and subsequent choice could also be explained by a separation between ‘liking’ and ‘wanting’. The duality between ‘liking’ and ‘wanting’, first described in rats (Berridge et al., 1989) and then confirmed extensively in humans (Brauer and De Wit, 1997; Leyton et al., 2002; Pool et al., 2015), distinguishes two aspects of reward. ‘Liking’ refers to acutely perceived hedonic reactions, while ‘wanting’ refers to motivation and desire for a goal. While ‘liking’ and ‘wanting’ usually co-vary, these are neurologically separate processes, and can, in vertebrates, be separately inactivated (Berridge et al., 1989) or enhanced (Berridge and Valenstein, 1991; Leyton et al., 2002; Treit and Berridge, 1990). A dedicated network for ‘wanting’ has been recently discovered in insects as well (Garcia and Dyer, 2022; Huang et al., 2022), making it very likely to be present in L. niger ants as well. Under this perspective, the immediate reaction to experiencing the reward (i.e. the pheromone deposited) may correspond to how much the animal ‘liked’ each option. The choice of which reward to head for in the Y-maze task may reflect how much ‘wants’ either option. However, neurological evidence would be required to support this speculation. Moreover, further experiments should test whether pheromone deposition and binary choice are indeed appropriate proxies of ‘liking’ and ‘wanting’, respectively.

Our work also demonstrates empirically that individual ants prefer near to distant food sources. Such a preference have been previously observed in nature (Frank and Linsenmair, 2017; Nyamukondiwa and Addison, 2014) but never directly tested. Ants also recruited significantly more to closer food source, as reported in this and other ant species as well (Devigne and Detrain, 2006; Fewell et al., 1992).

This study adds the bundling and segregation effect to the collection of value-distorting effects from behavioural economics which also affect animal value perception. Critically, by finding this effect in an invertebrate, we demonstrate that the complexities of the vertebrate brain are not required for these effects to manifest. Animal behaviour, much like human behaviour, can often be modelled as a value-maximizing system. However, deviations for strict economic rationality, such as the one demonstrated here, may play important roles in the animals’ ecology, especially in their foraging behaviour and biotic interactions.

Data availability

Raw data collected in the experiments and used for the analysis are available in Source data 1.

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Decision letter

  1. Eva Pool
    Reviewing Editor; University of Geneva, Switzerland
  2. Christian Rutz
    Senior Editor; University of St Andrews, United Kingdom
  3. Giorgia Silani
    Reviewer; University of Vienna, Austria
  4. Erik Thomas Frank
    Reviewer; University of Würzburg, Germany
  5. Yoann Stussi
    Reviewer; University of Geneva, Switzerland

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Bundling and segregation affects "liking", but not "wanting", in an insect" for consideration by eLife. Your article has been reviewed by 4 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Christian Rutz as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Giorgia Silani (Reviewer #1); Erik Frank (Reviewer #2); Yoann Stussi (Reviewer #4).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

Overall, the reviewers agreed that the manuscript is of broad interest for many behavioral scientists in the fields of behavioural economics, decision-making, and reward processing. We were also positively impressed by the open practices in openly sharing data and analysis code in a very clear and detailed manner. However, the manuscript requires substantial revisions of the interpretation and discussion of the data as well as supplementary data to rule out some important alternative explanations. While there seems to be a consensus concerning the value of the demonstrated bundling versus segregation effects, we also agree that the interpretation in terms of "wanting versus liking" appears too speculative and questionable given the data. Therefore, we strongly suggest removing the interpretation in terms of motivational and hedonic processes and staying closer to the actual bundling effects which are innovative by themselves. In terms of alternative interpretations of the data, there are some major confounds that we would like to see addressed with supplementary data or supplementary analysis.

1) Concerning the results from the Y maze behavior: For interpretation of the results of the Y maze in terms of choice behaviour it is necessary to demonstrate that the associations between the reward conditions and the runway scents were successfully learned. The pilot experiment differs in many ways from the main experiment, therefore one cannot rule out that the lack of preference in the Y maze simply reflects a lack of learning. Also related to the interpretation of the behavior in the Y maze, the choice test is administered under extinction, an analysis of the first choice only would allow to rule out that the absence of preference reflects extinction processes.

2) Concerning the results from the pheromone deposit: A possible alternative interpretation of the pheromone deposit measure would be that it simply reflects the caloric intake rather than food value, it is worth controlling that across the different reward conditions the total amount of food consumed does not systematically vary and that the pheromone release did not vary according to the quantity of food that was consumed. More generally, the manuscript needs to provide more information about how pheromone deposition was measured and on the specificities of this measure, such as its physiological bases, timing properties, and granularity. Importantly, the results from the pheromone deposit measure are interpreted as if they support the hypothesis that segregated rewards with bundled costs should be the most "liked" option relative to bundled rewards and costs and segregated rewards and costs. But the data reported does not fully support this: The difference between the 'segregated rewards' condition and the 'bundled' condition is not statistically significant when all ants are considered. More nuance is needed so that this statistically nonsignificant result does not appear as being treated as statistically significant.

3) Concerning main experimental manipulation: the experimental design relies on the assumption that ants can perceive drops spaced by 5 mm as being segregated (two of 0.2 µl and 1 ad libitum). Since this is the key manipulation on which the experimental manipulation relies, we would like to see data demonstrating that spacing two food drops by 0.5 mm induces a segregated perception in ants.

4) Concerning the statistical approach: The choice of the random-effects structure for the GLMM analyses should be clearly motivated and justified. In particular, random intercepts were modelled (despite repeated measurements) and an appropriate specification of the random-effects structure is pivotal to reaching correct inferences when using mixed-effects models.

Reviewer #4 (Recommendations for the authors):

1) Concerning the ethics statement, it is mentioned 'No' next to animal subjects. Does this mean that no ethical approval was required or submitted for this study? If so, is this a typical procedure for studying with ants?

2) Across the paper, there is a lot of emphasis that the study is the first to report a bundling effect and a "wanting" / "liking" dissociation in ants. However, I don't think such an emphasis is necessary or even warranted as it doesn't provide helpful information to evaluate the importance or robustness of the results. I feel that the manuscript would benefit from removing these mentions or at least keeping them to a minimum.

3) It would be beneficial to report how the sample size for the main experiment was established or provide some considerations about the power of the study (e.g., a power sensitivity analysis to indicate the smallest effect size in terms of the odds ratio that could be detected with a certain power threshold given the sample size).

4) The degrees of freedom of the t-tests associated with the effects from the generalised linear mixed models (GLMM) should be consistently reported.

5) The choice of the random-effects structure for the GLMM analyses should be clearly motivated and justified. This is particularly important because only random intercepts were modelled (despite repeated measurements) and an appropriate specification of the random-effects structure is pivotal to reaching correct inferences when using mixed-effects models (see Barr et al., 2013. https://doi.org/10.1016/j.jml.2012.11.001; Bates et al., 2015. https://arxiv.org/abs/1506.04967; Frossard & Renaud, 2019. https://arxiv.org/abs/1903.10766).

6) Complementing the results with confidence intervals (e.g., 95% for statistical tests reporting a t statistics) around the effect sizes of interest could provide valuable information and be a nice addition to the manuscript. A useful R package to calculate confidence intervals for effect sizes is effectsize (https://cran.r-project.org/web/packages/effectsize/index.html; https://easystats.github.io/effectsize/).

7) I would like to commend the authors for openly sharing their data and analysis code in a clear and detailed manner.

https://doi.org/10.7554/eLife.79314.sa1

Author response

Essential revisions:

Overall, the reviewers agreed that the manuscript is of broad interest for many behavioral scientists in the fields of behavioural economics, decision-making, and reward processing. We were also positively impressed by the open practices in openly sharing data and analysis code in a very clear and detailed manner. However, the manuscript requires substantial revisions of the interpretation and discussion of the data as well as supplementary data to rule out some important alternative explanations. While there seems to be a consensus concerning the value of the demonstrated bundling versus segregation effects, we also agree that the interpretation in terms of "wanting versus liking" appears too speculative and questionable given the data. Therefore, we strongly suggest removing the interpretation in terms of motivational and hedonic processes and staying closer to the actual bundling effects which are innovative by themselves. In terms of alternative interpretations of the data, there are some major confounds that we would like to see addressed with supplementary data or supplementary analysis.

1) Concerning the results from the Y maze behavior: For interpretation of the results of the Y maze in terms of choice behaviour it is necessary to demonstrate that the associations between the reward conditions and the runway scents were successfully learned. The pilot experiment differs in many ways from the main experiment, therefore one cannot rule out that the lack of preference in the Y maze simply reflects a lack of learning. Also related to the interpretation of the behavior in the Y maze, the choice test is administered under extinction, an analysis of the first choice only would allow to rule out that the absence of preference reflects extinction processes.

2) Concerning the results from the pheromone deposit: A possible alternative interpretation of the pheromone deposit measure would be that it simply reflects the caloric intake rather than food value, it is worth controlling that across the different reward conditions the total amount of food consumed does not systematically vary and that the pheromone release did not vary according to the quantity of food that was consumed. More generally, the manuscript needs to provide more information about how pheromone deposition was measured and on the specificities of this measure, such as its physiological bases, timing properties, and granularity. Importantly, the results from the pheromone deposit measure are interpreted as if they support the hypothesis that segregated rewards with bundled costs should be the most "liked" option relative to bundled rewards and costs and segregated rewards and costs. But the data reported does not fully support this: The difference between the 'segregated rewards' condition and the 'bundled' condition is not statistically significant when all ants are considered. More nuance is needed so that this statistically nonsignificant result does not appear as being treated as statistically significant.

3) Concerning main experimental manipulation: the experimental design relies on the assumption that ants can perceive drops spaced by 5 mm as being segregated (two of 0.2 µl and 1 ad libitum). Since this is the key manipulation on which the experimental manipulation relies, we would like to see data demonstrating that spacing two food drops by 0.5 mm induces a segregated perception in ants.

4) Concerning the statistical approach: The choice of the random-effects structure for the GLMM analyses should be clearly motivated and justified. In particular, random intercepts were modelled (despite repeated measurements) and an appropriate specification of the random-effects structure is pivotal to reaching correct inferences when using mixed-effects models.

We note that many of the comments were shared by multiple reviewers, and the editor effectively summarized the main, shared positions in the points below. To make our response easy to navigate, we added a numbering system to the different points made. We will include our main arguments as answers to the editors comments, and we will refer to those, while adding further specific comments, in the answers to reviewers below when these points are raised.

1. The liking vs wanting framework

While there seems to be a consensus concerning the value of the demonstrated bundling versus segregation effects, we also agree that the interpretation in terms of "wanting versus liking" appears too speculative and questionable given the data. Therefore, we strongly suggest removing the interpretation in terms of motivational and hedonic processes and staying closer to the actual bundling effects which are innovative by themselves.

Having seen how this is a shared opinion of multiple reviewers, we agree with greatly reducing our claims about liking vs wanting as an explanation to our results. Indeed, the experiments were not designed to test this specific segregation, and as such cannot make broad claims on its presence in our experimental subjects.

However, we still believe we can’t ignore the unusual split result between the binomial choice test and the pheromone deposition. This was unexpected for us, as in virtually all studies we ever carried out using this odor learning procedure we found a clear response in the Y-maze choice tests. Occasionally this was not mirrored in the pheromone data, but never the other way around. Note that it was never our intention to suggest that our paper was designed to test liking vs wanting. Indeed in the introduction we are very clear in saying that this reasoning came after observing the results (L99-106).

We however agree that our inclusion of this argument was too wide for this manuscript, given the ideas’ speculative nature. We agree that this paper does not demonstrate the presence of a separation in liking vs wanting in ants, but we feel we should at least mention it as a possible explanation to the difference in the effect between pheromone deposition and binomial choice. This position we believe is also sustained by the recently published paper by Huang, J. et al. (2022), that came out just a month after out submission to eLife.

Accordingly, we changed the title of the paper to “Bundling and segregation affects pheromone deposition, but not choice, in an ant” and removed every mention of the liking vs wanting literature from the introduction, focusing solely on economic theories and bundling vs segregation. In the discussion, we reduced greatly its contribution, but we mention liking vs wanting as a possible, yet speculative, explanation to our observation. We limit this discussion to one paragraph and refer to the recent Huang et al. (2022) paper as a demonstration of the existence of this dichotomy in insects, and our observation as a possible effect. We also make very clear that this is speculation, note statements of fact, by ending the paragraph with “However, neurological evidence would be required to support this speculation.” The mismatch between memory and perception is maintained as an alternative explanation, indicating also how this mismatch may cause a failure in memory itself, as was suggested by some reviewers.

In terms of alternative interpretations of the data, there are some major confounds that we would like to see addressed with supplementary data or supplementary analysis.

Regarding some of the requested additional data, intended to exclude alternative explanations, we believe we have sufficient evidence, stemming either from the literature or from other pilot experiments performed by us. We will present our argument below when brought up. We are of course open to discussing collecting more data if our position was to be found unsatisfactory.

2. The Y-maze results

2.1. Presence of learning

Concerning the results from the Y maze behavior: For interpretation of the results of the Y maze in terms of choice behaviour it is necessary to demonstrate that the associations between the reward conditions and the runway scents were successfully learned. The pilot experiment differs in many ways from the main experiment, therefore one cannot rule out that the lack of preference in the Y maze simply reflects a lack of learning.

Yes, this is an issue which affects all ‘negative results’ in behavior-based learning assays: it is impossible to distinguish lack of learning from learning but without a resultant behavioural response. Thus, as far as we can tell, is impossible to demonstrate learning under this exact bundling vs segregation setup as separate from preference.

However, we have good reasons to expect learning to have taken place, given their successful learning in other contexts using an identical odour-association choice assay. These ants learn such associations very well in a multi-reward context. In 2018 we performed a control experiment required for a different experiment. In that experiment, as in the experiment we report here, ants had to encounter multiple sucrose solution drops over the course of one visit, and associate all of those to an odor. We designed the pilot to demonstrate the ability of ants to associate a smell with a multi-reward visit. Each ant (n=20) would be allowed onto a 20cm long scented runway. At the end of it, the animal would encounter a small 0.2ul drop of sucrose solution. The solution could be either 1.5M or 0.25M, depending on the condition. After the ant had fully consumed the drop, a second one would be presented, and after that a third one, that the ant consumed until satiated. All the three drops had the same molarity. The ant was then allowed back to the nest, unloaded, and allowed to make a second visit. Here the ant would experience a differently scented runway, and presented with a different molarity drop (0.25M if 1.5M for the first visit and vice-versa). This procedure was repeated 8 times, for a total of 4 visits for each odor. In essence, this experiment is almost identical to the “Segregated reward” condition as presented in this manuscript, where we also presented a succession of three identical drops. At test, 90% of the ants chose the scent associated with high molarity for the first choice, 75% did so for the second choice, 70% for the third. This experiment demonstrates the ability of ants to associate an odor with a multi-reward experience. We are attaching the raw data for this pilot experiment to the response. If the reviewers and editor were to find this data crucial to the discussion of the current experiment, we can add the full pilot as a supplement.

Regardless, in the new version of the manuscript we give more space to the perception vs memory explanation (Figure 6) as an alternative explanation to the data. We raise the possibility that the ants simply didn’t learn the task, but the bundling vs segregation effect had an effect or weighting on their perception. We find this explanation unlikely, given the corroborating evidence we presented, and thus lean more towards an equal weight in memory but not in perception.

2.2. Results under extinction

Also related to the interpretation of the behavior in the Y maze, the choice test is administered under extinction, an analysis of the first choice only would allow to rule out that the absence of preference reflects extinction processes.

Regarding the test being administered under extinction, note that visit number was always included in the models in order to observe whether the subsequent testing had a decreasing effect of choice. Even when no difference was found between subsequent testing visits, we included the relevant post-hoc analyses too, regardless of the preference in the very first testing visit.

When looking only at the first choice, for condition 1, the ants chose the segregated option 51.6% of the times (p=1). For condition 2, they chose the segregated reward option 49.7% of the times (p=1). Lastly, in condition 3 they chose the segregated option 34.7% of the times (p=1). This data is available in the Supplementary file 1 (before supplement ESM2).

3. Pheromone deposition

3.1. The origin of pheromone deposited

Concerning the results from the pheromone deposit: A possible alternative interpretation of the pheromone deposit measure would be that it simply reflects the caloric intake rather than food value, it is worth controlling that across the different reward conditions the total amount of food consumed does not systematically vary and that the pheromone release did not vary according to the quantity of food that was consumed. More generally, the manuscript needs to provide more information about how pheromone deposition was measured and on the specificities of this measure, such as its physiological bases, timing properties, and granularity.

We feel fairly confident in excluding the possibility that pheromone deposited is linked to caloric consumption. First of all, ants participating in this experiment do not consume the liquid. The reward gets stored in the social stomach and not assimilated, and gets then passed on other members of the colony. As such, metabolic processes should not take place during the experiment.

More importantly, however, good evidence exists that, in this species, pheromone deposition is released after a specific volume threshold is consumed (A. C. Mailleux et al., 2000; A.-C. Mailleux et al., 2003, 2005). Critically, after passing this limit, ants which consume the same quality of food in the same manner deposit the same amount of pheromone, regardless of the specific volume drunk. Ants will not freely return to the nest before reaching this threshold, if allowed to feed further, and the gaster only becomes very visibly distended after this threshold. Thus, we are confident that all the ants in our experiment consumed more than this threshold food volume, and so even if the different treatments cause small changes in volume consumption, this should not affect their pheromone deposition.

Finally, regardless of the reported literature, we feel that even if the ants were to deposit more pheromone only as a byproduct of different caloric intake, the results would still support our interpretation. The only difference between the treatments is the segregation of rewards and costs itself. All the drops presented are of the same sizes across treatments, and they all carry the same molarity. As such, if the ants were to consume the food at a different rate or in a different amount, this variance would have to be caused by the segregation effect itself. Some evidence is available demonstrating that ants tend consume more overall volume for moderately high sucrose concentrations (Sola & Josens, 2016), which would indicate that the segregation has an effect on the perceived value. In other words, even if pheromone was linked to caloric consumption, the deposition would act as a proxy of consumption rate, just another modulated measure.

We also now provide, when first introducing the pheromone deposition measurement, some more detail on the behavior, and how it correlates with the (perceived) value of a resource.

3.2. The presence of not statistically significant results

Importantly, the results from the pheromone deposit measure are interpreted as if they support the hypothesis that segregated rewards with bundled costs should be the most "liked" option relative to bundled rewards and costs and segregated rewards and costs. But the data reported does not fully support this: The difference between the 'segregated rewards' condition and the 'bundled' condition is not statistically significant when all ants are considered. More nuance is needed so that this statistically nonsignificant result does not appear as being treated as statistically significant.

Apologies, we were not very clear in our discussion here, and didn’t sufficiently distinguish out original hypotheses from our interpretation of the results. Our interpretation was that Bundling vs Segregation has an effect only on costs, but not on gains (L428-435, original manuscript version). This idea is derived from the data as is, considering the Bundling vs Segregated reward as identical, the bundling as preferred to the segregated all, the segregated reward being preferred to segregated all. Under this interpretation, the segregated rewards condition should NOT result different from the bundled condition, as in both the costs are bundled, and they only differ in the gains (L435-439, original manuscript version). For the same reason, Segregated reward (that presents bundled cost) is preferred to segregated all (that presents segregated costs); bundled (bundled cost) is preferred to segregated all (segregated costs).

We understand that the confusion originates from us repeating our a priori hypothesis in the discussion, which instead expected to see a difference in segregated reward vs bundles. We feel that is more honest of us to declare our a priori hypothesis, and then specify what the data suggest, rather than to give the impression to having had the second hypothesis all along. We address this issue by explicitly distinguishing our a priori hypothesis from our interpretation. L391, L394, L398, L403-404

Another reason for confusion is that we discussed at length the nuance of the results. We felt that declaring a result to be definitely at chance level when the p-value is 0.059 would be careless, especially considering the pattern of preference when the initial encounter is included.

We have now reworded the discussion and made clearer the expectation for each condition depending on the working hypothesis.

4. The segregation effect

Concerning main experimental manipulation: the experimental design relies on the assumption that ants can perceive drops spaced by 5 mm as being segregated (two of 0.2 µl and 1 ad libitum). Since this is the key manipulation on which the experimental manipulation relies, we would like to see data demonstrating that spacing two food drops by 0.5 mm induces a segregated perception in ants.

This issue is related to point 2 – how can we distinguish a lack of response to a perceived stimulus from a stimulus that is not perceived? We cannot. Indeed, we were very careful not to make strong claims that ants do not respond to reward segregation. We now state explicitly “…either because the ants do not segregate gains, or because our manipulation failed to trigger segregation”.

Happily, for our main claim that bundling and segregation influences the behavior of ants, it does not matter: this treatment was added in case reward segregation affected the ants in an equal and opposite way to cost segregation, since they both co-occur in the “all segregated” treatment. Has the response to “all segregated” and “all bundled” been indistinguishable, we would have required the “reward segregated” treatment to interpret the result: a lower reward perception of “segregated all” compared to “segregated all” would have been taken as evidence that reward and cost segregation, when co-occurring, balance each other out. As it happens, however, there was a difference between “all segregated” and “all bundled”, showing that costs are experienced differently if bundled or segregated. Unclear is only whether rewards are perceived as segregated, but have a weaker effect that segregated costs, or whether the ants only perceived the costs, but not the rewards, as segregated.

In summary: we agree completely that we cannot know whether the reward segregation treatment was really perceived as segregated by the ants. However, given the results, it does not matter for our main message.

To make this clear, we have now added these considerations to the discussion. L410-416

5. The choice of random effect structure

Concerning the statistical approach: The choice of the random-effects structure for the GLMM analyses should be clearly motivated and justified. In particular, random intercepts were modelled (despite repeated measurements) and an appropriate specification of the random-effects structure is pivotal to reaching correct inferences when using mixed-effects models.

Our choice of random effect structure was informed by our knowledge of the study species. Different colonies can have an overall different tendency to deposit pheromone. In each colony, different individuals can have a different tendency to deposit overall more or less pheromone. For this reason, the individual ants nested in colonies are included as a random intercept in the model: the overall pheromone deposited may vary per each individual independently from the treatment. However, we do not have the same expectation for across trials changes: an over-performer ant will consistently over-perform in terms of pheromone deposited, with no expectation for it to differentially change behaviour (Beckers et al., 1992). However, the evidence is sparse, and we are mostly relying on our own experience. Given this, prompted by one reviewer comments, we tried including visit number as a random slope for models looking at pheromone deposition (Binomial choice models only presented 3 subsequent visits, all being of equal treatment. As such we only included a random intercept). This turned out to be not feasible all the time, causing a failure in model convergence. We believe that the reason is the low number of repetitions per condition per subject: each performs only 4 visits to each segregation option. Moreover, visit 1 almost always results in no pheromone deposition, due to the existence of pre-training and generally low deposition on the first 1-2 visits to a food source, leaving probably too few repetitions to estimate the random slope. Following the suggestions of the reviewer, we tried simplifying the random effect structure in those cases, including Visit number as random intercept. This sometimes fixed the convergence failure, in which case we maintained it in the model, while in others the model kept failing to converge. In those cases we were forced to remove it from the final model.

Reviewer #4 (Recommendations for the authors):

1) Concerning the ethics statement, it is mentioned 'No' next to animal subjects. Does this mean that no ethical approval was required or submitted for this study? If so, is this a typical procedure for studying with ants?

Indeed, ants and all invertebrates (notably excluding cephalopods, and recently some crustaceans) are not protected by European law (or by any other country as far as we know) in research. As such, no ethical approval is required to perform experiments on these animals.

2) Across the paper, there is a lot of emphasis that the study is the first to report a bundling effect and a "wanting" / "liking" dissociation in ants. However, I don't think such an emphasis is necessary or even warranted as it doesn't provide helpful information to evaluate the importance or robustness of the results. I feel that the manuscript would benefit from removing these mentions or at least keeping them to a minimum.

We agree with the reviewer. See point 1.

3) It would be beneficial to report how the sample size for the main experiment was established or provide some considerations about the power of the study (e.g., a power sensitivity analysis to indicate the smallest effect size in terms of the odds ratio that could be detected with a certain power threshold given the sample size).

Sample size was set a-priori, but based on pragmatic considerations: we knew how much research time we could allocate to data collection, and could make a good estimate of how many ants could be tested in this time. This was then used to set the sample size. Using power analyses can be tricky when we have poor a-priori estimates for the effect size. We consider our pragmatic a priori sample size setting approach to be functional while avoid the possibility of p-hacking.

4) The degrees of freedom of the t-tests associated with the effects from the generalised linear mixed models (GLMM) should be consistently reported.

DF are now reported in the text, thank you.

5) The choice of the random-effects structure for the GLMM analyses should be clearly motivated and justified. This is particularly important because only random intercepts were modelled (despite repeated measurements) and an appropriate specification of the random-effects structure is pivotal to reaching correct inferences when using mixed-effects models (see Barr et al., 2013. https://doi.org/10.1016/j.jml.2012.11.001; Bates et al., 2015. https://arxiv.org/abs/1506.04967; Frossard & Renaud, 2019. https://arxiv.org/abs/1903.10766).

We would like to thank the reviewer greatly for the clarification provided via email. It helped us immensely in navigating the development of a random effect structure. We answered fully to the comment in the general response, and the changes are now appreciable in the supplement.

6) Complementing the results with confidence intervals (e.g., 95% for statistical tests reporting a t statistics) around the effect sizes of interest could provide valuable information and be a nice addition to the manuscript. A useful R package to calculate confidence intervals for effect sizes is effectsize (https://cran.r-project.org/web/packages/effectsize/index.html; https://easystats.github.io/effectsize/).

Thank you for suggesting the package! We did not know it and we found it quite useful. We added a calculation of the effect size in the analysis provided in the supplement. We however did not add it in the main text, to maintain readability. Indeed, effect size calculated by the effectsize package is directly derived from DF and t value, and as such we felt it would be redundant.

7) I would like to commend the authors for openly sharing their data and analysis code in a clear and detailed manner.

Many thanks! Open science is very important to us, and we work hard on it. It is pleasing to see that it is sometimes noticed.

https://doi.org/10.7554/eLife.79314.sa2

Article and author information

Author details

  1. Massimo De Agrò

    1. Animal Comparative Economics laboratory, Department of Zoology, University of Regensburg, Regensburg, Germany
    2. Department of Biology, University of Florence, Florence, Italy
    Contribution
    Conceptualization, Data curation, Software, Formal analysis, Supervision, Visualization, Methodology, Writing – original draft, Writing – review and editing
    For correspondence
    massimo.deagro@gmail.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9284-5964
  2. Chiara Matschunas

    Animal Comparative Economics laboratory, Department of Zoology, University of Regensburg, Regensburg, Germany
    Contribution
    Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
  3. Tomer J Czaczkes

    Animal Comparative Economics laboratory, Department of Zoology, University of Regensburg, Regensburg, Germany
    Contribution
    Conceptualization, Supervision, Funding acquisition, Investigation, Writing – original draft, Project administration, Writing – review and editing
    Competing interests
    No competing interests declared

Funding

Deutsche Forschungsgemeinschaft (CZ234/4-1)

  • Tomer J Czaczkes

Universität Regensburg

  • Massimo De Agrò

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

TJC was supported by a Heisenberg fellowship from the Deutsche Forschungsgemeinschaft (CZ 237/4-1). MDA was funded by University of Regensburg 'Anreizsystem' funding to TJC.

Senior Editor

  1. Christian Rutz, University of St Andrews, United Kingdom

Reviewing Editor

  1. Eva Pool, University of Geneva, Switzerland

Reviewers

  1. Giorgia Silani, University of Vienna, Austria
  2. Erik Thomas Frank, University of Würzburg, Germany
  3. Yoann Stussi, University of Geneva, Switzerland

Publication history

  1. Received: April 6, 2022
  2. Preprint posted: May 26, 2022 (view preprint)
  3. Accepted: November 3, 2022
  4. Version of Record published: November 22, 2022 (version 1)

Copyright

© 2022, De Agrò et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Massimo De Agrò
  2. Chiara Matschunas
  3. Tomer J Czaczkes
(2022)
Bundling and segregation affect pheromone deposition, but not choice, in an ant
eLife 11:e79314.
https://doi.org/10.7554/eLife.79314
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