Response to olfactory cues

The experimental setup was adapted from previous experiments on reciprocal food exchange in rats [13,18,19] (Fig 1). A wire mesh divided the test cage (80 cm × 50 cm × 37.5 cm), with one compartment used for the focal rat and the other one for its experimental partner. A movable tray was installed in front of the cage, which could be pulled towards the cage by an attached stick accessible only to one of the two rats. A food reward was placed on the other side of the tray, so that it could be reached only by the experimental partner of the pulling rat, that is, the receiver. Thus, rats could not pull food for themselves but only for their respective partner. Pulling the tray towards the partner is costly for the focal subject, and it has been shown that they take these costs into account when deciding to donate food to a partner [13]. Experimental dyads consisted of unrelated and unfamiliar females. The 20 focal rats used in this experiment had all been made acquainted with reciprocal cooperation and were familiar with the setup (cf., the work by Rutte and Taborsky [17]). Furthermore, we used four rats that were specifically trained to always pull as cooperative partners of the focal test subjects (experimentally assigned ‘cooperators’).

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larger image TIFF original image Download: Fig 1. Experimental setup. (a) During the experience phase a cooperative partner (A) placed in a Plexiglas box in the neighbouring compartment of the focal rat (F) produced food for the latter by pulling a stick attached to a tray containing a food reward. The air of the Plexiglas box of cooperator A was removed from the experimental room by a pump (depicted by outgoing arrow). A second individual (B) was placed in a similar box in a different room, and the air from this box was pumped into the compartment of the focal rat (depicted by ingoing arrow). Individual B was satiated during this experience phase. (b) Experimental setup of the test phase. The focal rat now had the opportunity to reciprocate the help it had received during the experience phase by pulling food for the partner in the adjacent Plexiglass box, while receiving odour from a hungry or satiated individual (B) located in a Plexiglas box in another room. The arrows depict air flow as in panel (a). https://doi.org/10.1371/journal.pbio.3000628.g001

In order to block information transfer between the experimental rats in the adjacent compartments of the test cage, the partner of the focal rat (‘cooperator A’) was placed in a clear Plexiglas box (38 cm × 48 cm × 36 cm) from which a pump sucked out the air and blew it outside of the room. Hence the focal rat was unable to smell the partner in the neighbouring compartment. The air pump created noise in the audible and ultrasonic acoustic ranges of rats, thereby impeding acoustical communication, which was further hampered by the fact that the partner was contained in a sealed Plexiglas box [23]. We furthermore covered the side of the partner rat’s box adjacent to the compartment of the focal individual with opaque foil so that the rats could not see each other.

In the experience phase (Fig 1A), the partner (‘cooperator A’) pulled food for the focal rat during 7 minutes. We noted the latency until the first pulling, as well as the number of pulls. During this experience phase, the focal rat did not receive chemical information from its pulling partner in the neighbouring Plexiglas box but instead was provided with odour from a similar box located in a different room, which contained a second (satiated) rat (‘B’). On the next day, during the test phase (Fig 1B), the focal rat was enabled to reciprocate the help received on the previous day to ‘cooperator A’ while receiving odour from rat ‘B’ in the neighbouring room. Rat ‘B’ had been either food deprived (hungry) or not (satiated) overnight. The experience and test phases were repeated once with opposite hunger states of the individual (B) from which the odour was presented to the focal rat in the test phase. The partners as well as the sequence in which the focal rat received odour from hungry or satiated individuals were randomised. In the test phase, hungry partners were only presented in the morning because of ethical reasons, in order to avoid suffering from prolonged food restriction.

Focal rats started to pull earlier for the stooge if they received odour from a hungry rather than from a satiated rat (X2 = 413.5; p < 0.001; mean latencies for hungry rats: 29 s; for satiated rats: 85 s; N = 16; Fig 2), even when excluding the outlier (X2 = 191.93; p < 0.001; mean latencies for hungry rats: 29 s; for satiated rats: 63 s; N = 15; outlier defined as value exceeding mean + (SD × 2)), suggesting that they assess the hunger status of a partner using olfactory cues alone. An enhanced propensity to donate food to hungry as compared with satiated partners has been identified also in previous studies using the same setup, in which the hungry or satiated partners were located in the same cage as the test subject [6,13]. One might argue that olfactory cues emitted by hungry rats could lead to increased general agitation by the focal rat, thereby resulting in higher activity and consequently earlier pulling. However, rats also pull earlier for cooperative than for defective partners [12,13,17,19,24], and the latency to pull correlates negatively with pulling frequency, suggesting that this latency indeed represents the helping motivation of the focal subjects [6,18].

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larger image TIFF original image Download: Fig 2. Latency to the start of food provisioning of test rats for a stooge in the neighbouring compartment when receiving odour from a hungry or a satiated conspecific located in a different room (for raw data, see Latency to the start of food provisioning of test rats for a stooge in the neighbouring compartment when receiving odour from a hungry or a satiated conspecific located in a different room (for raw data, see S1 Data ). Boxes mark medians (bold line), means (dashed line), and interquartile ranges of the data. The asteriks indicate a significant difference at p < 0.001. https://doi.org/10.1371/journal.pbio.3000628.g002

In a previous study, in which focal rats had full access to all cues provided by the partner (visual, olfactory, and acoustic), the mean latency to pull was 196 s for hungry and 183 s for satiated rats [13]. Thus, in the current experiment, in which focal subjects received olfactory cues alone, they started to pull much earlier, and they differed in their latency to pull depending on the hunger state of the receiver. Confining the information received to one modality, olfaction, apparently triggered an earlier response. The lack of pronounced latency differences between pulling for hungry and satiated rats in the previous study, in which the partner was physically present nearby, might indicate that the behaviour of the partner can influence the decision of the focal rat, which has been shown to occur in a previous experiment [6]. We should like to stress that the previous data were obtained with a different batch of rats, and thus the absolute latency and number of pulls may not be directly comparable between experiments because of potential differences between populations (for example, in the general motivation and/or ability to pull).

The mean numbers of pulls by focal rats appeared to be higher when receiving odour from a hungry than from a satiated partner, but this was not significant, and thus there was no evidence that this difference was meaningful (X2 = 1.62; p = 0.20; mean number of pulls per 7 minutes for hungry rats: 7.56; for satiated rats: 6.37; N = 16). In a previous study in which the partner was physically present nearby and no sensory modality was excluded, body mass of the partners in connection with their hunger state played a crucial role in the decision of the focal rat on how much food to provide. When the receiver was hungry, focal rats pulled more often for light than for heavy partners. In contrast, when the receiver was satiated, focal rats pulled more often for heavy than for the light partners. As weight reflects dominance in rats [25], heavier partners may generally more readily receive food donations from focal rats simply because of dominance effects [26]. As the rats in our study were all of similar body weight, dominance can be ruled out as potential explanatory factor for our results.

The latencies until the first pull for the focal rat that the ‘cooperator A’ had shown on the experience day and that of the focal rat pulling for the stooge on the test day were not correlated with each other (rho = 0.027; p = 0.85) nor were their numbers of pulls (rho = 0.035; p = 0.89). This indicates that the test subjects did not merely copy the behaviour of their partner [23]. During the experience phase, experimental partners pulled earlier and more often for focal rats in the afternoon than in the morning (latency: X2 = 67.46; p < 0.001; number of pulls: X2 = 19.68; p < 0.001; N = 16), revealing an increase in the motivation of rats to donate food during the course of the day. This contrasts with the behaviour of the focal rats on respective test days; they showed shorter latencies before starting to pull for their partners in the morning (for hungry partners) than in the afternoon (for satiated partners; see above results). Thus, the discrimination between pulling for hungry and satiated partners might be even stronger than observed considering the apparently increasing motivation of rats to pull during the course of the day. Furthermore, we tested for a potential sequence effect by comparing for each individual the number of pulls and the latency to pull between the two test days, which did not reveal a significant effect (paired Wilcoxon signed rank test; number of pulls: V = 99.5; p = 0.110; latency: V = 50.5; p = 0.379).

A previous study of Norway rats showed that they reciprocate help according to the quality of help they previously received [18], indicating that the specific outcome of a helpful act for the receiver can subsequently benefit the donor. In our study, rats provided food faster when the partner was hungry than when it was satiated. ‘Gratitude’ is often argued to play a key role in the evolution of social behaviour in humans, with individuals being more willing to help somebody else if they are grateful for the help they have previously obtained [27]. If individuals reward cooperative conspecifics according to their perceived benefit of previously received help, assessment of the need of the partner might critically affect the probability to get help back in a future interaction.

In our experiment, the rat from which the odour derived was separated from the experimental room and placed in a box alone. Hence, the information transferred by the odour was not socially induced. Therefore, rats seem to use the partner’s inadvertent smell of hunger as a reliable indicator for its current need, adjusting their helping propensity accordingly. This supports the ‘index hypothesis’ of honest signalling, which assumes that honesty is enforced because of physical, developmental, or physiological constraints that cannot be cheated [5,20].