Recruitment in starved nests: the role of direct and indirect interactions between scouts and nestmates in the ant Lasius niger

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1 Insect. Soc. (2011) 58: DOI /s Insectes Sociaux RESEARCH ARTICLE Recruitment in starved nests: the role of direct and indirect interactions between scouts and nestmates in the ant Lasius niger A.-C. Mailleux A. Buffin C. Detrain J.-L. Deneubourg Received: 16 November 2010 / Revised: 24 May 2011 / Accepted: 1 June 2011 / Published online: 21 June 2011 Ó International Union for the Study of Social Insects (IUSSI) 2011 Abstract In social insects, the foraging activity usually increases with the length of food deprivation. In Lasius niger, a mass-recruiting ant species, the foraging adjustment to the level of food deprivation is regulated by the scout that fed at the food source and by the response of the nestmates to signals performed by the scout inside the nest. In this study, we look at the role of these direct interactions (antennations or trophallaxes) and indirect interactions (pheromonal emission) and how they are influenced by the level of food deprivation. At the beginning of recruitment, the relative number of nestmates leaving the nest to forage increases with the level of deprivation. The nestmates propensity to exit the nest is not influenced by a previous trophallactic and/or antennal contact with a scout. Our results strongly suggest that the exit of nestmates is triggered by a chemical signal emitted by a scout. Deprivation lowers the response threshold of nestmates to this chemical signal resulting in a more important exit from the nest. Surprisingly, 27% of starved nestmates that receive food from the scout relay the information by depositing a chemical signal before having discovered and drunk the food source. Both phenomena boost the recruitment process. Though successful foragers returning to the nest have a significant role in the recruitment to the food source, we observed that the response of the nestmates inside the nest also greatly influence regulation of the foraging activity. A.-C. Mailleux (&) Unit of Ecology and Biogeography, Catholic University of Louvain, Croix du Sud 4-5 (Carnoy), 1348 Louvain-la-Neuve, Belgium anne-catherine.mailleux@uclouvain.be A. Buffin C. Detrain J.-L. Deneubourg Service d Ecologie Sociale, Université libre de Bruxelles, CP231, Avenue F. D. Roosevelt, 50, 1050 Brussels, Belgium Keywords Ants Food transfer Trophallaxis Starvation Lasius niger Introduction Animals constantly face trade-off between their need for energy and the risk they take when foraging. Their ecological success depends on their ability to adjust their foraging strategies to the level of food deprivation. In societies, this problem is even more complex as the society s needs have to be met, not just the needs of one individual. Individuals thus have to exchange information and regulate their activity to the needs of the other members of the group/society (e.g. Gotceitas and Godin, 2004; Theraulaz et al., 1991; Cassill and Tschinkel, 1999a, b; Smiseth and Moore, 2004). In eusocial societies, the length of food deprivation is one of most important parameters affecting collective foraging behaviour, this has been observed in ants (Cosens and Toussaint, 1986; Roces and Hölldobler, 1996; Schafer et al., 2006; Sendova-Franks et al., 2010; Szlep-Fessel, 1970), honeybees (Schulz et al., 1998, 2002; Toth et al., 2005) and bumble bees (Cartar and Dill, 1990). For instance, in the honeybee Apis mellifera, after a short food deprivation period, the number of foragers searching for food does not markedly increase, with few foragers being engaged in food exploitation once a source is discovered (Toth et al., 2005). After a longer food deprivation period, colonies exhibit a higher number of foragers, a steeper recruitment of foragers towards exploited food sources, as well as higher amounts of food retrieved to the colony (Toth et al., 2005). The regulation of the recruitment in function of the level of food deprivation has been studied in species tending aphids such as Solenopsis invicta (Howard and Tschinkel, 1980), Pheidole pallidula (Hölldobler, 1971, 1985; Traniello, 1977;

2 560 A.-C. Mailleux et al. Baroni-Urbani et al. 1988; Roces and Nuñez, 1993; De Biseau and Pasteels, 1994; Stuart and Moffett, 1994; Liefke et al., 2001) and Lasius niger (Devigne and Detrain, 2002; Mailleux et al., 2003, 2006, 2010; Sakata, 1995; Völkl et al., 1999). These species of ants are good biological models to study the behavioural flexibility of colonies that face fluctuations in food supply. Indeed, honeydew produced by aphids is an important source of carbohydrates (Auclair, 1963; Hölldobler and Wilson, 1990; Kaplan and Eubanks, 2005) and its production undergoes important temporal variations throughout the year (Sakata, 1995; Sudd and Sudd, 1985; Völkl et al., 1999). In the beginning of spring, honeydew resources are limited since aphid s colonies are scarce. As the year goes by, some aphid colonies suddenly disappear (Devigne and Detrain, 2002), whereas others spread out and produce huge quantities of honeydew. Moreover, the weather (such as rain) can stop honeydew crop for a rather long time. Due to such fluctuations in honeydew availability, ant s species tending aphids can occasionally undergo long food deprivation time (Howard and Tschinkel, 1980). After a prolonged deprivation, these aphid-tending ants intensify their food retrieving activities (Howard and Tschinkel, 1980; Cosens and Toussaint, 1986; Josens and Roces, 2000; Mailleux et al., 2006; Wallis, 1962). These researches were mainly focused on the strategy of foraging followed by starved animals outside the nest (Stephens and Krebs, 1986) while the underlying behavioural mechanisms of communication and interactions inside the nest were less with some noticeable exceptions such as Solenopsis invicta. Inside the nest, foragers of S. invicta coming back from a food source use six discrete behaviours to inform nestmates of the location and quality of a food site (Cassill, 2003). They increase walking tempo, lay incoming trails, waggle their heads and push their way through the cluster of ants inside the entrance, stroke nestmates with their antennae, advertise with a brief food display and lead groups of nestmates to the food site by laying outgoing trails. In response, nestmates assess the food sample with taste buds on the tips of their antennae. The motivation of the recruit depends on the quality of food advertised by the foragers, its employment status (nurse versus unemployed) and its level of hunger. Therefore, in the S. invicta, recruitment is an emergent event based on supply and demand decisions that depends on direct contact inside the nest rather than on a pheromone trail or at the food site. The influence of deprivation on foraging activity is linked to the increased trophallactic activity of the recruiters (Howard and Tschinkel, 1980). In L. niger, the regulation of the recruitment behaviour in the nest, in function of the food deprivation is not well known and deserves further investigations. In L. niger, the first foragers finding a liquid food source outside the nest drink and come back to the nest (Beckers et al., 1993; Mailleux et al., 2003, 2006) while laying a trail from the food source to the nest (Mailleux et al., 2003, 2006). The ants coming from starved nests bring larger quantities of food back to the nest. Remarkably, food deprivation does not alter the quantity of recruitment pheromone laid on the substrate by L. niger foragers on their homeward trip. This means that the increased global recruitment after a period of food deprivation does not result from an increased intensity of individual trail marking by foragers (Mailleux et al., 2006). Contrary to S. invicta, after long deprivation periods, higher mobilisation is essentially based on the increased motivation of the recruits to leave the nest and forage (Mailleux et al., 2010). This leaves the question open on how this motivation of the recruits is increased. This paper is the continuation of previous studies (Mailleux et al., 2003, 2006, 2010). We followed some foragers that found the food source, drank from it and came back to the nest whilst laying a recruitment trail inside the nest. We observed their behaviour when they were confronted with nestmates in colonies subjected to different levels of food deprivation. One can imagine that interindividual direct interactions such as antennations or trophallaxes and/or indirect interactions such as pheromonal emission may differ depending on deprivation and may influence recruits motivation (Cassill and Tschinkel, 1999a, b; Mc Cabe et al., 2006; Farina and Grüter, 2009). This should result from a more rapid trophallactic activity, an increased stimulation mediated by antennal contact and/or by an increased emission of pheromones, all those hypotheses are not mutually exclusive. In this paper, we measured and compared, for different levels of food deprivation, how the trophallactic and antennal contacts between L. niger foragers and nestmates inside nests influence nest departure. These observations were made at the very beginning of recruitment, a crucial phase in a process following autocatalytic dynamics. Methods Mature queenless colonies (n = 3) of Lasius niger were collected from the slopes of earth banks in Brussels. They contained around 1,100 workers (range 800 1,400 workers) and were reared in plaster nests ( cm). As a first step in understanding the colony s behaviour to diminish the risks of inter-colony heterogeneities due to heterogeneous development of the brood, our colonies contained no larvae or brood. Each colony was subdivided into three connected sections ( cm) covered by a glass plate and placed in plastic arenas ( cm), the borders of which were coated with Fluon Ò. Fluon is a colloidal solution of Teflon Ò that creates a very smooth

3 Recruitment in starved nests: the role of direct and indirect interactions 561 surface that prevents ants from escaping from the arena. These nests were closed, except for the entrance (1 cm wide) located in the middle of the longer front side of the nest. The nests were regularly moistened and kept at a temperature of 22 ± 3 C with a 12 h photoperiod. The ants were fed thrice a week with brown sugar solution (0.6 M), cockroaches (Periplaneta americana) and maggots (Calliphora erythrocephala). The influence of the length of food deprivation on the behaviour of foragers and nestmates was studied at the beginning of food recruitment. We focused our observations on the first 20 min of food exploitation since this period greatly influences further collective behaviours that may rapidly snowball. We analysed the response of ants to 3 ll droplets of sucrose solution (0.6 M), which exceeded crop capacity (Mailleux et al., 2003). The concentration and volume of these experimental droplets were similar to those of the honeydew droplets produced by aphids (Auclair, 1963). During one experiment, the nests were connected by a cardboard bridge (length 20 cm, width 0.5 cm) for 1 h to a small foraging area (6 9 6 cm) that the ants were able to freely explore. After the exploration phase, a food source was placed in the centre of the foraging area. The first forager that discovered the food droplet returned to the nest. It was called Scout (S, see below) and we quantified its behaviour inside the nest. Once this scout had contacted some nestmates and left the nest, it was gently removed and the foraging area was also removed. In doing so, we prevented other ants from reaching the food and limited our study to the behaviour of only one scout and its contact with its nestmates. For each experiment, we video recorded the nest for 20 min (magnification 29), starting when the observed ant entered the nest. Overall, we observed 28, 25 and 26 scouts after 1, 4 and 8 days of food deprivation (called 1 dd,4 dd and 8 dd in the text, respectively). Each food deprivation level is imposed on each colony in a random order. After a food deprivation period, the colonies were fed ad libitum during a resting period of 7 days before another food deprivation period. The maximum food deprivation period (8 days) did not induce abnormal mortality (Lenoir, 1979). We studied all the possible interactions between scouts and nestmates: antennal, trophallactic contacts and/or pheromonal emission. A trophallactic contact (Wheeler, 1910) is the transfer of fluids between members of a society through mouth-to-mouth feeding. We considered different behavioural groups, as detailed in Table 1. Nestmates were defined according to the type of contact (antennal or trophallactic) they made with the scout. Some nestmates made several antennal contacts and/or trophallactic contacts and/or a mixture of both. The nestmates that made one or more trophallactic contacts with the scout, regardless of the antennal contacts they had with the scout, were classified a tc (for trophallactic contact). Table 1 defines behavioural groups we studied and the abbreviation we used to refer to them. The other nestmates that only made antennal contact with the scouts were classified as ac (antennal contact). As the results concerning the Ne, workers that were close to the entrance in control condition i.e. when no scout entered the nest were already published (Mailleux et al., 2010), we did not detail them here but we used them for comparison. The following parameters were measured: 1. The proportion that left the nest during a period of 20 min after the entrance of the scout. This was calculated for each food deprivation period for each ant Table 1 Description of the different behavioural groups S Scout Individual that explored a new foraging area, discovered food and returned to the nest while laying a recruitment trail. Scouts were tested only once. After their visit to the nest, they were kindly removed from the nest ac tc NC Ne Nestmate having an antennal contact with S Nestmate having an trophallactic contact with S Nestmate having no contact with S (scout having access to food) Nestmate having no contact with S (no food) Nestmate having an antennal contact with the scout inside the nest. Nestmates having antennal contact with an ac worker were called a? ac (a for antennal, C for contact) Nestmate having a trophallactic contact with the scout inside the nest and received sugar solution regurgitated by the returning fed scout. Nestmates having a secondary trophallactic contact with the tc worker were called t? tc.) Nestmates having an antennal contact with the tc worker were called (a? tc). Nestmate that stood motionless at 1 cm (±0.3 cm) from the entrance. It was not in direct contact with the scout although it could potentially perceive pheromones emitted by the scout. In these conditions, the departure of the NC from the nest was considered to be spontaneous and/or triggered by the emission of chemical signals emitted by the scout (Mailleux et al., 2010). We did not take account all ants close to the scout but chose one worker NC per scout. Nestmate chosen with similar criteria as NC (individuals situated around 1 cm from the entrance, motionless), but observed during the exploration phase when no food was offered and no chemical trail was laid. They were considered as control ants i.e. ants that left the nest spontaneously without being stimulated either by a contact or by a pheromone.

4 562 A.-C. Mailleux et al. category. For example, in all experiments, we counted the number of ac observed after 1 day of deprivation that left the nest within the 20 min period and divided this by the total number of ac observed after 1 day of deprivation. This was calculated for all behavioural groups (S, ac, tc, a? ac, t? tc, a? tc, NC, Ne) (Table 1). 2. The time to leave the nest. For the scouts (S), the time to leave the nest started once the last contact ended and lasted until the scout left the nest. For tc and ac, the time to leave the nest started at the end of the last contact with the scout and ended when the nestmates left the nest. For NC, the time to leave the nest began when a scout reached the vicinity of the NC nestmate (around 1 cm) and ended when the NC left the nest. This was calculated for all experiments and for each behavioural group of ants (S, ac, tc, a? ac, t? tc, a? tc, NC). 3. The duration and number of antennal or trophallactic contacts. For S, the total contact time summed the time one scout spent in antennal and/or trophallactic contact with other nestmates. The number of contacts was the total number of contacts one scout had before leaving the nest. For tc, the total contact time and the number of contacts were measured during the contacts between tc and all of the other nestmates. The total contact time was the sum of the time spent in contacts (antennal and/or trophallactic) by one tc with the other nestmates. Similarly, for ac, we measured the same two parameters, although since ac did not receive sugar solution from the scout, all acs only had antennal contact with the other nestmates. The parameters (total contact time, number of contacts, time to leave the nest) were measured only for ants that left the nest within 20 min after the entrance of the scout. Indeed, preliminary observations showed that ants that did not leave the nest within 20 min joined an aggregate of ants situated deep within the nest and stopped moving. Hence, we considered that these ants were not involved in the initiation of foraging and we did not measure their time to leave the nest nor the contacts they had with their nestmates. 4. The trail-laying behaviour of the scout. When laying trail pheromones a worker bends its gaster down towards the substrate (the normal position being nearly horizontal), interrupts its walk for a fraction of a second and backs up to amplify the movement of its gaster. This behaviour was taken as the criteria for trail laying (Beckers et al., 1993). As ants were observed in a low-angle shot, it was not possible to precisely quantify the trail-laying behaviour (Mailleux et al., 2003), but it was possible to qualify the ants as trail-layers or non-trail layers. The behavioural parameters of L. niger foragers and nestmates subjected to different levels of food deprivation were compared using GraphPad Instat version 3.05 for Win95/NT (GraphPad Software, 1998, Inc., San Diego, California, USA). Given that most data were not normally distributed, we used non-parametric statistics to compare behavioural parameters as a function of food deprivation period. All behavioural parameters were compared in function of the colony and showed no colony effects. We used the Kruskal Wallis test with Dunn s post hoc test to compare the time parameters (time to leave the nest, total contact time) and the numbers of contacts (trophallactic or antennal) as a function of the deprivation period. We used chi-square tests to compare the proportion of ants leaving the nest versus the one not leaving the nest. Results Scout (S) Regardless of the length of the food deprivation period, all scouts returned to the nest, unloaded their sugar freight and left the nest within 5 min. When food deprivation was prolonged, the time to leave the nest significantly decreased (Table 2). When food deprivation was prolonged, the total number of contacts between by scouts and nestmates significantly decreased (from five to two antennal contacts) while the number of trophallactic contacts remained unchanged (Table 3). Indeed, no matter the food deprivation period, a scout performed about two trophallaxis and spent about 33 s in each of them; 1 dd : 28 ± 20 s (median ± 25th percentile, t rophallaxes = 52); 4 dd : 39 ± 27 s (n t = 45); 8 dd : 35 ± 25 s (n t = 43) (Kruskal Wallis test, KW = 4.72, ). We observed that 91% of the scouts laid at least one chemical spot of trail pheromone near the nest entrance when they returned from the source (after 1 dd ; 85% (n = 28) after 4 dd ; 91% (n = 25) after 8 dd, 96% (n = 26), v 2 test: v = 2.2, p = 0.33). Ants that had antennal contact with a scout (ac) and ants that had antennal contact with ac (a? ac) The proportion of ac that left the nest within the 20 min observation period significantly increased from 0.2 to 0.55 as food deprivation increased (Fig. 1, v 2 test: v = 37.1, p \ 0.001). Apart from this, the other behavioural para-

5 Recruitment in starved nests: the role of direct and indirect interactions 563 Table 2 Comparison between the time to leave the nest and the total time of contact for the different ant groups after three food deprivation periods i.e. the scouts (S), nestmates having antennal contact with the scouts (ac) and nestmates having contact with these ac (a? ac), nestmates having trophallactic contact with the scout (tc), nestmates having trophallactic contact with tc (t? tc) or antennal contact with tc (a? tc) S ac tc a? ac a? tc t? tc Time to leave the nest (in s) Total time of contact (in s) 1 dd 125 ± 88 [28] 58 ± 27 [26] 314 ± 120 [11] 52 ± 12 [21] 42 ± 31 [9] 361 ± 73[2] 4 dd 94 ± 77 [25] a 26 ± 18 [25] 334 ± 188 [27] 45 ± 12 [23] 48 ± 27 [13] 187 ± 36[8] 8 dd 81 ± 61 [26] a 105 ± 34 [12] 368 ± 115 [25] 47 ± 21 [38] 45 ± 18 [29] 179 ± 102[8] KW = 9.03, p \ 0.01 KW = 4.69, KW = 4.81, KW = 0.91, KW = 0.82, KW = 0.09, 1 dd 80 ± 53 [28] 1 ± 1 [26] 130 ± 42 [11] 1 ± 1 [21] 3 ± 1 [9] 121 ± 11 [2] 4 dd 90 ± 63 [25] 1 ± 1 [25] 154 ± 41 [24] 1 ± 1 [23] 1 ± 1 [13] 47 ± 20 [8] 8 dd 61 ± 46 [26] 1 ± 1 [12] 80 ± 45 [25] 1 ± 1 [38] 1 ± 1 [29] 35 ± 30 [8] KW = 4.27 KW = 0.25 KW = 4.92 KW = 5.57 KW = 5.20 KW = 0.24 Median and 25% percentile are given for all parameters. Numbers in brackets indicate sample size. All parameters were compared using the Kruskal Wallis test. Values of p and the results of the statistical tests (: not significant) are given in the last row for each parameter. When a group of data was considered as statistically different in function of the deprivation period, we made a post hoc Dunn test at the 0.05 level of significance; In this case, groups sharing the same letter were not statistically different. The parameters were measured only for ants that left the nest within 20 min after the entrance of the scout Table 3 Comparison between the number of antennation and trophallaxis for the different ant groups after three food deprivation periods i.e. the scouts (S), nestmates having antennal contact with the scouts (ac), nestmates having trophallactic contact with the scout (tc) S ac tc Number of antennal contacts 1 dd 5 ± 3 [28] 1 ± 0 [26] 9 ± 7 [15] 4 dd 3 ± 2 [25] a 1 ± 0 [25] 11 ± 4 [24] 8 dd 2 ± 1 [26] a 3 ± 1 [12] 9 ± 8 [25] KW = 14.38, p \ KW = 0.16, KW = 0.36, Number of trophallaxes 1 dd 2 ± 2 [28] 0 ± 0 [26] 1 ± 1 [15] 4 dd 2 ± 1 [25] 0 ± 0 [24] 2 ± 1 [24] 8 dd 2 ± 1 [26] 0 ± 0 [12] 2 ± 2 [25] KW = 2.66, KW = 1.42, KW = 4.40, For S, these parameters concerned the contacts between S and C. For ac (or for tc), these parameters concerned the contacts between ac (or tc) and all the other nestmates that ac (or tc) met in the nest. Median and 25th percentile are given for all parameters. Numbers in brackets indicate sample size. All parameters were compared using the Kruskal Wallis test. Values of p and the results of the statistical tests (n.s: not significant) are given in the last row for each parameter. When a group of data was considered as statistically different in function of the deprivation period, we made a post hoc Dunn test at the 0.05 level of significance; In this case, groups sharing the same letter were not statistically different. The parameters were measured only for ants that left the nest within 20 min after the entrance of the scout meters (time to leave the nest, number and duration of contacts with other nestmates) did not differ with food deprivation levels (Tables 2 and 3). No matter how long the food deprivation period was, the acs that left the nest did so within 44 ± 25 s (n = 63) (Table 2) and, before leaving, these ac had antennal contacts with around two nestmates (a? ac individuals) for a total duration of around 1 s (n = 63, Table 2). The proportion of these a? ac leaving the nest significantly increased with food deprivation (Fig. 1, v 2 test: v = 19.95, p \ 0.001). Before leaving, a? ac ants (ants that had antennal contact with nestmates that had previously come in contact with the scout) for a total time of 1 s ± 1s (n = 81). This time was similar to the contact time between S and ac (Kruskal Wallis test, KW = 6.72, ). Ants that had trophallactic contact with a scout (tc), ants that had antennal contact with tc (a? tc) and that had trophallactic contact with tc (t? tc) The proportion of tc ants that left the nest after having exchanged food with scouts significantly increased from 0.2 to 0.6 with food deprivation (Fig. 1, v 2 test: v = 21.1, p \ 0.001). However, the percentage of those tc ants that engaged themselves into a secondary trophallaxis with other nestmates did not increase with food deprivation

6 564 A.-C. Mailleux et al. Proportions that left the nest after the entrance of the scout a 129 b 35 bc 22 a 56 be bd cdef 39 bg 37 chi 66 fij 121 afh a 168 (Table 3). The total time spent doing trophallaxis was not influenced by food deprivation (Table 2). Among the starved tc ants, 25% (n = 24) and 32% (n = 25) (after 4 dd and 8 dd, respectively) left the nest after laying chemical marks near the nest entrance. This percentage of ants that laid a trace was not statistically significant for 4 dd and 8 dd (Fisher s test, D = 0.07, p [ 0.05). This behaviour was not observed after 1 dd (n = 28) and it was not observed among ac nestmates (n = 63). The proportion of t? tc ants that received a secondary trophallaxis and left the nest increased with food deprivation (v 2 test: v = 10.99, p \ 0.001). These t? tc individuals left the nest in a similar proportion to tc ants having received food directly from the scout at least after 1 dd and 4 dd (v 2 test: v = 0.03 and 1.71, respectively, ). However, the proportion of departing t? tc was lower than that of tc after 8 dd (v = 5.89, p \ 0.05). The proportion of a? tc ants that left the nest after antennal contact with tc increased with the length of food deprivation (v 2 test: v = 31.1, p \ 0.001). Before leaving, a? tc had antennal contacts for a total time of 1 ± 1s (n = 63). In fact, concerning the time to leave the nest, none of the ants that had antennal contact (ac, a? ac, a? tc) b 35 acj 9 acdegj 20 ac tc NC a ac a tc t tc Behavioural groups 1 deprivation day 4 deprivation days 8 deprivation days Fig. 1 Proportions of ants that left the nest within the first 20 min in function of the food deprivation level (1, 4 and 8 food deprivation days). The behavioural groups were all composed of nestmates: (ac) nestmates having antennal contact with the scouts (a? ac) nestmates having contact with ac (tc) nestmates having trophallactic contact with the scout (t? tc) nestmates having trophallactic contact with tc or (a? tc) nestmates having antennal contact with tc and (NC) were the ones having no contact with the scout. The numbers on top of the bars were the number of ants in each group. Proportions were compared with v 2 test: groups with the same letter were not significantly different. All proportions after 1 food deprivation day differed from the proportion after 4 and 8 food deprivations days except in the group a? tc acj 27 were statistically different (Kruskal Wallis test, KW = ). Ants that had no contact with a scout (NC) When food deprivation was prolonged, the proportion of non-contacted ants (NC) that left the nest increased statistically from 0.38 to 0.72 (Fig. 1, v 2 test: v = 9.39, p \ 0.01). The time to leave the nest (s) decreased significantly with food deprivation (1 dd : 42 ± 35, n = 15; 4 dd : 12 ± 3, n = 24; 8 dd : 10 ± 1, n = 25, KW = 10.64, p \ 0.005). The NCs were closer to the nest entrance and did not receive food from the scouts. These ants did not spend time in contacts. This resulted in a shorter time to leave the nest than the other behavioural groups. It must be noted that the proportion of ants that left the nest was similarly influenced by length of food deprivation for NC, ac and tc after 4 dd and 8 dd. Several NC departed from the nest without having had contact with the scout or any other nestmates. Was the exit of these NC ants spontaneous or triggered by the emission of chemical signals by the scout? We compared the proportions of NC and Ne that left the nest. Ne were considered a control group as they were chosen with similar criteria as NC (Table 1) but observed during the exploration phase when no food was offered and no trail laid. No matter what the food deprivation period was, the proportion of NC that left the nest was higher than the proportion of Ne (these last results were presented in Mailleux et al., 2010, v 2 test: after 1 dd, v = 4.8, p = 0.03; 4 dd, v = 8.9, p \ 0.005; 8 dd, v = 9.3, p \ 0.005). Comparison between ants that had antennal contact with the scout (ac) or trophallactic contact with the scout (tc) When the food deprivation period was prolonged, the proportion of ants that left the nest increased for both groups of nestmates ac and tc: 1 dd : 0.20 (n = 185); 4 dd : 0.52 (n = 82); 8 dd : 0.58 (n = 64) (v 2 test: v = 43.43, p \ 0.001) (Fig. 1). For equal food deprivation periods, the proportion that left the nest was similar for tc and ac suggesting that food intake did not facilitate nor prevent the exit of recruited nestmates. The time to leave the nest, the duration of contact and the number of contacts were always higher for tc workers than for ac as tc spent time making trophallaxes with some t? tc to unload their crop. When the food deprivation period was prolonged, scouts had fewer antennal contacts with ac and these nestmates departed more frequently. Therefore, the number of exiting ac per scout did not change with food deprivation: 1 dd : 1.0 ± 0.0, n = 28; 4 dd : 1.0 ± 0.0, n = 25; 8 dd : 1.0 ± 0.3, n = 26, KW = 1.89, ).

7 Recruitment in starved nests: the role of direct and indirect interactions 565 Comparison between ants that had no contact with the scout (NC), ants that had a? ac, a? tc, t? tc) and the control group (Ne) No matter how long the food deprivation period was, the proportion of a? ac that left the nest was similar to the Ne group (the control group) (v = 0.73, 0.14, 0.75 after 1 dd,4 d, and 8 dd, respectively, for all). Similarly, the proportion of t? tc that left the nest was similar to that of the Ne group (the control group) (v = 0.17, 0.47, 0.41 after 1 dd,4 d, and 8 dd, respectively, for all). This suggests that the exit of a? ac and t? tc was spontaneous as it occurred at similar rates than spontaneous departure of ants in control conditions (Ne). Discussion This study of social interactions between ant foragers and nestmates has shed light on the mechanisms that increase recruitment in colonies that are highly deprived of food. It is known that L. niger scouts that discovered a food source deposited a chemical signal that stimulated the departure of nestmates located near the nest entrance (Beckers et al., 1993; Mailleux et al., 2006, 2010). Here, we show that the proportions of nestmates that left the nest increase similarly with the level of food deprivation, independently of any direct contact with a scout. Thus, the signal delivered by the scout that triggers the exit of recruits must be chemical and is probably the same pheromone trail as the one laid when scouts come back to the nest. When the colony is food deprived, this signal is more effective although not stronger (Mailleux et al., 2010). Our results strongly suggest that food deprivation lowers the response threshold of the nestmates to this signal resulting in a more important exit from the nest (de Biseau and Pasteels, 2000; Mailleux et al., 2006, 2010). It is noticeable that nestmates that received food from scouts laid a chemical mark on the nest substrate. Indeed, 27% of food deprived nestmates that received food through trophallaxis increased the arousal state of workers by depositing chemical marks before leaving the nest. This phenomenon has never been evidenced before. It is commonly thought that an ant must get in contact with the source to lay a trail. This unexpected behaviour is not observed after short food deprivation (1 day) and it was not observed for nestmates that only had antennal contacts with scouts. This phenomenon may contribute to the amplification process of recruitment when the colony is food deprived. Here, we observe that starved scouts take less time to leave the nest. This time-saving is not responsible for steeper recruitment after long food deprivation. Indeed, a starved scout drinks for a longer time at the food source (Mailleux et al., 2006). This increased drinking time counterbalances the decrease in the time taken to leave the nest observed in the present study. As a result, the duration of foraging cycles (i.e. the time between two visits to the food source) is similar whatever the food deprivation period: it lasts around 337 s after 1 dd ; 327 after 4 dd and 312 after 8 dd. Therefore, the influence of food deprivation level on the collective response is not mediated by differences in the time to upload food at the source and to share this load with its nestmates. Previous studies have evidenced the major role of trophallaxes in the regulation of foraging in social insects (e.g. in fire ants Cassill and Tschinkel, 1999b; Cassill, 2003). In honeybees and ants, trophallaxes govern the dynamics of forager s departure from the nest: a forager has to unload the food it ingested at the source before leaving for another trip (Anderson and Ratnieks, 1999; Buffin et al., 2009; Hart and Ratnieks, 2001). The availability of the receivers acts as negative feedback: if no recruit accepts the recruiter s load, the latter remains inside the nest and the exploitation of the food source will diminish and finally stop. Here, we show that L. niger scouts must also unload their freight before leaving the nest but that the number of trophallaxes remains unchanged whatever the level of food deprivation. However, food deprivation facilitates the unloading of food by the scout towards nestmates. Indeed, in L. niger, after a short food deprivation period, nestmates rarely accept trophallaxis, which forces scouts to contact a greater number of nestmates before unloading. After long food deprivation periods, scouts more easily find motivated nestmates to accept their sugar load (Mailleux et al., 2010). Trophallaxes seem to delay the departure of the L. niger. Ants that received sugar solution (directly from the scout or indirectly during a secondary trophallaxis) take more than 3 min to leave the nest. Therefore, food transfer between ants increases the dynamics of the first steps of the recruitment process in such a mass foraging ant. Our study of workers behaviour inside the nest confirms the role of chemical cues in the regulation of the foraging activity but also highlights the importance of nestmates motivation. Indeed, the foraging activity of the colony depends not only on the recruiting signals but also on the state of the individuals that perceive it (here below a certain threshold of hunger). Not only does the behaviour of the recruiting scouts change according to the food source (Wilson, 1962; Nonacs and Dill, 1990, De Marco, 2006) but the response of potential recruits will depend on their own needs. 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