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Seasonal variation in nestmate recognition in Paratrechina flavipes (Smith) worker ants (Hymenoptera: Formicidae)
Anita. Behav., 1991, 41, 1-6
Seasonal variation in nestmate recognition in Paratrechinaflavipes (Smith) worker ants (Hymenoptera: Formicidae) KATSUYA ICHINOSE The Institute of Low Temperature Science, Hokkaido University, N19W8 Kita-ku, Sapporo 060, Japan
(Received 7 March 1989; initial acceptance27 March 1989; final acceptance 20 December 1989; MS. number: 3368)
Abstract. Colonies of Paratrechinaflavipes were split into artificial nests, and placed at three different sites. About every 2 weeks for 2 years workers from one of these nests were paired with (related) workers from another nest from the same colony or with (unrelated) workers from a different colony. Aggression between workers of the paired nests varied seasonally. Workers were aggressive to related individuals only during the season when the nest was active, but they were always aggressive to unrelated workers. Such variability in aggression within a colony may be due to a seasonal change in the way nestmates are recognized, i.e. workers in active nests may discriminate their nestmates from non-nestmates by using both environmental and genetic odours, whereas when the nest is inactive they use genetic odour alone. The variable recognition mechanism of this ant may be involved in the evolution of more than one nest and one reproductive queen in a colony.
Nestmate recognition by social Hymenoptera can be based on genetic or environmental odours, or both (Carlin & H611dobler 1986). Whatever the source of the odours, however, workers must learn those occurring within their own nest (Carlin et al. 1987; Isingrini & Lenoir 1988; see also Waldman 1988). These workers will then attack ants with strange odours. F o r ant species such as Camponotus floridanus (Morel & Blum 1988), Solenopsis invicta (Obin & Vander Meer 1988) and Leptothorax spp. (Stuart 1988), there is no critical period for learning the odour of the nest. These workers can change the odours they use for nestmate recognition throughout their lives. Recently Vander Meer et al. (1989) showed clearly that patterns of hydrocarbons on the cuticle of S. invicta workers change periodically throughout the life of the colony. Since the workers recognize their nestmates from substances that are decided by both genetic and environmental factors in the colony, they must adjust their perception to the current milieu of odours. Thus their recognition of, and aggression towards, nestmates or nonnestmates varies according to the conditions within and around the colony. In many animals, both vertebrates and invertebrates, nestmate recognition involves them perceiving the odours (or labels) of other individuals and comparing them with those they have learnt previously (the template; see Waldman 1988). Among social insects, for example, 0003-3472/91/010001 + 06 $03.00/0
their response to another individual, whether aggressive or otherwise, depends on whether the label and template match. If they do not match, the insects will attack each other. If they match, but the template of, say, insect A, is much broader than the label of insect B, insect A will not be aggressive towards insect B but insect B will be aggressive towards insect A. Carlin & H611dobler (1986) reported such an asymmetry in aggression among workers of Camponotus spp. Flexibility among ants in learning recognition odours can explain how new nests split off and become independent (Gamboa et al. 1986; Breed et al. 1988; Morel & Blum 1988). For example, Lasius flavus sometimes establishes colonies composed of more than one nest (Pontin 1961). Some budded nests separate gradually from their maternal nest when they relocate. If they lose all contact with the maternal nest and become isolated from other nests, they become independent colonies. If workers of each of these colonies learn a distinctive nest odour, they may behave aggressively towards related workers of the other nests which have different odours. They cannot then resume intimate contact with each other, i.e. these isolated nests are unlikely to merge. Paratrechina flavipes also establishes colonies with more than one nest by budding (Ichinose 1986). Some budded nests become independent in @ 1991 The Association for the Study of Animal Behaviour
Animal Behaviour, 41, 1 summer but the majority merge with other nests in autumn (Ichinose 1987b). Flexibility in learning nest odours can explain how this ant forms independent nests but not how they merge so quickly. Such nest dynamics, however, may be explained if the aggression of workers varies seasonally as shown by Rosengren et al. (1985) for Formica truncorum: cool conditions between autumn and spring lower aggression between workers from different nests, while warm conditions in summer increase it. If temperature is involved in the nest dynamics of P.flavipes, aggression between workers should vary from season to season. In this study I tested whether there was a seasonal effect on worker aggression by rearing workers in artificial nests under semi-natural conditions. By using nests with and without queens and placing them in different habitats, I could test the seasonal changes in response of the ants to queen odours and environmental odours which remained constant throughout the year. The tests were designed to reveal the effects of odour differences, based on queen and habitat, on nestmate recognition in this ant species.
METHODS
I collected 10 colonies of P. flavipes during 15-20 June 1986 in the Tomakomai Experiment Forest, southern Hokkaido. Each of them occupied only one nest and had only one queen. I extracted all ants from each sample and until 29 June 1986 reared each colonyin a separate incubator at 5 __+1~ having put them in an artificial nest (measuring 137 x 65 • 35 ram) with 1 cm of moist plaster of Paris covering the bottom. I paired the colonies (A, B) at random making five replicates. In a replicate I prepared two artificial nests from the A colony and four from the B colony: A (forest/queen) and A (forest/no queen) from A; B (forest/queen), B (forest/no queen), B (grass/no queen) and B (spruce/no queen) from B. I provided each of these nests with 50 workers and 50 final instar but caste-labile larvae from the original colony. I added the queens of the A and B colonies to A (forest/queen) and B (forest/queen), respectively. These nests were the same size as the artificial nest in the incubator, but they were provided with three openings sealed with stainless steel mesh to allow ventilation and to prevent ants from escaping. One opening (15ram diameter) was on each of the longer sides and the other (5 mm) on the ceiling of the nest.
From 1 July 1986 to 30 September 1987 1 settled A (forest/queen), A (forest/no queen), B (forest/ queen) and B (forest/no queen) within 50 cm of each other on the ground in a broad-leaved forest, B (grass/no queen) in a grassland site and B (spruce/ no queen) in a broad-leaved forest containing about 30% Yedo spruces, Picea jezoensis. All the sites in the Experiment Forest were 0.5-8-0km from each other. From 1 December 1986 to 26 May 1987 these nests were buried deep enough (about 70 cm) to avoid freezing and to allow successful hibernation. Except during hibernation, each nest was given Drosophila virilis and sugar crystals every 1-2 days through its sliding lid. About every 2 weeks I carried these nests to the laboratory of the Experiment Forest and made the following observations over a day. Three workers (recipients) were randomly selected from a nest and introduced into an arena (measuring 61 x43 • 17 ram), the bottom of which was covered with moist filter paper. When these workers had calmed down, a worker (donor) from another nest was introduced into the arena, and I observed the behaviour of the recipient workers towards the donor for 5 rain to determine the degree of aggression between them. After removing the donor worker I repeated the same observation twice using other workers from the donor nest. After the observation, these workers were returned to their original nests. The same observation was made for all possible pairs of nests on every test day. All nests examined were rested in the laboratory at room temperature, and then settled again at their original sites in the evening of the same day. The test days were: 1, 17 and 31 July, 15 and 31 August, 15 and 30 September, 15 and 31 October and 30 November in 1986; 15 and 30 June, 15 and 30 July, 17 August and 25 September in 1987. Following De Vroey & Pasteels (1978), I graded worker aggression in a pair as follows: 0 (nonaggressive); 1 (persistently licking towards an opponent); 2 (tentatively biting or threatening with mandibles opened); and 3 (struggling severely with gaster flexed or chasing an opponent). Aggression between workers of Formica spp. decreases with time after they are mixed, and disappears eventually (Rosengren & Pamilo 1983). I observed that aggression of P. flavipes workers would also disappear within a day after they were mixed in an arena. Workers from the same colony, however, did not show aggression when I observed them intermittently over several days. The longer
Ichinose: Ant nestmate recognition one observes aggression between workers of different colonies, the more likely it is that amicable contacts between them will be seen. Thus long observations could reduce the average score of aggression, whereas the largest score recorded should be less influenced by how long the observation period lasts. The maximum aggression score observed in a test is thus likely to be the most moderate index for the likelihood that the nests of the observed workers would mingle with each other or that workers of one nest would recognize those of the other as nestmates. I therefore determined aggression between a pair of workers from two nests (e.g. A (forest/queen)= recipient and B (forest/queen)=donor) by the maximum score of aggression observed on any given test day. For example, the score is 2 when 0scored behaviour is elicited by the first B (forest/ queen) worker introduced to an arena containing three workers of A (forest/queen), 2 by the second and 1 by the third. I also noted aggression in the opposite direction, from donor to recipient. I made such reciprocal observations on all possible pairs on every test day. I observed and scored 1500 pairs in 1986 (150 pairs on a test d a y • 10 days), but 136 in 1987 because many nests collapsed after unsuccessful hibernation or from the deaths of workers during that period.
RESULTS I observed aggressive behaviour between workers from two nests 1636 times. Since this aggression involved two directions, e.g. from A (forest/queen) to B (forest/queen) and vice versa, I actually examined aggression between two nests 818 times (1636/2). The scores of reciprocal pairs differed from each other in 44 out of 818 cases (5.4%). I tested by A N O V A whether or not there was a significant difference between each reciprocal pair. This test was done 75 times (15 reciprocal pairs in a replicate (see below) x five replicates), but none of these tests showed significant differences between them (Fs was always less than 1, P>0-05). Since this means that there is no statistical reason to distinguish between the two directions of each reciprocal pair, I adopted the larger score of the two pairs as the aggression score between the two nests on any given day. Consequently, the number of scores was halved, 750 in 1986 and 68 in 1987. Thus I analysed aggression scores between the following
15 pairs: A (forest/queen) versus A (forest/no queen), B (forest/queen), B (forest/no queen), B (grass/no queen) and B (spruce/no queen); A (forest/no queen) versus B (forest/queen), B (forest/ no queen), B (grass/no queen) and B (spruce/no queen); B (forest/queen) versus B (forest/no queen), B (grass/no queen) and B (spruce/no queen); B (forest/no queen) versus B (grass/no queen) and B (spruce/no queen); B (grass/no queen) versus B (spruce/no queen). Because I observed too few pairs in 1987, I analysed only the results from 1986. To assess the effects of either the difference in colony (A or B) or the presence of a queen on scores of worker aggression, I calculated averages of aggression scores over the five replicates in four groups (Fig. la). Aggression scores were influenced more by which colony the workers came from than by the presence of a queen. I also averaged aggression scores in each of six groups to assess the effects of the difference in habitat (broad-leaved forest, grassland or spruce forest) on aggression (Fig. 1b). Aggression between colonies (A versus B) remained high over all seasons but aggression within colonies (A versus A, B versus B) varied seasonally (Fig. 1). A two-dimensional ANOVA was applied to the between-colony and within-colony groups by integrating the five replicates in order to detect differences in the score between pairs of nests (df=79 in the betweencolony group, df= 69 in the within-colony group) and dates (t/f= 9 in both groups). The difference between pairs was not significant in either group (Fs=0.404 for the between-colony group, F s = 0.967 for the within-colony group, P > 0 ' 0 5 for both). The difference between dates was significant in the within-colony group ( F s = 31.56, P<0.01) but not the between-colony group (Fs=0-064, P>0"05). In addition, the lack of a difference in aggression between pairs in each of the two groups suggests that there were no effects of the queen and habitat on changes in worker aggression. DISCUSSION Aggression of P. flavipes workers was not influenced by either the presence of a queen or the habitat, regardless of whether the aggression was between colonies or within colonies (Fig. 1). Since aggression between colonies remained high over all seasons, it seems that nestmate recognition depends primarily on the genetic makeup of a colony. Aggression between workers of the same colony
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Figure 1. Aggression between pairs from different colonies ( &, 0 , 9 and from the same colony (A, 9 []). (a) Queen effects. Either or both of the paired nests have a queen ( A, A ) or both are queenless ( 9 9 ): A (forest/queen) versus all nests from the B colony, A (forest/no queen) versus B (forest/queen); A (forest/no queen) versus B (forest, grass, spruce/ no queen); A (forest/queen) versus A (forest/no queen), B (forest/queen) versus B (forest, grass, spruce/no queen); B (forest/no queen) versus B (grass, spruce/no queen) and B (grass/no queen) versus B (spruce/no queen). (b) Environmental effects. Paired nests were settled at the broad-leaved forest (A, •), either of the paired nests at the grassland site ( 0 , O), or either of the paired nests at the spruce mixed forest ( 9 []): A (forest/queen) versus B (forest/queen, no queen); A (forest/queen, no queen) versus B (grass/no queen); A (forest/queen, no queen) versus B (spruce/no queen); A (forest/queen) versus A (forest/no queen) and B (forest/queen) versus B (forest/no queen); B (forest/queen, no queen) and B (spruce/no queen) versus B (grass/no queen); B (forest/queen, no queen) and B (grass/no queen) versus B (spruce/ no queen).
was negligiblejust before and after hibernation, but increased during the active season. So some seasonal modification is probably involved in nestmate recognition of P.flavipes workers. One possible means of change is that workers become more aggressive as they get older, as in Myrmica rubra (Cammaerts-Tricot 1975) or S. invicta (Sorensen & Fletcher 1985). In m a n y ant species callows are unlikely to attack or to be attacked by workers of different colonies (Cammaerts-Tricot 1975; Brian 1986; Carlin & Hflldobler 1986; 9 1986; Carlin
et al. 1987; Stuart 1987; Morel & Blum 1988). Callows of these ants will learn and acquire the odours of their own nests as they grow. With experience, they can discriminate their nestmates from non-nestmates and be recognized as familiar or unfamiliar workers by others. This means that when the callows replace m a n y or all of the older workers, the colony as a whole may be less aggressive towards other colonies. The replacement of P. flavipes workers used in the present study probably occurred in September
Ichinose: Ant nestmate recognition
or by early October of 1986 (cf. Ichinose 1987a). The expected change of callows and old workers in autumn cannot explain the reduction of aggression between colonies within a season. Alternatively, the recognition mechanism could be mediated by temperature-dependent changes in the physiology of workers rather than by their age. If their metabolism is always high enough to allow workers to detect genetic differences between kin and non-kin, aggression between colonies would be elicited at all seasons. In the active season, from June to September, workers are so sensitive that they can discriminate their nestmates from nonnestmates as aliens or enemies by their different environmentalas well as genetic odours. The microhabitats in the Tomakomai Experiment Forest (Higo 1986) may produce their own odours, causing artificial nests to acquire different odours. In the inactive season, October to May, lowered metabolism may allow the detection only of different genetic odours. Thus, aggression between colonies was evident all year. If addition of environmental odours triggered aggression of the workers within a colony, we can apply the 'cue similarity threshold' model for nestmate recognition which was first developed for paper wasps (Gamboa et al. 1986; Gamboa 1988). According to this model, workers have a threshold for nestmate recognition. When a worker encounters an individual she compares its odours with her own. If the difference in the odours exceeds the threshold she will behave aggressively towards the individual, while if it is below the threshold contact will be amicable. It is interesting that aggression was always lower within the colony than between colonies. This suggests that workers have two or more thresholds and environmental odours within the colony do not push the workers' perception over the final threshold. If so, environmental odours may influence worker aggression secondarily. If genetic odours on the body surface of workers had changed during the present study, aggression would appear even within the colony. Vander Meer et al. (1989) reported that colonies of S. invicta change recognition odours on the workers throughout their lives. However, it is hard to imagine that the odours of P.flavipes workers changed in such a way that within-colony aggression in all pairs of replicates disappeared until the inactive season. I therefore think that environmental odours added to genetic odours changed nestmate recognition in
P. flavipes only in the active season, while genetic odours prevailed in all seasons. The additive effect of environmental odours makes workers aggressive against non-nestmates and makes it easier for budded nests to become independent in summer. Disappearance of the effect in autumn allows the budded nests from a colony to merge again. On the other hand, permanent aggression against non-kin prevents them from merging with any nests budded from other colonies. In turn the mechanism of nestmate recognition in P.flavipes has kept it from evolving a multi-queened colony, although having a single queen in a colony, a multi-nested colony or a fragile nest structure (Ichinose 1986, 1987b) are consistent with the conditions necessary for a multi-queened colony to evolve (H61tdobler & Wilson 1977).
ACKNOWLEDGMENTS I thank Professor S. F. Sakagami for his critical comments and Professor K. Ishigaki for his permission to collect ants in the Tomakomai Experiment Forest. Miss J. B. Jacob kindly helped me prepare this manuscript.
REFERENCES Breed, M. D., Williams,K. R. & Fewell,J. H. 1988.Comb wax mediates the acquisition of nest-mate recognition cues in honey bees. Proc. natn. Acad. Sei. U.S.A., 85, 8766-8769. Brian, M. V. 1986.Bondingbetweenworkers and queensin the ant genus Myrmica. Anim. Behav., 34, 1I35 1145. Cammaerts-Tricot, M. C. 1975. Ontogenesis of the defence reactions in the workers of Myrmica rubra L. (Hymenoptera: Formicidae). Anim. Behav., 23, 124 130. Carlin, N. F. & H611dobler,B. 1986. The kin recognition system of carpenter ants (Camponotus spp.). I. Hierarchical cues in small colonies. Behav. Ecol. Sociobiol., 19, 123-134. Carlin, N. F., Halpern, R., H611dobler,B. & Schwartz, P. 1987. Early learning and the recognition of conspecific cocoonsby carpenter ants (Camponotus spp.). Ethology, 75, 306-316. De Vroey, C. & Pasteels,J. M. 1978.Agonistic behaviour of Myrmiea rubra L. Insectes Soe., 25, 24%265. Gamboa, G. J. 1988. Sisters, aunt-niece, and cousin recognition by social wasps. Behav. Genet., 18, 409-423. Gamboa, G. J., Reeve, H. K. & Pfennig, D. W. 1986.The evolution and ontogeny of nestmate recognition in social wasps. A. Rev. Entomol., 31, 431-454. Higo, M. 1986.Communitydynamicsof deciduousbroadleaved forests: tree growth in small gaps. Trans. Meeting Jap. For. Sot., 97, 327-328 (in Japanese).
Animal Behaviour, 41, 1 H611dobler, B. & Wilson, E. O. 1977. The number of queens: an important trait in ant evolution. Naturwissenschaften, 64, 8-15. Ichinose, K. 1986. Occurrence of polydomy in a monogynous ant, Paratrechina flavipes (Hymenoptera, Formicidae). Kontyft, 54, 208-217. Ichinose, K. 1987a. Annual life cycle of Paratrechina flavipes (Hymenoptera, Formicidae) in the Tomakomai Experiment Forest, southern Hokkaido. Konty~, 55, 9-20. Ichinose, K. 1987b. Seasonal changes in nest and colony densities of the formicine ant, Paratrechina flavipes (Hymenoptera, Formicidae), in Tomakomai, southern Hokkaido. Kontyft, 55, 565-572. Isingrini, M. & Lenoir, A. 1988. Colony recognition of larvae by young workers of Cataglyphis cursor (Hymenoptera, Formicidae). Behav. Proc., 17, 69-72. Morel, L. & Blum, M. S. 1986. Nestmate recognition in Camponotus floridanus callow worker ants: are sisters or nestmates recognized? Anita. Behav., 36, 718-725. Obin, M. S. 1986. Nestmate recognition cues in laboratory and field colonies of Solenopsis invicta Buren (Hymenoptera: Formicidae). Effect of environmental and role of cuticular hydrocarbons. J. chem. Ecol., 12, 1965-1976. Obin, M. S. & Vander Meer, R. K. 1988. Sources of nestmate recognition cues in the imported fire ant
Solenopsis invicta Buren (Hymenoptera: Formicidae). Anim. Behav., 36, 1361-1370. Pontin, A. J. 1961. Population stabilization and competition between the ants Lasiusflavus (F.) and L. niger (L.). J. Anita. Ecol., 30, 4%54. Rosengren, R., Herix, D. & Pamilo, P. 1985. Insular ecology of the red wood ant Formica truncorum Fabr. I. Polydomous nesting, population size and foraging. Mitt. Schw. Entomol. GeseL, 38, 147 175. Rosengren, R. & Pamilo, P. 1983. The evolution of polygyny and polydomy in mound-building Formica ants. Acta entomol.fenn., 42, 65-77. Sorensen, A. A. & Fletcher, D. J. C. 1985. Techniques for studying the execution of foreign queens by temporal subcastes in fire ants (Solenopsis invicta). Entomol. exp. appl., 37, 289-295. Stuart, R. J. I987. Transient nestmate recognition cues contribute to a multicolonial population structure in the ant, Leptothorax curvispinosus. Behav. Ecol. Sociobiol., 21,229-235. Stuart, R. J. 1988. Collective cues as a basis for nestmate recognition in polygynous leptothoracine ants. Proc. natn. Acad. Sci. U.S.A., 85, 4572-4575. Vander Meer, R. K., Saliwanchik, D. & Lavine, B. 1989. Temporal changes in colony cuticular hydrocarbon patterns of Solenopsis invicta. Implications for nestmate recognition. J. chem. EcoL, 15, 2115-2125. Waldman, B. 1988. The ecology of kin recognition. A. Rev. Ecol. Syst., 19, 543-571.
Seasonal variation in nestmate recognition in Paratrechinaflavipes (Smith) worker ants (Hymenoptera: Formicidae) KATSUYA ICHINOSE The Institute of Low Temperature Science, Hokkaido University, N19W8 Kita-ku, Sapporo 060, Japan
(Received 7 March 1989; initial acceptance27 March 1989; final acceptance 20 December 1989; MS. number: 3368)
Abstract. Colonies of Paratrechinaflavipes were split into artificial nests, and placed at three different sites. About every 2 weeks for 2 years workers from one of these nests were paired with (related) workers from another nest from the same colony or with (unrelated) workers from a different colony. Aggression between workers of the paired nests varied seasonally. Workers were aggressive to related individuals only during the season when the nest was active, but they were always aggressive to unrelated workers. Such variability in aggression within a colony may be due to a seasonal change in the way nestmates are recognized, i.e. workers in active nests may discriminate their nestmates from non-nestmates by using both environmental and genetic odours, whereas when the nest is inactive they use genetic odour alone. The variable recognition mechanism of this ant may be involved in the evolution of more than one nest and one reproductive queen in a colony.
Nestmate recognition by social Hymenoptera can be based on genetic or environmental odours, or both (Carlin & H611dobler 1986). Whatever the source of the odours, however, workers must learn those occurring within their own nest (Carlin et al. 1987; Isingrini & Lenoir 1988; see also Waldman 1988). These workers will then attack ants with strange odours. F o r ant species such as Camponotus floridanus (Morel & Blum 1988), Solenopsis invicta (Obin & Vander Meer 1988) and Leptothorax spp. (Stuart 1988), there is no critical period for learning the odour of the nest. These workers can change the odours they use for nestmate recognition throughout their lives. Recently Vander Meer et al. (1989) showed clearly that patterns of hydrocarbons on the cuticle of S. invicta workers change periodically throughout the life of the colony. Since the workers recognize their nestmates from substances that are decided by both genetic and environmental factors in the colony, they must adjust their perception to the current milieu of odours. Thus their recognition of, and aggression towards, nestmates or nonnestmates varies according to the conditions within and around the colony. In many animals, both vertebrates and invertebrates, nestmate recognition involves them perceiving the odours (or labels) of other individuals and comparing them with those they have learnt previously (the template; see Waldman 1988). Among social insects, for example, 0003-3472/91/010001 + 06 $03.00/0
their response to another individual, whether aggressive or otherwise, depends on whether the label and template match. If they do not match, the insects will attack each other. If they match, but the template of, say, insect A, is much broader than the label of insect B, insect A will not be aggressive towards insect B but insect B will be aggressive towards insect A. Carlin & H611dobler (1986) reported such an asymmetry in aggression among workers of Camponotus spp. Flexibility among ants in learning recognition odours can explain how new nests split off and become independent (Gamboa et al. 1986; Breed et al. 1988; Morel & Blum 1988). For example, Lasius flavus sometimes establishes colonies composed of more than one nest (Pontin 1961). Some budded nests separate gradually from their maternal nest when they relocate. If they lose all contact with the maternal nest and become isolated from other nests, they become independent colonies. If workers of each of these colonies learn a distinctive nest odour, they may behave aggressively towards related workers of the other nests which have different odours. They cannot then resume intimate contact with each other, i.e. these isolated nests are unlikely to merge. Paratrechina flavipes also establishes colonies with more than one nest by budding (Ichinose 1986). Some budded nests become independent in @ 1991 The Association for the Study of Animal Behaviour
Animal Behaviour, 41, 1 summer but the majority merge with other nests in autumn (Ichinose 1987b). Flexibility in learning nest odours can explain how this ant forms independent nests but not how they merge so quickly. Such nest dynamics, however, may be explained if the aggression of workers varies seasonally as shown by Rosengren et al. (1985) for Formica truncorum: cool conditions between autumn and spring lower aggression between workers from different nests, while warm conditions in summer increase it. If temperature is involved in the nest dynamics of P.flavipes, aggression between workers should vary from season to season. In this study I tested whether there was a seasonal effect on worker aggression by rearing workers in artificial nests under semi-natural conditions. By using nests with and without queens and placing them in different habitats, I could test the seasonal changes in response of the ants to queen odours and environmental odours which remained constant throughout the year. The tests were designed to reveal the effects of odour differences, based on queen and habitat, on nestmate recognition in this ant species.
METHODS
I collected 10 colonies of P. flavipes during 15-20 June 1986 in the Tomakomai Experiment Forest, southern Hokkaido. Each of them occupied only one nest and had only one queen. I extracted all ants from each sample and until 29 June 1986 reared each colonyin a separate incubator at 5 __+1~ having put them in an artificial nest (measuring 137 x 65 • 35 ram) with 1 cm of moist plaster of Paris covering the bottom. I paired the colonies (A, B) at random making five replicates. In a replicate I prepared two artificial nests from the A colony and four from the B colony: A (forest/queen) and A (forest/no queen) from A; B (forest/queen), B (forest/no queen), B (grass/no queen) and B (spruce/no queen) from B. I provided each of these nests with 50 workers and 50 final instar but caste-labile larvae from the original colony. I added the queens of the A and B colonies to A (forest/queen) and B (forest/queen), respectively. These nests were the same size as the artificial nest in the incubator, but they were provided with three openings sealed with stainless steel mesh to allow ventilation and to prevent ants from escaping. One opening (15ram diameter) was on each of the longer sides and the other (5 mm) on the ceiling of the nest.
From 1 July 1986 to 30 September 1987 1 settled A (forest/queen), A (forest/no queen), B (forest/ queen) and B (forest/no queen) within 50 cm of each other on the ground in a broad-leaved forest, B (grass/no queen) in a grassland site and B (spruce/ no queen) in a broad-leaved forest containing about 30% Yedo spruces, Picea jezoensis. All the sites in the Experiment Forest were 0.5-8-0km from each other. From 1 December 1986 to 26 May 1987 these nests were buried deep enough (about 70 cm) to avoid freezing and to allow successful hibernation. Except during hibernation, each nest was given Drosophila virilis and sugar crystals every 1-2 days through its sliding lid. About every 2 weeks I carried these nests to the laboratory of the Experiment Forest and made the following observations over a day. Three workers (recipients) were randomly selected from a nest and introduced into an arena (measuring 61 x43 • 17 ram), the bottom of which was covered with moist filter paper. When these workers had calmed down, a worker (donor) from another nest was introduced into the arena, and I observed the behaviour of the recipient workers towards the donor for 5 rain to determine the degree of aggression between them. After removing the donor worker I repeated the same observation twice using other workers from the donor nest. After the observation, these workers were returned to their original nests. The same observation was made for all possible pairs of nests on every test day. All nests examined were rested in the laboratory at room temperature, and then settled again at their original sites in the evening of the same day. The test days were: 1, 17 and 31 July, 15 and 31 August, 15 and 30 September, 15 and 31 October and 30 November in 1986; 15 and 30 June, 15 and 30 July, 17 August and 25 September in 1987. Following De Vroey & Pasteels (1978), I graded worker aggression in a pair as follows: 0 (nonaggressive); 1 (persistently licking towards an opponent); 2 (tentatively biting or threatening with mandibles opened); and 3 (struggling severely with gaster flexed or chasing an opponent). Aggression between workers of Formica spp. decreases with time after they are mixed, and disappears eventually (Rosengren & Pamilo 1983). I observed that aggression of P. flavipes workers would also disappear within a day after they were mixed in an arena. Workers from the same colony, however, did not show aggression when I observed them intermittently over several days. The longer
Ichinose: Ant nestmate recognition one observes aggression between workers of different colonies, the more likely it is that amicable contacts between them will be seen. Thus long observations could reduce the average score of aggression, whereas the largest score recorded should be less influenced by how long the observation period lasts. The maximum aggression score observed in a test is thus likely to be the most moderate index for the likelihood that the nests of the observed workers would mingle with each other or that workers of one nest would recognize those of the other as nestmates. I therefore determined aggression between a pair of workers from two nests (e.g. A (forest/queen)= recipient and B (forest/queen)=donor) by the maximum score of aggression observed on any given test day. For example, the score is 2 when 0scored behaviour is elicited by the first B (forest/ queen) worker introduced to an arena containing three workers of A (forest/queen), 2 by the second and 1 by the third. I also noted aggression in the opposite direction, from donor to recipient. I made such reciprocal observations on all possible pairs on every test day. I observed and scored 1500 pairs in 1986 (150 pairs on a test d a y • 10 days), but 136 in 1987 because many nests collapsed after unsuccessful hibernation or from the deaths of workers during that period.
RESULTS I observed aggressive behaviour between workers from two nests 1636 times. Since this aggression involved two directions, e.g. from A (forest/queen) to B (forest/queen) and vice versa, I actually examined aggression between two nests 818 times (1636/2). The scores of reciprocal pairs differed from each other in 44 out of 818 cases (5.4%). I tested by A N O V A whether or not there was a significant difference between each reciprocal pair. This test was done 75 times (15 reciprocal pairs in a replicate (see below) x five replicates), but none of these tests showed significant differences between them (Fs was always less than 1, P>0-05). Since this means that there is no statistical reason to distinguish between the two directions of each reciprocal pair, I adopted the larger score of the two pairs as the aggression score between the two nests on any given day. Consequently, the number of scores was halved, 750 in 1986 and 68 in 1987. Thus I analysed aggression scores between the following
15 pairs: A (forest/queen) versus A (forest/no queen), B (forest/queen), B (forest/no queen), B (grass/no queen) and B (spruce/no queen); A (forest/no queen) versus B (forest/queen), B (forest/ no queen), B (grass/no queen) and B (spruce/no queen); B (forest/queen) versus B (forest/no queen), B (grass/no queen) and B (spruce/no queen); B (forest/no queen) versus B (grass/no queen) and B (spruce/no queen); B (grass/no queen) versus B (spruce/no queen). Because I observed too few pairs in 1987, I analysed only the results from 1986. To assess the effects of either the difference in colony (A or B) or the presence of a queen on scores of worker aggression, I calculated averages of aggression scores over the five replicates in four groups (Fig. la). Aggression scores were influenced more by which colony the workers came from than by the presence of a queen. I also averaged aggression scores in each of six groups to assess the effects of the difference in habitat (broad-leaved forest, grassland or spruce forest) on aggression (Fig. 1b). Aggression between colonies (A versus B) remained high over all seasons but aggression within colonies (A versus A, B versus B) varied seasonally (Fig. 1). A two-dimensional ANOVA was applied to the between-colony and within-colony groups by integrating the five replicates in order to detect differences in the score between pairs of nests (df=79 in the betweencolony group, df= 69 in the within-colony group) and dates (t/f= 9 in both groups). The difference between pairs was not significant in either group (Fs=0.404 for the between-colony group, F s = 0.967 for the within-colony group, P > 0 ' 0 5 for both). The difference between dates was significant in the within-colony group ( F s = 31.56, P<0.01) but not the between-colony group (Fs=0-064, P>0"05). In addition, the lack of a difference in aggression between pairs in each of the two groups suggests that there were no effects of the queen and habitat on changes in worker aggression. DISCUSSION Aggression of P. flavipes workers was not influenced by either the presence of a queen or the habitat, regardless of whether the aggression was between colonies or within colonies (Fig. 1). Since aggression between colonies remained high over all seasons, it seems that nestmate recognition depends primarily on the genetic makeup of a colony. Aggression between workers of the same colony
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Figure 1. Aggression between pairs from different colonies ( &, 0 , 9 and from the same colony (A, 9 []). (a) Queen effects. Either or both of the paired nests have a queen ( A, A ) or both are queenless ( 9 9 ): A (forest/queen) versus all nests from the B colony, A (forest/no queen) versus B (forest/queen); A (forest/no queen) versus B (forest, grass, spruce/ no queen); A (forest/queen) versus A (forest/no queen), B (forest/queen) versus B (forest, grass, spruce/no queen); B (forest/no queen) versus B (grass, spruce/no queen) and B (grass/no queen) versus B (spruce/no queen). (b) Environmental effects. Paired nests were settled at the broad-leaved forest (A, •), either of the paired nests at the grassland site ( 0 , O), or either of the paired nests at the spruce mixed forest ( 9 []): A (forest/queen) versus B (forest/queen, no queen); A (forest/queen, no queen) versus B (grass/no queen); A (forest/queen, no queen) versus B (spruce/no queen); A (forest/queen) versus A (forest/no queen) and B (forest/queen) versus B (forest/no queen); B (forest/queen, no queen) and B (spruce/no queen) versus B (grass/no queen); B (forest/queen, no queen) and B (grass/no queen) versus B (spruce/ no queen).
was negligiblejust before and after hibernation, but increased during the active season. So some seasonal modification is probably involved in nestmate recognition of P.flavipes workers. One possible means of change is that workers become more aggressive as they get older, as in Myrmica rubra (Cammaerts-Tricot 1975) or S. invicta (Sorensen & Fletcher 1985). In m a n y ant species callows are unlikely to attack or to be attacked by workers of different colonies (Cammaerts-Tricot 1975; Brian 1986; Carlin & Hflldobler 1986; 9 1986; Carlin
et al. 1987; Stuart 1987; Morel & Blum 1988). Callows of these ants will learn and acquire the odours of their own nests as they grow. With experience, they can discriminate their nestmates from non-nestmates and be recognized as familiar or unfamiliar workers by others. This means that when the callows replace m a n y or all of the older workers, the colony as a whole may be less aggressive towards other colonies. The replacement of P. flavipes workers used in the present study probably occurred in September
Ichinose: Ant nestmate recognition
or by early October of 1986 (cf. Ichinose 1987a). The expected change of callows and old workers in autumn cannot explain the reduction of aggression between colonies within a season. Alternatively, the recognition mechanism could be mediated by temperature-dependent changes in the physiology of workers rather than by their age. If their metabolism is always high enough to allow workers to detect genetic differences between kin and non-kin, aggression between colonies would be elicited at all seasons. In the active season, from June to September, workers are so sensitive that they can discriminate their nestmates from nonnestmates as aliens or enemies by their different environmentalas well as genetic odours. The microhabitats in the Tomakomai Experiment Forest (Higo 1986) may produce their own odours, causing artificial nests to acquire different odours. In the inactive season, October to May, lowered metabolism may allow the detection only of different genetic odours. Thus, aggression between colonies was evident all year. If addition of environmental odours triggered aggression of the workers within a colony, we can apply the 'cue similarity threshold' model for nestmate recognition which was first developed for paper wasps (Gamboa et al. 1986; Gamboa 1988). According to this model, workers have a threshold for nestmate recognition. When a worker encounters an individual she compares its odours with her own. If the difference in the odours exceeds the threshold she will behave aggressively towards the individual, while if it is below the threshold contact will be amicable. It is interesting that aggression was always lower within the colony than between colonies. This suggests that workers have two or more thresholds and environmental odours within the colony do not push the workers' perception over the final threshold. If so, environmental odours may influence worker aggression secondarily. If genetic odours on the body surface of workers had changed during the present study, aggression would appear even within the colony. Vander Meer et al. (1989) reported that colonies of S. invicta change recognition odours on the workers throughout their lives. However, it is hard to imagine that the odours of P.flavipes workers changed in such a way that within-colony aggression in all pairs of replicates disappeared until the inactive season. I therefore think that environmental odours added to genetic odours changed nestmate recognition in
P. flavipes only in the active season, while genetic odours prevailed in all seasons. The additive effect of environmental odours makes workers aggressive against non-nestmates and makes it easier for budded nests to become independent in summer. Disappearance of the effect in autumn allows the budded nests from a colony to merge again. On the other hand, permanent aggression against non-kin prevents them from merging with any nests budded from other colonies. In turn the mechanism of nestmate recognition in P.flavipes has kept it from evolving a multi-queened colony, although having a single queen in a colony, a multi-nested colony or a fragile nest structure (Ichinose 1986, 1987b) are consistent with the conditions necessary for a multi-queened colony to evolve (H61tdobler & Wilson 1977).
ACKNOWLEDGMENTS I thank Professor S. F. Sakagami for his critical comments and Professor K. Ishigaki for his permission to collect ants in the Tomakomai Experiment Forest. Miss J. B. Jacob kindly helped me prepare this manuscript.
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