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Bonding between workers and queens in the ant genus Myrmica
Anirn. Behav., 1986, 34, 1135-1145
Bonding between workers and queens in the ant genus
Myrmica
M. V. B R I A N *
Department of Biological Sciences, University of Exeter, Exeter EX4 4PS, U.K.
Abstract. Bond formation between young workers and queens is described for three species of Myrmica. This caste bond inhibits aggression and develops cooperation. Workers have a bias towards queens of their own species and do not discriminate between a queen of their own colony and a novel conspecific one. They are able to develop bonds with queens of other species of the genus provided they meet them first and well before their state of queenless aggressiveness sets in. A bispecific group, in situations contrived to enable them to meet gradually, will fight a normal group. They will then develop their bond to include the conspecific queen without becoming hostile to the earlier substitute. Evidently a queen of another species is able to provide an outline set of stimuli that is later built up by the conspecific queen, a characteristic of the imprinting development process. Allospecific queens can establish new microsocieties in a way reminiscent of temporary social parasites. The fact that allospecifically bonded workers will fight their one-time litter-mates that have been normally bonded suggests that the two groups have acquired different smells from their specifically distinct queens.
The work described in this paper started as an attempt to understand how queens, even dead ones, influence the way workers treat sexual larvae (reviewed in Brian 1980). Carr (1962) showed that workers brought up with queens suppress sexualization in these larvae more effectively than if they meet them for the first time during the experiment itself, and Brian (1965) suggested that imprinting might be involved. Brian & Evesham (1982) confirmed this and Evesham (1982, 1984a) showed that the first few weeks are the most sensitive period in a worker's life for this queen experience to occur. Preliminary tests showed that workers of Myrmica rubra, when less than a week old, could be imprinted with false information about their associate brood and adults. They could be made to work with a different species of the same genus just as can Formica (Jaisson 1975; Le Moli & Passetti 1977, 1978; Jaisson & Fresneau 1978; Le Moli 1980; Errard 1984; Le Moli & Mori 1984). From this it was only a short step to conceiving the possibility that they could be made to work with queens of other species and that this would throw light on the nature of the postulated queen/worker bond, and so of queen influence over worker behaviour.
* Present address: Orchard Lands, Hilton, Blandford Forum, Dorset, U.K.
METHODS
AND RESULTS
Myrmica rubra L. was used as a base species. For comparison two other species: M. ruginodis Nylander subspecies mierogyna (Brian & Brian 1949) and M. sabuleti Meincrt were used. Samples of these species were collected from many different localities in the south of England to increase the variability of the experimental material but colonies were not mixed. During winter the colonies were stored at 5~ from this stock, cultures were set to produce worker pupae as required and during summer the pupae from wild colonies were used. All were allowed to mature alone so that new workers emerged without the help of older ones or queens. Pupae died if any attempt was made to store them, even at 5-10~ and it was necessary to start to bond them to a queen at once: the sooner this was done the more likely it was to succeed since young workers become more antagonistic to queens the longer they are queenless (Evesham 1982, 1984a). Bonding took place if the workers and queens were put together in a space that was small enough for them to make frequent contact. The workers formed a cluster which at first excluded the queen; they pulled her away if she tried to join them but she seemed to be able to overcome this hostility by persistently approaching with stealth. A careful approach was especially important if the workers and queens were of
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Animal Behaviour, 34, 4
different species for, quite often, bonding failed not just because of heightened worker hostility but because the queen sensed the worker species difference and avoided contact. Soon after the queen was allowed into the group she was groomed and fed and allowed to stand on the brood. This state of acceptance was taken to indicate that bonding was complete but it was usually confirmed when queenlaid eggs appeared. At this stage the queens were painted individually and the workers as a group. 'Silver' enamel was best for visibility through the red covers used to watch the ants without disturbing them. Once bonded and marked, the ants were put in standard cellular nests and allowed to settle down again. The cellular structure made it easy to plot the size and shape of a group. These nests were of the Janet type, made of dental plaster held in a plastic frame. Each had 14 identical cells indented in the plaster and covered with a glass slide that left room for the ants to walk over the tops of the walls. The red cover lay on top of the glass. Each cell was a hemispherical depression made by sinking a glass marble as far as its equator into the plaster whilst it was still soft. The cell diameter was 1.5 cm, its depth half this, and its volume 1 ml. Fourteen of these cells were arranged in a rhomboidal matrix with three columns of five, four and five cells. Each cell held 30 resting workers but only 10-12 active ones. The nests were kept in a cabinet at 20~ and 80~ relative humidity, with a day:night regime of 16 h: 8 h. Drosophila and i 0 ~ sucrose were supplied ad libitum. Aggressive behaviour is often seen between workers and has been graded in Myrmica rubra both by De Vroey & Pasteels (1978) and Winterbottom (1980). In this paper three of their six grades were used: most intense but rarely seen was an attempt to sting; moderately intense and often seen was biting and dragging; least intense and rare was mandible opening. The last has been called a 'threat' and no doubt it seems to be so, but no signal, chemical or tactile, has yet been shown to pass. It usually resulted in avoidance, or 'apotrepsis' to follow Barnett (1981). The method used to assess the bond is due to Provost (1979). After an age-group or 'litter' of new workers had been divided into two parts, each bonded differently and the workers and queens marked and put in a cellular nest, two such nests were joined by removing a plug in the connecting tube. The ensuing interaction was monitored by
samples in the morning and afternoon or as often as was useful. This tested the viability and independence of the microsocieties that had been synthesized, by allowing them to meet gradually, at first in a common run and later, in the two nests that were potential nest territories. These paired tests can be classified into balanced and unbalanced. All had Myrmiea rubra workers on both sides but, whilst the former had the same species of queen on each side, the latter had queens of different species on each side. First, four balanced tests are described: three had a single species (homospecific) and used Myrmica rubra queens, while one had two species (heterospecific) and used M. sabuleti queens. Then, four unbalanced tests are described: two had M. rubra queens against M. ruginodis queens and two had M. rubra queens against M. sabuleti queens. Throughout this paper upper case letters represent queens and lower case letters represent workers, e.g. b workers associate with B queens.
Balanced One- and Two-species Tests In the first three of the four experiments, the workers and queens were both M. rubra. In test 1, a concolonial queen was compared with an allocolonial one; in test 2, two allocolonial ones were compared; and in test 3 the sizes of the two groups that supported allocolonial queens were unequal.
Test 1 New workers, all from one colony, were divided equally and given either a queen (A) from the same colony, or a queen (B) from another colony. The two colonies were collected from valleys 75 km apart. Group a (with queen A) was formed in the left nest of the pair and group b (with queen B) in the right. Both queens settled and within 7 days entered the brood cluster; even the allocolonial queen met no resistance. Three weeks later the separating plug was removed and the workers began to explore. After 2 days group a moved as a body to the right and joined the heterogenous b culture without hostility. Clearly their identical origin outweighed any differences acquired from their brief sojourn with different queens. The B queen in group b was not attacked by the incoming a workers, in fact they fed and groomed her even though they had not met her before. The situation was not quite reciprocal, however, for queen A was at first attacked
Brian." Bonding between workers and queens mildly by the resident b workers and she attracted fewer workers than B for some time: for example, 12 twice-daily samples gave a total of 133 with A compared with 190 with B ( A N O V A , P<0.05). This asymmetrical response was probably due to the fact that a moved to join b and not the other way round, but whether this was chance or because the workers with a concolonial queen (a) were more firmly bonded, more socially complete and so more venturesome remains a problem. The two queens rarely met each other and when they did they only made a brief antennary contact; they were probably avoiding each other, i.e. were apotreptic. Once, when B ran into the same cell as A and jigged round, A ran off though she was not attacked directly. A few days later, B spent several days in a cell, the vestibule, next to the run often used as an assembly point by groups before crossing to the other nest. This suggests that B was trying to escape but would not leave by herself. She only returned to the core of the group (still on the right) after A was taken away. When a few days later she was returned to the nest, the workers clustered round her as though she was superstimulatory and B was completely ignored; clearly A was more attractive to the workers. Thus, throughout this trial the queens appeared to compete passively for worker attention. Ascendancy passed from the resident queen of the right B, to the immigrant queen A, originally on the left. A was, of course, concolonial with the workers but unknown to them as adults before the experiment. Though the queens were for the most part apotreptic, the rotary jig, centred on the brood group, comes near to being an offensive, possessive dance. As far as bonding is concerned, the important conclusions are as follows: first, that the two sets of litter-mates coalesced without hostility in spite of being bonded to different queens; second, that the workers clearly preferred the concolonial queen and were not exclusively bonded to the queen that they first encountered. They had the potential in fact, as would be expected with this species, of being polygyne.
Test 2 N e w workers from one colony were divided between two novel allocolonial queens (B and C). This test was similar to test 1 except that neither queen was concolonial with the workers. A set of 120 callows 2 weeks older than before, was divided into two equal parts; one part was given queen B
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and the other queen C. B and C were obtained from sources 75 km apart which were not the same as those used in the first test. Both queens were at first attacked; this was probably because the workers had been queenless for longer than in test 1 (Evesham 1982, 1984a). G r o u p b settled on the left and group c on the right of the double cellular nest; a week later they were joined together. After only a short period of mutual investigation, group c moved as a body to the left. They joined the other group peacefully. Whilst queen C then stayed on the left, B moved with 11 workers to the right; these workers were a random mix of the two types. After a further 3 days, B returned but this time she was attacked by workers and forced to remain peripheral. This hostility waned and then ceased but the two queens were never equal socially. This can be illustrated by the distribution matrices shown in Table I. The core cells, i.e. the ones that contained most workers over the whole period and which most often held the brood and the queens, were three adjacent cells in the right nest. Whilst C was seen here 30/34 times, B was only seen 18/34 times and only twice with C. So B both avoided C and was ranked below C by the workers; in fact she was probably starved for she became quite weak. This test thus shows that even 3-week-old workers can be bonded to totally strange queens. Table I. The distribution of two queens;~ of Myrmica rubra after two groups of callows each containing one had fused Number of occurrences Queen B 0
0
Queen C
Cumulative worker number
0
26
t
5*
l
16"
6* 7*
1
1
Ill*
0
0 t
1
1 0
11 68*
59*
3 0
4
0
5*
6 31
9*
3 4
0 0
19 19
0 t
19
0
2
II
9 ? 4
One-species series, test 2: the distribution in 14 rhomboidally arranged cells on the left side of the double nest, of queens B and C and the summed positions of the workers. * Core cells; ? the entrance to the nest which is the left one of the pair. $ Both queens were allocolonial to the workers.
Animal Behaviour, 34,4
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Though equally unrelated to the workers, one of the queens was able to settle in the core whilst the other remained peripheral and unsettled. Worker choice was exerted even amongst allocolonial queens. This unequal relationship lasted the whole period of the trial.
Test 3 A worker group was divided unequally between two novel allocolonial queens (B and C). In this test the workers were less than a week old. The groups were intentionally of different sizes so that the possibility that queen establishment is affected by the number of workers in their support could be explored. The group on the left, c, consisted of 50 workers and the queen C and that on the right, b, of only 10 workers and queen B (Table II). After the plug had been taken out, the two lots of workers explored and then started to mix without hostility. At first workers moved both ways, but this movement back and forth stopped after a mass movement to the right which resulted in a single mixed group. Queen C then joined B but the latter moved away into a corner with 10 c and three b workers. The eggs were left with queen C who moved 3 days later with a very mixed retinue and fused with the group associated with the B queen. The structure of this new group was then as follows: in the core, 26 c and five b with queen C; on the fringe, 19 c and four b workers with queen B. The slightly greater proportion of b on the fringe, which might be thought to imply that, though workers, they were excluded from the core, is not statistically significant.
Table II. The fusion of two groups of Myrmica rubra workers, each with an allocolonial queen On left Days after nest joined 0 1 2 4
c
C
b
On right B
c
5 0 1 0 0 0 2 8 1 4 0 1 3 5 1 1 0 0 0 0 0 0 4
0 6 9 4
1 0 0 1
C
b
0 6 0 9
1 1 1 1
B
One-species series, test 3: the numbers of workers and queens on the left and right sides of the double nest on four occasions; b and c workers with queens B and C respectively.
This test thus confirms that worker groups sooner or later coalesce and mix freely and randomly. It also shows that workers within these groups do not associate with the queens to which they were originally bonded. They take up additional queens peacefully. The queen with least initial support (10 workers) attracted less support later, but degree of sponsorship seems unlikely to be a cause of priority since the workers did not associate with their bonded queens once they had joined a bigger group. All three of these single-species balanced tests indicate that Myrmica rubra workers can be bonded to any queen of their own species; differently bonded groups will soon coalesce. Within these worker-made groups the queens are apotreptic, but one sometimes uses a 'rotary jig' dance to dislodge another. The dislodged queen may try to leave but will only go if a retinue of workers accompanies her, not alone. In test 1, the workers favoured a concolonial queen but when she was experimentally removed, they switched their attention to the other queen; in test 2, one of the two allocolonial queens was favoured and the other only just tolerated; and in test 3, one allocolonial queen was again favoured out of the two offered. The one chosen had more bonded workers at the start but this was probably irrelevant. The workers form a group around a preferred queen and attract other workers. They then attempt to capture and incorporate any splinter groups that shield low priority unattractive queens. The latter find difficulty in becoming independent, at any rate in the conditions provided in the experimental nest.
Test 4 To complete these balanced tests, a test was carried out using a two-species culture (Myrmica rubra workers with M. sabuleti queens on both sides). A group of 60 workers of M. rubra less than a week old was divided equally and half put on each side of the double nest, each with two queens of M. sabuleti. Twenty-four days were allowed for bonding owing to the uncertain interaction between this pair of species. During this period one queen died so that, when the two parts were joined, there were two queens on the left (B 1, B2) and one on the right (B3). At this time no eggs had been laid. The day after the connecting tube was opened, workers passed in both directions but, in spite of a close watch, no fighting was seen. At the end of the
Brian: Bonding between workers and queens first day, there were six b3 o n the left a n d three b 1b2 on the right, but still no hostility. T w o days later, all the b r o o d including some new eggs were t a k e n to the left a n d queen B3 remained with only two workers, one of each sort. Queens B 1 a n d B2 were in separate cells, one with eight b l b 2 and two b3 a n d the o t h e r with 11 b l b 2 a n d five b3. T h u s the two sets mixed peacefully. B3 m o v e d over to j o i n the o t h e r two queens but they all used different cells. Q u e e n B3 was a c c o m p a n i e d by 10 b l b 2 a n d three b3 workers, showing no bias against her; in fact, 6 days later, all three queens were in the same cell. This also confirms their effective b o n d i n g to the Myrmica rubra workers. Later the queens m o v e d about, sometimes singly sometimes together, b u t with n o sign o f apotrepsis, a n d the experiment was stopped. So, M. rubra workers will a d o p t M. sabuleti queens a n d form a viable microsociety t h o u g h the species are not considered close taxonomically. Moreover, two such groups will join peacefully. Queens of this species may p r o v e on further study to be less apotreptic than those of
M. rubra.
Unbalanced Two-species Tests
These four tests involved Myrmica rubra workers with a queen of the same species o n one side a n d a queen o f M . ruginodis a n d M. sabuleti on the other. Thus, allospecific queens were competing with conspecific queens for the s u p p o r t of a divided g r o u p of workers, h a l f b o n d e d to one and h a l f to the other.
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Test 1 A litter of 72 workers, less t h a n a week old, was divided into two equal parts, one o f which was given a queen of M. rubra (A) a n d the other a microgyne queen of M. ruginodis (B). The former queen was from a different colony from the workers but they settled together after only mild hostility. The M. ruginodis queen on the c o n t r a r y was heavily bitten a n d pulled a b o u t for at least 10 days. With the a culture o n the left a n d the b on the right, the two were joined a n d the distribution recorded regularly for 6 weeks. The situation each week is given in Table III. As usual, workers started to cross over to the opposite side and explore at once b u t this time fighting b r o k e out a n d went on for 3 weeks. The m o v e m e n t o f b to the left stopped a n d they began to return. Between the third a n d fifth week, a m o v e d to the right, leaving only three b a n d one a on the left. All the rest, with the two queens, were on the right, Thus it t o o k as long as 5 weeks for the two groups to fuse. This delay in the r e u n i o n of one time litter-mates was caused by the hostility between a a n d b workers; they behaved as t h o u g h they belonged to different colonies. A v o i d a n c e was the rule but, when this was impossible, the workers bit a n d tugged at each o t h e r for a long time. Very few fights were reciprocal, so it was possible to tell w h o was the aggressor. The sequence o f fights has been tabulated in Table IV. It should be noted that the first 30 interactions were between workers, for b o t h queens were inaccessible behind their screens of defenders. O f
Table lIl. The interaction of two groups of Myrmica rubra callows, one
with a queen of the same species and one with a queen of M. ruginodis Left nest
Right nest
Workers
Queens
Workers
Queens
Week of observation
a
b
A
B
a
b
A
0
36
0
A
O
0
36
O
B
1
35
1
A
O
1
34
O
B
2 3 4 5 6
31 28 16 1 27
7 14 13 3 24
A A A O A
O O O O O
4 6 18 32 5
28 21 22 30 8
O O O A O
B B B B B
B
Unbalanced two-species series, test 1: a: the workers with a conspecific queen (A) starting on the left; b: the workers with an allospecific queen (B) starting on the right.
Animal Behaviour, 34,4
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Table IV.-The sequence of fights when two groups of Myrmica rubra callows were joined, one with a conspecific queen and the other with an allospecific queen (M. ruginodis) Between workers and workers On the left Sequenceoffights a > b
Between queens and queens
On the right
On the left
On the right
b>a
a>b
b>a
A>b
a>B
B>a
b>B
1 10 11-20
4 5
0 l
6 4
0 0
0 0
0 0
0 0
0 0
21-30 3140 41-50 51 60 61-70 71-80
3 0 1 2 0 0
1 0 0 0 0 0
5 4 4 1 0 0
1 0 0 0 2 0
0 0 1 0 0 0
0 3 2 5 7 10
0 3 2 1 0 0
0 0 0 1 1 0
Total
15
2
24
3
1
27
6
2
Unbalanced two-species series, test 1: a, the worker group with conspecific queen A; b, the worker group with allospecific queen B. The first 80 fights after connection have been classified and listed after grouping in 10s: a > b means that a worker of the a group attacked one of the b group and so on. these 30 fights, 27 were defensive a n d three offensive. Both sides were a b o u t equally concerned to defend their nest territory. In detail, the three offensive actions were: two on the left by a n i m m i g r a n t b against a resident a a n d one on the right by a n i m m i g r a n t a against a resident b. D u r i n g the next 30 events (31 60) defensive actions by workers diminished from 12 to 3 on the left a n d from 15 to 9 o n the right b u t offensive actions did n o t replace them. However, the workers were still crossing over (Table IV). M o s t p r o b a b l y the explanation for the decline in inter-worker hostility was due to the discovery o f the M. ruginodis queen by the a workers, w h o were seen to attack her 10 times. Her ' o w n ' b workers did n o t help at all but she herself fought the a workers with her mandibles. This struggle was n o t balanced by a similar action against the A queen, but once she attacked a b worker t h a t ' t h r e a t e n e d ' her in her cell. The attack against the core of the M. ruginodis queen increased so t h a t 17 of the next 20 events were fights initiated by a workers against the queen. The other queen, A, was left alone again a n d later m o v e d over with 32 of ' h e r ' workers to the right. This sealed the fate o f B who, with her a n t e n n a l tips cut off, died. The A queen did not stay, b u t returned to the left with m o s t o f the workers, which were, by now, well mixed.
This battle started with a n exploration t h a t led to defensive retaliation. Later, as the workers from the two sides mixed, they h a b i t u a t e d to each o t h e r a n d avoided open hostility. W h e n the atypical queen, B, was discovered she was attacked, but only by the n o r m a l l y - b o n d e d workers. The queens were never seen to fight directly. T h o u g h a workers at first dragged the B queen out into the run, she always got back; later the workers were less aggressive a n d only 'arrested' her if she tried to move away. A l t h o u g h she defended herself, her b o n d e d workers did not help directly b u t they groomed a n d fed her at the same time as the others, a, bit and held her down. She appears to have been too unusual to settle in the fringe as the M. rubra queens did in the early series. Evidently, after she h a d been killed, the workers previously b o n d e d to her assimilated into the m a i n group and followed the n o r m a l conspecific queen. It is nevertheless astonishing h o w long the workers attached to B remained supportive, if n o t defensive, towards her. This inhibition o f aggression t h a t transcends species differences is p r o b a b l y the most i m p o r t a n t feature of the caste b o n d ; it clearly demonstrates the nature a n d strength of the imprinting. P r o b a b l y the conspecific queen w o n the c o m p e t i t i o n for the workers' s u p p o r t because she was able to supply workers with m o r e detailed a n d clearer data with
Brian." Bonding between workers and queens Table V. The distribution changes in two joined nests containing groups of Myrmica rubra callows, one with a conspecific queen and the other a queen of M. ruginodis Le~
Right
Date
a
b
a
b
17 July 24 July 31 July 7August 14 August* 21 August
25 21 20 10 12 16
0 0 I 8 4 3
25 25 24 16 16 15
0 4 5 6 10 4
Unbalanced two-species series, test 2: those starting on the left (a) were bonded to concolonial queen A, those on the right (b) were bonded to an M. ruginodis queen, B. The queens did not change sides at all. * At this time, 10 new M. rubra workers were formed on the left and 10 new M. ruginodis on the right.
which to develop the fine structure of the imprinting.
Test 2 This repeated test 1 using different material. Fifty newly prepared workers of Myrmica rubra were divided equally and given either a queen of the same species but from a different colony (A) or a microgyne queen of M. ruginodis (B). The group a was put on the left, and the group b on the right. When the plug was taken out, the a group began to move to the right (Table V) but were resisted by the b group. The a workers found the B queen much more quickly than in the first test and attacked her. She defended herself and was once seen to bite at the neck of a worker but without severing its head; several b workers joined in and defended the queen by attacking this and another a worker. Yet, next day, B had been dragged to the vestibule by an a worker with a b worker attached. This queen continued to defend herself effectively and was once seen to apply a droplet with her sting to the m o u t h parts of an a worker, which had an immediate repellant effect. Meanwhile B's own workers (b) groomed and fed her and, although she escaped this time, she was attacked again (Table VI). The b workers were still quite active in territorial defence
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on the right and some even spread to the left where they attacked the base of the a group and set off defence reactions. The invading b workers, when they met the A queen, were not hostile. This is understandable if imprinting is progressive and starts with a general impression of a queen as a distinct caste: the queen M. ruginodis must satisfactorily provide this input in spite of her wrong species smell. M o r e details, such as only an M. rubra queen can provide, are built in later. The vigorous b workers in this test were able to protect 'their' queen B from constant harrying by a workers to such a degree that she managed to lay eggs that the b workers protected from the thieving a workers. These hatched and were brought up to the adult stage as 10 workers after 2 months. All this was achieved in spite o f a fresh offensive by the a workers and their simultaneous production of 10 new M. rubra workers. The new M. ruginodis workers bonded normally with queen B. This stabilized the interaction and the number of a workers on the b side declined from then on. Nest territories were thus reinforced, migrations between the two nests stopped and the M. ruginodis microsociety became potentially independent. If microgyne queens normally start new societies by using the routine o f a temporary social parasite then they may well have the social and structural adaptations needed for success.
Test 3 Queens of M. sabuleti can also be bonded to M. rubra workers. Starting with the usual litter of equal-aged workers, one half was given to two M. rubra queens, A1 and A2, and the other to three M. sabuleti queens, B 1, B2 and B3. After 2 weeks, the former were put on the left and the latter on the right of the pair of nests. The first 90 fights are summarized in Table VII. The first 40 of these were once again between workers from different sides and were mostly defensive. On the right, in B territory, the resident b group set upon the invading a group, and reciprocally on the left. O f these fights, 12 were on the left by a against b and 18 were on the right by b against a. Some offensive actions occurred and comprised four on the left (b against a) and six on the right (a against b). Besides being more frequent on the right, the fights lasted longer and so supported the earlier idea that conspecific bonding gives a stronger social base than allospecific bonding. The a workers were first seen attacking the B
Animal Behaviour, 34, 4
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Table Vl. The sequence of fights after two groups of Myrmiea rubra catlows were joined; one had a conspecific queen and the other an M. ruginodis queen
Workers with workers Left Sequenceoffights a > b 1 20 21-- 40 41 60 61 80 81-100 101-120
Total
Queens with workers
Right
Right
b>a
b>a
a>b
a>B
B>a
0 0 7 4 9 3
0 0 2 3 4 1
12 10 8 8 6 9
1 l 1 3 0 0
5 8 1 1 1 6
2 1 1 1 0 1
23
I0
53
6
22
6
Unbalanced two-species series, test 2; the first 120 fights classed as offensive and defensive and grouped in 20s. A and B were queens of Myrmica rubra and M. ruginodis respectively; a and b were the worker groups containing them; a > b means that a attacked b and so on. Group a was on the left and b on the right.
Table VII. The sequence of fights after two groups of Myrmica rubra callows were joined; one had two conspecific queens and the other three M. sabuleti queens
Workers with workers Left Sequenceoffights a > b
Right
Queens with workers Right
b>a
b>a
a>b
a>B
1-10 11-20 21 30 3140 41-50 51-60 61 70 71-80 81-90
2 2 3 5 4 3 3 3 1
2 I l 0 0 0 0 0 2
5 5 4 4 3 0 1 0 0
1 2 2 1 2 2 3 1 0
0 0 0 0 1 5 3 6 7
Total
26
6
22
14
22
Unbalanced two-species series, test 3; the first 90 fights classed as offensive and defensive and grouped in 10s. A and B were the Myrmica rubra and M. sabuleti queens respectively; a and b were the worker groups containing them; a > b means that a attacked b and so on. G r o u p a was on the left and b on the right.
Brian: Bonding between workers and queens queens on the 47th occasion and, from then on, these queens were attacked whenever they were encountered. The b workers were also on both sides and they gave up attacking the a group, even defensively. By the third day, a workers had reached and entered all the peripheral cells on the right and were beginning to invade the core. One B queen had already been pulled out of the core and was held by the antennae in the next cell. The other two were still free and had seven of their bonded workers with them but one a worker was seen to enter their cell and adopt a 'threat' stance. The displaced B queen soon returned to the core and was fed and groomed by one of the seven workers. This state lasted for another 3 days but, by constant pressure, the a workers gradually broke up the b core and dissipated the resistance of the defending workers. On day 13 one of the A queens travelled over to the right and herself entered the b core. At the same time, one of the B queens was held by two a workers; seven b workers who were present took no action at all. In fact, in spite of many opportunities, the A queens were never attacked by b workers. The second B queen was carried away by an a worker and put in a marginal cell where five b workers fed her. After 1 day on the right, the A queen went back to her own side where she proved to be super-attractive; over 25 workers collected around her (cf. the similar event in the one-species series, test 1). So again, though the b workers succoured their queens they only once, after the initial defensive phase, attacked a workers on the right, or b side. N o t surprisingly, their aggressiveness depends on the structural integrity of their group. Only 15 workers remained on the right by day 20, though all three B queens were still there and appeared to be free; at least they were only lightly restricted when they tried to move. They attracted very little lasting attention and were able to go into the run to drink. All of them were in fact starving and could only climb the walls of the cells with difficulty. Most of the b workers that might have fed them had joined the A queens on the left. N o M. sabuleti queens laid any eggs and no prospect ever occurred of a social parasitic nucleus developing. After some dragging about they were starved to death rather than being dismembered. Test 4 A litter of 96 Myrmica rubra workers less than a week old was divided into two equal parts and one
1143
group given an M. rubra queen, A, and the other an M. sabuleti queen, B. They were put in the left and right sides of a double nest. The first homospecific 'graft', a, was quickly formed, but the second heterospecific one, b, took longer. Unexpectedly, 1 day after the separating plug had been removed, the b workers moved over to the left, taking the B queen with them and joined the a group. The cause of this remarkably sudden migration, which was entirely peaceful, is unknown; no workers fought each other. Later, the A queen was attacked by the b and the B by the a workers. Perhaps the unusually quick fusion of the two groups prevented the usual habituation between b and A. Hostility soon passed off and A established herself in the core whilst B remained peripheral, arrested by two or three a workers who dragged her into the run if she moved. So the workers selected a conspecific queen and tried to expel the alien, only failing in this because of her persistent return from the run. During the interaction the queens were only seen in the same cell once. This was after a slight jolt to the nest caused B to run about wildly and she blundered into the core cell where A was established with the egg cluster. Here she grabbed a few eggs in her mandibles but A gave the menace gesture and several of the a workers dragged queen B out. Opening the mandibles may well release some pheromone and cause an alarm recruitment of workers as in foraging. After this, to test for interaction between the queens, a special test was set up. A was taken away for 6 days; this let the B queen back into the brood chamber, where she was soon surrounded by attentive workers who clearly preferred a queen of a different species to no queen at all. Although she did not spend much time in the core, she was quite free to move and was once seen in the vestibule with a worker cluster. Next day, when A was put back, the workers at first refused to accept her; they even left B and came over to attack A. This phase only lasted 1 h, after which A was allowed to settle in a new cell where the workers pooled all the eggs. The situation was still unstable however, for 20 rain later the eggs and all the brood had been taken away and A was pinned down. w h e n , a few minutes later, A was again released, she rejoined the eggs and settled again, this time for good. B, although arrested, managed to escape and settle on the fringe of A's group. Throughout this behaviour sequence, as long as the M. rubra queen was present, the M. sabuleti
Animal Behaviour, 34, 4
1144
queen was treated with low priority; though actively kept out of the core, she was not destroyed. Removal of A, however, enabled B to develop some degree of acceptance, if not quite of establishment (though only 6 days was allowed for this). As a result, when A was returned, she needed a period of readaptation before she could again enter the core. Although a little mutual hostility may have occurred, the main channel through which the queens interacted had to be the workers. Worker support was essential; they chose their own species of queen if possible and, even within conspecific queens, they showed preferences. The absence of an inter-worker defensive phase in this test is interesting, for in this it resembles the earlier series of balanced tests in which opposing queens were of the same species. The sudden migration to the other side, whatever its cause, was possible because the workers at that time had not developed any method for distinguishing their groups. If the inter-worker fighting depends on acquiring a distinction (presumably from the queens) then it had not then spread to the workers.
DISCUSSION It has been shown that the bond that has been investigated links the queen with the workers in such a way as to create a viable system that is capable of defending and reproducing itself. The workers feed and clean the queen and defend her against intruders that they are able to recognize. The queens in return satisfy a need in the workers and produce female eggs. Bonding is most easily established soon after emergence, which is roughly equivalent to birth or hatching in vertebrates. This sensitive period fades and gives way to overt hostility, even against conspecific queens, if no bondable object presents itself. Yet, if bonded normally, such workers will accept more normal queens. Workers can be misled into adopting a wrong species of queen, for only a general outline of queen characteristics appears to be used at first; the details of species and individuality are filled in later. Presumably queens of the same genus have enough in common to provide satisfactory initial bond objects and they can even carry it as far as the production of offspring as long as no normal conspecific queen appears. However, the workers evidently have a bias, which is of course adaptive, towards a conspecific queen, and do not reject such
queens even if they have started to form an allospecific bond. The immediate cause of this bias that occurs in workers which, as adults, have not met a queen at all, is probably a species characteristic template but their conditioning as larvae by contact with the workers that nurse them cannot yet be ruled out; they could be given the correct species information at this stage. In correcting these mistakes by rebonding to conspecific queens, workers do not erase their original information; their superseded queens are just kept out of the breeding core in reserve, to be restored if the preferred queen is removed (cf. queen supersedure in honeybees). The individuality of the bonded queen, if it is appreciated 'by the workers, is not allowed to interfere and workers at this stage of the colony cycle are prepared to take in and adopt any conspecific queen. An interesting result of this research is that workers develop an antagonism towards littermates that have become experimentally bonded to queens of a different species. They are able to recognize this deviation, and recognition is reciprocal. The most obvious suggestion is that these have acquired a veneer of the strange species smell; such workers would then smell to the normal workers as though they were of a different species. Whether this idea can be extended to 'colony odour' remains to be seen. This aspect of the work on bispecific groups complements the results which Carlin & H611dobler (1983) obtained using mixed species groups of Camponotus; they found that the length of time that was spent in the company of individuals of a different species increased their similarity and they suggested that the queen probably produces a pheromone that coats the workers strongly enough to obscure their genetic relatedness. Using species of Leptothorax, in a system of nests with a common arena, Provost (1979) found that, although worker groups would coalesce, the queens fought to the death. In Nothomyrmecia a status interaction appears to exist between queens, which leads to the eventual extrusion of the loser queen (H611dobler & Taylor 1983). In Monomorium pharaonis, workers treat queens that are not in phase with the population cycle aggressively and they are eliminated (Petersen-Braun 1982). In Myrmica, queens have been described as fighting when they could not avoid each other (Evesham 1982; 1984b) and old queens were accompanied to the run to die. In this study, unwanted queens were
Brian: Bonding between workers and queens extruded. Elmes (1982) has p r o d u c e d evidence in the field t h a t this h a p p e n s , t h o u g h the queens he found w a n d e r i n g a b o u t were not necessarily superfluous. Clearly such queens are u n d e r a higher risk of d e a t h f r o m accident a n d p r e d a t i o n t h a n those living in the nests.
ACKNOWLEDGMENTS This work was s u p p o r t e d by a g r a n t f r o m the Royal Society whilst h o l d i n g a n H o n o r a r y Fellowship at the D e p a r t m e n t o f Biological Sciences, Exeter University. It is pleasant to t h a n k Professor J o h n W e b s t e r and Professor D a v i d Nichols for the hospitality o f their d e p a r t m e n t a n d to t h a n k D r D a v i d Stradling for his help a n d e n c o u r a g e m e n t d u r i n g this period.
REFERENCES Barnett, S. A. 198l. Modern Ethology: The Science o f Animal Behaviour. New York and Oxford: Oxford University Press. Brian, M. V. 1965. Caste differentiation in social insects. Syrup. Zool. Soc. Lond., 14, 13-38. Brian, M. V. 1980. Social control over sex and caste in bees, wasps and ants. Biol. Rev., 55, 379-415. Brian, M. V. & Brian, A. D. 1949. Observations on the taxonomy of the ants Myrmica rubra L and M. laevinodis Nylander. Trans. R. entomol. Soe. Lond., 100, 393-409. Brian, M. V. & Evesham, E. J. M. 1982. The role of young workers in Myrmiea colony development. In: Proceedings o f the IXth LU.S.S.L International Congress, Colorado (Ed, by M. D. Breed, C. T. Michener & H. E. Evans), pp. 228-232. Boulder, Colorado: Westview Press. Carlin, N. F. & H611dobler, B. 1983. Nestmate and kin recognition in interspecific mixed colonies of ants. Science, N. Y., 222, 1027-1029. Carr, C. A. H. 1962. Further studies on the influence of the queen in ants of the genus Myrmica. Insectes Sot., 9, 197-211. De Vroey, C. & Pasteels, J. M. 1978. Agonistic behaviour of Myrmica rubra. Insectes Soc., 25, 247-265.
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Elmes, G. W. 1982. The phenology of five species Of Myrmica from south Dorset, England. Insectes Soc., 29, 548-559. Errard, C. 1984. Evolution en fonction de l'~ge des relations sociales dans les colonies mixtes heterospbcifiques chez les fourmis des genres Camponotus et Pseudomyrmex. Insectes. Sot., 31, 185-198. Evesham, E. J. M. 1982. Regulation of the production of female sexual morphs in the ant Myrmiea rubra. Ph.D. thesis, University of Southampton. Evesharn, E. J. M. 1984a. The sensitization of young workers to queens in the ant Myrmiea rubra. Anim. Behav., 32, 782-789. Evesham, E. J. M. 1984b. Queen distribution, movements and interactions in semi-natural nests of the ant Myrmica rubra. Insectes Soc., 31, 5-19. H611dobler, B.'& Taylor, R. W. 1983. A behavioural study of the primitive ant Nothomyrmecia macrops Clark. Insectes Soc., 30, 384-401. Jaisson, P. 19751 L'impregnation dans l'ontogenese des comportements de soins aux cocons chcz ta jeune fourmi rousse~ Behaoiour, 52, 1-37. Jaisson, P. & Fresneau, D. 1978. The sensitivity and responsiveness of ants to their cocoons in relation to age and methods of measurement. Anim. Behav., 26, 1064-1071. Le Moll, F. 1980. On the origin of slaves in dulotic ant societies. Boll. ZooL, 47, 207-212. Le Moli, F. & Mori, A. 1984. The effect of early experience on the development of aggressive behaviour in Formica lugubris. Z. Tierpsychol., 65, 241-249. Le Moli, F. & Passetti, M. 1977. The effect of early learning on recognition acceptance and care of cocoons in the ant Formica rufa. Atti Soc. Bal. Sci. Nat., 118, 49-64. Le Moli, F. & Passetti, M. 1978. Olfactory learning phenomena and cocoon nursing behaviour in the ant Formica rufa L. Boll. Zool., 45, 389-397. Petersen-Braun, M. 1982. Intraspezifisches Aggressionsverhalten bei der Pharaoameise Monomorium pharaonis. Insectes Soc., 29, 25-33. Provost, E. 1979. t~tude de la fermeture de la soci~t6 de fourmis chez diverses esp6ces de Leptothorax et chez Camponotus lateralis. C.R. Acad. Sei. Paris, 288, 429432. Winterbottom, S. 1980, The chemical basis for species and colony recognition in three species of myrmicine ants. Ph.D. thesis, Southampton University. (Received 29 March 1985; revised 14 June 1985; MS. number: 2681)
Bonding between workers and queens in the ant genus
Myrmica
M. V. B R I A N *
Department of Biological Sciences, University of Exeter, Exeter EX4 4PS, U.K.
Abstract. Bond formation between young workers and queens is described for three species of Myrmica. This caste bond inhibits aggression and develops cooperation. Workers have a bias towards queens of their own species and do not discriminate between a queen of their own colony and a novel conspecific one. They are able to develop bonds with queens of other species of the genus provided they meet them first and well before their state of queenless aggressiveness sets in. A bispecific group, in situations contrived to enable them to meet gradually, will fight a normal group. They will then develop their bond to include the conspecific queen without becoming hostile to the earlier substitute. Evidently a queen of another species is able to provide an outline set of stimuli that is later built up by the conspecific queen, a characteristic of the imprinting development process. Allospecific queens can establish new microsocieties in a way reminiscent of temporary social parasites. The fact that allospecifically bonded workers will fight their one-time litter-mates that have been normally bonded suggests that the two groups have acquired different smells from their specifically distinct queens.
The work described in this paper started as an attempt to understand how queens, even dead ones, influence the way workers treat sexual larvae (reviewed in Brian 1980). Carr (1962) showed that workers brought up with queens suppress sexualization in these larvae more effectively than if they meet them for the first time during the experiment itself, and Brian (1965) suggested that imprinting might be involved. Brian & Evesham (1982) confirmed this and Evesham (1982, 1984a) showed that the first few weeks are the most sensitive period in a worker's life for this queen experience to occur. Preliminary tests showed that workers of Myrmica rubra, when less than a week old, could be imprinted with false information about their associate brood and adults. They could be made to work with a different species of the same genus just as can Formica (Jaisson 1975; Le Moli & Passetti 1977, 1978; Jaisson & Fresneau 1978; Le Moli 1980; Errard 1984; Le Moli & Mori 1984). From this it was only a short step to conceiving the possibility that they could be made to work with queens of other species and that this would throw light on the nature of the postulated queen/worker bond, and so of queen influence over worker behaviour.
* Present address: Orchard Lands, Hilton, Blandford Forum, Dorset, U.K.
METHODS
AND RESULTS
Myrmica rubra L. was used as a base species. For comparison two other species: M. ruginodis Nylander subspecies mierogyna (Brian & Brian 1949) and M. sabuleti Meincrt were used. Samples of these species were collected from many different localities in the south of England to increase the variability of the experimental material but colonies were not mixed. During winter the colonies were stored at 5~ from this stock, cultures were set to produce worker pupae as required and during summer the pupae from wild colonies were used. All were allowed to mature alone so that new workers emerged without the help of older ones or queens. Pupae died if any attempt was made to store them, even at 5-10~ and it was necessary to start to bond them to a queen at once: the sooner this was done the more likely it was to succeed since young workers become more antagonistic to queens the longer they are queenless (Evesham 1982, 1984a). Bonding took place if the workers and queens were put together in a space that was small enough for them to make frequent contact. The workers formed a cluster which at first excluded the queen; they pulled her away if she tried to join them but she seemed to be able to overcome this hostility by persistently approaching with stealth. A careful approach was especially important if the workers and queens were of
1135
1136
Animal Behaviour, 34, 4
different species for, quite often, bonding failed not just because of heightened worker hostility but because the queen sensed the worker species difference and avoided contact. Soon after the queen was allowed into the group she was groomed and fed and allowed to stand on the brood. This state of acceptance was taken to indicate that bonding was complete but it was usually confirmed when queenlaid eggs appeared. At this stage the queens were painted individually and the workers as a group. 'Silver' enamel was best for visibility through the red covers used to watch the ants without disturbing them. Once bonded and marked, the ants were put in standard cellular nests and allowed to settle down again. The cellular structure made it easy to plot the size and shape of a group. These nests were of the Janet type, made of dental plaster held in a plastic frame. Each had 14 identical cells indented in the plaster and covered with a glass slide that left room for the ants to walk over the tops of the walls. The red cover lay on top of the glass. Each cell was a hemispherical depression made by sinking a glass marble as far as its equator into the plaster whilst it was still soft. The cell diameter was 1.5 cm, its depth half this, and its volume 1 ml. Fourteen of these cells were arranged in a rhomboidal matrix with three columns of five, four and five cells. Each cell held 30 resting workers but only 10-12 active ones. The nests were kept in a cabinet at 20~ and 80~ relative humidity, with a day:night regime of 16 h: 8 h. Drosophila and i 0 ~ sucrose were supplied ad libitum. Aggressive behaviour is often seen between workers and has been graded in Myrmica rubra both by De Vroey & Pasteels (1978) and Winterbottom (1980). In this paper three of their six grades were used: most intense but rarely seen was an attempt to sting; moderately intense and often seen was biting and dragging; least intense and rare was mandible opening. The last has been called a 'threat' and no doubt it seems to be so, but no signal, chemical or tactile, has yet been shown to pass. It usually resulted in avoidance, or 'apotrepsis' to follow Barnett (1981). The method used to assess the bond is due to Provost (1979). After an age-group or 'litter' of new workers had been divided into two parts, each bonded differently and the workers and queens marked and put in a cellular nest, two such nests were joined by removing a plug in the connecting tube. The ensuing interaction was monitored by
samples in the morning and afternoon or as often as was useful. This tested the viability and independence of the microsocieties that had been synthesized, by allowing them to meet gradually, at first in a common run and later, in the two nests that were potential nest territories. These paired tests can be classified into balanced and unbalanced. All had Myrmiea rubra workers on both sides but, whilst the former had the same species of queen on each side, the latter had queens of different species on each side. First, four balanced tests are described: three had a single species (homospecific) and used Myrmica rubra queens, while one had two species (heterospecific) and used M. sabuleti queens. Then, four unbalanced tests are described: two had M. rubra queens against M. ruginodis queens and two had M. rubra queens against M. sabuleti queens. Throughout this paper upper case letters represent queens and lower case letters represent workers, e.g. b workers associate with B queens.
Balanced One- and Two-species Tests In the first three of the four experiments, the workers and queens were both M. rubra. In test 1, a concolonial queen was compared with an allocolonial one; in test 2, two allocolonial ones were compared; and in test 3 the sizes of the two groups that supported allocolonial queens were unequal.
Test 1 New workers, all from one colony, were divided equally and given either a queen (A) from the same colony, or a queen (B) from another colony. The two colonies were collected from valleys 75 km apart. Group a (with queen A) was formed in the left nest of the pair and group b (with queen B) in the right. Both queens settled and within 7 days entered the brood cluster; even the allocolonial queen met no resistance. Three weeks later the separating plug was removed and the workers began to explore. After 2 days group a moved as a body to the right and joined the heterogenous b culture without hostility. Clearly their identical origin outweighed any differences acquired from their brief sojourn with different queens. The B queen in group b was not attacked by the incoming a workers, in fact they fed and groomed her even though they had not met her before. The situation was not quite reciprocal, however, for queen A was at first attacked
Brian." Bonding between workers and queens mildly by the resident b workers and she attracted fewer workers than B for some time: for example, 12 twice-daily samples gave a total of 133 with A compared with 190 with B ( A N O V A , P<0.05). This asymmetrical response was probably due to the fact that a moved to join b and not the other way round, but whether this was chance or because the workers with a concolonial queen (a) were more firmly bonded, more socially complete and so more venturesome remains a problem. The two queens rarely met each other and when they did they only made a brief antennary contact; they were probably avoiding each other, i.e. were apotreptic. Once, when B ran into the same cell as A and jigged round, A ran off though she was not attacked directly. A few days later, B spent several days in a cell, the vestibule, next to the run often used as an assembly point by groups before crossing to the other nest. This suggests that B was trying to escape but would not leave by herself. She only returned to the core of the group (still on the right) after A was taken away. When a few days later she was returned to the nest, the workers clustered round her as though she was superstimulatory and B was completely ignored; clearly A was more attractive to the workers. Thus, throughout this trial the queens appeared to compete passively for worker attention. Ascendancy passed from the resident queen of the right B, to the immigrant queen A, originally on the left. A was, of course, concolonial with the workers but unknown to them as adults before the experiment. Though the queens were for the most part apotreptic, the rotary jig, centred on the brood group, comes near to being an offensive, possessive dance. As far as bonding is concerned, the important conclusions are as follows: first, that the two sets of litter-mates coalesced without hostility in spite of being bonded to different queens; second, that the workers clearly preferred the concolonial queen and were not exclusively bonded to the queen that they first encountered. They had the potential in fact, as would be expected with this species, of being polygyne.
Test 2 N e w workers from one colony were divided between two novel allocolonial queens (B and C). This test was similar to test 1 except that neither queen was concolonial with the workers. A set of 120 callows 2 weeks older than before, was divided into two equal parts; one part was given queen B
1137
and the other queen C. B and C were obtained from sources 75 km apart which were not the same as those used in the first test. Both queens were at first attacked; this was probably because the workers had been queenless for longer than in test 1 (Evesham 1982, 1984a). G r o u p b settled on the left and group c on the right of the double cellular nest; a week later they were joined together. After only a short period of mutual investigation, group c moved as a body to the left. They joined the other group peacefully. Whilst queen C then stayed on the left, B moved with 11 workers to the right; these workers were a random mix of the two types. After a further 3 days, B returned but this time she was attacked by workers and forced to remain peripheral. This hostility waned and then ceased but the two queens were never equal socially. This can be illustrated by the distribution matrices shown in Table I. The core cells, i.e. the ones that contained most workers over the whole period and which most often held the brood and the queens, were three adjacent cells in the right nest. Whilst C was seen here 30/34 times, B was only seen 18/34 times and only twice with C. So B both avoided C and was ranked below C by the workers; in fact she was probably starved for she became quite weak. This test thus shows that even 3-week-old workers can be bonded to totally strange queens. Table I. The distribution of two queens;~ of Myrmica rubra after two groups of callows each containing one had fused Number of occurrences Queen B 0
0
Queen C
Cumulative worker number
0
26
t
5*
l
16"
6* 7*
1
1
Ill*
0
0 t
1
1 0
11 68*
59*
3 0
4
0
5*
6 31
9*
3 4
0 0
19 19
0 t
19
0
2
II
9 ? 4
One-species series, test 2: the distribution in 14 rhomboidally arranged cells on the left side of the double nest, of queens B and C and the summed positions of the workers. * Core cells; ? the entrance to the nest which is the left one of the pair. $ Both queens were allocolonial to the workers.
Animal Behaviour, 34,4
1138
Though equally unrelated to the workers, one of the queens was able to settle in the core whilst the other remained peripheral and unsettled. Worker choice was exerted even amongst allocolonial queens. This unequal relationship lasted the whole period of the trial.
Test 3 A worker group was divided unequally between two novel allocolonial queens (B and C). In this test the workers were less than a week old. The groups were intentionally of different sizes so that the possibility that queen establishment is affected by the number of workers in their support could be explored. The group on the left, c, consisted of 50 workers and the queen C and that on the right, b, of only 10 workers and queen B (Table II). After the plug had been taken out, the two lots of workers explored and then started to mix without hostility. At first workers moved both ways, but this movement back and forth stopped after a mass movement to the right which resulted in a single mixed group. Queen C then joined B but the latter moved away into a corner with 10 c and three b workers. The eggs were left with queen C who moved 3 days later with a very mixed retinue and fused with the group associated with the B queen. The structure of this new group was then as follows: in the core, 26 c and five b with queen C; on the fringe, 19 c and four b workers with queen B. The slightly greater proportion of b on the fringe, which might be thought to imply that, though workers, they were excluded from the core, is not statistically significant.
Table II. The fusion of two groups of Myrmica rubra workers, each with an allocolonial queen On left Days after nest joined 0 1 2 4
c
C
b
On right B
c
5 0 1 0 0 0 2 8 1 4 0 1 3 5 1 1 0 0 0 0 0 0 4
0 6 9 4
1 0 0 1
C
b
0 6 0 9
1 1 1 1
B
One-species series, test 3: the numbers of workers and queens on the left and right sides of the double nest on four occasions; b and c workers with queens B and C respectively.
This test thus confirms that worker groups sooner or later coalesce and mix freely and randomly. It also shows that workers within these groups do not associate with the queens to which they were originally bonded. They take up additional queens peacefully. The queen with least initial support (10 workers) attracted less support later, but degree of sponsorship seems unlikely to be a cause of priority since the workers did not associate with their bonded queens once they had joined a bigger group. All three of these single-species balanced tests indicate that Myrmica rubra workers can be bonded to any queen of their own species; differently bonded groups will soon coalesce. Within these worker-made groups the queens are apotreptic, but one sometimes uses a 'rotary jig' dance to dislodge another. The dislodged queen may try to leave but will only go if a retinue of workers accompanies her, not alone. In test 1, the workers favoured a concolonial queen but when she was experimentally removed, they switched their attention to the other queen; in test 2, one of the two allocolonial queens was favoured and the other only just tolerated; and in test 3, one allocolonial queen was again favoured out of the two offered. The one chosen had more bonded workers at the start but this was probably irrelevant. The workers form a group around a preferred queen and attract other workers. They then attempt to capture and incorporate any splinter groups that shield low priority unattractive queens. The latter find difficulty in becoming independent, at any rate in the conditions provided in the experimental nest.
Test 4 To complete these balanced tests, a test was carried out using a two-species culture (Myrmica rubra workers with M. sabuleti queens on both sides). A group of 60 workers of M. rubra less than a week old was divided equally and half put on each side of the double nest, each with two queens of M. sabuleti. Twenty-four days were allowed for bonding owing to the uncertain interaction between this pair of species. During this period one queen died so that, when the two parts were joined, there were two queens on the left (B 1, B2) and one on the right (B3). At this time no eggs had been laid. The day after the connecting tube was opened, workers passed in both directions but, in spite of a close watch, no fighting was seen. At the end of the
Brian: Bonding between workers and queens first day, there were six b3 o n the left a n d three b 1b2 on the right, but still no hostility. T w o days later, all the b r o o d including some new eggs were t a k e n to the left a n d queen B3 remained with only two workers, one of each sort. Queens B 1 a n d B2 were in separate cells, one with eight b l b 2 and two b3 a n d the o t h e r with 11 b l b 2 a n d five b3. T h u s the two sets mixed peacefully. B3 m o v e d over to j o i n the o t h e r two queens but they all used different cells. Q u e e n B3 was a c c o m p a n i e d by 10 b l b 2 a n d three b3 workers, showing no bias against her; in fact, 6 days later, all three queens were in the same cell. This also confirms their effective b o n d i n g to the Myrmica rubra workers. Later the queens m o v e d about, sometimes singly sometimes together, b u t with n o sign o f apotrepsis, a n d the experiment was stopped. So, M. rubra workers will a d o p t M. sabuleti queens a n d form a viable microsociety t h o u g h the species are not considered close taxonomically. Moreover, two such groups will join peacefully. Queens of this species may p r o v e on further study to be less apotreptic than those of
M. rubra.
Unbalanced Two-species Tests
These four tests involved Myrmica rubra workers with a queen of the same species o n one side a n d a queen o f M . ruginodis a n d M. sabuleti on the other. Thus, allospecific queens were competing with conspecific queens for the s u p p o r t of a divided g r o u p of workers, h a l f b o n d e d to one and h a l f to the other.
1139
Test 1 A litter of 72 workers, less t h a n a week old, was divided into two equal parts, one o f which was given a queen of M. rubra (A) a n d the other a microgyne queen of M. ruginodis (B). The former queen was from a different colony from the workers but they settled together after only mild hostility. The M. ruginodis queen on the c o n t r a r y was heavily bitten a n d pulled a b o u t for at least 10 days. With the a culture o n the left a n d the b on the right, the two were joined a n d the distribution recorded regularly for 6 weeks. The situation each week is given in Table III. As usual, workers started to cross over to the opposite side and explore at once b u t this time fighting b r o k e out a n d went on for 3 weeks. The m o v e m e n t o f b to the left stopped a n d they began to return. Between the third a n d fifth week, a m o v e d to the right, leaving only three b a n d one a on the left. All the rest, with the two queens, were on the right, Thus it t o o k as long as 5 weeks for the two groups to fuse. This delay in the r e u n i o n of one time litter-mates was caused by the hostility between a a n d b workers; they behaved as t h o u g h they belonged to different colonies. A v o i d a n c e was the rule but, when this was impossible, the workers bit a n d tugged at each o t h e r for a long time. Very few fights were reciprocal, so it was possible to tell w h o was the aggressor. The sequence o f fights has been tabulated in Table IV. It should be noted that the first 30 interactions were between workers, for b o t h queens were inaccessible behind their screens of defenders. O f
Table lIl. The interaction of two groups of Myrmica rubra callows, one
with a queen of the same species and one with a queen of M. ruginodis Left nest
Right nest
Workers
Queens
Workers
Queens
Week of observation
a
b
A
B
a
b
A
0
36
0
A
O
0
36
O
B
1
35
1
A
O
1
34
O
B
2 3 4 5 6
31 28 16 1 27
7 14 13 3 24
A A A O A
O O O O O
4 6 18 32 5
28 21 22 30 8
O O O A O
B B B B B
B
Unbalanced two-species series, test 1: a: the workers with a conspecific queen (A) starting on the left; b: the workers with an allospecific queen (B) starting on the right.
Animal Behaviour, 34,4
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Table IV.-The sequence of fights when two groups of Myrmica rubra callows were joined, one with a conspecific queen and the other with an allospecific queen (M. ruginodis) Between workers and workers On the left Sequenceoffights a > b
Between queens and queens
On the right
On the left
On the right
b>a
a>b
b>a
A>b
a>B
B>a
b>B
1 10 11-20
4 5
0 l
6 4
0 0
0 0
0 0
0 0
0 0
21-30 3140 41-50 51 60 61-70 71-80
3 0 1 2 0 0
1 0 0 0 0 0
5 4 4 1 0 0
1 0 0 0 2 0
0 0 1 0 0 0
0 3 2 5 7 10
0 3 2 1 0 0
0 0 0 1 1 0
Total
15
2
24
3
1
27
6
2
Unbalanced two-species series, test 1: a, the worker group with conspecific queen A; b, the worker group with allospecific queen B. The first 80 fights after connection have been classified and listed after grouping in 10s: a > b means that a worker of the a group attacked one of the b group and so on. these 30 fights, 27 were defensive a n d three offensive. Both sides were a b o u t equally concerned to defend their nest territory. In detail, the three offensive actions were: two on the left by a n i m m i g r a n t b against a resident a a n d one on the right by a n i m m i g r a n t a against a resident b. D u r i n g the next 30 events (31 60) defensive actions by workers diminished from 12 to 3 on the left a n d from 15 to 9 o n the right b u t offensive actions did n o t replace them. However, the workers were still crossing over (Table IV). M o s t p r o b a b l y the explanation for the decline in inter-worker hostility was due to the discovery o f the M. ruginodis queen by the a workers, w h o were seen to attack her 10 times. Her ' o w n ' b workers did n o t help at all but she herself fought the a workers with her mandibles. This struggle was n o t balanced by a similar action against the A queen, but once she attacked a b worker t h a t ' t h r e a t e n e d ' her in her cell. The attack against the core of the M. ruginodis queen increased so t h a t 17 of the next 20 events were fights initiated by a workers against the queen. The other queen, A, was left alone again a n d later m o v e d over with 32 of ' h e r ' workers to the right. This sealed the fate o f B who, with her a n t e n n a l tips cut off, died. The A queen did not stay, b u t returned to the left with m o s t o f the workers, which were, by now, well mixed.
This battle started with a n exploration t h a t led to defensive retaliation. Later, as the workers from the two sides mixed, they h a b i t u a t e d to each o t h e r a n d avoided open hostility. W h e n the atypical queen, B, was discovered she was attacked, but only by the n o r m a l l y - b o n d e d workers. The queens were never seen to fight directly. T h o u g h a workers at first dragged the B queen out into the run, she always got back; later the workers were less aggressive a n d only 'arrested' her if she tried to move away. A l t h o u g h she defended herself, her b o n d e d workers did not help directly b u t they groomed a n d fed her at the same time as the others, a, bit and held her down. She appears to have been too unusual to settle in the fringe as the M. rubra queens did in the early series. Evidently, after she h a d been killed, the workers previously b o n d e d to her assimilated into the m a i n group and followed the n o r m a l conspecific queen. It is nevertheless astonishing h o w long the workers attached to B remained supportive, if n o t defensive, towards her. This inhibition o f aggression t h a t transcends species differences is p r o b a b l y the most i m p o r t a n t feature of the caste b o n d ; it clearly demonstrates the nature a n d strength of the imprinting. P r o b a b l y the conspecific queen w o n the c o m p e t i t i o n for the workers' s u p p o r t because she was able to supply workers with m o r e detailed a n d clearer data with
Brian." Bonding between workers and queens Table V. The distribution changes in two joined nests containing groups of Myrmica rubra callows, one with a conspecific queen and the other a queen of M. ruginodis Le~
Right
Date
a
b
a
b
17 July 24 July 31 July 7August 14 August* 21 August
25 21 20 10 12 16
0 0 I 8 4 3
25 25 24 16 16 15
0 4 5 6 10 4
Unbalanced two-species series, test 2: those starting on the left (a) were bonded to concolonial queen A, those on the right (b) were bonded to an M. ruginodis queen, B. The queens did not change sides at all. * At this time, 10 new M. rubra workers were formed on the left and 10 new M. ruginodis on the right.
which to develop the fine structure of the imprinting.
Test 2 This repeated test 1 using different material. Fifty newly prepared workers of Myrmica rubra were divided equally and given either a queen of the same species but from a different colony (A) or a microgyne queen of M. ruginodis (B). The group a was put on the left, and the group b on the right. When the plug was taken out, the a group began to move to the right (Table V) but were resisted by the b group. The a workers found the B queen much more quickly than in the first test and attacked her. She defended herself and was once seen to bite at the neck of a worker but without severing its head; several b workers joined in and defended the queen by attacking this and another a worker. Yet, next day, B had been dragged to the vestibule by an a worker with a b worker attached. This queen continued to defend herself effectively and was once seen to apply a droplet with her sting to the m o u t h parts of an a worker, which had an immediate repellant effect. Meanwhile B's own workers (b) groomed and fed her and, although she escaped this time, she was attacked again (Table VI). The b workers were still quite active in territorial defence
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on the right and some even spread to the left where they attacked the base of the a group and set off defence reactions. The invading b workers, when they met the A queen, were not hostile. This is understandable if imprinting is progressive and starts with a general impression of a queen as a distinct caste: the queen M. ruginodis must satisfactorily provide this input in spite of her wrong species smell. M o r e details, such as only an M. rubra queen can provide, are built in later. The vigorous b workers in this test were able to protect 'their' queen B from constant harrying by a workers to such a degree that she managed to lay eggs that the b workers protected from the thieving a workers. These hatched and were brought up to the adult stage as 10 workers after 2 months. All this was achieved in spite o f a fresh offensive by the a workers and their simultaneous production of 10 new M. rubra workers. The new M. ruginodis workers bonded normally with queen B. This stabilized the interaction and the number of a workers on the b side declined from then on. Nest territories were thus reinforced, migrations between the two nests stopped and the M. ruginodis microsociety became potentially independent. If microgyne queens normally start new societies by using the routine o f a temporary social parasite then they may well have the social and structural adaptations needed for success.
Test 3 Queens of M. sabuleti can also be bonded to M. rubra workers. Starting with the usual litter of equal-aged workers, one half was given to two M. rubra queens, A1 and A2, and the other to three M. sabuleti queens, B 1, B2 and B3. After 2 weeks, the former were put on the left and the latter on the right of the pair of nests. The first 90 fights are summarized in Table VII. The first 40 of these were once again between workers from different sides and were mostly defensive. On the right, in B territory, the resident b group set upon the invading a group, and reciprocally on the left. O f these fights, 12 were on the left by a against b and 18 were on the right by b against a. Some offensive actions occurred and comprised four on the left (b against a) and six on the right (a against b). Besides being more frequent on the right, the fights lasted longer and so supported the earlier idea that conspecific bonding gives a stronger social base than allospecific bonding. The a workers were first seen attacking the B
Animal Behaviour, 34, 4
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Table Vl. The sequence of fights after two groups of Myrmiea rubra catlows were joined; one had a conspecific queen and the other an M. ruginodis queen
Workers with workers Left Sequenceoffights a > b 1 20 21-- 40 41 60 61 80 81-100 101-120
Total
Queens with workers
Right
Right
b>a
b>a
a>b
a>B
B>a
0 0 7 4 9 3
0 0 2 3 4 1
12 10 8 8 6 9
1 l 1 3 0 0
5 8 1 1 1 6
2 1 1 1 0 1
23
I0
53
6
22
6
Unbalanced two-species series, test 2; the first 120 fights classed as offensive and defensive and grouped in 20s. A and B were queens of Myrmica rubra and M. ruginodis respectively; a and b were the worker groups containing them; a > b means that a attacked b and so on. Group a was on the left and b on the right.
Table VII. The sequence of fights after two groups of Myrmica rubra callows were joined; one had two conspecific queens and the other three M. sabuleti queens
Workers with workers Left Sequenceoffights a > b
Right
Queens with workers Right
b>a
b>a
a>b
a>B
1-10 11-20 21 30 3140 41-50 51-60 61 70 71-80 81-90
2 2 3 5 4 3 3 3 1
2 I l 0 0 0 0 0 2
5 5 4 4 3 0 1 0 0
1 2 2 1 2 2 3 1 0
0 0 0 0 1 5 3 6 7
Total
26
6
22
14
22
Unbalanced two-species series, test 3; the first 90 fights classed as offensive and defensive and grouped in 10s. A and B were the Myrmica rubra and M. sabuleti queens respectively; a and b were the worker groups containing them; a > b means that a attacked b and so on. G r o u p a was on the left and b on the right.
Brian: Bonding between workers and queens queens on the 47th occasion and, from then on, these queens were attacked whenever they were encountered. The b workers were also on both sides and they gave up attacking the a group, even defensively. By the third day, a workers had reached and entered all the peripheral cells on the right and were beginning to invade the core. One B queen had already been pulled out of the core and was held by the antennae in the next cell. The other two were still free and had seven of their bonded workers with them but one a worker was seen to enter their cell and adopt a 'threat' stance. The displaced B queen soon returned to the core and was fed and groomed by one of the seven workers. This state lasted for another 3 days but, by constant pressure, the a workers gradually broke up the b core and dissipated the resistance of the defending workers. On day 13 one of the A queens travelled over to the right and herself entered the b core. At the same time, one of the B queens was held by two a workers; seven b workers who were present took no action at all. In fact, in spite of many opportunities, the A queens were never attacked by b workers. The second B queen was carried away by an a worker and put in a marginal cell where five b workers fed her. After 1 day on the right, the A queen went back to her own side where she proved to be super-attractive; over 25 workers collected around her (cf. the similar event in the one-species series, test 1). So again, though the b workers succoured their queens they only once, after the initial defensive phase, attacked a workers on the right, or b side. N o t surprisingly, their aggressiveness depends on the structural integrity of their group. Only 15 workers remained on the right by day 20, though all three B queens were still there and appeared to be free; at least they were only lightly restricted when they tried to move. They attracted very little lasting attention and were able to go into the run to drink. All of them were in fact starving and could only climb the walls of the cells with difficulty. Most of the b workers that might have fed them had joined the A queens on the left. N o M. sabuleti queens laid any eggs and no prospect ever occurred of a social parasitic nucleus developing. After some dragging about they were starved to death rather than being dismembered. Test 4 A litter of 96 Myrmica rubra workers less than a week old was divided into two equal parts and one
1143
group given an M. rubra queen, A, and the other an M. sabuleti queen, B. They were put in the left and right sides of a double nest. The first homospecific 'graft', a, was quickly formed, but the second heterospecific one, b, took longer. Unexpectedly, 1 day after the separating plug had been removed, the b workers moved over to the left, taking the B queen with them and joined the a group. The cause of this remarkably sudden migration, which was entirely peaceful, is unknown; no workers fought each other. Later, the A queen was attacked by the b and the B by the a workers. Perhaps the unusually quick fusion of the two groups prevented the usual habituation between b and A. Hostility soon passed off and A established herself in the core whilst B remained peripheral, arrested by two or three a workers who dragged her into the run if she moved. So the workers selected a conspecific queen and tried to expel the alien, only failing in this because of her persistent return from the run. During the interaction the queens were only seen in the same cell once. This was after a slight jolt to the nest caused B to run about wildly and she blundered into the core cell where A was established with the egg cluster. Here she grabbed a few eggs in her mandibles but A gave the menace gesture and several of the a workers dragged queen B out. Opening the mandibles may well release some pheromone and cause an alarm recruitment of workers as in foraging. After this, to test for interaction between the queens, a special test was set up. A was taken away for 6 days; this let the B queen back into the brood chamber, where she was soon surrounded by attentive workers who clearly preferred a queen of a different species to no queen at all. Although she did not spend much time in the core, she was quite free to move and was once seen in the vestibule with a worker cluster. Next day, when A was put back, the workers at first refused to accept her; they even left B and came over to attack A. This phase only lasted 1 h, after which A was allowed to settle in a new cell where the workers pooled all the eggs. The situation was still unstable however, for 20 rain later the eggs and all the brood had been taken away and A was pinned down. w h e n , a few minutes later, A was again released, she rejoined the eggs and settled again, this time for good. B, although arrested, managed to escape and settle on the fringe of A's group. Throughout this behaviour sequence, as long as the M. rubra queen was present, the M. sabuleti
Animal Behaviour, 34, 4
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queen was treated with low priority; though actively kept out of the core, she was not destroyed. Removal of A, however, enabled B to develop some degree of acceptance, if not quite of establishment (though only 6 days was allowed for this). As a result, when A was returned, she needed a period of readaptation before she could again enter the core. Although a little mutual hostility may have occurred, the main channel through which the queens interacted had to be the workers. Worker support was essential; they chose their own species of queen if possible and, even within conspecific queens, they showed preferences. The absence of an inter-worker defensive phase in this test is interesting, for in this it resembles the earlier series of balanced tests in which opposing queens were of the same species. The sudden migration to the other side, whatever its cause, was possible because the workers at that time had not developed any method for distinguishing their groups. If the inter-worker fighting depends on acquiring a distinction (presumably from the queens) then it had not then spread to the workers.
DISCUSSION It has been shown that the bond that has been investigated links the queen with the workers in such a way as to create a viable system that is capable of defending and reproducing itself. The workers feed and clean the queen and defend her against intruders that they are able to recognize. The queens in return satisfy a need in the workers and produce female eggs. Bonding is most easily established soon after emergence, which is roughly equivalent to birth or hatching in vertebrates. This sensitive period fades and gives way to overt hostility, even against conspecific queens, if no bondable object presents itself. Yet, if bonded normally, such workers will accept more normal queens. Workers can be misled into adopting a wrong species of queen, for only a general outline of queen characteristics appears to be used at first; the details of species and individuality are filled in later. Presumably queens of the same genus have enough in common to provide satisfactory initial bond objects and they can even carry it as far as the production of offspring as long as no normal conspecific queen appears. However, the workers evidently have a bias, which is of course adaptive, towards a conspecific queen, and do not reject such
queens even if they have started to form an allospecific bond. The immediate cause of this bias that occurs in workers which, as adults, have not met a queen at all, is probably a species characteristic template but their conditioning as larvae by contact with the workers that nurse them cannot yet be ruled out; they could be given the correct species information at this stage. In correcting these mistakes by rebonding to conspecific queens, workers do not erase their original information; their superseded queens are just kept out of the breeding core in reserve, to be restored if the preferred queen is removed (cf. queen supersedure in honeybees). The individuality of the bonded queen, if it is appreciated 'by the workers, is not allowed to interfere and workers at this stage of the colony cycle are prepared to take in and adopt any conspecific queen. An interesting result of this research is that workers develop an antagonism towards littermates that have become experimentally bonded to queens of a different species. They are able to recognize this deviation, and recognition is reciprocal. The most obvious suggestion is that these have acquired a veneer of the strange species smell; such workers would then smell to the normal workers as though they were of a different species. Whether this idea can be extended to 'colony odour' remains to be seen. This aspect of the work on bispecific groups complements the results which Carlin & H611dobler (1983) obtained using mixed species groups of Camponotus; they found that the length of time that was spent in the company of individuals of a different species increased their similarity and they suggested that the queen probably produces a pheromone that coats the workers strongly enough to obscure their genetic relatedness. Using species of Leptothorax, in a system of nests with a common arena, Provost (1979) found that, although worker groups would coalesce, the queens fought to the death. In Nothomyrmecia a status interaction appears to exist between queens, which leads to the eventual extrusion of the loser queen (H611dobler & Taylor 1983). In Monomorium pharaonis, workers treat queens that are not in phase with the population cycle aggressively and they are eliminated (Petersen-Braun 1982). In Myrmica, queens have been described as fighting when they could not avoid each other (Evesham 1982; 1984b) and old queens were accompanied to the run to die. In this study, unwanted queens were
Brian: Bonding between workers and queens extruded. Elmes (1982) has p r o d u c e d evidence in the field t h a t this h a p p e n s , t h o u g h the queens he found w a n d e r i n g a b o u t were not necessarily superfluous. Clearly such queens are u n d e r a higher risk of d e a t h f r o m accident a n d p r e d a t i o n t h a n those living in the nests.
ACKNOWLEDGMENTS This work was s u p p o r t e d by a g r a n t f r o m the Royal Society whilst h o l d i n g a n H o n o r a r y Fellowship at the D e p a r t m e n t o f Biological Sciences, Exeter University. It is pleasant to t h a n k Professor J o h n W e b s t e r and Professor D a v i d Nichols for the hospitality o f their d e p a r t m e n t a n d to t h a n k D r D a v i d Stradling for his help a n d e n c o u r a g e m e n t d u r i n g this period.
REFERENCES Barnett, S. A. 198l. Modern Ethology: The Science o f Animal Behaviour. New York and Oxford: Oxford University Press. Brian, M. V. 1965. Caste differentiation in social insects. Syrup. Zool. Soc. Lond., 14, 13-38. Brian, M. V. 1980. Social control over sex and caste in bees, wasps and ants. Biol. Rev., 55, 379-415. Brian, M. V. & Brian, A. D. 1949. Observations on the taxonomy of the ants Myrmica rubra L and M. laevinodis Nylander. Trans. R. entomol. Soe. Lond., 100, 393-409. Brian, M. V. & Evesham, E. J. M. 1982. The role of young workers in Myrmiea colony development. In: Proceedings o f the IXth LU.S.S.L International Congress, Colorado (Ed, by M. D. Breed, C. T. Michener & H. E. Evans), pp. 228-232. Boulder, Colorado: Westview Press. Carlin, N. F. & H611dobler, B. 1983. Nestmate and kin recognition in interspecific mixed colonies of ants. Science, N. Y., 222, 1027-1029. Carr, C. A. H. 1962. Further studies on the influence of the queen in ants of the genus Myrmica. Insectes Sot., 9, 197-211. De Vroey, C. & Pasteels, J. M. 1978. Agonistic behaviour of Myrmica rubra. Insectes Soc., 25, 247-265.
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Elmes, G. W. 1982. The phenology of five species Of Myrmica from south Dorset, England. Insectes Soc., 29, 548-559. Errard, C. 1984. Evolution en fonction de l'~ge des relations sociales dans les colonies mixtes heterospbcifiques chez les fourmis des genres Camponotus et Pseudomyrmex. Insectes. Sot., 31, 185-198. Evesham, E. J. M. 1982. Regulation of the production of female sexual morphs in the ant Myrmiea rubra. Ph.D. thesis, University of Southampton. Evesharn, E. J. M. 1984a. The sensitization of young workers to queens in the ant Myrmiea rubra. Anim. Behav., 32, 782-789. Evesham, E. J. M. 1984b. Queen distribution, movements and interactions in semi-natural nests of the ant Myrmica rubra. Insectes Soc., 31, 5-19. H611dobler, B.'& Taylor, R. W. 1983. A behavioural study of the primitive ant Nothomyrmecia macrops Clark. Insectes Soc., 30, 384-401. Jaisson, P. 19751 L'impregnation dans l'ontogenese des comportements de soins aux cocons chcz ta jeune fourmi rousse~ Behaoiour, 52, 1-37. Jaisson, P. & Fresneau, D. 1978. The sensitivity and responsiveness of ants to their cocoons in relation to age and methods of measurement. Anim. Behav., 26, 1064-1071. Le Moll, F. 1980. On the origin of slaves in dulotic ant societies. Boll. ZooL, 47, 207-212. Le Moli, F. & Mori, A. 1984. The effect of early experience on the development of aggressive behaviour in Formica lugubris. Z. Tierpsychol., 65, 241-249. Le Moli, F. & Passetti, M. 1977. The effect of early learning on recognition acceptance and care of cocoons in the ant Formica rufa. Atti Soc. Bal. Sci. Nat., 118, 49-64. Le Moli, F. & Passetti, M. 1978. Olfactory learning phenomena and cocoon nursing behaviour in the ant Formica rufa L. Boll. Zool., 45, 389-397. Petersen-Braun, M. 1982. Intraspezifisches Aggressionsverhalten bei der Pharaoameise Monomorium pharaonis. Insectes Soc., 29, 25-33. Provost, E. 1979. t~tude de la fermeture de la soci~t6 de fourmis chez diverses esp6ces de Leptothorax et chez Camponotus lateralis. C.R. Acad. Sei. Paris, 288, 429432. Winterbottom, S. 1980, The chemical basis for species and colony recognition in three species of myrmicine ants. Ph.D. thesis, Southampton University. (Received 29 March 1985; revised 14 June 1985; MS. number: 2681)