Francesco Le Moli and Alessandra Mori.
Laboratory experiments on environmental sources of nestmate and non-nestmate discrimination in three species of Formica ants (Hymenoptera: Formicidae).
Psyche 97:147-170, 1990.

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LABORATORY EXPERIMENTS ON ENVIRONMENTAL
SOURCES OF NESTMATE AND NON-NESTMATE
DISCRIMINATION IN THREE SPECIES OF
FORMICA ANTS (HYMENOPTERA: FORMICIDAE)
Nestmate recognition in social insects is a central issue of sociobiological theory on kin selection (Holldobler and Michener 1980; Getz 1981, 1982; Holmes and Sherman 1983), because it pro- vides an important trait of altruistic behaviour towards kin. In fact, the ability to recognize kin from non-kin enables the individuals to achieve greater benefits by directing their altruism towards their relatives (Wilson 1975; Gadagkar 1985; Sherman and Holmes 1985; Hepper 1986; Breed and Bennett 1987). This ability is consistent with the major genetic mechanisms hypothesized for the evolution of sociality (cf. Shellman and Gamboa 1982). In this last decade several researches have demonstrated that ants possess a great ability to discriminate between colony members (including queens) and non-colony members (Gamboa et al. 1986; Carlin and Holldobler 1986, 1987; Bennett 1988; Keller and Passera 1989) on the basis of olfactory cues which determine the distinctive colony odour of these eusocial insects (Wilson 1971; Parry and Morgan 1979; Stuart 1987a; Obin and Vander Meer 1989). These chemical labels of kin recognition are likely surface pheromones, mainly epicuticular hydrocarbons (Howard and Blomquist 1982; Bonavita-Cougourdan et al. 1987; Blum 1987), so that alien colony odour is the main olfactory stimulus evoking attack in such insects (Jutsum 1979; Jutsum el al. 1979; Mabelis 1984; Le Moli and Mori 1986).
Generally, genetically produced (Ribbands 1965; Haskins and Haskins 1979; Mintzer 1982; Carlin and Holldobler 1986, 1987; Mintzer and Vinson 1985) or environmentally derived factors (Lange 1960; Le Moli and Mori 1985; Obin 1986; Obin and Vander 'Istituto di Zoologia, Universita'degli Studi, Via Eke di Sotto, 06100 Perugia, Italy. 21stituto di Zoologia, Universita' degli Studi, Viale delle Scienze, 43100 Parma, Italy. Manuscript received by the editor March 28, 1990. Pu&e 97:147-169 t HIM), http:llpsyche cnlclub or@W-147 hlml



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148 Psyche [vo~. 97
Meer 1988) or both (Wallis 1962; Jutsum et al. 1979; Crosland 1989) contribute to colony odour, but their relative importance may vary among different species. Other researches (Hangartner et al. 1970; Hubbard 1974) showed that in some species of ants the workers affect the odour of the soil in which they live, suggesting that odorants from the nest materials can be absorbed into the epicuticle to serve as colony odours. More recently, Stuart (19870) demonstrated that colony segregation within local populations of polydomous colonies of Leptothorax curvispinosus is largely main- tained by transient environmentally-based nestmate recognition cues.
The evident role exercised by environmental factors in determin- ing the distinctive colony odour in some ant species, as well as the existence of early olfactory learning mechanisms in the ontogeny of nestmate and brood recognition (for a review see Jaisson 1985; Le Moli and Mori 1987; Carlin 1988), recently allowed ethologists (Jaisson 1987; Le Moli and Mori 1987) to advance a new hypothesis on fellowship (or living together) that, with the kinship hypothesis, contributes to the sociobiological theory on kin selection and inclu- sive fitness in such insects. More recently, in some species of ants we demonstrated (Le Moli and Mori 1989) that following the same diet, which is an important consequence of group living, is essential for the production, maintenance and evolution of the common olfac- tory signals on which individual recognition in these eusocial insects is based. In fact, field experiments carried out both in Formica lugubris and in Formica rufa, showed that individuals reintroduced to their natal colony after a 30-day period of laboratory housing (with nest substratum and materials) were almost always attacked by resident nestmates. This result firstly indicates that metabolic differences induced by a laboratory diet alter the distinctive colony odour in both these related species when workers are tested in the field. This implies that workers of these two Formica species con- stantly learn to recognize the odour of the colony, emphasizing the significance of environmental sources on ant nestmate identifica- tion. Secondly, this means that fellowship is essential to maintain the common olfactory signals of nestmate recognition and accep- tance (worker-worker), since a lack of living together generally releases an overt aggression towards the reintroduced kin.



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19901 Le Moli & Mori-Formica 149
In the present work we have attempted to confirm the above- mentioned result obtained in the field, studying by a laboratory aggression test, the effect of the diet alone on nestmate recognition (homocolonial interactions) by workers of F. lugubris, F. rufa, and F. cunicularia, after a 30-day period of their maintenance in artifi- cial colonies. Moreover, to evaluate the possible degree of aggres- sion recorded in the homospecific tests, we analysed and quantified also the heterospecific relationships (worker-worker) of F. lugubris contrasted respectively with F. rufa and F. cunicularia, after a sim- ilar period of laboratory housing under artificial diet. For this heterospecific test, we considered the trials involving F. lugubris, because it is well known as a very efficient predatory species which shows a high level of aggression in its interspecific relationships (Le Moli and Parmigiani 1981; Le Moli et al. 1984; Le Moll and Mori 1986). Heterospecific trials were preferred to heterocolonial trials, because overt aggression is almost always recorded between such species (Le Moli et al. 1984). On the contrary, at least within wood ant species, the social conflict among heterocolonial members usu- ally takes the form of ritualized aggression (Le Moli and Parmigiani 1982; Le Moli et al. 1982).
Natural populations
To avoid any previous contact among members of F. lugubris (Fl), F. rufa (Fr), and F. cunicularia (Fc), we selected colonies which were geographically isolated.
We used a polygynous colony of F. lugubris situated in the Apen- nine mountains (Mount Alpe), about 1200 m high in the province of Pavia. The anthill consisted of Larch leaves (Larix decidua) and Austrian pine needles (Pinus nigra var. austriaca). The polygynous colony of F. rufa selected was located on the Prealps (Gulter Wood), at about 1100 m in the province of Ber- gamo. The materials of this anthill were mainly spruce fir (Picea excelsa) needles.
Finally, the F. cunicularia individuals came from a polydomous colony with multiple queens dug into the ground in the Apennine mountains (Mount Caio), at about 1200 m in the province of Parma.




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150 Psyche [vo~. 97
Laboratory conditions
Several thousand ants (workers and some homocolonial queens) with nest substratum and materials from the natural colonies were collected in the field and kept for all the experimental period in the laboratory in separate artificial nests (one for each species) made of glass (45X60X30 cm) under controlled conditions. The relative humidity was at 60-80%, and the room temperature was about 22-24OC. These artificial nests provided the ant-workers paired in the homocolonial and heterospecific interactions of the control test (see forward).
After the control test, the remaining ants were divided into 2 fragments (A, B) for each of the original colonies, and submitted for 30 days to different diets, consisting respectively of yolk, acacia- honey and water (diet a) or fly tissue, eucalyptus-honey and water (diet B). We selected this period of time because it is probably long enough to induce metabolic differences due to the diet in workers of such colony fragments, without causing a decrease of their vigour (cf. also Le Moli and Mori 1989). According to this procedure, the ants of the three species (Fl, Fr, Fc) belonging to the fragments A and B were fed either diet a (FlAa, FrAa, FcAa, FlBa, FrBa, FcBa) or diet B (FlAfi, FrAfi, FcAfi, FlBfi, FrBfi, FcBfi). Thus, we set up 12 colony fragments, 4 for each species. All the fragments of the original colonies were provided with at least one homocolonial queen, nest substratum and materials.
Control test
As a control, 48 hours after the arrival of ants in the laboratory, the homocolonial (Fl vs Fl, Fr vs Fr, and Fc vs Fc trials) and the heterospecific (Fl vs Fr, Fl vs Fc trials) interactions of the species considered were analysed and quantified by the aggression test (see Le Moli and Mori 1986). To this purpose, workers of the three species were tested in pairs in an experimental apparatus, namely the fighting box described by Le Moli and Parmigiani (1981). The behaviour of the dyads was observed and recorded for 15 minutes during which the following indices were measured in seconds:
mutual investigation (MI), i.e. the time spent in reciprocal inspection;
-latency to attack (LA), i.e. the time from the first contact to the attack (when no attack occurred, 15 minute latency was allocated);




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19901 Le Moli & Mori- Formica 151
-accumulated attacking time (AAT), i.e. the time spent in the attack/ s.
The frequencies of the following elements of aggressive behaviour were also recorded: startle response, upright posture, threat with open mandibles, gaster flexing, seizing, dragging, and carrying (cf. Le Moli and Mori 1986). Startle response, upright posture and threat are patterns peculiar to a ritualized fighting mainly involved in intraspecific interactions (agonistic behaviour), whereas the other items characterize the overt aggression typical of interspecific encounters (conflict behaviour) in such species of ants (Parmigiani and Le Moli 1987). Moreover, the proportion of fighting pairs was assessed, the number of attacks delivered counted, the parts of the body attacked noted, and the number of injured and/or killed ants recorded.
After the 15-minute test, the opponents were left free to move and contact each other for a further hour and then reobserved to deter- mine the proportion of injured and/or killed individuals by this time.
Treatment test
After the 30-day period of laboratory housing, workers belonging to the 12 fragments of the colonies were tested in pairs to analyse and quantify their intra- and interspecific interactions, using the same technique and apparatus of the controls. Ants of the three species were tested as follows: a) homocolonial interactions between workers of two different colony fragments fed the same laboratory diet (a or @, i.e.: - F. lugubris interactions: FlAa vs FlBa, FlAfi vs FlBfi; - F. rufa interactions: FrAa vs FrBa, FrAfi vs FrBfi; - F. cunicularia interactions: FcAa vs FcBa, FcAfi vs FcBfi; b) homocolonial interactions between workers of two different colony fragments fed a different laboratory diet (a or fi), i.e.: - F. lugubris interaction: FlAfi vs FlBa; - F. rufa interaction: FrAfi vs FrBa;
- F. cunicularia interaction: FcAfi vs FcBa; c) heterospecific interactions of F. lugubris workers paired with F. rufa or F. cunicularia workers fed the same laboratory diet (a or fi), i.e.:
- FlAa vs FrAa, FlBfi vs FrBfi, FlAfi vs FcAfi, FlBa vs FcBa; d) heterospecific interactions of F. lugubris workers paired with



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152 Psyche [VOI. 97
F. rufa or F. cunicularia workers fed a different laboratory diet (a or ft), i-e.:
- FlAft vs FrBa, FlBa vs FrAft, FlAfi vs FcBa, FlBa vs FcAfi. Control test
No attack was recorded in the encounters between homocolonial members within the three considered species (cf. interactions Fl vs Fl, Fr vs Fr, Fc vs Fc, respectively in Tables I, 11, and 111) after a week of laboratory housing. The paired ants performed prolonged antenna1 and foreleg inspections (see MI values), sometimes from an upright posture (10%) in the Fl vs Fl trial (Table I). No sign of aggression was observed in the Fr vs Fr context (see Table 11). Nevertheless, startle responses were displayed in Fc vs Fc (100%; see Table 111), and Fl vs Fl(60%; see Table I) trials. Moreover, in this last context, threat with open mandibles occurred in 4 (20%) dyads, confirming the high level of tendency to attack of this very efficient predatory species (cf. Le Moli and Parmigiani 1981; Le Moli and Mori 1986). One hour after the end of the observation period, all the ants of the three considered species were engaged in self-cleaning or exploration of the fighting box.
Concerning the heterospecific interactions, the encounters of F. lugubris were almost always characterized by overt attack (95% in Fl vs Fr; 100% in Fl vs Fc trials; cf. respectively Tables IV and V). In fact, in both interspecific contexts, there was no (Fl vs Fc) or low (Fl vs Fr) mutual inspection, but immediate, prolonged and repeated attacks, during which 2 (10%) Fl, 2 Fr, and 3 (15%) Fc individuals died. Moreoever, all the elements of aggressive behaviour were shown in Fl vs Fr trial (see Table IV); whereas in the Fl vs Fc context, the items characteristic of overt combats were mainly pres- ent (see Table V). Moreover, in this last case, the combats were particularly fierce and prolonged (see AAT values), according to the higher phylogenetic relationship existing between F. lugubris and F. rufa (both belonging to the F. rufa group of species) than with F. cunicularia (Le Moli and Mori 1986). One hour after the end of the experimental period, 2 (10%) Fl vs Fr and 1 (5%) Fl vs Fc dyads were still engaged in combats; moreover, 9 (45%) F. cunicularia were dead.




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19901 Le Moli & Mori-Formica 153
Table I. Median (with ranges) measures and proportion of elements of aggressive behaviour for Formica lugubris homocolonial workers paired in a 15-minute labora- tory test. The control test was set up after 48 h of laboratory conditions. The treat- ment test involved ants coming from colony fragment A or B, and fed the same or different diet (a, p) for a 30-day period in the laboratory. Control Test Treatment Test
Proportion
of fighting
pairs
MI
(s)
LA
6)
AAT
6)
No. of
Attacks
Startle
Response
Threat
Upright
Posture
Gaster
Flexing
Seizing
(1) (2) (3) (4)
Fl vs Fl FlAa vs FlBa FlAp vs F1Bp FlAP vs FlBa 0120 O/ 20 0120 3/20
(15%)
92 80.3 47.7 180
(20-280) (9-489) (2.3-155.4) (28.3-250.4) +900
+900
+900 900
(900-900) (900-900)
(900-900) (1 -900)
0 0
0 0
(0-0) (0-0)
(0-0) (0-19.7)
0
0
0 0
(0-0) (0-0) (0-0) (0-1)
12/20 3/20 4/20 18/20
(60%) (1 5%) (20%) (90%)
4/20 - 5/20 201 20
(20%)
(25%)
2/20 1/20 - 14/20
( 10%) (5%)
(70%)
- - -
14/20
(70%)
- - -
3/20
(15%)
MI values: (1) differs from (4) (p< 0.002); (1) does not differ from (2) and (3). LA, AAT, and No. of Attacks values: (1) does not differ from (2), (3), and (4). (Two-tailed Mann-Whitney "U" test throughout) Treatment test
When homocolonial individuals of F. lugubris, F. rufa and F. cunicularia coming from different colony fragments following the same laboratory diet (a or fi) for 30 days, were paired in the fighting box (see interactions FlAa vs FlBa, FlAfi vs FlBfi in Table I; FrAa vs FrBa, FrAfi vs FrBfi in Table 11; FcAa vs FcBa, FcAfi vs FcBfi



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154 Psyche [VOI. 97
in Table 111), they showed a behaviour not very different from that of the related control. No overt attack occurred in any case, but only quiet mutual inspection, eventually accompanied by some aggres- sive items, was displayed. In fact, all the elements of ritualized aggression (startle response, threat and upright posture) were gener- ally present in FlAa vs FlBa, FlAfi vs FlBfi, FrAa vs FrBa, and FrAfi vs FrBfi contexts (see Tables I and 11) whereas in both FcAa vs FcBfi, and FcAfi vs FcBP trials (see Table 111) only startle responses occurred. The only statistically significant difference con- cerns the MI values in the case of the encounters involving F. cuni- cularia individuals. In fact, they were significantly less prolonged in the dyads FcAa vs FcBa, and FcAP vs FcBP than in the Fc vs Fc context (cf. Table 111). One hour later, concerning the interactions FlAa vs FlBa, FlAfi vs FlBfi, FcAa vs FcBa, FcAfi vs FcBfi, and FrAa vs FrBa respectively 7 (35%), 8 (40%), 3 (15%), 3, and 2 (10%) dyads were engaged in MI activity. The remaining pairs were inves- tigating the fighting box apparatus or self-cleaning. The behavioural response was completely different from that recorded in the related control, when homocolonial individuals of F. lugubris, F. rufa and F. cunicularia coming from different colony fragments were paired after a 30-day period of following a different (a or fi) laboratory diet (see interactions FlAP vs FlBa in Table I; FrAfi vs FrBa in Table 11; FcAfi vs FcBa in Table 111). In fact, in 3 (15%) of FlAfi vs FlBa, and in 5 (25%) of FrAfi vs FrBa trials, the two ants showed mutual attack immediately after the first contact (see LA values), but the combat was frequently interrupted by exploratory or self-cleaning behaviour (see low AAT values). Morever, in these above-cited trials the mutual investigation was not very quiet, but excited and accompanied by some elements of overt aggression (see in particular gaster flexing and seizing proportions in Tables I and 11). The aggressive response was particularly evident in FcAfi vs FcBa context (Table 111), where all the encounters (100%) quickly led to overt attacks, and the combat was more pro- longed (see AAT value) because of the seizing behaviour of this species. There is a statistically significant difference between the proportions of fighting pairs (p< 0.001, Fisher exact probability test), as well as between MI, LA, AAT, and No. of attacks values in FcAfi vs FcBa compared with Fc vs Fc (control) trials reported in Table 111. In FcAfi vs FcBa interaction, contests were very vigor- ous, as shown by the high proportion of all the elements of overt



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19901 Le Moli & Mori- Formica 155
Table 11. Median (with ranges) measures and proportion of elements of aggressive behaviour for Formica rufa homocolonial workers paired in a 15-minute laboratory test. The control test was set up after 48 h of laboratory conditions. The treatment test involved ants coming from colony fragment A or B, and fed the same or different diet (a, fi) for a 30-day period in the laboratory. Control Test Treatment Test
(5) (6) (7) (8)
Fr vs Fr FrAa vs FrBa FrAfl vs FrBfi FrAfl vs FrBa Proportion
of fighting
pairs
MI
(s)
LA
(s)
AAT
6)
No. of
Attacks
Startle
Response
Threat
Upright
Posture
Gaster
Flexing
Seizing
(25%)
MI, LA, AAT, and No. of Attacks values: (5) does not differ from (6), (7), and (8). (Two-tailed Mann-Whitney "U" test throughout) aggression. Particularly evident were gaster flexing (100%) accom- panied by formic acid squirting, and seizing (100%). Sometimes, in such interaction the conflict was to the death, 2 F. cunicularia being killed during the experimental time. One hour after the end of the experimental period, concerning the interactions FlAB vs FlBa and FcAP vs FcBa, respectively 1 (5%) and 3 (15%) dyads were still fighting; on the contrary, all the F. rufa individuals (interaction FrAD vs FrBa) were engaged in the exploration of the fighting box. Moreover, 2 (5%) F. cunicularia were dead, and the legs and the



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Psyche [vo~. 97
Table 111. Median (with ranges) measures and proportion of elements of aggressive behaviour for Formica cunicularia homocolonial workers paired in a 15-minute laboratory test. The control test was set up after 48 h of laboratory conditions. The treatment test involved ants coming from colony fragment A or B, and fed the same or different diet (a, fi) for a 30-day period in the laboratory. p- - - - -
- --
Control Test Treatment Test
(9) (10) (1 1) (12)
Fc vs Fc FcAa vs FcBa FcAfl vs FcBfi FcAfl vs FcBa Proportion
of fighting
pairs
MI
(s)
LA
(s)
AAT
(s)
No. of
Attacks
Startle
Response
Threat
Gaster
Flexing
Seizing
Dragging
Carrying
No. of
killing
MI values; (9) differs from (10) (p< 0.002), from (1 1) and (12) (p< 0.02). LA, AAT, and No. of Attacks values: (9) does not differ from (10) and (1 1); (9) differs from (12) (p< 0.002).
(Two-tailed Mann-Whitney "U" test throughout)



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19901 Le Moli & Mori- Formica 157
antennae of other 2 (5%) individuals of this species were seriously injured.
As previously described (see the METHODS), heterospecific en- counters involved F. lugubris ant-workers respectively paired with F. rufa and F. cunicularia individuals.
When heterospecific members fed the same diet (a or p) for 30 days in the laboratory faced each other in the fighting box (see interactions FlAa vs FrAa, FlBfi vs FrBfi in Table IV; FlAfi vs FcAfi, FlBa vs FcBa in Table V), there was a significant decrease in the proportion of fighting pairs of FlAfi vs FcAfi (p= 0.01, Fisher exact probability test) and FlBa vs FcBa (p< 0.001, Fisher exact probability test) trials compared with Fl vs Fc control (see Table V). Also in FlAa vs FrAa and FlBfi vs FrBfi contexts there was a decrease in the proportion of fighting pairs compared with Fl vs Fr control situation (see Table IV), but it was not statistically signifi- cant. However, in general, in such heterospecific experimental trials, the ants contacted to a significantly greater degree than the related