ANIMAL BEHAVIOUR, 2004, 68, 1209–1219 doi:10.1016/j.anbehav.2003.11.026
Fertility signalling and reproductive skew in queenless ants
VIRGINIE CUVILLIER-HOT*†, ALAIN LENOIR†, ROBIN CREWE‡, CHRISTIAN MALOSSE§ & CHRISTIAN PEETERS* *Laboratoire d’E´cologie, Universite´ Pierre et Marie Curie, Paris yInstitut de Recherche sur la Biologie de l’Insecte, Faculte´ des Sciences, Tours zFaculty of Natural & Agricultural Sciences, University of Pretoria xUnite´ de Phytopharmacie, Institut National de Recherche Agronomique de Versailles
(Received 27 May 2003; initial acceptance 17 September 2003; final acceptance 24 November 2003; published online 29 September 2004; MS. number: A9622)
Social insects often show an extreme reproductive skew. In queenless ants, colonies consist of morphologically identical workers that can all potentially reproduce sexually. Similarly to that in social vertebrates, aggression in these ants functions to select the reproductive(s). We investigated the mechanisms underlying reproductive skew in the monogynous queenless ant Streblognathus peetersi. Behavioural observations of disturbed hierarchies were integrated with physiological measures of fertility (vitellogenin titre in the haemolymph) and chemical analysis of cuticular hydrocarbons, which are putative fertility pheromones. This multifaceted approach revealed that the colony reproductive is determined as a result of aggression between high-ranking workers, but once an alpha is established, chemical signalling is enough to maintain reproductive skew. As already reported in several species of ants and also in a social wasp, egg layers have distinct profiles of cuticular hydrocarbons compared with infertile workers. Importantly, ‘high rankers’ who are unable to lay eggs also have a specific cuticular profile; this is consistent with their intermediate state of fertility indicated by vitellogenin levels. Rather than just ovarian activity, the cuticular hydrocarbon profile thus reveals the individual hormonal state that underlies reproductive activity. We compare the fertility signal in queenless ants with the ‘badge of status’ reported in various birds. We discuss the evolutionary stability of this communication system and give special emphasis to ecological constraints and the high degree of intracolonial relatedness typical of social insects. Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Animal societies vary in the extent to which reproduction capacity by means of pheromones. Unlike previous is shared among group members. In various social birds explanations based on the inhibition of ovarian activity, and mammals, reproduction is almost equally distributed, honest signals would benefit all the parties (Keller & whereas reproductive skew is extreme in most insect Nonacs 1993), in line with biological signalling theory societies, even in species lacking morphologically special- (Grafen 1990). This raises the question of whether chem- ized queens and workers. Many studies have focused on ical communication alone is enough to regulate skew, and the role of behavioural interactions in the regulation of how such regulation can be evolutionarily stable. reproductive skew in vertebrates (e.g. Hrdy & Hrdy 1976; Some ponerine ants have lost the queen caste, and all the Creel et al. 1997; Clutton-Brock et al. 2001; Koenig & morphologically similar workers can potentially mate and Haydock 2002). Breeders are selected on the basis of either reproduce (Peeters 1993). Agonistic interactions between aggressive encounters or conventions (linked to body size, nestmates lead to a dominance hierarchy that regulates age or ranks of relatives: e.g. Frank 1986; Creel et al. 1992). reproduction (e.g. Monnin & Peeters 1999): according In either case, the asymmetry that underlies the domi- to species, only one or a few high-ranking workers mate nance relationships is thought to be a good indicator of (they are then called ‘gamergates’) and produce diploid reproductive potential. Keller & Nonacs (1993) proposed (female) offspring; the other workers remain infertile and another alternative to aggression in social insects: repro- are responsible for brood care and nest maintenance. This ductives can signal their presence and/or reproductive kind of social organization is comparable to that found in some primitively eusocial wasps (e.g. Polistes dominulus: Pardi 1948; Ro¨seler et al. 1986; Polistes fuscatus: West- Correspondence and present address: V. Cuvillier-Hot, Universite´ des Eberhard 1967; Parischnogaster nigricans serrei: Turillazzi Sciences et Techniques de Lille, baˆtimeent SN3, 59655 Villeneure 1991; Ropalidia marginata: Premnath et al. 1996), as well as d’Ascq Cedex, France (email: [email protected]). some mammals such as naked mole-rats, Heterocephalus 1209 0003–3472/03/$30.00/0 Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. 1210 ANIMAL BEHAVIOUR, 68, 5
glaber (Jarvis et al. 1994). However, in sharp contrast to a reliable reflection of hormonal parameters associated these species, in queenless ants, the gamergates cannot with dominance and oogenesis. breed independently. Accordingly, in a monogynous spe- We studied fertility signalling in Streblognathus peetersi, cies, the sole chance for a worker to gain direct fitness is a species whose regulation of reproduction makes it a good either to reach the top of the hierarchy and replace model for all queenless ants. Only one gamergate occurs a senescent gamergate, or wait for opportunities created in small colonies of about 100 workers. The different when the colony divides by fission (Peeters 1991). Aggres- social categories (top ranker (alpha), high ranker, low sive interactions within colonies are then particularly ranker) are clearly recognized by behavioural observa- intense. However, as soon as a new alpha is established, tions. The alpha is the only individual to mate and to the rate of aggression decreases and she begins to lay eggs. lay eggs (even when she is still virgin). In Dinoponera This has been found in a number of species: Pachycondyla quadriceps, the alpha mates with one foreign male sublaevis (Ito & Higashi 1991); Amblyopone sp. (Ito 1993); (Monnin & Peeters 1998), and the closely related Streblog- Dinoponera quadriceps (Monnin & Peeters 1999); Diacamma nathus (Haskins & Zahl 1971) is also very likely to be ceylonense (Cuvillier-Hot et al. 2002). The presence of monandrous. Unlike D. quadriceps, high-ranking S. peetersi a fertile alpha thus seems sufficient to ensure a period of workers are unable to lay eggs. We conducted observations stability during which conflicts are resolved by means and social manipulations to document the behavioural other than direct physical confrontations. regulation of reproduction in this species. We measured In vertebrates, honest signals are mainly used in the cuticular hydrocarbons and levels of vitellogenin (used as context of competition for food or territories; they are a fertility index) in workers of known social status to seldom involved in the resolution of reproductive con- determine whether particular cuticular profiles reflect just flicts (Rasmussen & Schulte 1998). In contrast, specific egg-laying ability or whether they give additional in- traits of insect societies may have allowed the resolution formation about hormonal state and fertility potential. of their conflicts over reproductive sharing to be based on We discuss the importance of balancing the roles of honest olfactory signals. Workers of several species of ants aggression and fertility signalling in societies with totipo- and social wasps can detect the presence of egg layers in tent nestmates. the nest, probably through contact pheromones (e.g. for ants: Gnamptogenys menadensis: Gobin et al. 1999; Diacamma sp.: Kikuta & Tsuji 1999; Tsuji et al. 1999; METHODS Harpegnathos saltator: Liebig et al. 1999; Formica fusca: Hannonen et al. 2002; for wasps: Polistes dominulus: Housing Conditions and Bonavita-Cougourdan et al. 1991; Sledge et al. 2001). A Behavioural Observations pheromone that reveals fertility could thus be an impor- tant component of the regulation of reproduction in such We collected 22 colonies of Streblognathus peetersi G Z societies. Moreover, within a colony of queenless ants, (Robertson 2002) near Magoebaskloof (mean SD G recognition goes far beyond the detection of egg layers. In 94 48 workers, range 32–204), Limpopo Province, particular, high-ranking ants unambiguously recognize South Africa, and three colonies from Vernon Crookes each other prior to interacting aggressively. In Dinoponera (26, 53 and 123 workers), Kwazulu-Natal Province. In the quadriceps, dominance interactions are highly directed laboratory, colonies were reared in plaster nests, at 25 C, (Monnin & Peeters 1999): a worker focuses her aggression under 12:12 h light:dark cycles and were fed daily with towards the few ants just below her in the hierarchy (also mealworm, Tenebrio molitor, pupae and crickets, Gryllus in Polistes wasps, Pardi 1948). How do workers recognize bimaculatus. Twelve of these colonies were used for infertile nestmates having a similar rank in the hierarchy? behavioural observations, whereas the others provided One hypothesis is that ants advertise not only their individuals for physiological and chemical investigations. fertility as defined by egg production, but also physiolog- We individually marked workers with small coloured ical traits that can enable them to reproduce in the plastic numbers (Opalithpla¨ttchen used for Apis) glued immediate future. A high-ranking ant would then be onto the thorax and noted the age of workers emerging in recognized as being physiologically close to an egg layer, the laboratory. Colonies were regularly checked for eggs. even though she may currently be unable to lay eggs. Large colonies were split into groups of about 50 ants, Cuticular hydrocarbons are involved in nestmate recog- taking care to distribute all age classes evenly, to increase nition in social insects (review by Lenoir et al. 1999), but the number of alphas studied (a total of 16 experimental a growing number of studies show that cuticular hydro- groups were thus used). carbon profiles also correlate with ovarian activity in ants (Dinoponera quadriceps: Monnin et al. 1998; Peeters et al. Behaviour of the alpha worker 1999; Harpegnathos saltator: Liebig et al. 2000; Diacamma Ten small groups (mean Z 25 workers, range 11–38) ceylonense: Cuvillier-Hot et al. 2001; Formica fusca: showing a stable reproductive structure (i.e. the alpha had Hannonen et al. 2002; Pachycondyla cf. inversa: Heinze been dominant for several weeks and had started to lay et al. 2002; Myrmecia gulosa: Dietemann et al. 2003) and in eggs) were each videotaped for 10 h continuously (1400– other primitively eusocial insect societies (Bombus hypno- 2400 hours; LD 5:5 h). In three cases, the alpha worker rum: Ayasse et al. 1995; Polistes dominulus: Sledge et al. was mated (Zgamergate), as revealed by dissection at the 2001). A distinctive blend of these long-chained molecules end of the experiment. We focused on the behaviour of could function as the fertility signal, provided that it is the alpha: the number of aggressive acts she performed CUVILLIER-HOTETAL.:FERTILITY SIGNALLING IN ANTS 1211 and received, and the time she spent self-grooming. The behaviour of a control subordinate (a randomly chosen 0.8 low-ranking worker active inside the nest) was also 5.4 4.7 G G G measured for comparison, as was the total number of aggressive acts in the nest (Table 1). Results were com- 3.7 0.6 pared with exact Wilcoxon tests. 3.4 3.6 3.6 2.7 G G G
Replacement of the alpha worker 3.5 4.1 We irregularly observed 10 other groups (mean Z 53 8.9 7.2 7.9 6.2 G workers, range 23–101) showing a stable reproductive G G Mean number structure over several days for a total time of 17.5 h. We per hour in the colony counted all agonistic interactions and identified all the of agonistic interactions 8.8 3.8 ants involved (Table 2). In five groups, we then removed 13.8 9.3 12.2 7.7 G G G the alpha and conducted a series of 15-min observations that were each observed continuously for 10 h 13.3 for the next 50 days (for each group: 6–10 observation 17.7 16.4
periods in the 50 days before the removal; 3–4 observa- a
tion periods in the 5 days after the removal; 4–5 observa- a a 0 0 0.12 G G tion periods between the 6th and the 50th day after the G removal). We counted all aggressive acts performed by the 0 0 3.6 workers during the three periods (day 50–0; day 0–5; day 6–50; Fig. 1a) and by the new alpha during the last a a a 0 0 0.07 two periods (Fig. 1b). For each group of ants and each Streblognathus peetersi G G G period, we calculated the mean number of aggressive acts 0 0 SD. Statistical tests: exact Wilcoxon two-sample tests; significance level: performed per individual and used this value for statistical 6.9 G a comparisons. b a 0.0 0 0.0 G G G 0 0.1 Titre of the Circulating Vitellogenin 5.1 a b a 0.0 0 To show a link between cuticular hydrocarbons and 0.0 G G G social status, we needed detailed information about an 0 0.4 5.6 ant’s capacity for egg production. Following Martinez & a a b 0 0 Wheeler (1991) and Vargo & Laurel (1994), we estimated 0 G G G fertility using a physiological marker: the circulating level 0 of vitellogenin, a yolk precursor synthesised in the fat 100 100 body, secreted in the haemolymph and taken into the a a b 0.03 0.30 oocytes by receptor-mediated endocytosis to form vitellins 0 G G G
(the major storage proteins in the eggs). The level of 0
vitellogenin in the haemolymph provides a continuous 98.3 82.3 b measure of readiness for egg production, irrespective of the gaster curling. Values are given as mean values a a
current degree of ovary development. Moreover, it gives a Z 0.3 0.3 more upstream and thus updated measure of the physio- 0.02 G G G logical state of the individual, informing about recent 0.9 changes in social status that will be translated into changes 34.3 59.7 % Aggressions performed % Aggressions received a a in ovarian activity only several days later. This technique b
is thus far more sensitive than the traditionally used dis- gaster rise; GC 0.06 0.29 0.03 Z AB B GR GC AB B GR GC AB B GR GC G section and visual description of ovarian development, G G which involves the subjective integration of various para- meters (i.e. number and size of oocytes, presence of yolk). Techniques of haemolymph storage and antibody pro- 10) 3.3 biting; GR
duction were mostly derived from Wheeler et al. (1999). Z Z N Ants of known social status were sacrificed to measure 7) 44.5 Z
their levels of circulating vitellogenin. Haemolymph was N 3) 20.4
collected by decapitation of cooled ants and centrifuga- Z tion of the body, and immediately mixed with 15 mlof N a cocktail of antiproteases (Sigma-Aldrich, Lyon, France)
after measuring the volume. Samples were then stored at Aggressive behaviours of gamergates or unmated alphas compared to control subordinates in 10 colonies of