In social animals, reproductive activity and ageing patterns are influenced by group composition. A well-documented phenomenon in monogynous (one-queen) insect societies is that queen presence affects worker fecundity and longevity. Little is known about whether and how workers respond to queen number variation in polygynous (multi-queen) species and how their queens age. We created queenless, one-queen, and two-queen colonies of the invasive, polygynous ant Tapinoma magnum to examine worker survival, ovary and oocyte development, oxidative stress resistance, and fat body gene expression. Additionally, we compared fecundity, and brain and fat body transcriptomes of young and old queens. Queenless workers experienced the highest mortality, contrasting with monogynous species where queen removal typically extends lifespan. Workers in single-queen colonies lived longer with a higher egg-laying potential than in two-queen colonies. Queen number did not directly affect oxidative stress resistance or fat body transcription, though the latter depended on an interaction with worker task. Furthermore, younger nurses demonstrated increased oocyte development, oxidative stress resistance and upregulated antioxidant genes compared to older foragers. Absent or minor shifts in fecundity and transcription with queen age, respectively, indicated physiological stability with age. Our research highlights distinct caste- and tissue-specific ageing patterns in this supercolonial species, deviating from typical monogynous ants.

Exceptional longevity of social insect queens despite their lifelong high fecundity remains poorly understood in ageing biology. To gain insights into the mechanisms that might underlie ageing in social insects, we compared gene expression patterns between young and old castes (both queens and workers) across different lineages of social insects (two termite, two bee and two ant species). After global analyses, we paid particular attention to genes of the insulin/insulin-like growth factor 1 signalling (IIS)/target of rapamycin (TOR)/juvenile hormone (JH) network, which is well known to regulate lifespan and the trade-off between reproduction and somatic maintenance in solitary insects. Our results reveal a major role of the downstream components and target genes of this network (e.g. JH signalling, vitellogenins, major royal jelly proteins and immune genes) in affecting ageing and the caste-specific physiology of social insects, but an apparently lesser role of the upstream IIS/TOR signalling components. Together with a growing appreciation of the importance of such downstream targets, this leads us to propose the TI–J–LiFe (TOR/IIS–JH–Lifespan and Fecundity) network as a conceptual framework for understanding the mechanisms of ageing and fecundity in social insects and beyond. This article is part of the theme issue ‘Ageing and sociality: why, when and how does sociality change ageing patterns?’.

Tandem-running is a recruitment behavior in ants that has been described as a form of teaching, where spatial information possessed by a leader is conveyed to following nestmates. Within Temnothorax ants, tandem-running is used within a variety of contexts, from foraging and nest relocation to – in the case of slavemaking species – slave raiding. Here, we elucidate the transcriptomic basis of scouting, tandem-leading, and tandem-following behavior across two species with divergent lifestyles: the slavemaking Temnothorax americanus and its primary, non-parasitic host T. longispinosus. Analysis of gene expression data from brains revealed that only a small number of unique differentially-expressed genes are responsible for scouting and tandem-running. Comparison of orthologous genes between T. americanus and T. longispinosus suggests that tandem-running is characterized by species-specific patterns of gene usage. However, within both species, tandem-leaders showed gene expression patterns median to those of scouts and tandem-followers, which was expected, as leaders can be recruited from either of the other two behavioral states. Most importantly, a number of differentially-expressed behavioral genes were found, with functions relating to learning and memory formation in other social and non-social insects. This includes a number of up-regulated receptor genes such as a glutamate and dopamine receptor, as well as serine/threonine protein phosphatases and kinases. Learning and memory genes were specifically up-regulated within scouts and tandem-followers, not only reinforcing previous behavioral studies into how Temnothorax navigate novel environments and share information, but also providing insight into the molecular underpinnings of teaching and learning within social insects.

The ecological success of social insects is based on division of labour, not only between queens and workers, but also among workers. Whether a worker tends the brood or forages is influenced by age, fertility and nutritional status, with brood carers being younger, more fecund and more corpulent. Here, we experimentally disentangle behavioural specialisation from age and fertility in Temnothorax longispinosus ant workers and analyse how these parameters are linked to whole-body gene expression. A total of 3644 genes were associated with behavioural specialisation which is ten times more than associated with age and 50 times more than associated with fertility. Brood carers were characterized by an upregulation of three Vitellogenin (Vg) genes, one of which, Vg-like A, was the most differentially expressed gene that was recently shown experimentally to control the switch from brood- to worker-care. The expression of Conventional Vg was unlinked to behavioural specialisation, age or fertility, which contrasts to studies on bees and some ants. Diversity in Vg/Vg-like copy number and expression bias across ants supports subfunctionalisation of vitellogenin genes and indicates that some regulatory mechanisms of division of labour diverged in different ant lineages. Simulations revealed that our experimental dissociation of co-varying factors reduced transcriptomic noise, suggesting that confounding factors could potentially explain inconsistencies across transcriptomic studies of behavioural specialisation in ants. Thus, our study reveals that worker gene expression is mainly linked to the worker’s function for the colony and provides novel insights into the evolution of sociality in ants.

Division of labor and task specialization explain the success of human and insect societies. Social insect colonies are characterized by division of labor with workers specializing on brood care early and foraging later in life. Theory posits that this task switching requires shifts in responsiveness to task-related cues, yet experimental evidence is weak. Here we show that a Vitellogenin (Vg) ortholog identified in a RNAseq study on the ant Temnothorax longispinosus is involved in this process: Using phylogenetic analyses of Vg and Vg-like genes, we firstly show that this candidate gene does not cluster with the intensively studied honey bee Vg, but falls into a separate Vg-like A cluster. Secondly, an experimental knockdown of Vg-like A in the fat body caused a reduction in brood care and an increase in nestmate care in young ant workers. Nestmate care is normally exhibited by older workers. We demonstrate experimentally that this task switch is at least partly based on Vg-like-A-associated shifts in responsiveness from brood to worker cues. We thus reveal a novel mechanism leading to early behavioral maturation via changes in social cue responsiveness mediated by Vg-like A and associated pathways, which proximately play a role in regulating division of labor.

Reduction in heterozygosity can lead to inbreeding depression. This loss of genetic variability especially affects diverse loci, such as immune genes or those encoding recognition cues. In social insects, nestmates are recognized by their odor, i.e. their cuticular hydrocarbon profile. Genes underlying hydrocarbon production are thought to be under balancing selection. If so, inbreeding should result in a loss of chemical diversity. We show here that cuticular hydrocarbon diversity decreases with inbreeding. Studying an ant with a facultative inbreeding lifestyle we found inbred workers to exhibit both a lower number of hydrocarbons and less diverse, that is, less evenly-proportioned profiles. The association with inbreeding was strong for methyl-branched alkanes, which play a major role in nestmate recognition, and for n-alkanes, whereas unsaturated compounds were unaffected. Shifts in allocation strategies with inbreeding in our focal species indicate that these ants can detect their inbreeding level and use this information to adjust their reproductive strategy. Our study is the first to demonstrate that odor profiles can encode information on inbreeding, with broad implications not only for social insects, but for sexual selection and mate choice in general. Odor profiles may constitute an honest signal of inbreeding, a fitness-relevant trait in many species.

Division of labour is of fundamental importance for the success of societies, yet little is known about how individual specialization affects the fitness of the group as a whole. While specialized workers may be more efficient in the tasks they perform than generalists, they may also lack the flexibility to respond to rapid shifts in task needs. Such rigidity could impose fitness costs when societies face dynamic and unpredictable events, such as an attack by socially parasitic slavemakers. Here, we experimentally assess the colony-level fitness consequences of behavioural specialization in Temnothorax longispinosus ants that are attacked by the slavemaker ant T. americanus. We manipulated the social organization of 102 T. longispinosus colonies, based on the behavioural responses of all 3842 workers. We find that strict specialization is disadvantageous for a colony's annual reproduction and growth during slave raids. These fitness costs may favour generalist strategies in dynamic environments, as we also demonstrate that societies exposed to slavemakers in the field show a lower degree of specialization than those originating from slavemaker-free populations. Our findings provide an explanation for the ubiquity of generalists and highlight their importance for the flexibility and functional robustness of entire societies.

Many parasites manipulate their hosts’ phenotype. In particular, parasites with complex life cycles take control of their intermediate hosts’ behaviour and life history to increase transmission to their definitive host. The proximate mechanisms underlying these parasite-induced alterations are poorly understood. The cestode Anomotaenia brevis affects the behaviour, life history and morphology of parasitized Temnothorax nylanderi ants and indirectly of their unparasitized nestmates. To gain insights on how parasites alter host phenotypes, we contrast brain gene expression patterns of T. nylanderi workers parasitized with the cestode, their unparasitized nestmates and unparasitized workers from unparasitized colonies. Over 400 differentially expressed genes between the three groups were identified, with most uniquely expressed genes detected in parasitized workers. Among these are genes that can be linked to the increased lifespan of parasitized workers. Furthermore, many muscle (functionality) genes are downregulated in these workers, potentially causing the observed muscular deformations and their inactive behaviour. Alterations in lifespan and activity could be adaptive for the parasite by increasing the likelihood that infected workers residing in acorns are eaten by their definitive host, a woodpecker. Our transcriptome analysis reveals numerous gene expression changes in parasitized workers and their uninfected nestmates and indicates possible routes of parasite manipulation. Although causality still needs to be established, parasite-induced alterations in lifespan and host behaviour appear to be partly explained by morphological muscle atrophy instead of central nervous system interference, which is often the core of behavioural regulation. Results of this study will shed light upon the molecular basis of antagonistic species interactions.

Parasites can induce alterations in host phenotypes in order to enhance their own survival and transmission. Parasites of social insects might not only benefit from altering their individual hosts, but also from inducing changes in uninfected group members. Temnothorax nylanderi ant workers infected with the tapeworm Anomotaenia brevis are known to be chemically distinct from nestmates and do not contribute to colony fitness, but are tolerated in their colonies and well cared-for. Here, we investigated how infected workers affect colony aggression by manipulating the presence of tapeworm-infected workers and analysing whether their absence or presence resulted in behavioural alterations in their nestmates. We report a parasite-induced shift in colony aggression, shown by lower aggression of uninfected nestmates from parasitized colonies towards conspecifics, potentially explaining the tolerance towards infected ants. We also demonstrate that tapeworm-infected workers showed a reduced flight response and higher survival, while their presence caused a decrease in survival of uninfected nestmates. This anomalous behaviour within infected ants, coupled with their increased survival, could facilitate the parasites’ transmission to its definitive hosts, woodpeckers. We conclude that parasites exploiting individuals that are part of a society not only induce phenotypic changes within their individual hosts, but in uninfected group members as well.

Host defences become increasingly costly as parasites breach successive lines of defence. Because selection favours hosts that successfully resist parasitism at the lowest possible cost, escalating coevolutionary arms races are likely to drive host defence portfolios towards ever more expensive strategies. We investigated the interplay between host defence portfolios and social parasite pressure by comparing 17 populations of two Temnothorax ant species. When successful, collective aggression not only prevents parasitation but also spares host colonies the cost of searching for and moving to a new nest site. However, once parasites breach the host's nest defence, host colonies should resort to flight as the more beneficial resistance strategy. We show that under low parasite pressure, host colonies more likely responded to an intruding Protomognathus americanus slavemaker with collective aggression, which prevented the slavemaker from escaping and potentially recruiting nest-mates. However, as parasite pressure increased, ant colonies of both host species became more likely to flee rather than to fight. We conclude that host defence portfolios shift consistently with social parasite pressure, which is in accordance with the degeneration of frontline defences and the evolution of subsequent anti-parasite strategies often invoked in hosts of brood parasites.