Brain and antennal transcriptomes of host ants reveal potential links between behaviour and the functioning of socially parasitic colonies.

Insect social parasites are characterized by exploiting the hosts' social behaviour. Why exactly hosts direct their caring behaviour towards these parasites and their offspring remains largely unstudied. One hypothesis is that hosts do not perceive their social environment as altered and accept the parasitic colony as their own. We used the ant Leptothorax acervorum, host of the dulotic, obligate social parasite Harpagoxenus sublaevis, to shed light on molecular mechanisms underlying behavioural exploitation by contrasting tissue-specific transcriptomes in young host workers. Host pupae were experimentally (re-)introduced into fragments of their original, another conspecific, heterospecific or parasitic colony. Brain and antennal mRNA was extracted and sequenced from adult ants after they had lived in the experimental colony for at least 50 days after eclosion. The resulting transcriptomes of L. acervorum revealed that ants were indeed affected by their social environment. Host brain transcriptomes were altered by the presence of social parasites, suggesting that the parasitic environment influences brain activity, which may be linked to behavioural changes. Transcriptional activity in the antennae changed most with the presence of unrelated individuals, regardless of whether they were conspecifics or parasites. This suggests early priming of odour perception, which was further supported by sensory perception of odour as an enriched function of differentially expressed genes. Furthermore, gene expression in the antennae, but not in the brain corresponded to ant worker behaviour before sampling. Our study demonstrated that the exploitation of social behaviours by brood parasites correlates with transcriptomic alterations in the central and peripheral nervous systems.

The influence of parasite load on transcriptional activity and morphology of a cestode and its ant intermediate host.

Parasites with complex life cycles are known to induce phenotypic changes in their intermediate hosts to increase transmission to the final host. The magnitude of these changes could increase with the number of parasites, which would be beneficial to co-infecting parasites. Yet, adverse effects of high parasite load (i.e. many parasites in a single host) might stress both hosts and parasites (e.g. through an increased immune response). We investigated the consequences of parasite load on the transcriptional activity and morphology of the cestode Anomotaenia brevis and its intermediate host, the ant Temnothorax nylanderi. We demonstrated that many differentially expressed host genes shifted with parasite load, and their functions indicate a stronger immune response and fight against oxidative stress in heavily infected hosts. The expression of other host genes responded to infection in an all-or-nothing manner, as did the morphology of the host workers. However, the cestodes became smaller when they competed with other parasites for resources from a single host. Their expression profile further indicated shifts in host immune avoidance, starvation resistance and vesicle-mediated transport. In summary, our study reveals clear consequences of parasite load and highlights specific processes and traits affected by this.

Long live the host! Proteomic analysis reveals possible strategies for parasitic manipulation of its social host.

Parasites with complex life cycles often manipulate the phenotype of their intermediate hosts to increase the probability of transmission to their definitive hosts. Infection with Anomotaenia brevis, a cestode that uses Temnothorax nylanderi ants as intermediate hosts, leads to a multiple-fold extension of host lifespan and to changes in behaviour, morphology and colouration. The mechanisms behind these changes are unknown, as is whether the increased longevity is achieved through parasite manipulation. Here, we demonstrate that the parasite releases proteins into its host with functions that might explain the observed changes. These parasitic proteins make up a substantial portion of the proteome of the hosts' haemolymph, and thioredoxin peroxidase and superoxide dismutase, two antioxidants, exhibited the highest abundances among them. The largest part of the secreted proteins could not be annotated, indicating they are either novel or severely altered during recent coevolution to function in host manipulation. We also detected shifts in the hosts' proteome with infection, in particular an overabundance of vitellogenin-like A in infected ants, a protein that regulates division of labour in Temnothorax ants, which could explain the observed behavioural changes. Our results thus suggest two different strategies that might be employed by this parasite to manipulate its host: secreting proteins with immediate influence on the host's phenotype and altering the host's translational activity. Our findings highlight the intricate molecular interplay required to influence the phenotype of a host and point to potential signalling pathways and genes involved in parasite-host communication.

Social isolation causes downregulation of immune and stress response genes and behavioural changes in a social insect.

Humans and other social mammals experience isolation from their group as stressful, triggering behavioural and physiological anomalies that reduce fitness. While social isolation has been intensely studied in social mammals, it is less clear how social insects, which evolved sociality independently, respond to isolation. Here we examined whether the typical mammalian responses to social isolation, e.g., an impaired ability to interact socially and immune suppression are also found in social insects. We studied the consequences of social isolation on behaviour and brain gene expression in the ant Temnothorax nylanderi. Following isolation, workers interacted moderately less with adult nestmates, increased the duration of brood contact, and reduced the time spent self-grooming, an important sanitary behaviour. Our brain transcriptome analysis revealed that only a few behaviour-related genes had altered their expression with isolation time. Rather, many genes linked to immune system functioning and stress response had been downregulated. This probably sensitizes isolated individuals to various stressors, in particular because isolated workers exhibit reduced sanitary behaviour. We provide evidence of the diverse consequences of social isolation in social insects, some of which resemble those found in social mammals, suggesting a general link between social well-being, stress tolerance, and immune competence in social animals.

Social organization and the evolution of life-history traits in two queen morphs of the ant Temnothorax rugatulus.

During the evolution of social insects, not only did life-history traits diverge, with queens becoming highly fecund and long lived compared with their sterile workers, but also individual traits lost their importance compared with colony-level traits. In solitary animals, fecundity is largely influenced by female size, whereas in eusocial insects, colony size and queen number can affect the egg-laying rate. Here, we focused on the ant Temnothorax rugatulus, which exhibits two queen morphs varying in size and reproductive strategy, correlating with their colony's social organization. We experimentally tested the influence of social structure, colony and body size on queen fecundity and investigated links between body size, metabolic rate and survival under paraquat-induced oxidative stress. To gain insight into the molecular physiology underlying the alternative reproductive strategies, we analysed fat body transcriptomes. Per-queen egg production was lower in polygynous colonies when fecundity was limited by worker care. Colony size was a determinant of fecundity rather than body size or queen number, highlighting the super-organismal properties of these societies. The smaller microgynes were more frequently fed by workers and exhibited an increase in metabolic activity, yet they were similarly resistant to oxidative stress. Small queens differentially expressed metabolic genes in the fat body, indicating that shifts in molecular physiology and resource availability allow microgyne queens to compensate for their small size with a more active metabolism without paying increased mortality costs. We provide novel insights into how life-history traits and their associations were modified during social evolution and adapted to queen reproductive strategies.

Tandem-running and scouting behaviour are characterized by up-regulation of learning and memory formation genes within the ant brain.

Tandem-running is a recruitment behaviour 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 behaviours across two species with divergent lifestyles: the slavemaking Temnothorax americanus and its primary, nonparasitic 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 behavioural states. Most importantly, a number of differentially expressed behavioural genes were found, with functions relating to learning and memory formation in other social and nonsocial 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 behavioural 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.

Transcriptomic Signature of Spatial Navigation in Brains of Desert Ants.

Navigation is crucial for central-place foragers to locate food and return to the nest. Cataglyphis ants are renowned for their advanced navigation abilities, relying on landmark cues and path integration. This study aims to uncover the transcriptomic basis of exceptional spatial learning in the central nervous system of Cataglyphis niger. Ants navigated a maze with a food reward, and we examined expression changes linked to correct decisions in subsequent runs. Correct decisions correlated with expression changes in the optic lobes, but not the central brain, showing a downregulation of genes associated with sucrose response and Creb3l1. The latter gene is homologous to Drosophila crebA, which is essential for long-term memory formation. To understand how ants use distance information during path integration, we analyzed expression shifts associated with the last distance traveled. We uncovered a transcriptomic footprint in the central brain, but not in the optic lobes, with genes enriched for energy consumption and neurological functions, including neuronal projection development, synaptic target inhibition, and recognition processes. This suggests that transcriptional activity in the central brain is necessary for estimating distance traveled, which is crucial for path integration. Our study supports the distinct roles of different brain parts for navigation in Cataglyphis ants.

Molecular (co)evolution of hymenopteran social parasites and their hosts.

Social parasitism describes a fascinating way of life in which species exploit the altruistic behaviour of closely related, social species. Social parasites have repeatedly evolved in the social Hymenoptera, including ants, bees, and wasps. The common ancestry and shared (social) environment with their hosts facilitates the study of molecular adaptations to the parasitic lifestyle. Moreover, when social parasites are widespread and virulent, they exert strong selection pressure on their hosts, leading to the evolution of defense mechanisms and triggering a coevolutionary arms race. Recent advances in sequencing technology now make it possible to study the molecular basis of this coevolutionary process. In addition to describing the latest developments, we highlight open research questions that could be tackled with genomic, transcriptomic, or epigenetic data.

Annotation and Analysis of 3902 Odorant Receptor Protein Sequences from 21 Insect Species Provide Insights into the Evolution of Odorant Receptor Gene Families in Solitary and Social Insects.

The gene family of insect olfactory receptors (ORs) has expanded greatly over the course of evolution. ORs enable insects to detect volatile chemicals and therefore play an important role in social interactions, enemy and prey recognition, and foraging. The sequences of several thousand ORs are known, but their specific function or their ligands have only been identified for very few of them. To advance the functional characterization of ORs, we have assembled, curated, and aligned the sequences of 3902 ORs from 21 insect species, which we provide as an annotated online resource. Using functionally characterized proteins from the fly Drosophila melanogaster, the mosquito Anopheles gambiae and the ant Harpegnathos saltator, we identified amino acid positions that best predict response to ligands. We examined the conservation of these predicted relevant residues in all OR subfamilies; the results showed that the subfamilies that expanded strongly in social insects had a high degree of conservation in their binding sites. This suggests that the ORs of social insect families are typically finely tuned and exhibit sensitivity to very similar odorants. Our novel approach provides a powerful tool to exploit functional information from a limited number of genes to study the functional evolution of large gene families.

Molecular regulation of lifespan extension in fertile ant workers.

The evolution of sociality in insects caused a divergence in lifespan between reproductive and non-reproductive castes. Ant queens can live for decades, while most workers survive only weeks to a few years. In most organisms, longevity is traded-off with reproduction, but in social insects, these two life-history traits are positively linked. Once fertility is induced in workers, e.g. by queen removal, worker lifespan increases. The molecular regulation of this positive link between fecundity and longevity and generally the molecular underpinnings of caste-specific senescence are not well understood. Here, we investigate the transcriptomic regulation of lifespan and reproduction in fat bodies of three worker groups in the ant Temnothorax rugatulus. In a long-term experiment, workers that became fertile in the absence of the queen showed increased survival and upregulation of genes involved in longevity and fecundity pathways. Interestingly, workers that re-joined their queen after months exhibited intermediate ovary development, but retained a high expression of longevity and fecundity genes. Strikingly, the queen's presence causes a general downregulation of genes in worker fat bodies. Our findings point to long-term consequences of fertility induction in workers, even after re-joining their queen. Moreover, we reveal longevity genes and pathways modulated during insect social evolution. This article is part of the theme issue 'Ageing and sociality: why, when and how does sociality change ageing patterns?'

Parasite Presence Induces Gene Expression Changes in an Ant Host Related to Immunity and Longevity.

Most species are either parasites or exploited by parasites, making parasite-host interactions a driver of evolution. Parasites with complex life cycles often evolve strategies to facilitate transmission to the definitive host by manipulating their intermediate host. Such manipulations could explain phenotypic changes in the ant Temnothorax nylanderi, the intermediate host of the cestode Anomotaenia brevis. In addition to behavioral and morphological alterations, infected workers exhibit prolonged lifespans, comparable to that of queens, which live up to two decades. We used transcriptomic data from cestodes and ants of different castes and infection status to investigate the molecular underpinnings of phenotypic alterations in infected workers and explored whether the extended lifespan of queens and infected workers has a common molecular basis. Infected workers and queens commonly upregulated only six genes, one of them with a known anti-aging function. Both groups overexpressed immune genes, although not the same ones. Our findings suggest that the lifespan extension of infected workers is not achieved via the expression of queen-specific genes. The analysis of the cestodes' transcriptome revealed dominant expression of genes of the mitochondrial respiratory transport chain, which indicates an active metabolism and shedding light on the physiology of the parasite in its cysticercoid stage.

Comparative transcriptomic analysis of the mechanisms underpinning ageing and fecundity in social insects.

The 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?'

Comparative analyses of caste, sex, and developmental stage-specific transcriptomes in two Temnothorax ants.

Social insects dominate arthropod communities worldwide due to cooperation and division of labor in their societies. This, however, makes them vulnerable to exploitation by social parasites, such as slave-making ants. Slave-making ant workers pillage brood from neighboring nests of related host ant species. After emergence, host workers take over all nonreproductive colony tasks, whereas slavemakers have lost the ability to care for themselves and their offspring. Here, we compared transcriptomes of different developmental stages (larvae, pupae, and adults), castes (queens and workers), and sexes of two related ant species, the slavemaker Temnothorax americanus and its host Temnothorax longispinosus. Our aim was to investigate commonalities and differences in group-specific transcriptomes, whereupon across-species differences possibly can be explained by their divergent lifestyles. Larvae and pupae showed the highest similarity between the two species and upregulated genes with enriched functions of translation and chitin metabolism, respectively. Workers commonly upregulated oxidation-reduction genes, possibly indicative of their active lifestyle. Host workers, but not workers of the slavemaker, upregulated a "social behavior" gene. In slavemaker queens and workers, genes associated with the regulation of transposable elements were upregulated. Queens of both species showed transcriptomic signals of anti-aging mechanisms, with hosts upregulating various DNA repair pathways and slavemaker queens investing in trehalose metabolism. The transcriptomes of males showed enriched functions for quite general terms realized in different genes and pathways in each species. In summary, the strong interspecific commonalities in larvae, pupae, and workers were reflected in the same enriched Gene Ontology (GO) terms. Less commonalities occurred in the transcriptomes of queens and males, which apparently utilize different pathways to achieve a long life and sperm production, respectively. We found that all analyzed groups in this study show characteristic GO terms, with similar patterns in both species.

Ant behaviour and brain gene expression of defending hosts depend on the ecological success of the intruding social parasite.

The geographical mosaic theory of coevolution predicts that species interactions vary between locales. Depending on who leads the coevolutionary arms race, the effectivity of parasite attack or host defence strategies will explain parasite prevalence. Here, we compare behaviour and brain transcriptomes of Temnothorax longispinosus ant workers when defending their nest against an invading social parasite, the slavemaking ant Temnothorax americanus. A full-factorial design allowed us to test whether behaviour and gene expression are linked to parasite pressure on host populations or to the ecological success of parasite populations. Albeit host defences had been shown before to covary with local parasite pressure, we found parasite success to be much more important. Our chemical and behavioural analyses revealed that parasites from high prevalence sites carry lower concentrations of recognition cues and are less often attacked by hosts. This link was further supported by gene expression analysis. Our study reveals that host-parasite interactions are strongly influenced by social parasite strategies, so that variation in parasite prevalence is determined by parasite traits rather than the efficacy of host defence. Gene functions associated with parasite success indicated strong neuronal responses in hosts, including long-term changes in gene regulation, indicating an enduring impact of parasites on host behaviour. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.