Frequent horizontal chromosome transfer between asexual fungal insect pathogens.

Entire chromosomes are typically only transmitted vertically from one generation to the next. The horizontal transfer of such chromosomes has long been considered improbable, yet gained recent support in several pathogenic fungi where it may affect the fitness or host specificity. To date, it is unknown how these transfers occur, how common they are, and whether they can occur between different species. In this study, we show multiple independent instances of horizontal transfers of the same accessory chromosome between two distinct strains of the asexual entomopathogenic fungus Metarhizium robertsii during experimental co-infection of its insect host, the Argentine ant. Notably, only the one chromosome-but no other-was transferred from the donor to the recipient strain. The recipient strain, now harboring the accessory chromosome, exhibited a competitive advantage under certain host conditions. By phylogenetic analysis, we further demonstrate that the same accessory chromosome was horizontally transferred in a natural environment between M. robertsii and another congeneric insect pathogen, Metarhizium guizhouense. Hence, horizontal chromosome transfer is not limited to the observed frequent events within species during experimental infections but also occurs naturally across species. The accessory chromosome that was transferred contains genes that may be involved in its preferential horizontal transfer or support its establishment. These genes encode putative histones and histone-modifying enzymes, as well as putative virulence factors. Our study reveals that both intra- and interspecies horizontal transfer of entire chromosomes is more frequent than previously assumed, likely representing a not uncommon mechanism for gene exchange.

Early queen infection shapes developmental dynamics and induces long-term disease protection in incipient ant colonies.

Infections early in life can have enduring effects on an organism's development and immunity. In this study, we show that this equally applies to developing 'superorganisms'--incipient social insect colonies. When we exposed newly mated Lasius niger ant queens to a low pathogen dose, their colonies grew more slowly than controls before winter, but reached similar sizes afterwards. Independent of exposure, queen hibernation survival improved when the ratio of pupae to workers was small. Queens that reared fewer pupae before worker emergence exhibited lower pathogen levels, indicating that high brood rearing efforts interfere with the ability of the queen's immune system to suppress pathogen proliferation. Early-life queen pathogen exposure also improved the immunocompetence of her worker offspring, as demonstrated by challenging the workers to the same pathogen a year later. Transgenerational transfer of the queen's pathogen experience to her workforce can hence durably reduce the disease susceptibility of the whole superorganism.

Ants avoid superinfections by performing risk-adjusted sanitary care.

Being cared for when sick is a benefit of sociality that can reduce disease and improve survival of group members. However, individuals providing care risk contracting infectious diseases themselves. If they contract a low pathogen dose, they may develop low-level infections that do not cause disease but still affect host immunity by either decreasing or increasing the host's vulnerability to subsequent infections. Caring for contagious individuals can thus significantly alter the future disease susceptibility of caregivers. Using ants and their fungal pathogens as a model system, we tested if the altered disease susceptibility of experienced caregivers, in turn, affects their expression of sanitary care behavior. We found that low-level infections contracted during sanitary care had protective or neutral effects on secondary exposure to the same (homologous) pathogen but consistently caused high mortality on superinfection with a different (heterologous) pathogen. In response to this risk, the ants selectively adjusted the expression of their sanitary care. Specifically, the ants performed less grooming and more antimicrobial disinfection when caring for nestmates contaminated with heterologous pathogens compared with homologous ones. By modulating the components of sanitary care in this way the ants acquired less infectious particles of the heterologous pathogens, resulting in reduced superinfection. The performance of risk-adjusted sanitary care reveals the remarkable capacity of ants to react to changes in their disease susceptibility, according to their own infection history and to flexibly adjust collective care to individual risk.

Social immunity modulates competition between coinfecting pathogens.

Coinfections with multiple pathogens can result in complex within-host dynamics affecting virulence and transmission. While multiple infections are intensively studied in solitary hosts, it is so far unresolved how social host interactions interfere with pathogen competition, and if this depends on coinfection diversity. We studied how the collective disease defences of ants - their social immunity - influence pathogen competition in coinfections of same or different fungal pathogen species. Social immunity reduced virulence for all pathogen combinations, but interfered with spore production only in different-species coinfections. Here, it decreased overall pathogen sporulation success while increasing co-sporulation on individual cadavers and maintaining a higher pathogen diversity at the community level. Mathematical modelling revealed that host sanitary care alone can modulate competitive outcomes between pathogens, giving advantage to fast-germinating, thus less grooming-sensitive ones. Host social interactions can hence modulate infection dynamics in coinfected group members, thereby altering pathogen communities at the host level and population level.

Oxytocin-like signaling in ants influences metabolic gene expression and locomotor activity.

Ants are emerging model systems to study cellular signaling because distinct castes possess different physiologic phenotypes within the same colony. Here we studied the functionality of inotocin signaling, an insect ortholog of mammalian oxytocin (OT), which was recently discovered in ants. In Lasius ants, we determined that specialization within the colony, seasonal factors, and physiologic conditions down-regulated the expression of the OT-like signaling system. Given this natural variation, we interrogated its function using RNAi knockdowns. Next-generation RNA sequencing of OT-like precursor knock-down ants highlighted its role in the regulation of genes involved in metabolism. Knock-down ants exhibited higher walking activity and increased self-grooming in the brood chamber. We propose that OT-like signaling in ants is important for regulating metabolic processes and locomotion.-Liutkeviciute, Z., Gil-Mansilla, E., Eder, T., Casillas-Pérez, B., Di Giglio, M. G., Muratspahic, E., Grebien, F., Rattei, T., Muttenthaler, M., Cremer, S., Gruber, C. W. Oxytocin-like signaling in ants influences metabolic gene expression and locomotor activity.

Correction to: The introduction history of invasive garden ants in Europe: integrating genetic, chemical and behavioural approaches.

Reinvestigation of the raw data revealed an unfortunate error in Ugelvig et al. 2008 [1].

Social Immunity: Emergence and Evolution of Colony-Level Disease Protection.

Social insect colonies have evolved many collectively performed adaptations that reduce the impact of infectious disease and that are expected to maximize their fitness. This colony-level protection is termed social immunity, and it enhances the health and survival of the colony. In this review, we address how social immunity emerges from its mechanistic components to produce colony-level disease avoidance, resistance, and tolerance. To understand the evolutionary causes and consequences of social immunity, we highlight the need for studies that evaluate the effects of social immunity on colony fitness. We discuss the roles that host life history and ecology have on predicted eco-evolutionary dynamics, which differ among the social insect lineages. Throughout the review, we highlight current gaps in our knowledge and promising avenues for future research, which we hope will bring us closer to an integrated understanding of socio-eco-evo-immunology.

Co-founding ant queens prevent disease by performing prophylactic undertaking behaviour.

BACKGROUND: Social insects form densely crowded societies in environments with high pathogen loads, but have evolved collective defences that mitigate the impact of disease. However, colony-founding queens lack this protection and suffer high rates of mortality. The impact of pathogens may be exacerbated in species where queens found colonies together, as healthy individuals may contract pathogens from infectious co-founders. Therefore, we tested whether ant queens avoid founding colonies with pathogen-exposed conspecifics and how they might limit disease transmission from infectious individuals. RESULTS: Using Lasius niger queens and a naturally infecting fungal pathogen Metarhizium brunneum, we observed that queens were equally likely to found colonies with another pathogen-exposed or sham-treated queen. However, when one queen died, the surviving individual performed biting, burial and removal of the corpse. These undertaking behaviours were performed prophylactically, i.e. targeted equally towards non-infected and infected corpses, as well as carried out before infected corpses became infectious. Biting and burial reduced the risk of the queens contracting and dying from disease from an infectious corpse of a dead co-foundress. CONCLUSIONS: We show that co-founding ant queens express undertaking behaviours that, in mature colonies, are performed exclusively by workers. Such infection avoidance behaviours act before the queens can contract the disease and will therefore improve the overall chance of colony founding success in ant queens.

Individual and social immunisation in insects.

Immune systems are able to protect the body against secondary infection with the same parasite. In insect colonies, this protection is not restricted to the level of the individual organism, but also occurs at the societal level. Here, we review recent evidence for and insights into the mechanisms underlying individual and social immunisation in insects. We disentangle general immune-protective effects from specific immune memory (priming), and examine immunisation in the context of the lifetime of an individual and that of a colony, and of transgenerational immunisation that benefits offspring. When appropriate, we discuss parallels with disease defence strategies in human societies. We propose that recurrent parasitic threats have shaped the evolution of both the individual immune systems and colony-level social immunity in insects.

Anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host.

The fitness effects of symbionts on their hosts can be context-dependent, with usually benign symbionts causing detrimental effects when their hosts are stressed, or typically parasitic symbionts providing protection towards their hosts (e.g. against pathogen infection). Here, we studied the novel association between the invasive garden ant Lasius neglectus and its fungal ectosymbiont Laboulbenia formicarum for potential costs and benefits. We tested ants with different Laboulbenia levels for their survival and immunity under resource limitation and exposure to the obligate killing entomopathogen Metarhizium brunneum. While survival of L. neglectus workers under starvation was significantly decreased with increasing Laboulbenia levels, host survival under Metarhizium exposure increased with higher levels of the ectosymbiont, suggesting a symbiont-mediated anti-pathogen protection, which seems to be driven mechanistically by both improved sanitary behaviours and an upregulated immune system. Ants with high Laboulbenia levels showed significantly longer self-grooming and elevated expression of immune genes relevant for wound repair and antifungal responses (ß-1,3-glucan binding protein, Prophenoloxidase), compared with ants carrying low Laboulbenia levels. This suggests that the ectosymbiont Laboulbenia formicarum weakens its ant host by either direct resource exploitation or the costs of an upregulated behavioural and immunological response, which, however, provides a prophylactic protection upon later exposure to pathogens.

Social transfer of pathogenic fungus promotes active immunisation in ant colonies.

Due to the omnipresent risk of epidemics, insect societies have evolved sophisticated disease defences at the individual and colony level. An intriguing yet little understood phenomenon is that social contact to pathogen-exposed individuals reduces susceptibility of previously naive nestmates to this pathogen. We tested whether such social immunisation in Lasius ants against the entomopathogenic fungus Metarhizium anisopliae is based on active upregulation of the immune system of nestmates following contact to an infectious individual or passive protection via transfer of immune effectors among group members--that is, active versus passive immunisation. We found no evidence for involvement of passive immunisation via transfer of antimicrobials among colony members. Instead, intensive allogrooming behaviour between naive and pathogen-exposed ants before fungal conidia firmly attached to their cuticle suggested passage of the pathogen from the exposed individuals to their nestmates. By tracing fluorescence-labelled conidia we indeed detected frequent pathogen transfer to the nestmates, where they caused low-level infections as revealed by growth of small numbers of fungal colony forming units from their dissected body content. These infections rarely led to death, but instead promoted an enhanced ability to inhibit fungal growth and an active upregulation of immune genes involved in antifungal defences (defensin and prophenoloxidase, PPO). Contrarily, there was no upregulation of the gene cathepsin L, which is associated with antibacterial and antiviral defences, and we found no increased antibacterial activity of nestmates of fungus-exposed ants. This indicates that social immunisation after fungal exposure is specific, similar to recent findings for individual-level immune priming in invertebrates. Epidemiological modeling further suggests that active social immunisation is adaptive, as it leads to faster elimination of the disease and lower death rates than passive immunisation. Interestingly, humans have also utilised the protective effect of low-level infections to fight smallpox by intentional transfer of low pathogen doses ("variolation" or "inoculation").

Fungal disease dynamics in insect societies: optimal killing rates and the ambivalent effect of high social interaction rates.

Entomopathogenic fungi are potent biocontrol agents that are widely used against insect pests, many of which are social insects. Nevertheless, theoretical investigations of their particular life history are scarce. We develop a model that takes into account the main distinguishing features between traditionally studied diseases and obligate killing pathogens, like the (biocontrol-relevant) insect-pathogenic fungi Metarhizium and Beauveria. First, obligate killing entomopathogenic fungi produce new infectious particles (conidiospores) only after host death and not yet on the living host. Second, the killing rates of entomopathogenic fungi depend strongly on the initial exposure dosage, thus we explicitly consider the pathogen load of individual hosts. Further, we make the model applicable not only to solitary host species, but also to group living species by incorporating social interactions between hosts, like the collective disease defences of insect societies. Our results identify the optimal killing rate for the pathogen that minimises its invasion threshold. Furthermore, we find that the rate of contact between hosts has an ambivalent effect: dense interaction networks between individuals are considered to facilitate disease outbreaks because of increased pathogen transmission. In social insects, this is compensated by their collective disease defences, i.e., social immunity. For the type of pathogens considered here, we show that even without social immunity, high contact rates between live individuals dilute the pathogen in the host colony and hence can reduce individual pathogen loads below disease-causing levels.

Unconditional versus condition-dependent social immunity.

Socially living animals can counteract disease through cooperative defences, leading to social immunity that collectively exceeds the sum of individual defences. In superorganismal colonies of social insects with permanent caste separation between reproductive queen(s) and nonreproducing workers, workers are obligate altruists and thus engage in unconditional social immunity, including highly specialised and self-sacrificial hygiene behaviours. Contrastingly, cooperation is facultative in cooperatively breeding families, where all members are reproductively totipotent but offspring transiently forgo reproduction to help their parents rear more siblings. Here, helpers should either express condition-dependent social immunity or disperse to pursue independent reproduction. We advocate inclusive fitness theory as a framework to predict when and how indirect fitness gains may outweigh direct fitness costs, thus favouring conditional social immunity.

Fungal infection alters collective nutritional intake of ant colonies.

In animals, parasitic infections impose significant fitness costs.1,2,3,4,5,6 Infected animals can alter their feeding behavior to resist infection,7,8,9,10,11,12 but parasites can manipulate animal foraging behavior to their own benefits.13,14,15,16 How nutrition influences host-parasite interactions is not well understood, as studies have mainly focused on the host and less on the parasite.9,12,17,18,19,20,21,22,23 We used the nutritional geometry framework24 to investigate the role of amino acids (AA) and carbohydrates (C) in a host-parasite system: the Argentine ant, Linepithema humile, and the entomopathogenic fungus, Metarhizium brunneum. First, using 18 diets varying in AA:C composition, we established that the fungus performed best on the high-amino-acid diet 1:4. Second, we found that the fungus reached this optimal diet when given various diet pairings, revealing its ability to cope with nutritional challenges. Third, we showed that the optimal fungal diet reduced the lifespan of healthy ants when compared with a high-carbohydrate diet but had no effect on infected ants. Fourth, we revealed that infected ant colonies, given a choice between the optimal fungal diet and a high-carbohydrate diet, chose the optimal fungal diet, whereas healthy colonies avoided it. Lastly, by disentangling fungal infection from host immune response, we demonstrated that infected ants foraged on the optimal fungal diet in response to immune activation and not as a result of parasite manipulation. Therefore, we revealed that infected ant colonies chose a diet that is costly for survival in the long term but beneficial in the short term-a form of collective self-medication.

Pupal cocoons affect sanitary brood care and limit fungal infections in ant colonies.

BACKGROUND: The brood of ants and other social insects is highly susceptible to pathogens, particularly those that penetrate the soft larval and pupal cuticle. We here test whether the presence of a pupal cocoon, which occurs in some ant species but not in others, affects the sanitary brood care and fungal infection patterns after exposure to the entomopathogenic fungus Metarhizium brunneum. We use a) a comparative approach analysing four species with either naked or cocooned pupae and b) a within-species analysis of a single ant species, in which both pupal types co-exist in the same colony. RESULTS: We found that the presence of a cocoon did not compromise fungal pathogen detection by the ants and that species with cocooned pupae increased brood grooming after pathogen exposure. All tested ant species further removed brood from their nests, which was predominantly expressed towards larvae and naked pupae treated with the live fungal pathogen. In contrast, cocooned pupae exposed to live fungus were not removed at higher rates than cocooned pupae exposed to dead fungus or a sham control. Consistent with this, exposure to the live fungus caused high numbers of infections and fungal outgrowth in larvae and naked pupae, but not in cocooned pupae. Moreover, the ants consistently removed the brood prior to fungal outgrowth, ensuring a clean brood chamber. CONCLUSION: Our study suggests that the pupal cocoon has a protective effect against fungal infection, causing an adaptive change in sanitary behaviours by the ants. It further demonstrates that brood removal-originally described for honeybees as "hygienic behaviour"-is a widespread sanitary behaviour in ants, which likely has important implications on disease dynamics in social insect colonies.

Trade-offs between immunity and competitive ability in fighting ant males.

BACKGROUND: Fighting disease while fighting rivals exposes males to constraints and trade-offs during male-male competition. We here tested how both the stage and intensity of infection with the fungal pathogen Metarhizium robertsii interfere with fighting success in Cardiocondyla obscurior ant males. Males of this species have evolved long lifespans during which they can gain many matings with the young queens of the colony, if successful in male-male competition. Since male fights occur inside the colony, the outcome of male-male competition can further be biased by interference of the colony's worker force. RESULTS: We found that severe, but not yet mild, infection strongly impaired male fighting success. In late-stage infection, this could be attributed to worker aggression directed towards the infected rather than the healthy male and an already very high male morbidity even in the absence of fighting. Shortly after pathogen exposure, however, male mortality was particularly increased during combat. Since these males mounted a strong immune response, their reduced fighting success suggests a trade-off between immune investment and competitive ability already early in the infection. Even if the males themselves showed no difference in the number of attacks they raised against their healthy rivals across infection stages and levels, severely infected males were thus losing in male-male competition from an early stage of infection on. CONCLUSIONS: Males of the ant C. obscurior have a well-developed immune system that raises a strong immune response very fast after fungal exposure. This allows them to cope with mild pathogen exposures without compromising their success in male-male competition, and hence to gain multiple mating opportunities with the emerging virgin queens of the colony. Under severe infection, however, they are weak fighters and rarely survive a combat already at early infection when raising an immune response, as well as at progressed infection, when they are morbid and preferentially targeted by worker aggression. Workers thereby remove males that pose a future disease threat by biasing male-male competition. Our study thus reveals a novel social immunity mechanism how social insect workers protect the colony against disease risk.

Dynamic pathogen detection and social feedback shape collective hygiene in ants.

Cooperative disease defense emerges as group-level collective behavior, yet how group members make the underlying individual decisions is poorly understood. Using garden ants and fungal pathogens as an experimental model, we derive the rules governing individual ant grooming choices and show how they produce colony-level hygiene. Time-resolved behavioral analysis, pathogen quantification, and probabilistic modeling reveal that ants increase grooming and preferentially target highly-infectious individuals when perceiving high pathogen load, but transiently suppress grooming after having been groomed by nestmates. Ants thus react to both, the infectivity of others and the social feedback they receive on their own contagiousness. While inferred solely from momentary ant decisions, these behavioral rules quantitatively predict hour-long experimental dynamics, and synergistically combine into efficient colony-wide pathogen removal. Our analyses show that noisy individual decisions based on only local, incomplete, yet dynamically-updated information on pathogen threat and social feedback can lead to potent collective disease defense.

Pathogen evasion of social immunity.

Treating sick group members is a hallmark of collective disease defence in vertebrates and invertebrates alike. Despite substantial effects on pathogen fitness and epidemiology, it is still largely unknown how pathogens react to the selection pressure imposed by care intervention. Using social insects and pathogenic fungi, we here performed a serial passage experiment in the presence or absence of colony members, which provide social immunity by grooming off infectious spores from exposed individuals. We found specific effects on pathogen diversity, virulence and transmission. Under selection of social immunity, pathogens invested into higher spore production, but spores were less virulent. Notably, they also elicited a lower grooming response in colony members, compared with spores from the individual host selection lines. Chemical spore analysis suggested that the spores from social selection lines escaped the caregivers' detection by containing lower levels of ergosterol, a key fungal membrane component. Experimental application of chemically pure ergosterol indeed induced sanitary grooming, supporting its role as a microbe-associated cue triggering host social immunity against fungal pathogens. By reducing this detection cue, pathogens were able to evade the otherwise very effective collective disease defences of their social hosts.

Antiviral immune response reveals host-specific virus infections in natural ant populations.

Hosts can carry many viruses in their bodies, but not all of them cause disease. We studied ants as a social host to determine both their overall viral repertoire and the subset of actively infecting viruses across natural populations of three subfamilies: the Argentine ant (Linepithema humile, Dolichoderinae), the invasive garden ant (Lasius neglectus, Formicinae) and the red ant (Myrmica rubra, Myrmicinae). We used a dual sequencing strategy to reconstruct complete virus genomes by RNA-seq and to simultaneously determine the small interfering RNAs (siRNAs) by small RNA sequencing (sRNA-seq), which constitute the host antiviral RNAi immune response. This approach led to the discovery of 41 novel viruses in ants and revealed a host ant-specific RNAi response (21 vs. 22 nt siRNAs) in the different ant species. The efficiency of the RNAi response (sRNA/RNA read count ratio) depended on the virus and the respective ant species, but not its population. Overall, we found the highest virus abundance and diversity per population in Li. humile, followed by La. neglectus and M. rubra. Argentine ants also shared a high proportion of viruses between populations, whilst overlap was nearly absent in M. rubra. Only one of the 59 viruses was found to infect two of the ant species as hosts, revealing high host-specificity in active infections. In contrast, six viruses actively infected one ant species, but were found as contaminants only in the others. Disentangling spillover of disease-causing infection from non-infecting contamination across species is providing relevant information for disease ecology and ecosystem management.

The dynamics of male-male competition in Cardiocondyla obscurior ants.

BACKGROUND: The outcome of male-male competition can be predicted from the relative fighting qualities of the opponents, which often depend on their age. In insects, freshly emerged and still sexually inactive males are morphologically indistinct from older, sexually active males. These young inactive males may thus be easy targets for older males if they cannot conceal themselves from their attacks. The ant Cardiocondyla obscurior is characterised by lethal fighting between wingless ("ergatoid") males. Here, we analyse for how long young males are defenceless after eclosion, and how early adult males can detect the presence of rival males. RESULTS: We found that old ergatoid males consistently won fights against ergatoid males younger than two days. Old males did not differentiate between different types of unpigmented pupae several days before emergence, but had more frequent contact to ready-to-eclose pupae of female sexuals and winged males than of workers and ergatoid males. In rare cases, old ergatoid males displayed alleviated biting of pigmented ergatoid male pupae shortly before adult eclosion, as well as copulation attempts to dark pupae of female sexuals and winged males. Ergatoid male behaviour may be promoted by a closer similarity of the chemical profile of ready-to-eclose pupae to the profile of adults than that of young pupae several days prior to emergence. CONCLUSION: Young ergatoid males of C. obscurior would benefit greatly by hiding their identity from older, resident males, as they are highly vulnerable during the first two days of their adult lives. In contrast to the winged males of the same species, which are able to prevent ergatoid male attacks by chemical female mimicry, young ergatoids do not seem to be able to produce a protective chemical profile. Conflicts in male-male competition between ergatoid males of different age thus seem to be resolved in favour of the older males. This might represent selection at the colony level rather than the individual level.