Immune challenges increase network centrality in a queenless ant.

Social animals display a wide range of behavioural defences against infectious diseases, some of which increase social contacts with infectious individuals (e.g. mutual grooming), while others decrease them (e.g. social exclusion). These defences often rely on the detection of infectious individuals, but this can be achieved in several ways that are difficult to differentiate. Here, we combine non-pathogenic immune challenges with automated tracking in colonies of the clonal raider ant to ask whether ants can detect the immune status of their social partners and to quantify their behavioural responses to this perceived infection risk. We first show that a key behavioural response elicited by live pathogens (allogrooming) can be qualitatively recapitulated by immune challenges alone. Automated scoring of interactions between all colony members reveals that this behavioural response increases the network centrality of immune-challenged individuals through a general increase in physical contacts. These results show that ants can detect the immune status of their nest-mates and respond with a general 'caring' strategy, rather than avoidance, towards social partners that are perceived to be infectious. Finally, we find no evidence that changes in cuticular hydrocarbon profiles drive these behavioural effects.

Patterns of host use by brood parasitic Maculinea butterflies across Europe.

The range of hosts exploited by a parasite is determined by several factors, including host availability, infectivity and exploitability. Each of these can be the target of natural selection on both host and parasite, which will determine the local outcome of interactions, and potentially lead to coevolution. However, geographical variation in host use and specificity has rarely been investigated. Maculinea (= Phengaris) butterflies are brood parasites of Myrmica ants that are patchily distributed across the Palæarctic and have been studied extensively in Europe. Here, we review the published records of ant host use by the European Maculinea species, as well as providing new host ant records for more than 100 sites across Europe. This comprehensive survey demonstrates that while all but one of the Myrmica species found on Maculinea sites have been recorded as hosts, the most common is often disproportionately highly exploited. Host sharing and host switching are both relatively common, but there is evidence of specialization at many sites, which varies among Maculinea species. We show that most Maculinea display the features expected for coevolution to occur in a geographic mosaic, which has probably allowed these rare butterflies to persist in Europe. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

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].

Camponotusfloridanus Ants Incur a Trade-Off between Phenotypic Development and Pathogen Susceptibility from Their Mutualistic Endosymbiont Blochmannia.

Various insects engage in microbial mutualisms in which the reciprocal benefits exceed the costs. Ants of the genus Camponotus benefit from nutrient supplementation by their mutualistic endosymbiotic bacteria, Blochmannia, but suffer a cost in tolerating and regulating the symbiont. This cost suggests that the ants face secondary consequences such as susceptibility to pathogenic infection and transmission. In order to elucidate the symbiont's effects on development and disease defence, Blochmannia floridanus was reduced in colonies of Camponotus floridanus using antibiotics. Colonies with reduced symbiont levels exhibited workers of smaller body size, smaller colony size, and a lower major-to-minor worker caste ratio, indicating the symbiont's crucial role in development. Moreover, these ants had decreased cuticular melanisation, yet higher resistance to the entomopathogen Metarhizium brunneum, suggesting that the symbiont reduces the ants' ability to fight infection, despite the availability of melanin to aid in mounting an immune response. While the benefits of improved growth and development likely drive the mutualism, the symbiont imposes a critical trade-off. The ants' increased susceptibility to infection exacerbates the danger of pathogen transmission, a significant risk given ants' social lifestyle. Thus, the results warrant research into potential adaptations of the ants and pathogens that remedy and exploit the described disease vulnerability.

Destructive disinfection of infected brood prevents systemic disease spread in ant colonies.

In social groups, infections have the potential to spread rapidly and cause disease outbreaks. Here, we show that in a social insect, the ant Lasius neglectus, the negative consequences of fungal infections (Metarhizium brunneum) can be mitigated by employing an efficient multicomponent behaviour, termed destructive disinfection, which prevents further spread of the disease through the colony. Ants specifically target infected pupae during the pathogen's non-contagious incubation period, utilising chemical 'sickness cues' emitted by pupae. They then remove the pupal cocoon, perforate its cuticle and administer antimicrobial poison, which enters the body and prevents pathogen replication from the inside out. Like the immune system of a metazoan body that specifically targets and eliminates infected cells, ants destroy infected brood to stop the pathogen completing its lifecycle, thus protecting the rest of the colony. Hence, in an analogous fashion, the same principles of disease defence apply at different levels of biological organisation.

Opposing effects of allogrooming on disease transmission in ant societies.

To prevent epidemics, insect societies have evolved collective disease defences that are highly effective at curing exposed individuals and limiting disease transmission to healthy group members. Grooming is an important sanitary behaviour--either performed towards oneself (self-grooming) or towards others (allogrooming)--to remove infectious agents from the body surface of exposed individuals, but at the risk of disease contraction by the groomer. We use garden ants (Lasius neglectus) and the fungal pathogen Metarhizium as a model system to study how pathogen presence affects self-grooming and allogrooming between exposed and healthy individuals. We develop an epidemiological SIS model to explore how experimentally observed grooming patterns affect disease spread within the colony, thereby providing a direct link between the expression and direction of sanitary behaviours, and their effects on colony-level epidemiology. We find that fungus-exposed ants increase self-grooming, while simultaneously decreasing allogrooming. This behavioural modulation seems universally adaptive and is predicted to contain disease spread in a great variety of host-pathogen systems. In contrast, allogrooming directed towards pathogen-exposed individuals might both increase and decrease disease risk. Our model reveals that the effect of allogrooming depends on the balance between pathogen infectiousness and efficiency of social host defences, which are likely to vary across host-pathogen systems.

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.

Ants disinfect fungus-exposed brood by oral uptake and spread of their poison.

To fight infectious diseases, host immune defenses are employed at multiple levels. Sanitary behavior, such as pathogen avoidance and removal, acts as a first line of defense to prevent infection before activation of the physiological immune system. Insect societies have evolved a wide range of collective hygiene measures and intensive health care toward pathogen-exposed group members. One of the most common behaviors is allogrooming, in which nestmates remove infectious particles from the body surfaces of exposed individuals. Here we show that, in invasive garden ants, grooming of fungus-exposed brood is effective beyond the sheer mechanical removal of fungal conidiospores; it also includes chemical disinfection through the application of poison produced by the ants themselves. Formic acid is the main active component of the poison. It inhibits fungal growth of conidiospores remaining on the brood surface after grooming and also those collected in the mouth of the grooming ant. This dual function is achieved by uptake of the poison droplet into the mouth through acidopore self-grooming and subsequent application onto the infectious brood via brood grooming. This extraordinary behavior extends the current understanding of grooming and the establishment of social immunity in insect societies.

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").

Reconstructing eight decades of genetic variation in an isolated Danish population of the large blue butterfly Maculinea arion.

BACKGROUND: Fragmentation of terrestrial ecosystems has had detrimental effects on metapopulations of habitat specialists. Maculinea butterflies have been particularly affected because of their specialized lifecycles, requiring both specific food-plants and host-ants. However, the interaction between dispersal, effective population size, and long-term genetic erosion of these endangered butterflies remains unknown. Using non-destructive sampling, we investigated the genetic diversity of the last extant population of M. arion in Denmark, which experienced critically low numbers in the 1980s. RESULTS: Using nine microsatellite markers, we show that the population is genetically impoverished compared to nearby populations in Sweden, but less so than monitoring programs suggested. Ten additional short repeat microsatellites were used to reconstruct changes in genetic diversity and population structure over the last 77 years from museum specimens. We also tested amplification efficiency in such historical samples as a function of repeat length and sample age. Low population numbers in the 1980s did not affect genetic diversity, but considerable turnover of alleles has characterized this population throughout the time-span of our analysis. CONCLUSIONS: Our results suggest that M. arion is less sensitive to genetic erosion via population bottlenecks than previously thought, and that managing clusters of high quality habitat may be key for long-term conservation.

Rapid anti-pathogen response in ant societies relies on high genetic diversity.

Social organisms are constantly exposed to infectious agents via physical contact with conspecifics. While previous work has shown that disease susceptibility at the individual and group level is influenced by genetic diversity within and between group members, it remains poorly understood how group-level resistance to pathogens relates directly to individual physiology, defence behaviour and social interactions. We investigated the effects of high versus low genetic diversity on both the individual and collective disease defences in the ant Cardiocondyla obscurior. We compared the antiseptic behaviours (grooming and hygienic behaviour) of workers from genetically homogeneous and diverse colonies after exposure of their brood to the entomopathogenic fungus Metarhizium anisopliae. While workers from diverse colonies performed intensive allogrooming and quickly removed larvae covered with live fungal spores from the nest, workers from homogeneous colonies only removed sick larvae late after infection. This difference was not caused by a reduced repertoire of antiseptic behaviours or a generally decreased brood care activity in ants from homogeneous colonies. Our data instead suggest that reduced genetic diversity compromises the ability of Cardiocondyla colonies to quickly detect or react to the presence of pathogenic fungal spores before an infection is established, thereby affecting the dynamics of social immunity in the colony.

The evolution of invasiveness in garden ants.

It is unclear why some species become successful invaders whilst others fail, and whether invasive success depends on pre-adaptations already present in the native range or on characters evolving de-novo after introduction. Ants are among the worst invasive pests, with Lasius neglectus and its rapid spread through Europe and Asia as the most recent example of a pest ant that may become a global problem. Here, we present the first integrated study on behavior, morphology, population genetics, chemical recognition and parasite load of L. neglectus and its non-invasive sister species L. turcicus. We find that L. neglectus expresses the same supercolonial syndrome as other invasive ants, a social system that is characterized by mating without dispersal and large networks of cooperating nests rather than smaller mutually hostile colonies. We conclude that the invasive success of L. neglectus relies on a combination of parasite-release following introduction and pre-adaptations in mating system, body-size, queen number and recognition efficiency that evolved long before introduction. Our results challenge the notion that supercolonial organization is an inevitable consequence of low genetic variation for chemical recognition cues in small invasive founder populations. We infer that low variation and limited volatility in cuticular hydrocarbon profiles already existed in the native range in combination with low dispersal and a highly viscous population structure. Human transport to relatively disturbed urban areas thus became the decisive factor to induce parasite release, a well established general promoter of invasiveness in non-social animals and plants, but understudied in invasive social insects.

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

BACKGROUND: The invasive garden ant, Lasius neglectus, is the most recently detected pest ant and the first known invasive ant able to become established and thrive in the temperate regions of Eurasia. In this study, we aim to reconstruct the invasion history of this ant in Europe analysing 14 populations with three complementary approaches: genetic microsatellite analysis, chemical analysis of cuticular hydrocarbon profiles and behavioural observations of aggression behaviour. We evaluate the relative informative power of the three methodological approaches and estimate both the number of independent introduction events from a yet unknown native range somewhere in the Black Sea area, and the invasive potential of the existing introduced populations. RESULTS: Three clusters of genetically similar populations were detected, and all but one population had a similar chemical profile. Aggression between populations could be predicted from their genetic and chemical distance, and two major clusters of non-aggressive groups of populations were found. However, populations of L. neglectus did not separate into clear supercolonial associations, as is typical for other invasive ants. CONCLUSION: The three methodological approaches gave consistent and complementary results. All joint evidence supports the inference that the 14 introduced populations of L. neglectus in Europe likely arose from only very few independent introductions from the native range, and that new infestations were typically started through introductions from other invasive populations. This indicates that existing introduced populations have a very high invasive potential when the ants are inadvertently spread by human transport.

Social prophylaxis: group interaction promotes collective immunity in ant colonies.

Life in a social group increases the risk of disease transmission. To counteract this threat, social insects have evolved manifold antiparasite defenses, ranging from social exclusion of infected group members to intensive care. It is generally assumed that individuals performing hygienic behaviors risk infecting themselves, suggesting a high direct cost of helping. Our work instead indicates the opposite for garden ants. Social contact with individual workers, which were experimentally exposed to a fungal parasite, provided a clear survival benefit to nontreated, naive group members upon later challenge with the same parasite. This first demonstration of contact immunity in Social Hymenoptera and complementary results from other animal groups and plants suggest its general importance in both antiparasite and antiherbivore defense. In addition to this physiological prophylaxis of adult ants, infection of the brood was prevented in our experiment by behavioral changes of treated and naive workers. Parasite-treated ants stayed away from the brood chamber, whereas their naive nestmates increased brood-care activities. Our findings reveal a direct benefit for individuals to perform hygienic behaviors toward others, and this might explain the widely observed maintenance of social cohesion under parasite attack in insect societies.

Density-dependence and within-host competition in a semelparous parasite of leaf-cutting ants.

BACKGROUND: Parasite heterogeneity and within-host competition are thought to be important factors influencing the dynamics of host-parasite relationships. Yet, while there have been many theoretical investigations of how these factors may act, empirical data is more limited. We investigated the effects of parasite density and heterogeneity on parasite virulence and fitness using four strains of the entomopathogenic fungus, Metarhizium anisopliae var. anisopliae, and its leaf-cutting ant host Acromyrmex echinatior as the model system. RESULTS: The relationship between parasite density and infection was sigmoidal, with there being an invasion threshold for an infection to occur (an Allee effect). Although spore production was positively density-dependent, parasite fitness decreased with increasing parasite density, indicating within-host scramble competition. The dynamics differed little between the four strains tested. In mixed infections of three strains the infection-growth dynamics were unaffected by parasite heterogeneity. CONCLUSIONS: The strength of within-host competition makes dispersal the best strategy for the parasite. Parasite heterogeneity may not have effected virulence or the infection dynamics either because the most virulent strain outcompeted the others, or because the interaction involved scramble competition that was impervious to parasite heterogeneity. The dynamics observed may be common for virulent parasites, such as Metarhizium, that produce aggregated transmission stages. Such parasites make useful models for investigating infection dynamics and the impact of parasite competition.