Ectoparasitic fungi of Myrmica ants alter the success of parasitic butterflies.

Exploitation of organisms by multiple parasite species is common in nature, but interactions among parasites have rarely been studied. Myrmica ants are rich in parasites. Among others, the ectoparasitic Rickia wasmannii fungus and the parasitic caterpillars of myrmecophilous Phengaris butterflies often infect the same Myrmica colonies. In this study, we examined the effects of R. wasmannii on the adoption, long-term development, and survival of P. alcon. In laboratory conditions, caterpillars introduced into nests of Myrmica scabrinodis uninfected with R. wasmannii survived significantly longer compared to caterpillars introduced into infected nests. In the field, joint infection was less common than expected if both parasites exploited M. scabrinodis colonies independently. Pre-pupal caterpillars of P. alcon were somewhat larger in nests infected with R. wasmannii than those found in uninfected nests. Based on these results it seems that R. wasmannii infection of M. scabrinodis affects the survival and development of P. alcon caterpillars, suggesting competition between these two ant parasites.

Host Ant Change of a Socially Parasitic Butterfly (Phengaris alcon) through Host Nest Take-Over.

The socially parasitic Alcon blue butterfly (Phengaris alcon) starts its larval stage by feeding on the seeds of gentians, after which it completes development in the nests of suitable Myrmica ant species. The host plant and host ant species can differ at the population level within a region, and local adaptation is common, but some host switches are observed. It has been suggested that one mechanism of change is through the re-adoption of caterpillars by different ant species, either through occupation of abandoned nests or take-over of established nests by competitively superior colonies. To test this question in the lab we introduced relatively strong colonies (50 workers) of alien Myrmica species to the arenas of weaker colonies (two caterpillars with six workers), and to orphaned caterpillars (two caterpillars without ants). We used caterpillars from a xerophylic population of P. alcon, and both local hosts, M. sabuleti and M. scabrinodis, testing the possibility of host switch between these two host ant species during larval development. Most of the caterpillars were successfully readopted by alien ants, and survived well. Our results suggest higher ecological plasticity in host ant usage of this butterfly than generally thought.

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'.

The evolution of abdominal microbiomes in fungus-growing ants.

The attine ants are a monophyletic lineage that switched to fungus farming ca. 55-60 MYA. They have become a model for the study of complex symbioses after additional fungal and bacterial symbionts were discovered, but their abdominal endosymbiotic bacteria remain largely unknown. Here, we present a comparative microbiome analysis of endosymbiotic bacteria spanning the entire phylogenetic tree. We show that, across 17 representative sympatric species from eight genera sampled in Panama, abdominal microbiomes are dominated by Mollicutes, α- and γ-Proteobacteria, and Actinobacteria. Bacterial abundances increase from basal to crown branches in the phylogeny reflecting a shift towards putative specialized and abundant abdominal microbiota after the ants domesticated gongylidia-bearing cultivars, but before the origin of industrial-scale farming based on leaf-cutting herbivory. This transition coincided with the ancestral single colonization event of Central/North America ca. 20 MYA, documented in a recent phylogenomic study showing that almost the entire crown group of the higher attine ants, including the leaf-cutting ants, evolved there and not in South America. Several bacterial species are located in gut tissues or abdominal organs of the evolutionarily derived, but not the basal attine ants. The composition of abdominal microbiomes appears to be affected by the presence/absence of defensive antibiotic-producing actinobacterial biofilms on the worker ants' cuticle, but the significance of this association remains unclear. The patterns of diversity, abundance and sensitivity of the abdominal microbiomes that we obtained explore novel territory in the comparative analysis of attine fungus farming symbioses and raise new questions for further in-depth research.

Ecological specialization is associated with genetic structure in the ant-associated butterfly family Lycaenidae.

The role of specialization in diversification can be explored along two geological axes in the butterfly family Lycaenidae. In addition to variation in host-plant specialization normally exhibited by butterflies, the caterpillars of most Lycaenidae have symbioses with ants ranging from no interactions through to obligate and specific associations, increasing niche dimensionality in ant-associated taxa. Based on mitochondrial sequences from 8282 specimens from 967 species and 249 genera, we show that the degree of ecological specialization of lycaenid species is positively correlated with genetic divergence, haplotype diversity and an increase in isolation by distance. Nucleotide substitution rate is higher in carnivorous than phytophagous lycaenids. The effects documented here for both micro- and macroevolutionary processes could result from increased spatial segregation as a consequence of reduced connectivity in specialists, niche-based divergence or a combination of both. They could also provide an explanation for the extraordinary diversity of the Lycaenidae and, more generally, for diversity in groups of organisms with similar multi-dimensional ecological specialization.

Eavesdropping on cooperative communication within an ant-butterfly mutualism.

Signalling is necessary for the maintenance of interspecific mutualisms but is vulnerable to exploitation by eavesdropping. While eavesdropping of intraspecific signals has been studied extensively, such exploitation of interspecific signals has not been widely documented. The juvenile stages of the Australian lycaenid butterfly, Jalmenus evagoras, form an obligate association with several species of attendant ants, including Iridomyrmex mayri. Ants protect the caterpillars and pupae, and in return are rewarded with nutritious secretions. Female and male adult butterflies use ants as signals for oviposition and mate searching, respectively. Our experiments reveal that two natural enemies of J. evagoras, araneid spiders and braconid parasitoid wasps, exploit ant signals as cues for increasing their foraging and oviposition success, respectively. Intriguingly, selection through eavesdropping is unlikely to modify the ant signal.

Reciprocal genomic evolution in the ant-fungus agricultural symbiosis.

The attine ant-fungus agricultural symbiosis evolved over tens of millions of years, producing complex societies with industrial-scale farming analogous to that of humans. Here we document reciprocal shifts in the genomes and transcriptomes of seven fungus-farming ant species and their fungal cultivars. We show that ant subsistence farming probably originated in the early Tertiary (55-60 MYA), followed by further transitions to the farming of fully domesticated cultivars and leaf-cutting, both arising earlier than previously estimated. Evolutionary modifications in the ants include unprecedented rates of genome-wide structural rearrangement, early loss of arginine biosynthesis and positive selection on chitinase pathways. Modifications of fungal cultivars include loss of a key ligninase domain, changes in chitin synthesis and a reduction in carbohydrate-degrading enzymes as the ants gradually transitioned to functional herbivory. In contrast to human farming, increasing dependence on a single cultivar lineage appears to have been essential to the origin of industrial-scale ant agriculture.

Host plant use drives genetic differentiation in syntopic populations of Maculinea alcon.

The rare socially parasitic butterfly Maculinea alcon occurs in two forms, which are characteristic of hygric or xeric habitats and which exploit different host plants and host ants. The status of these two forms has been the subject of considerable controversy. Populations of the two forms are usually spatially distinct, but at Rascruci in Romania both forms occur on the same site (syntopically). We examined the genetic differentiation between the two forms using eight microsatellite markers, and compared with a nearby hygric site, Sardu. Our results showed that while the two forms are strongly differentiated at Rascruci, it is the xeric form there that is most similar to the hygric form at Sardu, and Bayesian clustering algorithms suggest that these two populations have exchanged genes relatively recently. We found strong evidence for population substructuring, caused by high within host ant nest relatedness, indicating very limited dispersal of most ovipositing females, but not association with particular host ant species. Our results are consistent with the results of larger scale phylogeographic studies that suggest that the two forms represent local ecotypes specialising on different host plants, each with a distinct flowering phenology, providing a temporal rather than spatial barrier to gene flow.

Reduced entomopathogen abundance in Myrmica ant nests-testing a possible immunological benefit of myrmecophily using Galleria mellonella as a model.

Social insects such as ants have evolved collective rather than individual immune defence strategies against diseases and parasites at the level of their societies (colonies), known as social immunity. Ants frequently host other arthropods, so-called myrmecophiles, in their nests. Here, we tested the hypothesis that myrmecophily may partly arise from selection for exploiting the ants' social immunity. We used larvae of the wax moth Galleria mellonella as 'model myrmecophiles' (baits) to test this hypothesis. We found significantly reduced abundance of entomopathogens in ant nests compared with the surrounding environment. Specific entomopathogen groups (Isaria fumosorosea and nematodes) were also found to be significantly less abundant inside than outside ant nests, whereas one entomopathogen (Beauveria brongniartii) was significantly more abundant inside nests. We therefore hypothesize that immunological benefits of entering ant nests may provide us a new explanation of why natural selection acts in favour of such a life-history strategy.

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants.

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.

Interaction specificity between leaf-cutting ants and vertically transmitted Pseudonocardia bacteria.

BACKGROUND: The obligate mutualism between fungus-growing ants and microbial symbionts offers excellent opportunities to study the specificity and stability of multi-species interactions. In addition to cultivating fungus gardens, these ants have domesticated actinomycete bacteria to defend gardens against the fungal parasite Escovopsis and possibly other pathogens. Panamanian Acromyrmex echinatior leaf-cutting ants primarily associate with actinomycetes of the genus Pseudonocardia. Colonies are inoculated with one of two vertically transmitted phylotypes (Ps1 or Ps2), and maintain the same phylotype over their lifetime. We performed a cross-fostering experiment to test whether co-adaptations between ants and bacterial phylotypes have evolved, and how this affects bacterial growth and ant prophylactic behavior after infection with Escovopsis. RESULTS: We show that Pseudonocardia readily colonized ants irrespective of their colony of origin, but that the Ps2 phylotype, which was previously shown to be better able to maintain its monocultural integrity after workers became foragers than Ps1, reached a higher final cover when grown on its native host than on alternative hosts. The frequencies of major grooming and weeding behaviors co-varied with symbiont/host combinations, showing that ant behavior also was affected when cuticular actinomycete phylotypes were swapped. CONCLUSION: These results show that the interactions between leaf-cutting ants and Pseudonocardia bear signatures of mutual co-adaptation within a single ant population.

Sperm mixing in the polyandrous leaf-cutting ant Acromyrmex echinatior.

The insemination of queens by sperm from multiple males (polyandry) has evolved in a number of eusocial insect lineages despite the likely costs of the behavior. The selective advantages in terms of colony fitness must therefore also be significant and there is now good evidence that polyandry increases genetic variation among workers, thereby improving the efficiency of division of labor, resistance against disease, and diluting the impact of genetically incompatible matings. However, these advantages will only be maximized if the sperm of initially discrete ejaculates are mixed when stored in queen spermathecae and used for egg fertilization in a "fair raffle." Remarkably, however, very few studies have addressed the level of sperm mixing in social insects. Here we analyzed sperm use over time in the highly polyandrous leaf-cutting ant Acromyrmex echinatior. We genotyped cohorts of workers produced either 2 months apart or up to over a year apart, and batches of eggs laid up to over 2 years apart, and tested whether fluctuations in patriline distributions deviated from random. We show that the representation of father males in both egg and worker cohorts does not change over time, consistent with obligatorily polyandrous queens maximizing their fitness when workers are as genetically diverse as possible.

Development and implementation of a pharmacist-managed, neonatal and pediatric, opioid-weaning protocol.

OBJECTIVES: To compare the length of wean and abstinence severity in neonatal and pediatric patients with neonatal abstinence syndrome or iatrogenic opioid dependence treated with a pharmacist-managed, methadone-based protocol compared with physician-managed patients treated with either methadone or dilute tincture of opium (DTO). METHODS: This was a prospective, single-centered, interventional evaluation of 54 pharmacist-managed patients versus 53 retrospective, physician-managed patients. Wean duration and severity of neonatal abstinence syndrome were compared between groups using the Student t test. RESULTS: Significantly shorter wean duration in in utero-exposed pharmacist-managed patients compared with patients on physician-managed DTO (11.7 days vs 24.2 days, p < 0.001), but not compared with patients on physician-managed methadone (11.7 days vs 47 days, p = 0.101). No statistically significant difference was seen in wean duration in iatrogenic-exposed pharmacist-managed patients compared with patients on either physician-managed DTO or methadone (8.69 days vs 14 days, p = 0.096) and (8.69 days vs 9.82 days, p = 0.34), respectively. There were significantly fewer abstinence scores >12 in pharmacist-managed patients versus physician-managed DTO, but not physician-managed methadone (2.05 vs 17.3, p = 0.008 and 2.05 vs 74.3, p = 0.119, respectively). Significantly fewer abstinence scores ≥8 × 3 consecutively were seen in pharmacist-managed patients compared with patients on either physician-managed DTO or methadone (2.89 vs 11.9, p = 0.01 and 2.89 vs 24, p < 0.001, respectively). CONCLUSIONS: Use of a pharmacist-managed, methadone-based weaning protocol standardizes patient care and has the potential to decrease abstinence severity and shorten duration of wean versus physician-managed patients exposed to opioids in utero. Additionally, a methadone wean of 10% to 20% per day was well tolerated in both neonatal and pediatric patients.

Evolution: sympatric speciation the eusocial way.

Sympatric speciation normally requires particular conditions of ecological niche differentiation. However, ant social parasites have been suspected to arise sympatrically, because (dis)loyalty to eusocial kin-structures induces disruptive selection for dispersal and inbreeding. A new study documents this process in unprecedented detail.

Chemically armed mercenary ants protect fungus-farming societies.

The ants are extraordinary in having evolved many lineages that exploit closely related ant societies as social parasites, but social parasitism by distantly related ants is rare. Here we document the interaction dynamics among a Sericomyrmex fungus-growing ant host, a permanently associated parasitic guest ant of the genus Megalomyrmex, and a raiding agro-predator of the genus Gnamptogenys. We show experimentally that the guest ants protect their host colonies against agro-predator raids using alkaloid venom that is much more potent than the biting defenses of the host ants. Relatively few guest ants are sufficient to kill raiders that invariably exterminate host nests without a cohabiting guest ant colony. We also show that the odor of guest ants discourages raider scouts from recruiting nestmates to host colonies. Our results imply that Sericomyrmex fungus-growers obtain a net benefit from their costly guest ants behaving as a functional soldier caste to meet lethal threats from agro-predator raiders. The fundamentally different life histories of the agro-predators and guest ants appear to facilitate their coexistence in a negative frequency-dependent manner. Because a guest ant colony is committed for life to a single host colony, the guests would harm their own interests by not defending the host that they continue to exploit. This conditional mutualism is analogous to chronic sickle cell anemia enhancing the resistance to malaria and to episodes in human history when mercenary city defenders offered either net benefits or imposed net costs, depending on the level of threat from invading armies.

Dynamic disease management in Trachymyrmex fungus-growing ants (Attini: Formicidae).

Multipartner mutualisms have potentially complex dynamics, with compensatory responses when one partner is lost or relegated to a minor role. Fungus-growing ants (Attini) are mutualistic associates of basidiomycete fungi and antibiotic-producing actinomycete bacteria; the former are attacked by specialized fungi (Escovopsis) and diverse generalist microbes. Ants deploy biochemical defenses from bacteria and metapleural glands (MGs) and express different behaviors to control contaminants. We studied four Trachymyrmex species that differed in relative abundance of actinomycetes to understand interactions among antimicrobial tactics that are contingent on the nature of infection. MG grooming rate and actinomycete abundance were negatively correlated. The two species with high MG grooming rates or abundant actinomycetes made relatively little use of behavioral defenses. Conversely, the two species with relatively modest biochemical defenses relied heavily on behavior. Trade-offs suggest that related species can evolutionarily diverge to rely on different defense mechanisms against the same threat. Neither bacterial symbionts nor MG secretions thus appear to be essential for mounting defenses against the specialized pathogen Escovopsis, but reduced investment in one of these defense modes tends to increase investment in the other.

Regulation and specificity of antifungal metapleural gland secretion in leaf-cutting ants.

Ants have paired metapleural glands (MGs) to produce secretions for prophylactic hygiene. These exocrine glands are particularly well developed in leaf-cutting ants, but whether the ants can actively regulate MG secretion is unknown. In a set of controlled experiments using conidia of five fungi, we show that the ants adjust the amount of MG secretion to the virulence of the fungus with which they are infected. We further applied fixed volumes of MG secretion of ants challenged with constant conidia doses to agar mats of the same fungal species. This showed that inhibition halos were significantly larger for ants challenged with virulent and mild pathogens/weeds than for controls and Escovopsis-challenged ants. We conclude that the MG defence system of leaf-cutting ants has characteristics reminiscent of an additional cuticular immune system, with specific and non-specific components, of which some are constitutive and others induced.

Testing the adjustable threshold model for intruder recognition on Myrmica ants in the context of a social parasite.

Social insect colonies are like fortresses, well protected and rich in shared stored resources. This makes them ideal targets for exploitation by predators, parasites and competitors. Colonies of Myrmica rubra ants are sometimes exploited by the parasitic butterfly Maculinea alcon. Maculinea alcon gains access to the ants' nests by mimicking their cuticular hydrocarbon recognition cues, which allows the parasites to blend in with their host ants. Myrmica rubra may be particularly susceptible to exploitation in this fashion as it has large, polydomous colonies with many queens and a very viscous population structure. We studied the mutual aggressive behaviour of My. rubra colonies based on predictions for recognition effectiveness. Three hypotheses were tested: first, that aggression increases with distance (geographical, genetic and chemical); second, that the more queens present in a colony and therefore the less-related workers within a colony, the less aggressively they will behave; and that colonies facing parasitism will be more aggressive than colonies experiencing less parasite pressure. Our results confirm all these predictions, supporting flexible aggression behaviour in Myrmica ants depending on context.

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.

Immune defense in leaf-cutting ants: a cross-fostering approach.

To ameliorate the impact of disease, social insects combine individual innate immune defenses with collective social defenses. This implies that there are different levels of selection acting on investment in immunity, each with their own trade-offs. We present the results of a cross-fostering experiment designed to address the influences of genotype and social rearing environment upon individual and social immune defenses. We used a multiply mating leaf-cutting ant, enabling us to test for patriline effects within a colony, as well as cross-colony matriline effects. The worker's father influenced both individual innate immunity (constitutive antibacterial activity) and the size of the metapleural gland, which secretes antimicrobial compounds and functions in individual and social defense, indicating multiple mating could have important consequences for both defense types. However, the primarily social defense, a Pseudonocardia bacteria that helps to control pathogens in the ants' fungus garden, showed a significant colony of origin by rearing environment interaction, whereby ants that acquired the bacteria of a foster colony obtained a less abundant cover of bacteria: one explanation for this pattern would be co-adaptation between host colonies and their vertically transmitted mutualist. These results illustrate the complexity of the selection pressures that affect the expression of multilevel immune defenses.