Competition-based screening helps to secure the evolutionary stability of a defensive microbiome.

BACKGROUND: The cuticular microbiomes of Acromyrmex leaf-cutting ants pose a conundrum in microbiome biology because they are freely colonisable, and yet the prevalence of the vertically transmitted bacteria Pseudonocardia, which contributes to the control of Escovopsis fungus garden disease, is never compromised by the secondary acquisition of other bacterial strains. Game theory suggests that competition-based screening can allow the selective recruitment of antibiotic-producing bacteria from the environment, by providing abundant resources to foment interference competition between bacterial species and by using Pseudonocardia to bias the outcome of competition in favour of antibiotic producers. RESULTS: Here, we use RNA-stable isotope probing (RNA-SIP) to confirm that Acromyrmex ants can maintain a range of microbial symbionts on their cuticle by supplying public resources. We then used RNA sequencing, bioassays, and competition experiments to show that vertically transmitted Pseudonocardia strains produce antibacterials that differentially reduce the growth rates of other microbes, ultimately biassing the bacterial competition to allow the selective establishment of secondary antibiotic-producing strains while excluding non-antibiotic-producing strains that would parasitise the symbiosis. CONCLUSIONS: Our findings are consistent with the hypothesis that competition-based screening is a plausible mechanism for maintaining the integrity of the co-adapted mutualism between the leaf-cutting ant farming symbiosis and its defensive microbiome. Our results have broader implications for explaining the stability of other complex symbioses involving horizontal acquisition.

A Visual Guide for Studying Behavioral Defenses to Pathogen Attacks in Leaf-Cutting Ants.

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies have described the behaviors connected with disease defense, and the associated use of chemicals and antimicrobials, no common visual reference has been made. The main aim of this study was to record short clips of behaviors involved in disease defense, both prophylactically and directly targeted towards an antagonist of the colony following infection. To do so we used an infection experiment, with sub-colonies of the leaf-cutting ant species Acromyrmex echinatior, and the most significant known pathogenic threat to the ants' fungal crop (Leucoagaricus gongylophorus), a specialized pathogenic fungus in the genus Escovopsis. We filmed and compared infected and uninfected colonies, at both early and more advanced stages of infection. We quantified key defensive behaviors across treatments and show that the behavioral response to pathogen attack likely varies between different castes of worker ants, and between early and late detection of a threat. Based on these recordings we have made a library of behavioral clips, accompanied by definitions of the main individual defensive behaviors. We anticipate that such a guide can provide a common frame of reference for other researchers working in this field, to recognize and study these behaviors, and also provide greater scope for comparing different studies to ultimately help better understand the role these behaviors play in disease defense.

Chemical warfare between leafcutter ant symbionts and a co-evolved pathogen.

Acromyrmex leafcutter ants form a mutually beneficial symbiosis with the fungus Leucoagaricus gongylophorus and with Pseudonocardia bacteria. Both are vertically transmitted and actively maintained by the ants. The fungus garden is manured with freshly cut leaves and provides the sole food for the ant larvae, while Pseudonocardia cultures are reared on the ant-cuticle and make antifungal metabolites to help protect the cultivar against disease. If left unchecked, specialized parasitic Escovopsis fungi can overrun the fungus garden and lead to colony collapse. We report that Escovopsis upregulates the production of two specialized metabolites when it infects the cultivar. These compounds inhibit Pseudonocardia and one, shearinine D, also reduces worker behavioral defenses and is ultimately lethal when it accumulates in ant tissues. Our results are consistent with an active evolutionary arms race between Pseudonocardia and Escovopsis, which modifies both bacterial and behavioral defenses such that colony collapse is unavoidable once Escovopsis infections escalate.

Genome Analysis of Two Pseudonocardia Phylotypes Associated with Acromyrmex Leafcutter Ants Reveals Their Biosynthetic Potential.

The attine ants of South and Central America are ancient farmers, having evolved a symbiosis with a fungal food crop >50 million years ago. The most evolutionarily derived attines are the Atta and Acromyrmex leafcutter ants, which harvest fresh leaves to feed their fungus. Acromyrmex and many other attines vertically transmit a mutualistic strain of Pseudonocardia and use antifungal compounds made by these bacteria to protect their fungal partner against co-evolved fungal pathogens of the genus Escovopsis. Pseudonocardia mutualists associated with the attines Apterostigma dentigerum and Trachymyrmex cornetzi make novel cyclic depsipeptide compounds called gerumycins, while a mutualist strain isolated from derived Acromyrmex octospinosus makes an unusual polyene antifungal called nystatin P1. The novelty of these antimicrobials suggests there is merit in exploring secondary metabolites of Pseudonocardia on a genome-wide scale. Here, we report a genomic analysis of the Pseudonocardia phylotypes Ps1 and Ps2 that are consistently associated with Acromyrmex ants collected in Gamboa, Panama. These were previously distinguished solely on the basis of 16S rRNA gene sequencing but genome sequencing of five Ps1 and five Ps2 strains revealed that the phylotypes are distinct species and each encodes between 11 and 15 secondary metabolite biosynthetic gene clusters (BGCs). There are signature BGCs for Ps1 and Ps2 strains and some that are conserved in both. Ps1 strains all contain BGCs encoding nystatin P1-like antifungals, while the Ps2 strains encode novel nystatin-like molecules. Strains show variations in the arrangement of these BGCs that resemble those seen in gerumycin gene clusters. Genome analyses and invasion assays support our hypothesis that vertically transmitted Ps1 and Ps2 strains have antibacterial activity that could help shape the cuticular microbiome. Thus, our work defines the Pseudonocardia species associated with Acromyrmex ants and supports the hypothesis that Pseudonocardia species could provide a valuable source of new antimicrobials.

Lethal combat over limited resources: testing the importance of competitors and kin.

Although most animals employ strategies to avoid costly escalation of conflict, the limitation of critical resources may lead to extreme contests and fatal fighting. Evolutionary theories predict that the occurrence and intensity of fights can be explained by resource value and the density and relatedness of competitors. However, the interaction between these factors and their relative importance often remains unclear; moreover, few systems allow all variables to be experimentally investigated, making tests of these theoretical predictions rare. Here, we use the parasitoid wasp Melittobia to test the importance of all these factors. In contrast to predictions, variation in contested resource value (female mates) and the relatedness of competitors do not influence levels of aggression. However, as predicted, fight intensity increased with competitor density and was not influenced by the greater cost of fighting at high density. Our results suggest that in the absence of kin recognition, indirectly altruistic behavior (spite) is unlikely to evolve, and in such circumstances, the scale of competition will strongly influence the amount of kin discrimination in the form of level of aggression as observed in Melittobia species.

Virginity and the clutch size behavior of a parasitoid wasp where mothers mate their sons.

Theoretical and empirical research on the evolution of clutch size has proved to be an extremely productive area of evolutionary biology. A general prediction is that individuals should produce a smaller number of offspring when resources are more limited, such as when multiple individuals compete for the same resources for their development. However, we expect that the opposite prediction arises with virgin females of haplodiploid species, which are subject to extreme local mate competition. We test the key assumption and predictions of this theory with the parasitoid wasp Melittobia australica. Our data demonstrate that there is a trade-off between the size of the first and subsequent clutches and that virgin females adjust their production of sons according to the mating status (mated or not) of cofounding females. We also found that mated females facultatively change their offspring sex ratio in response to the mating status of cofoundresses. We discuss the potential mechanisms used to recognize the mating status and the implications of our results in the context of the extremely female-biased sex ratios observed across Melittobia species..

Lethal combat and sex ratio evolution in a parasitoid wasp.

Sex allocation theory provides excellent opportunities for testing how behavior and life histories are adjusted in response to environmental variation. One of the most successful areas from this respect is Hamilton's local mate competition theory. As predicted by theory, a large number of animal species have been shown to adjust their offspring sex ratios (proportion male) conditionally, laying less female-biased sex ratios as the number of females that lay eggs on a patch increases. However, recent studies have shown that this predicted pattern is not followed by 2 parasitoid species in the genus Melittobia, which always produce extremely female-biased sex ratios. A possible explanation for this is that males fight fatally and that males produced by the first female to lay eggs on a patch have a competitive advantage over later emerging males. This scenario would negate the advantage of later females producing a less female-biased sex ratio. Here we examine fatal fighting and sex ratio evolution in another species, Melittobia acasta. We show that females of this species also fail to adjust their offspring sex ratio in response to the number of females laying eggs on a patch. We then show that although earlier emerging males do have an advantage in winning fights, this advantage 1) can be reduced by an interaction with body size, with larger males more likely to win fights and 2) only holds for a brief period around the time at which the younger males emerge from their pupae. This suggests that lethal male combat cannot fully explain the lack of sex ratio shift observed in Melittobia species. We discuss alternative explanations.

Social evolution: cooperation by conflict.

A recent study suggests that aggression between wasps depends upon the costs and benefits of fighting, as determined by the position of individuals in a dominance hierarchy.