The effects of social environment and the metapleural gland on disease resistance in acorn ants

Eusocial species differ in living conditions when compared to solitary species primarily due to the dense living conditions of genetically related individuals. Consequently, these crowded conditions can induce a high rate of pathogen transmission and pathogen susceptibility. To resist an epidemic, individuals rely on sets of behaviors, known as social immunity, to decrease pathogen transmission among nestmates. Alongside social immunity, ants have a pair of secretory metapleural glands (MG), thought to secrete antimicrobial compounds important for antisepsis, that are believed to be transferred among nestmates by social immune behaviors such as grooming. To investigate the effects of social immunity on pathogen resistance, we performed a series of experiments by inoculating acorn ants Temnothorax curvispinosus with a lethal spore concentration of the entomopathogenic fungus Metarhizium brunneum . After inoculation ant survival was monitored in two environments: solitary and in groups. Additionally, the MG role in pathogen resistance was evaluated for both solitary and grouped living ants, by sealing the MG prior to inoculations. Individuals within a group environment had a higher survival compared to those in a solitary environment, and individuals with sealed glands had significantly decreased survival than ants with non-sealed-MG in both solitary and social environments. We observed the lowest survival for solitary-sealed-MG individuals. Although sealing the MG reduced survival probability, sealing the MG did not remove the benefits of grouped living. We show here that social living plays a crucial role in pathogen resistance and that the MG has an important role in pathogen resistance of individual T. curvispinosus ants. Although important for an individual's pathogen resistance, our data show that the MG does not play a strong role in social immunity as previously believed. Overall, this study provides insights into mechanisms of social immunity and the role of MG in disease resistance.

Queens of the inquiline social parasite Acromyrmex insinuator can join nest-founding queens of its host, the leaf-cutting ant Acromyrmex echinatior

Queens of the inquiline social parasite Acromyrmex insinuator are known to infiltrate mature colonies of Acromyrmex echinatior and to exploit the host’s perennial workforce by producing predominantly reproductive individuals while suppressing host reproduction. Here we report three cases of an A. insinuator queen having joined an incipient colony of A. echinatior that contained only the founding host-queen and her small symbiotic fungus garden. We conjectured that 1:1 host-inquiline co-founding—a phenomenon that has only rarely been reported in ants—may imply that the presence of an A. insinuator queen may incur benefits to the host by increasing survival of its incipient colonies. We observed that the parasite queens neither foraged nor defended the nest against intruders. However, the parasite queens interacted with the host and fungus in a way that could be consistent with grooming and/or with contributing eggs. These observations may help explain why A. insinuator queens have maintained metapleural glands, even though they are smaller than those of host queens, and why A. insinuator has lost the large foraging worker caste but not the small worker caste.

Hygiene Defense Behaviors Used by a Fungus-Growing Ant Depend on the Fungal Pathogen Stages

Parasites and their hosts use different strategies to overcome the defenses of the other, often resulting in an evolutionary arms race. Limited animal studies have explored the differential responses of hosts when challenged by differential parasite loads and different developmental stages of a parasite. The fungus-growing ant Trachymyrmex sp. 10 employs three different hygienic strategies to control fungal pathogens: Grooming the antibiotic-producing metapleural glands (MGs) and planting or weeding their mutualistic fungal crop. By inoculating Trachymyrmex colonies with different parasite concentrations (Metarhizium) or stages (germinated conidia or ungermianted conidia of Metarhizium and Escovopsis), we tested whether ants modulate and change hygienic strategies depending on the nature of the parasite challenge. There was no effect of the concentration of parasite on the frequencies of the defensive behaviors, indicating that the ants did not change defensive strategy according to the level of threat. However, when challenged with conidia of Escovopsis sp. and Metarhizium brunneum that were germinated or not-germinated, the ants adjusted their thygienic behavior to fungal planting and MG grooming behaviors using strategies depending on the conidia germination status. Our study suggests that fungus-growing ants can adjust the use of hygienic strategies based on the nature of the parasites.

A carbohydrate-rich diet increases social immunity in ants

Increased potential for disease transmission among nest-mates means living in groups has inherent costs. This increased potential is predicted to select for disease resistance mechanisms that are enhanced by cooperative exchanges among group members, a phenomenon known as social immunity. One potential mediator of social immunity is diet nutritional balance because traits underlying immunity can require different nutritional mixtures. Here, we show how dietary protein–carbohydrate balance affects social immunity in ants. When challenged with a parasitic fungus Metarhizium anisopliae , workers reared on a high-carbohydrate diet survived approximately 2.8× longer in worker groups than in solitary conditions, whereas workers reared on an isocaloric, high-protein diet survived only approximately 1.3× longer in worker groups versus solitary conditions. Nutrition had little effect on social grooming, a potential mechanism for social immunity. However, experimentally blocking metapleural glands, which secrete antibiotics, completely eliminated effects of social grouping and nutrition on immunity, suggesting a causal role for secretion exchange. A carbohydrate-rich diet also reduced worker mortality rates when whole colonies were challenged with Metarhizium . These results provide a novel mechanism by which carbohydrate exploitation could contribute to the ecological dominance of ants and other social groups.

The metapleural glands of fungus-growing and non-fungus-growing ants: Ultrastructural study

The metapleural glands are considered an autapomorphic structure to ants and probable have an antibiotic or antifungal function. The present study was aimed at investigating the ultrastructural morphology of the metapleural glands in ants which have different feeding types: from fungus-growing ants, the higher and lower attine, and non-fungus-growing ants from the tribes Blepharidattini and Ectatommini analyzed by transmission electron microscopy. Plasma membrane invaginations in secretory cells of both fungus-growing and non-fungus-growing ants facilitate absorption of extracellular material from hemolymph. Higher and lower attines differed slightly from non-fungus-growing ants, by the presence of oval secretory cells and well-developed RER in the metapleural glands, which indicates a higher production of secretion in attines. Also, well-developed Golgi regions in the leaf-cutting ants and Ectatommini probably modify the secretions, produced by the secretory cell or coming of the hemolymph, into pheromone or antimicrobial compounds, the latter mainly in leaf-cutting ants. Still, the secretory cells of the metapleural gland of leaf-cutting ants exhibited several mitochondria near microvilli of the intracytoplasmic portion of the canaliculus, indicating an important role of the metapleural gland in the production and transport of secretion in metapleural gland of leaf-cutting ants. Thus, our work corroborates other findings, however our results add that the slight ultrastructural difference in the metapleural glands of leaf-cutting ants can be due to the feeding type (fungus-growing ants), resulting in greater secretory capacity and antimicrobial properties to combat pathogens (for example, micro-fungi parasites Escovopsis).

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.

Reduced biological control and enhanced chemical pest management in the evolution of fungus farming in ants

To combat disease, most fungus-growing ants (Attini) use antibiotics from mutualistic bacteria ( Pseudonocardia ) that are cultured on the ants' exoskeletons and chemical cocktails from exocrine glands, especially the metapleural glands (MG). Previous work has hypothesized that (i) Pseudonocardia antibiotics are narrow-spectrum and control a fungus ( Escovopsis ) that parasitizes the ants' fungal symbiont, and (ii) MG secretions have broad-spectrum activity and protect ants and brood. We assessed the relative importance of these lines of defence, and their activity spectra, by scoring abundance of visible Pseudonocardia for nine species from five genera and measuring rates of MG grooming after challenging ants with disease agents of differing virulence. Atta and Sericomyrmex have lost or greatly reduced the abundance of visible bacteria. When challenged with diverse disease agents, including Escovopsis , they significantly increased MG grooming rates and expanded the range of targets. By contrast, species of Acromyrmex and Trachymyrmex maintain abundant Pseudonocardia. When challenged, these species had lower MG grooming rates, targeted primarily to brood. More elaborate MG defences and reduced reliance on mutualistic Pseudonocardia are correlated with larger colony size among attine genera, raising questions about the efficacy of managing disease in large societies with chemical cocktails versus bacterial antimicrobial metabolites.