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.
Kay et al RSPB-2013-2374 allogrooming data
In June 2012, we assessed whether diet affects allogrooming, an established mechanism of social immunity in ants (Hughes et al. 2002). We assigned 24 colonies to either the 1P:3C or 3P:1C diet, as above. After an 18-day rearing period, we created solitary ant and worker group sets and treated them with either Metarhizium or the control Triton-X solution. We then immediately began assaying grooming behaviour after Walker & Hughes (2011). We observed behaviour for 30-s periods at every 10 min for 60 min, as most grooming occurs immediately after solution application (pers. obs., also Walker & Hughes, 2009). We recorded the number of antennal self-grooming and allo-grooming events. Behaviours that occurred for more than a 5-s period were split into multiple events.
Kay et al RSPB-2013-2374 selfgrooming data
In June 2012, we assessed whether diet affects allogrooming, an established mechanism of social immunity in ants (Hughes et al. 2002). We assigned 24 colonies to either the 1P:3C or 3P:1C diet, as above. After an 18-day rearing period, we created solitary ant and worker group sets and treated them with either Metarhizium or the control Triton-X solution. We then immediately began assaying grooming behaviour after Walker & Hughes (2011). We observed behaviour for 30-s periods at every 10 min for 60 min, as most grooming occurs immediately after solution application (pers. obs., also Walker & Hughes, 2009). We recorded the number of antennal self-grooming and allo-grooming events. Behaviours that occurred for more than a 5-s period were split into multiple events.
Kay et al RSPB-2013-2374 controls for metapleural blocking technique
We used workers sampled from field colonies to show that blocking metapleural glands per se did not affect survivorship. We blocked the metapleural gland on ants (using nail polish). We housed solitary ants and worker groups in 3 cm-diameter Petri dishes with moistened cotton (re-moistened every 2 days) and checked daily for mortality. We removed dead ants, sterilized them (with 1% NaClO), placed them on filter paper, and monitored them for 7 days for signs of Metarhizium infection (characteristic conidia growth that occurs within 2-3 days).
Kay et al RSPB-2013-2374 evidence of infection in metapleural blockage experiment
In survivorship experiments, we removed dead ants, sterilized them (with 1% NaClO), placed them on filter paper, and monitored them for 7 days for signs of Metarhizium infection (characteristic conidia growth that occurs within 2-3 days).
Kay et al RSPB-2013-2374 food intake in the worker survivorship experiment
We assigned 52 colonies (26 in June 2011, 26 in January 2012) to diet treatment after ordering them by size (sum of principal components from analysis of worker, pupa, and larva numbers); we split 5 nests with >60 workers (3 in June 2011, 2 in Jan 2012) and assigned halves to different diets. We reared colonies for 18 days by providing ~1g blocks of food every 2nd day (=ad lib feeding), and quantified food dry mass loss as in Kay et al. (2012).
Kay et al RSPB-2013-2374 food intake in the colony level experiment
For this test, we used 16 colonies in June 2011 and 10 in January 2012. We assigned colonies (blocked by size as above) to the 1P:3C or 3P:1C diet, and removed larvae from half of the colonies in each treatment; we removed and returned larvae for control colonies. We reared colonies for 21 days. During the rearing period, we challenged colonies with Metarhizium by placing headless ant corpses covered in Metarhizium spores in each nest every 2 days.
Kay et al RSPB-2013-2374 worker survivorship experiment, metarhizium treated ants
For survival assays, we measured responses of solitary and social ant groupings to treatment with M. anisopliae (strain KVL02-73 collected at this site (Hughes et al. 2004)). After the 18-day diet manipulation, we created 1-ant (solitary ants) or 5-ant (worker groups) sets from each colony. In June 2011, we assigned 10 solitary ants and 2 worker groups per colony to a Metarhizium treatment; in Jan 2012, we assigned 5 solitary ants and 1 worker group per colony to a Metarhizium treatment
Kay et al RSPB-2013-2374 worker survivorship experiment, untreated ants
For survival assays, we measured responses of solitary and social ant groupings to treatment with M. anisopliae (strain KVL02-73 collected at this site (Hughes et al. 2004)). After the 18-day diet manipulation, we created 1-ant (solitary ants) or 5-ant (worker groups) sets from each colony. In Jan 2012, we assigned 5 solitary ants and 1 worker group per colony to a control treatment (not challenged with Metarhizium).
Kay et al RSPB-2013-2374 worker survival, metapleural blockage experiment, solitary
In January 2012, we tested whether secretions from the metapleural gland (a specialized gland on the thorax of ants that produces antibiotic secretions) influences diet-related immunity. To do this, we reared 12 colonies on either the 1P:3C or 3P:1C diet for 18 days. We then blocked the metapleural gland (using nail polish) on 10 ants per colony, created 5 solitary-ant or 1 worker-group sets, and treated all ants with Metarhizium as described above. We assessed survivorship as above. We used workers sampled from field colonies to show that blocking metapleural glands per se did not affect survivorship (Cox Proportional Hazards: L-R χ2 = 0.087, d.f. = 1, p = 0.762, n=40).
Kay et al RSPB-2013-2374 worker survival, metapleural blockage experiment, 5-ant groups
In January 2012, we tested whether secretions from the metapleural gland (a specialized gland on the thorax of ants that produces antibiotic secretions) influences diet-related immunity. To do this, we reared 12 colonies on either the 1P:3C or 3P:1C diet for 18 days. We then blocked the metapleural gland (using nail polish) on 10 ants per colony, created 5 solitary-ant or 1 worker-group sets, and treated all ants with Metarhizium as described above. We assessed survivorship as above. We used workers sampled from field colonies to show that blocking metapleural glands per se did not affect survivorship (Cox Proportional Hazards: L-R χ2 = 0.087, d.f. = 1, p = 0.762, n=40).
Kay et al RSPB-2013-2374 mortality in colony level experiment
In June 2011 and January 2012, we tested how nutrition affects colony-level immunity, and whether nutritional immunity depended on the presence of larvae in colonies. Given that nutritional feedbacks between workers and larvae influence protein:carbohydrate regulation in ant colonies (Dussutour & Simpson 2009), we predicted that larval presence would enhance nutritional effects on colony immunity.
For this test, we used 16 colonies in June 2011 and 10 in January 2012. We assigned colonies (blocked by size as above) to the 1P:3C or 3P:1C diet, and removed larvae from half of the colonies in each treatment; we removed and returned larvae for control colonies. We reared colonies for 21 days. During the rearing period, we challenged colonies with Metarhizium by placing headless ant corpses covered in Metarhizium spores in each nest every 2 days. We used this approach to challenge colonies (rather than direct application to workers of spores in solution, as above) because we wanted to sufficiently challenge colonies to test for differences among diet treatments; spore-covered corpses could be added at regular intervals, whereas periodic solution application to workers would have considerably disturbed colonies. We counted the number of dead ants in each colony (easily distinguishable from headless ant additions) every 2 days, and monitored each for signs of infection.