DOI: 10.5061/dryad.865s4
title: Data from: A carbohydrate-rich diet increases social immunity in ants

description: 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.
description: Kay et al RSPB-2013-2374
 allogrooming dataIn 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 dataIn 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 techniqueWe 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 experimentIn 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 experimentWe 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 &gt;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 experimentFor 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 antsFor 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 treatmentKay et al RSPB-2013-2374
 worker survivorship experiment, untreated antsFor 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,
 solitaryIn 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
 groupsIn 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 experimentIn 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.