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.