Long-term assessment reveals the hidden and hiding effects of experimental stress on ant colonies

Social insects react to stress at both the individual and colonial level by modifying their physiology, behavior, offspring morphology, and colonial productivity. Experimental protocols involve treatments that potentially increase the stress load and may lead to misleading conclusions if not appropriately evaluated. We examined the long-term consequences of an experimental treatment (in vivo cuticular hydrocarbon sampling) and of the exposure to a fungal pathogen (Metarhizium anisopliae) on queen mortality and colonial development in Crematogaster scutellaris ants. Cuticular hydrocarbon sampling has been carried out through a widely used non-destructive procedure analogous to the solid-phase microextraction (SPME) technique. The exposure to pathogen clearly increased mortality but cuticular hydrocarbon sampling interacted with infection in determining survival. In fact, the manipulation increased the mortality of non-exposed queens but decreased the mortality of queens exposed to the pathogen. Queens subjected to cuticular hydrocarbon sampling also accelerated the emergence of the first worker thus shortening the critical claustral phase. On the other hand, the two treatments did not significantly affect the final number of successful colonies and the number and the morphology of the workers produced in the first season. Our results indicate that each manipulation may be followed by hidden effects becoming evident months after the application of the stress, and those immediate effects may disappear in the long term. We thus suggest that in pluriannual species, it is important to evaluate the effects of the stress covering at least an entire colonial season. Moreover, considering that manipulation can interact with pathogen exposure in determining queen mortality, immunological experiments on social insects should carefully take into account the potentially confounding effect of any experimentally induced stresses.