Despite the growing number of experimental studies on mechanisms of social immunity in ant societies, little is known about how social behavior relates to disease progression within the nests of ants. In fact, when empirically studying disease in ant societies, it is common to remove dead ants from experiments to confirm infection by the studied parasite. This unfortunately does not allow disease to progress within the nest as it may be assumed would happen under natural conditions. Therefore, the approach taken so far has resulted in a limited knowledge of diseases dynamics within the nest environment. Here we introduced a single infectious cadaver killed by the fungus Beauveria bassiana into small nests of the ant Camponotus castaneus. We then observed the natural progression of the disease by not removing the corpses of the ants that died following the first entry of the disease. Because some behaviors such as social isolation of sick individuals or the removal of cadavers by nestmates are considered social immune functions and thus adaptations at the colony level that reduce disease spread, we also experimentally confined some sub-colonies to one or two chamber nests to prevent the expression of such behaviors. Based on 51 small nests and survival studies in 1,003 ants we found that a single introduced infectious cadaver was able to transmit within the nest, and social immunity did not prevent the collapse of the small sub-colonies here tested. This was true whether ants did or did not have the option to remove the infectious cadaver. Therefore, we found no evidence that the typically studied social immunity behaviors can reduce disease spread in the conditions here tested.

Some parasites have evolved the ability to adaptively manipulate host behavior. One notable example is the fungus Ophiocordyceps unilateralis sensu lato, which has evolved the ability to alter the behavior of ants in ways that enable fungal transmission and lifecycle completion. Because host mandibles are affected by the fungi, we focused on understanding changes in the metabolites of muscles during behavioral modification. We used High-Performance Liquid Chromatography-Mass/Mass (HPLC-MS/MS) to detect the metabolite difference between controls and O. unilateralis-infected ants. There was a significant difference between the global metabolome of O. unilateralis-infected ants and healthy ants, while there was no significant difference between the Beauveria bassiana treatment ants group compared to the healthy ants. A total of 31 and 16 of metabolites were putatively identified from comparisons of healthy ants with O. unilateralis-infected ants and comparisons of B. bassiana with O. unilateralis-infected samples, respectively. This result indicates that the concentrations of sugars, purines, ergothioneine, and hypoxanthine were significantly increased in O. unilateralis-infected ants in comparison to healthy ants and B. bassiana-infected ants. This study provides a comprehensive metabolic approach for understanding the interactions, at the level of host muscles, between healthy ants and fungal parasites.

Environmental conditions exert strong selection on animal behavior. We tested the hypothesis that the altered behavior of hosts due to parasitic manipulation is also subject to selection imposed by changes in environmental conditions over time. Our model system is ants manipulated by parasitic fungi to bite onto vegetation. We analyzed the correlation between forest type (tropical vs. temperate) and biting substrate (leaf vs. twigs), the time required for the fungi to reach reproductive maturity, and the phylogenetic relationship among specimens from tropical and temperate forests in different parts of the globe. We show that the fungal development in temperate forest is longer than the period of time leaves are present and the ants are manipulated to bite twigs. When biting twigs, 90% of the we examined dead ants had their legs wrapped around twigs, which appears to provide better attachment to the plant. Ancestral state character reconstruction suggests that the leaf biting is the ancestral trait and that twig biting is a convergent trait in temperate regions of the globe. These three lines of evidence suggest that changes in environmental conditions have shaped the manipulative behavior of the host by its parasite.