Species-specific ant brain manipulation by a specialized fungal parasite

Abstract

Background: A compelling demonstration of adaptation by natural selection is the ability of parasites to manipulate host behavior. One dramatic example involves fungal species from the genus Ophiocordyceps that control their ant hosts by inducing a biting behavior. Intensive sampling across the globe of ants that died after being manipulated by Ophiocordyceps suggests that this phenomenon is highly species-specific. We advance our understanding of this system by reconstructing host manipulation by Ophiocordyceps parasites under controlled laboratory conditions and combining this with field observations of infection rates and a metabolomics survey.

Results: We report on a newly discovered species of Ophiocordyceps unilateralis sensu lato from North America that we use to address the species-specificity of Ophiocordyceps-induced manipulation of ant behavior. We show that the fungus can kill all ant species tested, but only manipulates the behavior of those it infects in nature. To investigate if this could be explained at the molecular level, we used ex vivo culturing assays to measure the metabolites that are secreted by the fungus to mediate fungus-ant tissue interactions. We show the fungus reacts heterogeneously to brains of different ant species by secreting a different array of metabolites. By determining which ion peaks are significantly enriched when the fungus is grown alongside brains of its naturally occurring host, we discovered candidate compounds that could be involved in behavioral manipulation by O. unilateralis s.l.. Two of these candidates are known to be involved in neurological diseases and cancer.

Conclusions: The integrative work presented here shows that ant brain manipulation by O. unilateralis s.l. is species-specific seemingly because the fungus produces a specific array of compounds as a reaction to the presence of the host brain it has evolved to manipulate. These studies have resulted in the discovery of candidate compounds involved in establishing behavioral manipulation by this specialized fungus and therefore represent a major advancement towards an understanding of the molecular mechanisms underlying this phenomenon.

Figure 1

Natural and lab infections with O. unilateralis s.l. . (A-B) C. castaneus (A) and C. americanus (B) infected with O. unilateralis s.l. collected in Donalds, SC. (C) O. unilateralis s.l. culture isolated from an infected C. castaneus specimen. (D-E) Manipulated C. castaneus (D) and C. americanus (E) upon infection with O. unilateralis s.l. in the lab.

Figure 2

Survival, fungal growth, and behavioral manipulation of three Camponotus species infected with O. unilateralis s.l. . (A) Kaplan-Meier survival curve for 3 different Camponotus species infected with O. unilateralis s.l.. The thickened lines represent the time period in which fungal growth was observed. The grey box indicates days post-infection during which behavioral manipulation was observed. (B) Mean proportion of observations outside for each species-treatment combination (data presented as mean +/− SD, P<0.001 Tukey post-hoc on two-way ANOVA).

Figure 3

PCA-DA analyses to determine the heterogeneity of O. unilateralis s.l. on different ant species’ brains. (A) PCA-DA plot showing the clustering of O. unilateralis s.l. secretion in the presence of different ant species’ brains kept ex vivo in Schneider’s insect medium and the medium without ant brains. (B) PCA-DA plot showing the clustering of different ant species’ brains kept ex vivo and the medium by itself without fungal growth that served as controls in this study. (C-F) PCA-DA plots showing the clustering per species of O. unilateralis s.l. secretion in the presence of ant brains of that species versus secretion in the medium without ant brains, ant brains kept ex vivo without fungal growth and the Schneider’s insect medium by itself.

Figure 4

Heterogeneous metabolite secretion by O. unilateralis s.l. on four ant species’ brains. (A) Venn-diagram comparing all ion features found to be significantly (P < 0.01) enriched in the medium of samples in which O.unilateralis s.l. was grown next to ex vivo kept ant brains of the species C. castaneus, C. americanus, C. pennsylvanicus and F. dolosa. (B) Bar chart visualizing how the ions, that were found to be significantly enriched (P < 0.01) due to O. unilateralis s.l.-C. castaneus brain interactions across two independent studies, overlap with ions enriched in interactions with other species’ brains.